key: cord- -t b j j authors: laufer, s.d; restle, t title: peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems date: - - journal: curr pharm des doi: . / sha: doc_id: cord_uid: t b j j cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. accordingly, laboratories worldwide focus on the development of suitable delivery systems. among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (cpps) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. while uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. as a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. in this review, we will concentrate on peptide-mediated delivery of sirnas and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. to illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments. oligonucleotide-based strategies which can be used to modulate a vast variety of cellular functions represent a promising alternative to conventional therapies (for a review see: [ , ] ). among the different oligonucleotides with therapeutic potential are aptamers, transcription factor-binding decoy oligonucleotides, ribozymes, triplex-forming oligonucleotides (tfo), immunostimulatory cpg motifs, antisense oligonucleotides, small interfering rnas (sirnas) and antagomirs. nowadays, these potential macromolecular drugs are generally either relatively easily derived by rational design (e.g. antisense or sirna) or straightforward selection processes (e.g. aptamers). one of their main advantages over protein-or peptide-based approaches comprises the high specificity for their target while being non-immunogenic. however, despite these advances, a major impediment to the development of nucleic acid-based strategies for treatment and prevention of diseases is the relatively inefficient means to effectively deliver these macromolecules into the desired target cells. although viral vectors have been widely used to transfer genetic material into cells [ , ] , they bear an inherent risk for the patient to encounter severe immunological responses or even develop cancer [ ] [ ] [ ] [ ] . as a result of these problems much attention has been paid in recent years to the development of non-viral delivery systems. this conception *address correspondence to this author at the institut für molekulare medizin, universität zu lübeck, ratzeburger allee , lübeck, germany; tel: + - - - ; fax: + - - - ; e-mail: restle@imm.uni-luebeck.de includes an assortment of fairly unrelated approaches yielding various degrees of enhanced cellular uptake of nucleic acids. currently, liposomes and cationic polymers are used as a standard tool to transfect cells in vitro. however, these procedures are characterized by a significant lack of efficiency accompanied by a high level of toxicity rendering them mostly inadequate for in vivo applications. in this context cell-penetrating peptides (see below) represent an interesting alternative as they generally are less toxic than liposomes or cationic polymers. moreover, they are commonly better suited to transfer cargo into different cell types like non-adherent cells and primary cells, which are hard to transfect using commercially available standard protocols. the most advanced approaches in the field, which are not subject of the present article, are complex carrier systems combining vantages of assorted strategies to generate nanoparticles with better defined properties aimed towards enhanced uptake as well as intracellular trafficking in combination with cell-specific functionalities. for example, there are attempts to combine peptides with cationic liposomes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or polyethyleneimine (pei) [ ] . other strategies are aimed towards the synthesis of high or low molecular weight branched polymers and/or peptides [ ] [ ] [ ] [ ] [ ] or dendrimers [ , ] . even more complex systems are particularly promising with respect to in vivo delivery [ ] [ ] [ ] [ ] [ ] . in this review we will report about particular aspects of non-viral oligonucleotide delivery in vitro, pinpointing the current limitations, and provide quantitative means for determining where the bottlenecks of such strategies at present are. the focus of this article is recent progress in the field of peptide-mediated cellular delivery of sirna and steric block oligonucleotides in cell tissue culture as a starting point for further developments illustrated by own experimental data. our intention is not to provide the reader with easy solutions on how to solve the existing problems encountered with such approaches but give some hints where to start optimizing a particular approach. the idea of using peptides as carriers goes back some twenty years when it was discovered that the hiv- transactivating protein tat is taken up by mammalian cells [ , ] . a few years later, the antennapedia homeodomain of drosophila melanogaster was shown to act similarly [ ] . later on, it could be shown that peptides derived from tat and antennapedia as well as other proteins are capable of transporting macromolecular cargo molecules into cells [ ] [ ] [ ] . based on such promising results, a rapidly expanding field focusing on the so-called cell-penetrating peptides (cpps), also referred to as protein transduction domains (ptd), began to develop. since the first reports about tat, a large number of naturally occurring as well as engineered cpps have been discovered [ ] [ ] [ ] [ ] [ ] [ ] [ ] . table gives an overview of selected "classical" cpps. generally, cpps are short polycationic sequences of less than amino acids that are able to translocate different cargoes (e.g. nucleic acids, peptides and even entire proteins) into cells. the only common characteristic of these peptides appears to be that they are amphipathic and net positively charged at physiological ph. frequently the cargo is covalently attached to the cpp which can be achieved by expression as a fusion construct or by chemical coupling (for a review see: [ ] ). in particular cases, cargo and carrier bind each other non-covalently through mainly ionic interactions [ , , ] . despite the widespread interest in peptide carriers, the mechanisms underlying the cellular translocation of cpps are poorly understood. early work relied upon fluorescence imaging or flow cytometry analysis of chemically fixed cells to examine intracellular localization of fluorescently labeled peptides in the absence or presence of cargo. according to these experiments peptides appeared to be internalized very rapidly within minutes even at °c. from such observations it was concluded that cpps penetrate cell membranes by an energy-independent mechanism [ ] [ ] [ ] [ ] [ ] . although it had been reported quite early on that certain fixation procedures may cause artefacts leading to an overestimation of the cellular uptake rates [ ] [ ] [ ] the dimension of this problem was not commonly recognized until a side by side comparison of fixed and living cells was published [ ] . based on these findings, many groups re-examined their data. however, despite considerable technical improvements, there are still puzzling controversial results concerning the exact mechanism of cpp uptake. though in most cases endocytosis has been suggested to be the main route of internalization (fig. ( a) ), substantial difficulties are encountered identifying the exact pathway ( [ , ] and references therein). prior to endocytosis cpps interact electrostatically with the extracellular matrix of the cell surface mostly through binding to negatively charged glycosaminoglycans, i.e. heparan sulfate proteoglycans [ ] [ ] [ ] [ ] . recent studies indicate that the uptake mechanism of cpps can be influenced by the attachment of cargos. for example, richard et al. [ , ] reported a colocalization of tat - with markers of clathrin-mediated endocytosis, whereas fittipaldi et al. [ ] found a caveolae/lipid raft-dependent process for a tat-gfp fusion protein and wadia et al. [ ] described a macropinocytotic uptake pathway for a fusion construct of tat peptide with cre recombinase. in summary, the precise mechanism of internalization remains elusive and strongly depends on the properties of both cpp and cargo as well as on the transfection conditions and the cell lines used [ ] [ ] [ ] [ ] [ ] [ ] . as opposed to the majority of cpp applications reported, which rely on covalent linkage of carrier and cargo, limiting their general use considerably as a new construct has to be generated as well as tested for any given nucleic acid cargo, we will focus in this article on a peptide termed mpg which forms highly stable non-covalent complexes with nucleic acids (fig. ( ) ). the peptide is a derivative of the original mpg peptide described by morris and coworkers [ ] and differs by five amino acids in the hydrophobic part. these changes result in an alteration of the overall structure of the peptide towards a higher tendency of adopting a helical conformation [ ] . accordingly, the two peptides behave penetratin (antp - ) rqikiwfqnrrmkwkk [ ] transportan gwtlnsagyllgkinlkalaalakkil [ ] tp agyllgkinlkalaalakkil [ ] oligoarginine (r ) rrrrrrrr [ ] map klalklalkalkaalkla [ ] mpg galflgflgaagstmgawsqpkkkrkv [ ] mpg galflaflaaalslmglwsqpkkkrkv [ ] differently with respect to their interaction with artificial lipids as well as xenopus oocytes [ , ] and most probably, their exact mechanism of uptake is not the same. besides ionic interactions responsible for the initial peptide/nucleic acid complex formation, hydrophobic peptide/peptide interactions drive the maturation of large nanoparticles in a sandwich-like assembly reaction (fig. ( b) and fig. ( ) ). in recent years, rna interference (rnai) has gained a lot of interest as a tool for functional genomics studies and probably equally important as a promising therapeutic approach for the treatment of various diseases [ , ] . rnai is a highly evolutionally conserved and specific process of post-transcriptional gene silencing (ptgs) by which double stranded rna (dsrna), when introduced into a cell, causes sequence-specific degradation of homologous mrna sequences [ , ] . mechanistically the process can be divided into two steps. an initiator step where dsrna is cleaved by dicer, a member of the rnase iii family, into - nt long small interfering rna (sirna) fragments [ ] . in a consecutive step, these fragments are transferred to risc (rnainduced silencing complex) where one of the strands, the so called guide strand, serves as a molecular template to recognize homologous mrna that is cleaved by argonaute [ , ] , a protein component of risc. once the guide strand is bound to risc this complex can undergo many rounds of mrna binding and cleavage ( [ ] , fig. ( a) ). to circumvent application of long double stranded rnas, which inevitably trigger an interferon response, it is sufficient to extracellularly supply nt long dsrnas [ , ] . alternatively, sirnas can be expressed endogenously using dna vectors which code for short hairpin (sh) rnas [ ] [ ] [ ] . these shrnas are than cleaved by dicer to sirnas. short hairpin rna constructs have advantages over sirna because the effects of these constructs can lead to a more stable and long-term result. on the other hand, besides the fact that shrnas might interfere with the microrna pathway [ , ] , this strategy requires a gene therapy approach in the long run [ ] . for this reason we will not cover shrnas. as described above, sirnas represent a valuable tool to inhibit the expression of a target gene in a sequence-specific manner. in the following section, selected examples of cppmediated sirna delivery will be presented which are summarized in table . only a few studies describe the covalent attachment of nucleic acid cargo and peptide carrier (confer table ). in one approach, simple mixing of sirna targeted against gfp or cdk and tat peptide did not generate any measurable rnai effect whereas cross-linked sirna-tat - led to a significant down-regulation of the target proteins. however, high concentrations of sirna (about nm) had to be used [ ] . both lf-and tat - -mediated transfections resulted in a perinuclear localization of sirna. in contrast, fluorescently labeled tat - without cargo was mainly found in the nucleolus, suggesting that interactions with risc influence subcellular localization. in another approach, significant uptake of sirnas targeted against luciferase or gfp could be observed after disulfide coupling the '-end of the sense strand to penetratin or transportan [ ] . compared to lf , slightly higher levels of transfection were achieved. interestingly, after lf -mediated transfection, basal luciferase activity returned to normal levels one day earlier than after cpp-mediated transfection although the same concentration of sirna was applied. a remarkably strong rnai effect in hard to transfect primary neuronal cells was reported by davidson et al. [ ] . here, sirnas directed against several endogenous proteins were coupled to penetratin via a disulfide bond. the observed down regulation of the target proteins after peptide-mediated sirna delivery was found to be far more effective compared to lf . this was in part attributed to the toxicity of the lipids. as one of the first groups to report on tat - -or penetratin-mediated sirna delivery in vivo, moschos et al. showed, that intratracheal administration of the conjugates did not lead to any intensification of the knockdown of the target gene p mitogen-activated protein kinase in mouse lungs in comparison to unmodified non-formulated sirna [ ] . strikingly, it was found that the peptides alone triggered a detectable decrease in target gene expression and that the penetratin-conjugate induced elevated levels of the immune markers ifn-, tnf-, and il- p in lung tissue. besides technical difficulties arising from the syntheses of conjugates consisting of short cationic or hydrophobic peptides and highly negatively charged sirnas, dowdy and his group [ ] present a rather critical point of view referring to previous studies with cpp-sirna-conjugates. they claim that the successful delivery described therein is solely the result of excess free peptide, which leads to additional complexation, and thereby cellular import of the sirna. this is in accordance with turner et al. [ ] , who were the first to observe that careful purification of cpp-antisense-conjugates abrogates their biological effect. among other things, this might be the reason why most of the studies reporting on successful peptide-mediated delivery of sirnas use a noncovalent complexation approach (confer table ). in , simeoni et al. [ ] were the first who noncovalently complexed sirna with the peptide mpg. at a : ratio of negative nucleic acid to positive peptide charges a decrease in luciferase activity of about % was detectable in hela or cos- cells. this effect was further enhanced to about % down-regulation by a mutation in the nls sequence of the carrier peptide (mpg nls ), presumably due to an increased delivery to the cytoplasm, where risc is localized. recently, veldhoen et al. [ ] used a derivative of the mpg peptide for the delivery of sirna, which will be described in the chapter "mpg -mediated delivery of sirna and steric block oligonucleotides". leng et al. [ ] presented promising results with a prospect for cell-specific sirna delivery. different versions of a branched histidine/ lysine-polymer (h k b) yielded up to % knockdown of the target gene in several cell types. structure-function studies revealed an important role of the composition of the histidine-rich domain as well as its position within the peptide and the branches for sirna delivery, whereas size and surface charge did not have any effect. furthermore, the toxicity was much lower than for the commercial cationic lipids oligofectamine and lf . finally, the attachment of the tripeptide rgd, an integrin-ligand, slightly enhanced sirna delivery and turned this carrier into a cell-specific system. a fig. ( ) . simplistic scheme of peptide-based nucleic acid delivery systems (a). interaction of cpp and cargo is either achieved by covalent attachment or by non-covalent complexation through mainly ionic interactions. in case of non-covalent complex formation, a further assembly of cargo/carrier complexes occurs, leading to the formation of large nanoparticles (confer fig. ( ) ). in case of covalently joined molecules a similar scenario is less likely, yet cannot be excluded. prior to the translocation process the particles attach to the cell surface by ionic interactions of positively charged cpp residues with negatively charged membrane components. subsequently, complexes are taken up via an endocytotic pathway. although less likely, direct penetration cannot be excluded and may occur simultaneously. once inside the cell, the cargo has to escape from vesicular compartments, otherwise it eventually gets degraded in the lysosome. red: negative charges, blue: positive charges, green: hydrophobic domains. three-dimensional model of mpg /sirna interactions (b). the model was generated by iterative rigid body docking cycles of sirna (pdb r f) and peptide using the program hex . [ ] . the pdb file of mpg was generated with the program icm (molsoft llc) taking into consideration different secondary structure predictions and energy minimization protocols. out of many docking solutions particular ones were picked for illustration purposes using the program chimera [ ] . the phosphate backbone of the sirna is shown in red, the nucleobases in light gray. aliphatic, aromatic and hydrophobic residues of the peptide are shown in green, positive charged residues in blue and the remaining amino acids in gray. it is assumed that formation of larger particles is driven by hydrophobic peptide/peptide interactions generating free positive charges where other sirna molecules can interact. this eventually drives complex formation in a sandwich or mesh like assembly reaction. in principle such a scenario holds true for any given nucleic acid cargo. similar concept has very recently been used by kumar et al. [ ] for a specific delivery approach into the brain. a peptide derived from rabies virus glycoprotein (rvg) interacts specifically with the nicotinic acetylcholine receptor (achr) on neuronal cells to enable viral entry. the authors could show that the biotinylated form of the -amino-acid peptide (ytiwmpenprpgtpcdiftnsrgkrasng) was taken up by neuronal cells. in order to transport nucleic acids with this vehicle, r was conjugated to rvg peptide. systemic treatment of mice with sirna in a non-covalent complex with this modified peptide promoted a highly specific cellular import of sirna only into cells expressing achr. even more important, an antiviral sirna treatment resulted in successful protection of mice against encephalitis caused by japanese encephalitis virus (jev). this is the first study to report on a non-toxic method to deliver sirna across the blood brain barrier which could help to circumvent dangerous and ineffective injections into the brain. to date it presents one of the most promising tissue-specific delivery approaches which might be expandable to other in vivo applications. along these lines, most studies today are performed with the aim of cpp-mediated sirna delivery in vivo. although many of them are already showing promising results, e.g. concerning tumor-targeting and ocular delivery [ ] [ ] [ ] , this is beyond the scope of this review and will be discussed elsewhere in this issue [ , ] . with the aim to increase the endosomal escape of sir-nas after peptide-mediated delivery, lundberg et al. [ ] rationally modified penetratin to form a cpp (termed eb ) with improved endosomolytic properties. they achieved a ph-dependent conformational change of the peptide to a higher degree of helicity by the replacement of two basic amino acids with histidines and the n-terminal addition of six amino acids. in this study, several cpps were compared in a non-covalent approach by measuring the overall cellular stearyl-r n-c egfp, map b primary rat hippocampal neurons [ ] r -mend (sirna/stearyl-r core) n-c luciferase hela [ ] uptake via fluorescence and biological effect of sirna targeted to luciferase mrna. penetratin-as well as tp mediated transfection did not lead to any silencing of luciferase gene expression, despite high amounts of intracellular sirna [ ] and in contrast to previous reports using sirna-penetratin-conjugates [ ] or tp /dna-complexes [ ] . eb -mediated delivery of nm sirna led to approximately % reduction of luciferase activity. this silencing effect was slightly better than for bprpp and in the same range as for mpg nls , but still not as pronounced as for lf -mediated transfection of nm sirna. as it was described earlier, that addition of a ph-sensitive peptide derived from hemagglutinin (ha ) can promote endosomal escape [ ] , the authors linked ha to penetratin [ ] . it turned out that although ha -penetratin improved the silencing effect when coincubated with penetratin, eb was more potent than this combination of peptides. together with confocal microscopy studies the authors concluded that the lack of biological effect after penetratin-mediated sirna delivery is due to a lack of endosomal escape and that eb has a superior endosomolytic activity in comparison to ha penetratin. endoh et al. [ , ] very recently presented an innovative strategy, called clip-rnai (i.e. cpp-linked rbpmediated rna internalization and photo-induced rnai) combining delivery of a specific rna sequence with enhanced photoinduced release of rna from endosomes. this goal was accomplished by fusing the u a rna-binding domain (rbd) to the tat peptide and extending the sirna with a short stretch of nucleotides specifically recognized by this rbd. these complexes were efficiently internalized but exhibited a punctuate cytoplasmic localization pattern, indicative of endosomal entrapment. however, photostimulation of a fluorophore attached to the peptide led to a redistribution of complex into the cytosol followed by efficient rnai-mediated gene silencing. human pre-mrnas contain on average eight expressed sequences (exons) with an average length of nt and up to intervening sequences (introns) which can vary in length between and , nt, therefore comprising up to % of each transcriptional unit. in the nucleus, ribonucleoprotein complexes called spliceosomes recognize exon-intron boundaries and catalyze the precise removal of introns and subsequent joining of exons in a process called rna splicing [ ] [ ] [ ] . additionally, each primary transcript can yield different mature rnas through alternative splicing, thereby expanding the information content and versatility of the transcriptome, e.g. through the production of protein isoforms. a recent study of , human genes revealed, that at least % of all multi-exon genes are alternatively spliced [ ] . there are several different types of alternative splicing, amongst others affecting transcription start sites, splice sites, polyadenylation sites or even whole introns and exons. disruptions of these intricate splicing patterns are tightly coupled with human pathophysiology, either as a determinant or a direct cause of disease or as a modifier of disease susceptibility and severity [ ] . among these diseases are -thalassemia, cystic fibrosis, muscular dystrophies, frasier syndrome, certain kinds of dementia and cancer. a more de-tailed description of the underlying mechanisms is beyond the focus of this article and can be found in a number of reviews [ ] [ ] [ ] [ ] . lópez-bigas et al. [ ] proposed that % of mutations that cause disease lead to splicing defects rather than changes in the amino acid sequence. two common forms of mutations are depicted in fig. ( b) . on the one hand, a mutation in the splice donor can favor recognition of a cryptic splice donor and result in a mutant mrna containing additional intronic sequences (part i). on the other hand, a mutation in the splice acceptor can lead to skipping of a whole exon and result in a shortened mrna (part ii). both scenarios have been used in the context of antisense oligonucleotide-mediated approaches targeting alternative splicing. the use of antisense oligonucleotides interacting with mrna to affect protein production goes back some years [ , ] . since then, three principle mechanisms have been exploited for this purpose (for a review see: [ , ] ): (i) the oligonucleotide/rna duplex forms a substrate for endogenous rnase h, leading to mrna cleavage; (ii) the oligonucleotide/rna duplex prevents the productive assembly of the ribosomal complex or arrests a ribosomal complex already engaged in translation, in both cases affecting protein biosynthesis; (iii) the oligonucleotide/rna duplex alters pre-mrna splicing in the nucleus. the following section will focus on the last approach with the aim to treat splicing disorders and give examples of possible applications for cpps in this context. different forms of human -thalassemia are caused by mutations within in the -globin intron , which activate cryptic splice sites and thus lead to the formation of nonfunctional transcripts (fig. ( b part i) ). those aberrantly used sites can be blocked by antisense steric block oligonucleotides, which leads to the synthesis of functional protein [ , ] . kole and his group adopted this principle for the development of a splice correction assay [ ] . in this model system, a firefly luciferase construct leads to the synthesis of inactive enzyme because the reporter gene pre-mrna is interrupted by the human -globin intron containing an aberrant splice site. upon binding of a steric block oligonucleotide, correct splicing is restored which in turn yields a functional luciferase protein. to achieve this, the oligonucleotide has to be delivered to the nucleus. furthermore, only oligonucleotides that don't activate rnase h are applicable [ ] , e.g. phosphorodiamidate morpholino oligomers (pmo, [ ] ), locked nucleic acids (lna, [ ] ), peptide nucleic acids (pna, [ ] ) or '-o-methyl-modified oligonucleotides (ome). in addition to their inability to activate rnase h, most of these modifications confer higher affinity to the target rna and increased resistance against enzymatic degradation than unmodified versions. compared to rnaibased model systems, this assay is less susceptible to side effects like cytotoxicity or off-target effects because the reporter gene activity is turned up rather than turned down. the splice correction assay has been successfully applied for the analysis of several carrier systems [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the following section, selected examples will be presented, which are summarized in table . in contrast to many noncovalent cpp-mediated sirna delivery approaches, efficient splice correction was only achieved with conjugates of peptide and steric block oligonucleotide. astriab-fisher et al. [ ] described delivery of ome rna phosphorothioate oligonucleotides linked via a disulfide bridge to tat peptide and penetratin. a few hours after transfection, the cpp-oligonucleotide conjugates were detected both in cytoplasmic vesicles and in the nucleus and caused a dose-dependent increase in luciferase activity. these findings are in contrast to results of turner et al. [ ] , who could not find a biological effect for several cpp-oligonucleotide conjugates in a hela cell assay for tat-mediated transactivation of the hiv- long terminal repeat. the authors observed vesicular uptake but no nuclear import for their highly pure conjugates. interestingly, the rate of uptake could be enhanced by addition of free cpp to the conjugates, though still no biological activity was detected. based on these findings, turner et al. [ ] concluded that these free cpps form complexes with cpp-cargo conjugates, which play a significant role in the uptake process. this is in accordance with observations by meade et al. [ ] for the uptake of cpp-sirna conjugates described above. moulton et al. [ ] achieved correction of missplicing at low micromolar concentrations of a r f -pmo conjugate but not with complexes of peptide and pmo. the steric block activity of the r f -pmo conjugates could be further increased with longer spacers whereas variations in the conjugation chemistry did not result in any differences. furthermore, transfection rates were higher than for conjugates with tat peptide, penetratin or a tat peptide analogue. using the hiv- transactivation assay mentioned above, turner et al. [ ] could show that most cpp-oligonucleotide conjugates attained biologic activity only through co-administration of the endosomolytic substance chloroquine. fluorescence microscopy analyses revealed that this treatment released fluorescently labeled conjugates from endosomal compartments into the nucleus. besides the addition of chloroquine, different endosome disrupting strategies have been evaluated using the splice correction assay, for example co-treatment with endosome-disruptive peptides [ ] or photochemical internalization [ ] (see chapter "strategies to enhance endosomal escape"). however, the most promising results have been achieved with two newly developed derivatives of classical cpps (reviewed in [ ] ). the modification of oligoarginines with non-natural, uncharged amino acids [ ] led, amongst others, to the peptide (r-ahx-r) , in which ahx represents a six-atom aminohexanoic acid spacer. abes et al. demonstrated that in contrast to tat or oligoargine, pmo-conjugates of this peptide led to dose-dependent splice correction at low micromolar concentrations in the absence of endosomolytic agents. the underlying mechanism for this superior activity is not clear yet, as the uptake of (r-ahx-r) constructs was less efficient than the uptake of tat or oligoarginine constructs and also involved endocytotic routes [ ] . the second peptide is a derivative of penetratin, to which six arginine residues were added at the n-terminus (r pen). r pen-pna conjugates were shown to promote efficient splice correction at low concentrations and in the absence of endosomolytic agents [ ] . again, uptake of r pen-conjugates seemed to involve endocytosis and there was hardly any difference in splice correcting activity regardless of the nature of the linker used for conjugation, e.g. a stable thioether versus a reducible disulfide linker [ ] . part ii of fig. ( b) illustrates a phenomenon that represents a strategy for the treatment of duchenne muscular dystrophy (dmd). dmd is a severe progressive neuromuscular disorder caused by several different mutations in the dystrophin gene that abolish the production of functional protein [ ] . depending on the location of the mutation, the corresponding exon is skipped by covering the responsible splice sites with steric block oligonucleotides. this allows the transcription of internally deleted, but largely functional, dystro-phin proteins and converts a severe dmd into a milder becker muscular dystrophy phenotype. a more detailed description of this approach and its application in a number of animal models can be found in several excellent recent reviews [ , ] . successful systemic delivery of splice switching oligonucleotides with or without chemical modifications (pmo, lna, ome) has been accomplished via injection of naked nucleic acids [ , ] , with the help of viral vectors [ ] , through re-implantation of ex vivo manipulated stem cells [ ] or in combination with cpps [ ] [ ] [ ] [ ] [ ] . for the latter purpose, several studies were carried out with novel derivatives of arginine-rich peptides containing different numbers of non-amino acids, e.g. aminohexanoic acid and/or -alanine. these cpp-pmo conjugates showed higher serum stability, less endosomal trapping and led to efficient exon skipping in myoblasts and mice at lower dosages than the splice switching pmo alone [ , ] . yin et al. [ ] used a pna-modified splice-switching oligonucleotide conjugated to tat, a muscle specific peptide (msp) or different functional domains of the adenovirus capsid protein vp (aav , aav ) and examined exon skipping efficiency in vitro and in vivo. surprisingly, both after transfection and intramuscular injection, the activity of these pna-peptide conjugates was not significantly better than that achieved by naked neutral pna, presumably due to endosomal trapping. intracellular trafficking represents one of the major limitations of current non-viral nucleic acid delivery approaches [ ] . in other words a large percentage of intracellular cargo molecules are entrapped in vesicular compartments and thus will not trigger the desired effect. moreover, degradation or retrograde transport might further reduce the number of active molecules. so in order to determine the overall efficacy of a given delivery approach it is essential to know the numbers of intact cargo molecules inside the cell along with the minimal numbers of molecules required to cause a particular effect. based on such information one can easily calculate the percentage of bioactive molecules. in the following chapter we will briefly describe selected examples of variable suitability for a quantitative determination of nucleic acids in a cellular context. in principle, either the peptide or the cargo can be labeled by a reporter group, e.g. a radioisotope [ ] or a fluorophore [ ] . fluorescent peptides or cargos have been quantitatively evaluated by facs [ ] or fluorescence correlation microscopy (fcs) [ ] or fret [ ] . in all cases, it is crucial to distinguish between internalized and membraneassociated signals. for this purpose, a simple wash step with just buffer is not sufficient to completely remove membranebound peptide/cargo-complexes [ , ] . extracellularly bound complexes can be efficiently removed for example by enzymatic digestion with trypsin [ ] , acid wash [ ] or heparin treatment [ , ] . alternatively, discrimination between intra-and extracellular material is possible through chemical modification of extracellular components [ ] or fluorescence quenching [ ] . having established that only intracellular signals are taken into account, it is still challenging to distinguish between intact and degraded forms of peptide or cargo. in two studies, a fluorescence-based quantification method was combined with either hplc analysis [ ] or "cell activity by capillary electrophoresis" [ ] to verify the integrity of cargo and carrier. recently, a technique to measure cellular uptake of cpps by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) was reported by the group of burlina [ ] [ ] [ ] . this quantification is based on the addition of an internal standard, i.e. a peptide with a stable isotope label. the method has been used to determine the amount and stability of intact internalized peptides, e.g. penetratin, r and several novel cpps, and can also be used for the quantification of peptidic cargoes, e.g. an inhibitor of protein kinase c [ ] . varga et al. [ ] [ ] [ ] developed an "integrative systems" approach, combining quantitative experiments and computational modeling studies of vector uptake and trafficking kinetics, with the aim to take multiple potentially rate-limiting cellular and molecular processes into account. by applying their mathematical model to plasmid delivery with either lipofectamine, several pei-based vector formulations or an adenoviral vector, they could successfully predict experimentally observed effects and identify endosomal escape as the most important rate-limiting intracellular barrier for non-viral vectors. recently, zhou et al. [ ] applied a similar strategy for the characterization of a novel lipopolymer (wlsp). this carrier shows an increased rate of endosomal escape compared to conventional pei-based carriers. with the aim to quantify rhodamine-labeled plasmid dna in cellular compartments while avoiding problems arising from subcellular fractionation, like recovery and leakage, akita et al. [ ] developed a novel quantitative strategy, the confocal image-assisted three-dimensionally integrated quantification (cidiq) method. to distinguish endosomes/lysosomes and the nucleus from the cytosol, they were stained with lysosensor dnd- and hoechst , respectively, and sequential z-series images were captured by clsm. by applying this quantification method, the authors could show that due to a rapid endosomal escape, lipofectamine plus delivered more plasmid into the nucleus than r or stearylated r . hama et al. [ ] used the same method in combination with taqman pcr to evaluate the uptake and intracellular distribution of plasmid dna after delivery with viral as well as non-viral vectors. due to superior cell surface binding, the efficiency of cellular uptake was significantly higher for lipofectamine plus than for adenovirus whereas intracellular trafficking, i.e. endosomal escape and nuclear transfer, were essentially the same. however, to achieve comparable transgene expression, -fold higher intranuclear plasmid numbers were required in case of lipofectamine plus. this finding suggests a difference in nuclear transcription efficiency after non-viral delivery. in another approach, jiang et al. [ ] extended the ' end of the sense strand of a sirna with a nuclease-resistant dna hairpin to obtain a so-called "crook" sirna. this modification had no effect on rnai-mediated reporter gene inhibition and served as a primer for a filling-in reaction followed by pcr. parameters were chosen so that the initial rate of template amplification correlates with the initial con-centration of the "crook" sirna. under these conditions, quantification of attomolar sirna levels per cell was possible after liposomal transfections with oligofectamine. a highly sensitive method was developed by overhoff et al. [ ] for the detection of sirna after phosphorothioatestimulated uptake [ ] and adapted for the quantification of sirna or steric block oligonucleotides after non-covalent peptide-mediated delivery ( [ ] and laufer et al., manuscript in preparation, see chapter "mpg -mediated delivery of sirna and steric block oligonucleotides"). this so-called liquid hybridization assay is based on the extraction of total cellular rna and the subsequent hybridization in solution of a radioactively labeled probe which is complementary to the oligonucleotide to be detected. finally, following page analysis, absolute amounts of internalized oligonucleotide can be quantified with high accuracy down to ~ molecules per cell using internal standards [ ] . in addition to this outstanding sensitivity, no amplification step is needed and only intact oligonucleotides are taken into account. considering the multitude of available cpps, nucleic acid cargos and cellular as well as animal model systems, a comparison of different delivery strategies seems nearly impossible. in this context, we have for the first time undertaken a detailed side by side comparison of two different model systems using the peptide mpg as delivery agent. in the following paragraph we present own experimental data to exemplarily illustrate particular aspects regarding current limitations of peptide-based delivery systems. in contrast to many other cpp procedures, which rely on covalent linkage of carrier and cargo, the peptide mpg forms highly stable non-covalent complexes with nucleic acids, displaying binding constants in the low nanomolar range ( [ ] , a. trampe, unpublished data). the high flexibility of this non-covalent approach can be exploited to easily transport a wide variety of nucleic acid cargos without having to synthesize a new construct for each oligonucleotide. we have used mpg for the delivery of sirnas in the context of an rnai-based reporter system and for the delivery of steric block oligonucleotides in the context of the splice correction assay described above [ ] . after mpg -mediated transfection of a luciferase-targeted sirna, we observed strong inhibition of reporter gene readout with an ic in the subnanomolar range [ ] . after mpg -mediated transfection of a luciferasetargeted steric block oligonucleotide (further on also referred to as on- ), we observed a moderate up-regulation of reporter gene readout, representative of low splice correcting activity (laufer et al., manuscript in preparation). one possible explanation for the different degree of reporter gene regulation could be the different intracellular target sites, i.e. the cytoplasm for sirnas and the nucleus for splice correction oligonucleotides. to attain more information about the subcellular localization of mpg -oligonucleotide complexes, we performed confocal laser scanning as well as conventional fluorescence microscopy studies with fluorescently labeled sirnas or steric block oligonucleotides. in both cases, a punctuate non-homogenous distribution of the nucleic acids inside the cells was observed. this pattern is indicative of an accumulation of nucleic acids in endocytotic vesicles, which was verified by coincubation with lysosensor (fig. ( a) ). a quantitative computational analysis of fluorescence microscopy data yielded an average of approximately % colocalization between endosomes and sirna (fig. ( b) ). in contrast to earlier assumptions that cpps directly traverse the lipid bilayer, it has commonly become accepted that for most peptide-cargo combinations endocytosis plays a major role in cellular uptake. as described above and in the chapter "strategies to enhance endosomal escape" below, administration of endosome disruptive substances like chloroquine can greatly increase endosomal release of trapped nucleic acids. chloroquine is a weak base, non-charged at neutral ph but charged at ph . [ ] . it is able to pass easily through membranes in its uncharged form, but becomes protonated and accumulates within acidic vesicles in its positively charged, membraneimpermeable form. although its exact mode of action has not yet been resolved, it is generally accepted that chloroquine works via prevention of endosome acidification which in turn increases the residence time of cargo within the endosomes eventually resulting in a higher probability of transfer to the cytoplasm. fluorescence microscopy analyses in the presence of m chloroquine yielded two quite contrary outcomes. while the localization of sirna did not change after addition of chloroquine (fig. ( c) ), for the steric block oligonucleotide the picture changed completely (fig. ( d) ). in the latter case, a diffuse fluorescence all over the cytoplasm with an accumulation of on- in the nucleus could be observed. nonetheless, considerable amounts of nucleic acid molecules were still visible as a punctual pattern, which indicates that the chloroquine treatment liberates only a certain fraction. on the whole, these qualitative observations are in full agreement with the observed biologic effects in the absence or presence of chloroquine (fig. ( a) ). for mpg -mediated transfection of sirna, even under conditions where the amount of bio-available sirna was severely limited, only a minor increase in rnai (ca. %) was measurable. for mpg -mediated transfection of steric block oligonucleotide, on the other hand, a dramatic increase of reporter gene up-regulation by a factor of - was observed. however, in both cases, the overall uptake did not change upon incubation with chloroquine ( fig. ( b) ), which proves that the endosomolytic substance does not interfere with uptake but leads to a re-distribution of internalized nucleic acids. the underlying mechanism for the different effects triggered by chloroquine in case of peptide/sirna and peptide/steric block oligonucleotide complexes remains unclear. though, this is a good example that the cargo can substantially affect the properties and thereby intracellular trafficking of a particular carrier system. ultimately, to assess the overall efficacy of this carrier system, we were interested to elucidate which percentage of molecules taken up after mpg -mediated delivery is biologically active. to derive such information, the exact intracellular amount of intact oligonucleotide, the corresponding reporter signal and the minimal number of molecules necessary to trigger a specific degree of reporter gene modulation have to be known. for the quantification of internalized cargo, we adapted a highly sensitive method first described by overhoff et al. [ ] , enabling us to detect intracellular oligonucleotide amounts down to copies per cell [ ] . the method is based on the liquid hybridization of a radioactively labeled probe with the corresponding oligonucleotide in cellular lysates. in this context it should be noted that a stringent heparin wash following the transfection procedure is crucial to avoid an overestimation of intracellular nucleic acid molecules due to complexes attached to the outside of the cell membrane [ , ] . in order to correlate the numbers derived from the quantification experiments with the minimal number of molecules essential to trigger the observed effect, an independent assay had to be established. the gold standard in this case is microinjection as this technique enables one to deliver definite amounts of nucleic acids with a high degree of bioavailability into the cytoplasm or the nucleus of a mammalian cell along with a low toxicity profile and great accuracy. considering that after cytoplasmic microinjection only sirna molecules are sufficient for halfmaximal inhibition of reporter gene expression, one can estimate that of the , molecules measured after mpgmediated transfection, only ca. . % are biologically active ( table ) . for the splice correction assay, the numbers are different in terms of absolute numbers but the outcome remains the same. compared to the , molecules sufficient for maximal splice correction after nuclear microinjection, of the , , molecules required following peptide-mediated delivery only ca. . % are biologically active ( table ). in both cases >> % of internalized oligonucleotides are most likely retained in endosomes and subsequently degraded in lysosomes after peptide-mediated delivery. frankly, this is a sobering result and puts in numbers how much room for improvement there actually is. though it certainly is not legitimate to generalize these findings, there are countless reports in the literature suggesting similar limitations for the majority of non-viral strategies. according to actual conceptions, sirna enters a multiple-turnover pathway with one sirna molecule capable of risc-mediated cleavage of or more mrna molecules [ ] . even though the fate of steric block oligonucleotides is not really clear, it can be assumed that per splicing event one molecule is used up and translated into a functional mrna molecule (e.g. single-turnover pathway). as a result the number of molecules needed to trigger an apparent effect is much higher in the splice correction assay compared to the rnai-based reporter system (confer table and table ). on the other hand, being a single-turnover pathway, the splice correction assay should be much more sensitive to even minor changes in intracellular steric block oligonucleotide concentrations whereas the catalytic nature of the multiple-turnover rnai mechanism might mask such small variations. this is in accordance with the data described above. taken together, based on the results presented as well as unpublished data (laufer et al., manuscript in preparation) , the splice correction assay appears to be the superior tool for a quantitative assessment of nucleic acid delivery strategies. as outlined above, endosomal release is one of the major rate-limiting steps for cellular delivery of macromolecules via cationic lipids, polyplexes and especially cpps. in the following chapter we will present some examples of how to increase endosomal release. transfections were performed with . m mpg and nm sirna or g/ml lf and . nm sirna, i.e. in the range of the ic value [ ] . quantification was performed after h according to the liquid hybridization protocol [ ] . molecules per cell were calculated based on the cell number seeded for transfection. for microinjection experiments, molecules per cell were calculated on the basis of the injection volume. endosome-disrupting substances, like chloroquine, calcium or sucrose, were used to significantly enhance the activity of antisense pna oligonucleotides conjugated to tat, oligoarginines or oligolysines [ , , ] . this effect did not result from increased uptake, but rather improved bioavailability in the cytoplasm or nucleus after endosomal escape. takeuchi et al. [ ] showed that by incubation of the target cells with pyrenebutyrate, delivery of arginine-rich peptides could be shifted from endocytic uptake to direct membrane translocation, yielding a rapid distribution of the peptide throughout the cytoplasm, even at °c. pyrenebutyrate acts as a counteranion and, by interacting with the positively charged peptide, increases the overall hydrophobicity, thereby facilitating a direct translocation through the lipid bilayer, as earlier shown with artificial membranes [ ] . this method, which works only in the absence of a medium or serum, was successfully applied for administration of a fluorescent protein and an apoptosis-inducing peptide into dividing as well as non-dividing cells [ ] . however, in general the strategies described above are not feasible for in vivo applications, due to high cytotoxicity or other undesirable secondary effects. the imidazole group of histidine (his) can absorb protons in the acidic environment of the endosome, leading to osmotic swelling, membrane disruption and eventually nucleic acid escape. accordingly, lo et al. [ ] modified tat, which can bind and condense dna through ionic interactions but has no acidic residues that can promote endosomal release, with different numbers of his residues. highest reporter gene expression could be achieved after plasmid delivery with a tat peptide covalently fused to his residues (tat- h). insertion of two additional cysteine residues into tat- h further enhanced stability of peptide/dna complexes and transgene expression through formation of interpeptide disulfide bonds. youngblood et al. [ ] evaluated the influence of the stability of arginine-rich peptide pmo conjugates on cellular uptake and antisense activity. they could show that the stability is affected by the amino acid composition and the type of linkage to the cargo. moreover, they found that degraded fragments could not escape anymore from endosomal or lysosomal compartments. another concept makes use of photosensitive substances, which induce the release of macromolecules from vesicles by light exposure. this so-called photochemical internalization (pci) has, in the past, been used for intracellular delivery of a large variety of macromolecules (reviewed in [ ] ) and, more recently, for the endosomal release of nucleic acids after delivery mediated by liposomes [ , ] , polyplexes [ ] or cpps [ , ] . shiraishi et al. [ ] investigated the biological activity of pnas conjugated either to tat, r or kla-peptide in combination with a pci treatment. depending on the peptide, nuclear as well as cytosolic antisense effects could be enhanced by up to two orders of magnitude. similar results were presented by folini et al. [ ] for a pna targeting human telomerase reverse transcriptase conjugated to tat. in both studies, lower nucleic acid doses were sufficient, thereby reducing the probability of off-target effects. in light of encouraging data from ongoing anticancer clinical trials employing photodynamic therapy [ , ] , an in vivo application of pci seems feasible and will be discussed in more detail by oliveira et al. [ ] in this issue. furthermore, target specificity could be increased by local illumination of cells or tissue. viral fusion proteins drive the fusion process between the viral membrane and the endosomal host cell membrane in a ph-dependent manner, which is required to translocate the viral genome into the cytoplasm after receptor-mediated endocytosis. fusogenic peptides, usually hydrophobic, rich in glycine residues and found at the amino terminus of these proteins, were shown to have membrane perturbing and lipid mixing activities [ ] . many well studied representatives of this group are derived from the fusion sequence of influenza virus ha or hiv- gp [ ] and have been used to improve the transfection efficiency of non-viral delivery systems [ ] . addition of the influenza-derived dimeric peptide diinf- to lf /sirna complexes had no effect on the particle size of ca. nm, but significantly improved gene silencing activity of sirnas targeting the epidermal growth factor receptor or the k-ras oncogene [ ] . similar results were obtained for plasmid delivery through addition of a fusogenic peptide derived from herpes simplex virus glycoprotein h to lipofectamine/dna complexes [ ] . to the same end, futaki et al. [ ] used the peptide gala, which was specially designed to mimic the function of viral fusion sequences, together with various commercially available cationic liposomes. although they could not detect significant differences in cellular localization, plasmid transfection efficiency was increased and liposomal dosage could be reduced. pei covalently modified with the hiv- gp -derived peptide hgp led to a -fold increase of gene expression after plasmid dna delivery and also enhanced sirnamediated knockdown of gapdh by approximately -fold [ ] . % of cells incubated with pei-hgp polyplexes showed not only the punctuate plasmid dna staining observed with pei polyplexes alone, but also a diffuse fluorescence throughout the cell, indicative of endosomal release of vectors. this would explain the observed increase in transfection efficiency, since the overall uptake was unaffected. wadia et al. [ ] were the first to use the influenza hemagglutinin-derived fusogenic peptide ha in combination with a cpp. delivery of a tat-ha conjugate with increasing concentrations of a tat-cre conjugate enhanced reporter protein activity, presumably through an increased release from macropinosomes. the same fusogenic peptide, linked to a polyarginine-p conjugate, promoted release of p from macropinosomes and subsequent translocation to the nucleus, accompanied by an enhanced anti-cancer effect [ ] . the development of delivery systems for therapeutic oligonucleotides is a fast growing field. owing to the enormous potential of short nucleic acids as alternative drugs such a growth is not unexpected. besides viral vectors there is a highly diverse and constantly increasing number of non-viral systems evolving. however, despite considerable progress achieved in recent years, even the most advanced systems either lack the efficiencies required for downstream drug development or do show a substantial degree of toxicity or both. of the many factors which limit their use, cellular uptake of the cargo/carrier complexes and subsequent intracellular trafficking to reach the target site are the most important. in addition to such essential considerations there are various additional parameters to be taken into account like serum stability, pharmacokinetic features and tissue barriers as well as target cell specificity. now the question arises where to start optimizing a given delivery system. currently, there is a clear trend towards in vivo testing. in principle such a development is a step in the right direction since many of the experimental data derived from artificial cell tissue culture systems with established cell lines are not applicable to the in vivo situation. on the other hand, it is questionable to what extent such animal experiments will eventually pay off as long as important fundamental problems remain largely unsolved. as outlined above, our quantitative studies along with microscopic analyses of sirnas and steric block oligonucleotides using either a peptide or a commercially availably cationic lipid as carrier clearly show that less than . % - % of molecules taken up are involved in a biological response, i.e. rnaimediated down regulation or splice correction-mediated up regulation of reporter gene activity. evidently, the vast majority of internalized cargo never reaches the target. this implies that uptake per se is not the limiting factor here. although there are no such detailed quantitative numbers available in the literature for other systems, there are countless reports showing that to various degrees this applies to almost any non-viral delivery approach currently available. taken together, if one would succeed to optimize intracellular trafficking this holds the potential to boost overall efficacy by up to orders of magnitude. moreover, it is reasonable to assume that such fundamental cellular restrictions can be adequately investigated in tissue culture without the use of animal models. so it might be worthwhile to reconsider the concept of maybe premature in vivo testing by moving backwards one step and first optimizing the systems with regard to intracellular limitations before dealing with the next level of complexity. accordingly, in vitro model systems like the ones described above for sirnas or steric block oligonucleotides are valuable tools to study particular aspects of nucleic acid delivery. however, currently available data are based on studies using a variety of different cell lines and techniques, which renders a direct comparison of different delivery approaches impossible. in this context it would be highly desirable to introduce standardized protocols for in vitro testing (e.g. the splice correction system developed by kole and coworkers [ ] ) together with methods for detailed quantitative analyses (e.g. the liquid hybridization protocol [ , ] ). this would facilitate a direct quantitative comparison of exceedingly diverse approaches on at least the cellular level. one reason for the problem with intracellular trafficking of oligonucleotides arises from the mode carrier/cargo complexes are taken up by cells. today it is well established that the majority of these complexes are taken up via endosomal pathways and therefore end up in vesicular compartments from which they have to escape in order to reach their target. although there are many attempts reported to trigger endosomal escape by various strategies, they either proved to be toxic or did not achieve sustained success. additionally, there might be a further reason for the encountered difficulties to overcome these intracellular barriers. it is not unreasonable to speculate that during evolution cells might have evolved mechanisms to avoid large amounts of foreign nucleic acids freely floating in the cytoplasm and thus safely contain them in vesicular compartments where they are eventually degraded. alternatively they might be exported by retrograde transport. co-evoluting viruses evidently have developed strategies to circumvent such defense mechanisms. so it might be somewhat naive to expect that a rather simple man-made carrier system is capable to efficiently overcome such an intrinsic barrier. current developments towards more complex and elaborate carrier systems take into account such considerations. in any case it appears there is no simple solution for this problem. in conclusion, despite significant progress in the field of nucleic acid delivery in vitro as well as in vivo, there still is a long way to go before this will become a standard procedure in the clinic. certain problems like endosomal escape are known for more than twenty years and still far from being resolved. in order to develop new strategies, more information about intracellular processes involved in nucleic acid trafficking is needed. moreover, it would be desirable if the field would move from a qualitative description towards a quantitative evaluation preferentially using standardized model systems. this would allow for comparison of different approaches with one another. while animal studies are inevitable in the long run, there still is a lot of room for improvement on the cellular level. so it might be worthwhile to fathom how far we can push the different systems on this level. nucleic-acid therapeutics: basic principles and recent applications the versatility of oligonucleotides as potential therapeutics gene therapy with viral vectors gene therapy: twenty-first century medicine a pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency lmo -associated clonal t cell proliferation in two patients after gene therapy for scid-x fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer gene therapy put on hold as third child develops 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correlation between sirna localization, cellular uptake, and rnai in living cells conjugate for efficient delivery of short interfering rna (sirna) into mammalian cells highly efficient small interfering rna delivery to primary mammalian neurons induces microrna-like effects before mrna degradation lung delivery studies using sirna conjugated to tat( - ) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity enhancing the cellular uptake of sirna duplexes following noncovalent packaging with protein transduction domain peptides synthesis, cellular uptake and hiv- tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides insight into the mechanism of the peptide-based gene delivery system mpg: implications for delivery of sirna into mammalian cells transvascular delivery of small interfering rna to the central nervous system cell-penetrating peptide for enhanced delivery of nucleic acids and drugs to ocular tissues including retina and cornea cholesteryl oligoarginine delivering vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma cell-penetrating-peptidemediated sirna lung delivery cellular delivery in vivo of sirna-based therapeutics targeting the lung using sirna and antisense based oligonucleotides delivery of short interfering rna using endosomolytic cellpenetrating peptides tp , a delivery vector for decoy oligonucleotides targeting the myc protein photo inducible rna interference using cell permeable protein carrier cellular sirna delivery mediated by a cell-permeant rna-binding protein and photoinduced rna interference split genes and rna splicing the spliceosome: the most complex macromolecular machine in the cell? understanding alternative splicing: towards a cellular code genome-wide survey of human alternative pre-mrna splicing with exon junction microarrays splicing in disease: disruption of the splicing code and the decoding machinery alternative splicing: multiple control mechanisms and involvement in human disease pre-mrna splicing and human disease alternative splicing in disease and therapy alternative splicing in disease are splicing mutations the most frequent cause of hereditary disease? inhibition of rous sarcoma viral rna translation by a specific oligodeoxyribonucleotide inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide progress in antisense technology antisense oligonucleotides: from design to therapeutic application restoration of hemoglobin a synthesis in erythroid cells from peripheral blood of thalassemic patients therapeutic potential of antisense oligonucleotides as modulators of alternative splicing up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development morpholino antisense oligomers: design, preparation, and properties lna (locked nucleic acid): high-affinity targeting of complementary rna and dna endosome trapping limits the efficiency of splicing correction by pna-oligolysine conjugates vectorization of morpholino oligomers by the (r-ahx-r) peptide allows efficient splicing correction in the absence of endosomolytic agents efficient splicing correction by pna conjugation to an r -penetratin delivery peptide conjugates of antisense oligonucleotides with the tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions induction of splice correction by cell-penetrating peptide nucleic acids cell penetrating peptide conjugates of steric block oligonucleotides evaluation of transfection protocols for unmodified and modified peptide nucleic acid (pna) oligomers cellular delivery of polyheteroaromate-peptide nucleic acid conjugates mediated by cationic lipids comparison of basic peptides-and lipid-based strategies for the delivery of splice correcting oligonucleotides antisense-mediated redirection of mrna splicing structural requirements for cellular uptake and antisense activity of peptide nucleic acids conjugated with various peptides cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides cell-penetrating peptide conjugates of peptide nucleic acids (pna) as inhibitors of hiv- tat-dependent transactivation in cells photochemically enhanced cellular delivery of cell penetrating peptide-pna conjugates arginine-rich molecular transporters for drug delivery: role of backbone spacing in cellular uptake dystrophin: the protein product of the duchenne muscular dystrophy locus modification of pre-mrna processing: application to dystrophin expression antisense-mediated exon skipping: a versatile tool with therapeutic and research applications clinical approaches in the treatment of duchenne muscular dystrophy (dmd) using oligonucleotides the therapeutic potential of antisense-mediated exon skipping potential of oligonucleotide-mediated exon-skipping therapy for duchenne muscular dystrophy systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology efficient and persistent splice switching by systemically delivered lna oligonucleotides in mice rescue of dystrophic muscle through u snrnamediated exon skipping restoration of human dystrophin following transplantation of exon-skipping-engineered dmd patient stem cells into dystrophic mice morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of dmd cell-penetrating peptide-morpholino conjugates alter pre-mrna splicing of dmd (duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice effective exon skipping and restoration of dystrophin expression by peptide nucleic acid antisense oligonucleotides in mdx mice cellular uptake and intracellular release are major obstacles to the therapeutic application of sirna: novel options by phosphorothioate-stimulated delivery quantitative analysis of permeation peptide complexes labeled with technetium- m: chiral and sequence-specific effects on net cell uptake quantitative assessment of the cell penetrating properties of ri-tat- : evidence for a cell type-specific barrier at the plasma membrane of epithelial cells a quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import cargo delivery kinetics of cell-penetrating peptides translocation properties of novel cell penetrating transportan and penetratin analogues acid wash in determining cellular uptake of fab/cellpermeating peptide conjugates cationic tat peptide transduction domain enters cells by macropinocytosis cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically physico-chemical requirements for cellular uptake of pantp peptide. role of lipid-binding affinity characterization of tat-mediated transport of detachable kinase substrates quantification of the efficiency of cargo delivery by peptidic and pseudopeptidic trojan carriers using maldi-tof mass spectrometry quantification of the cellular uptake of cell-penetrating peptides by maldi-tof mass spectrometry a direct approach to quantification of the cellular uptake of cell-penetrating peptides using maldi-tof mass spectrometry quantitative analysis of synthetic gene delivery vector design properties quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes intracellular kinetics of non-viral gene delivery using polyethylenimine carriers quantitative three-dimensional analysis of the intracellular trafficking of plasmid dna transfected by a nonviral gene delivery system using confocal laser scanning microscopy quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems a bi-functional sirna construct induces rna interference and also primes pcr amplification for its own quantification quantitative detection of sirna and single-stranded oligonucleotides: relationship between uptake and biological activity of sirna phosphorothioate-stimulated cellular uptake of sirna: a cell culture model for mechanistic studies weak bases and ionophores rapidly and reversibly raise the ph of endocytic vesicles in cultured mouse fibroblasts calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate direct observation of anion-mediated translocation of fluorescent oligoarginine carriers into and across bulk liquid and anionic bilayer membranes an endosomolytic tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for dna transfection stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells photochemical internalization: a new tool for drug delivery photochemically induced gene silencing using small interfering rna molecules in combination with lipid carriers photochemical internalization enhances silencing of epidermal growth factor receptor through improved endosomal escape of sirna photochemical enhancement of dna delivery by egf receptor targeted polyplexes photochemically enhanced delivery of a cell-penetrating peptide nucleic acid conjugate targeting human telomerase reverse transcriptase: effects on telomere status and proliferative potential of human prostate cancer cells photodynamic therapies: principles and present medical applications the present and future role of photodynamic therapy in cancer treatment delivery of small interfering rna to the target cell cytoplasm: photochemical internalization facilitates endosomal escape and imprves silencing efficiency, in vitro and in vivo fusion peptides and the mechanism of viral fusion properties and structures of the influenza and hiv fusion peptides on lipid membranes: implications for a role in fusion application of membrane-active peptides for drug and gene delivery across cellular membranes fusogenic peptides enhance endosomal escape improving sirnainduced silencing of oncogenes a fusogenic segment of glycoprotein h from herpes simplex virus enhances transfection efficiency of cationic liposomes unique features of a ph-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes application of an hiv gp -derived peptide for enhanced intracellular trafficking of synthetic gene and sirna delivery vehicles the nh terminus of influenza virus hemagglutinin- subunit peptides enhances the antitumor potency of polyargininemediated p protein transduction docking essential dynamics eigenstructures ucsf chimera--a visualization system for exploratory research and analysis the third helix of the antennapedia homeodomain translocates through biological membranes cell penetration by transportan deletion analogues of transportan stearylated octaarginine and artificial virus-like particles for transfection of sirna into primary rat neurons octaargininemodified multifunctional envelope-type nano device for sirna we apologize to those authors whose work was not cited directly owing to space limitations. we thank alexander key: cord- -o duxhs authors: chandramouli, kondethimmanahalli; qian, pei-yuan title: proteomics: challenges, techniques and possibilities to overcome biological sample complexity date: - - journal: hum genomics proteomics doi: . / / sha: doc_id: cord_uid: o duxhs proteomics is the large-scale study of the structure and function of proteins in complex biological sample. such an approach has the potential value to understand the complex nature of the organism. current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. in addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. however, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. the quantity of data that is acquired with new techniques places new challenges on data processing and analysis. this article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. some solutions to circumvent technical difficulties are proposed. the term proteomics describes the study and characterization of complete set of proteins present in a cell, organ, or organism at a given time [ ] . in general, proteomic approaches can be used (a) for proteome profiling, (b) for comparative expression analysis of two or more protein samples, (c) for the localization and identification of posttranslational modifications, and (d) for the study of protein-protein interactions. the human genome harbors - protein encoding genes [ ] ; whereas the total number of human protein products, including splice variants and essential posttranslational modifications (ptms), has been estimated to be close to one million [ , ] . it is evident that most of the functional information on the genes resides in the proteome, which is the sum of multiple dynamic processes that include protein phosphorylation, protein trafficking, localization, and protein-protein interactions [ ] . moreover, the proteomes of mammalian cells, tissues, and body fluids are complex and display a wide dynamic range of proteins concentration one cell can contain between one and more than copies of a single protein [ ] . in spite of new technologies, analysis of complex biological mixtures, ability to quantify separated protein species, sufficient sensitivity for proteins of low abundance, quantification over a wide dynamic range, ability to analyze protein complexes, and high throughput applications is not yet fulfilled [ ] . biomarker discovery remains a very challenging task due to the complexity of the samples (e.g., serum, other bodily fluids, or tissues) and the wide dynamic range of protein concentrations [ ] . most of the serum biomarker studies performed to date seem to have converged on a set of proteins that are repeatedly identified in many studies and that represent only a small fraction of the entire blood proteome [ ] . processing and analysis of proteomics data is indeed a very complex multistep process [ , ] . the consistent and transparent analysis of lc/ms and lc-ms/ms data requires multiple stages [ ] , and this process remains the main bottleneck for many larger proteomics studies. to overcome these issues, effective sample preparation (to reduce complexity and to enrich for lower abundance components while depleting the most abundant ones), state-of-the-art mass spectrometry instrumentation, and extensive data processing and data analysis are required. a wide range of proteomic approaches are available such as gel-based applications include one-dimensional and twodimensional polyacrylamide gel electrophoresis [ , ] , and gel-free high throughput screening technologies are equally available, including multidimensional protein identification technology [ ] , isotope-coded affinity tag icat [ ] ; silac [ ] ; isobaric tagging for relative and absolute quantitation (itraq) [ ] . shotgun proteomics [ ] and de dige [ ] as well as protein microarrays [ , ] are applied to obtain overviews of protein expression in tissues, cells, and organelles. large-scale western blot assays [ ] , multiple reaction monitoring assay (mrm) [ ] , and label-free quantification of high mass resolution lc-ms data [ ] are being explored for high throughput analysis. many different bioinformatics tools have been developed to aid research in this field such as optimizing the storage and accessibility of proteomic data or statistically ascertaining the significance of protein identifications made from a single peptide match [ ] . in this review we attempt to provide a overview of the major developments in the field of proteomics, some success stories as well as challenges that are currently being faced. about - % of all genes in an organism encode integral membrane proteins, which are involved in numerous cellular processes [ ] . membrane proteins constitute % of the typical proteome, yet their propensity to aggregate and precipitate in solution confounds their analysis [ ] . the target residues for tryptic cleavage (i.e., lysine and arginine) are mainly absent in transmembrane helices and preferentially found in the hydrophilic part of these lipid bilayer-incorporated proteins. because of the protein aggregation step of ief, de is unsuitable for the separation of integral membrane proteins and is limited to detection of membraneassociated proteins and membrane proteins with a low hydrophobicity [ ] . membrane solubilization methods have been deployed to analyze enriched membrane fractions and address the solubility issue by using detergents [ ] , organic solvents [ ] , and organic acids [ ] compatible with subsequent proteolytic digestion/chemical cleavage, separation and analysis by lc/ms. in this approach, ( ) an enriched yeast membrane fraction is solubilized with % formic acid in the presence of cyanogens bromide. the concentrated organic acid provides the solubilization agent, and cyanogen bromide, functional under acidic conditions, allows many embedded membrane proteins to be cleaved, ( ) a membrane-enriched microsomal fraction is solubilized by boiling in . % sds and, following isotope-coded affinity tag (icat) labeling, is diluted to reduce the concentration of sds, and ( ) by using an enriched membrane sample, the proteins are thermally denatured and sonicated in % organic solvent (methanol) in the presence of trypsin. the resultant peptide mixture is then analyzed by lc/ms. all three of these methods are effective and optimize the identifications of membrane proteins. another method using high ph and protenase k is optimized specifically for the global analysis of both membrane and soluble proteins [ ] . high ph favors the formation of membrane sheets, while proteinase k cleaves exposed hydrophilic domains of membrane proteins. commercially available nonionic detergents, dodecyl maltoside, and decaethylene glycol mono hexadecyl are proved most efficient membrane protein solubilizers [ ] . another more successful approach to isolate membrane proteins relies on cell surface labeling in combination with high resolution two-dimensional ( d) lc-ms/ms [ ] . in addition, improved analytical tools should be developed, that is, multidimensional liquid chromatography of peptide mixtures generated from membrane proteins, nanoflow chromatographic techniques for hydrophobic transmembrane peptides, and native electrophoresis of membrane protein complexes, which, in combination with mass spectrometry, should lead to the identification of the majority of proteins in the membrane proteome of simple microorganisms. it is important to quantify not only the identified membrane proteins but also to determine the levels of interacting partners. subcellular fractionation techniques that employ a combination of centrifugation steps are a common choice for preparing plasma membrane-(pm-) enriched fractions including detergent-resistant membrane fractions, commonly known as lipid rafts. these methods can offer a significant improvement in specificity for pm proteins over approaches that do not perform any subcellular fractionation, but rather use whole-cell or tissue preparations [ ] . chemical-tagging methods [ ] have been a more applied technique used to enrich for pm proteins and are often used in conjunction with physical separation strategies. this method allows for a specific class of protein or modification of interest to be physically separated from other nontagged proteins. importantly, when chemical tags are attached to the extracellular domain of pm proteins on intact cells, they offer an unrivaled specificity for pm proteins, because they offer a manner to distinguish true pm proteins from intracellular contaminants. cell-surface biotinylation, the covalent attachment of a biotin tag to the extracellular domain of pm proteins, is also a popular choice [ ] [ ] [ ] . serum is a complex body fluid, containing a large diversity of proteins. more than different proteins are present in the human serum and many of them are secreted or shed by cells during different physiology or pathology processes [ ] . serum is expected to be an excellent source of protein biomarkers because it circulates through, or comes in contact all tissues. consequently, serum proteomics has raised great expectations for the discovery of biomarkers to improve diagnosis or classification of a wide range of diseases, including cancers [ ] . however, serum has been termed as the most complex human proteome [ ] with considerable differences in the concentrations of individual proteins, ranging from several milligrams to less than one pictogram per milliliter [ ] . the analytical challenge for biomarker discovery arises from the high variability in the concentration and state of modification of some human plasma proteins between different individuals [ ] . albumin is a protein of very high abundance in serum ( - mg/ml) that would be a prime candidate for complete selective removal prior to performing a proteomic analysis of lower abundance proteins. thus, removal of albumin from serum may also result in the specific removal of low abundance cytokines, peptide hormones, and lipoproteins of interest. immunoglobulins, and antibodies are also abundant proteins in serum that function by recognizing "foreign" antigens in blood and initiating their destruction [ ] . the presence of higher abundance proteins interferes with the identification and quantification of lower abundance proteins (lower than ng/ml in serum). complexity and dynamic range of protein concentrations can be addressed with a combination of prefractionation techniques that deplete highly abundant proteins and fractionate. heparin chromatography coupled with protein g appears to be an efficient and economical strategy to pretreat serum for serum proteomics [ ] . protein prefractionation by immunodepletion and reversed-phase separation of the depleted plasma on mrp-c column provide methods compatible with lc-ms-based analysis. a polyclonal antibody-based system to rapidly deplete multiple high abundant proteins in serum, plasma, csf, and other biological fluids. individual antibody materials are mixed in selected percentages and packed into a column format. albumin can be removed by immunoaffinity columns [ ] , isoelectric trapping [ ] , dye-ligand chromatography [ ] , and peptide affinity chromatography [ ] . another approach involves the removal of igg by affinity chromatography using immobilized protein a or protein g [ ] . a recently developed depletion method that mixes high-specificity polyclonal antibodies (mars) to remove the top proteins in a single purification step is commercially available [ ] . human- multiple affinity removal column depletes the top abundant proteins from human serum, plasma, csf, and other biological fluids. to address d limitations several types of mass spectrometry, in conjunction with various separation and analysis methods, are increasingly being adopted for proteomic measurements [ ] . in contrast, d-page analysis, seldi-tof ms is a rather new method which is especially valuable for the identification of serum-derived biomarkers [ ] . this method is based on proteinchip arrays which carry various chromatographic properties, such as anion exchange, cation exchange, and hydrophilic or hydrophobic surfaces [ ] . for the analysis of serum, only - μl of serum sample is applied to these surfaces; after washing off unbound material, the protein fingerprint can be determined and visualized by time-of-flight mass spectrometry. the advantages of this method are the low amount of sample necessary for analysis, its speed, and high throughput capability. many different groups have used this method and related methods based on prefractionation of serum proteins by beads and subsequent maldi analysis for the identification of biomarkers in serum, urine, pancreatic juice, and other biological fluids [ ] . the necessity of this removal or separation is also illustrated that many proteins found useful as biomarkers [ ] . different fractionation steps (such as electrophoresis, seldi, and liquid chromatography) have been developed to reduce the complexity of serum proteome and to allow the detection and the identification of single proteins [ ] . de and maldi ms had applied to identify candidate biomarkers at early and late stages of lung cancer disease. this method identified proteins in tumor bearing mice this included disease regulated expression of orosomucoid- , a- -macroglobulin, apolipoprotein-a , apolipoprotein-c , glutathione peroxidase- , plasma retinol-binding protein, and transthyretin [ ] . recently proteins were identified by stable isotope labeled proteome (silap) standard coupled with extensive multidimensional separation with tandem mass spectrometry of which proteins were present at . -fold or greater concentrations in the sera of patients with pancreatic cancer [ ] . specimen collection (blood, serum, plasma samples) is an integral component of clinical research. access to high-quality specimens, collected and handled in standardized ways that minimize potential bias or confounding factors, is key to the "bench to bedside" aim of translational research [ ] . variables that may impact analytic outcomes include ( ) the type of additive in the blood collection tubes; ( ) sample processing times or temperatures; ( ) hemolysis of the sample; ( ) sample storage parameters; ( ) the number of freeze-thaw cycles [ , ] . the key variable in any analysis is that the case and control samples are handled in the exact same manner throughout the entire analytical process from study design and collection of samples to data analysis [ , ] . these types of differences between samples could have a significant impact on the stability of proteins or other molecules of interest in the specimens. small differences in the processing or handling of a specimen can have dramatic effects in analytical reliability and reproducibility, especially when multiplex methods are used. a representative working group, standard operating procedures internal working group, comprised of members from across early detection research network should be formed to develop standard operating procedures (sops) for various types of specimens collected and managed for biomarker discovery and validation work. limitations of two-dimensional electrophoresis. figure gives the general work flow in proteomics and table addresses their strengths and limitations. two-dimensional electrophoresis ( de) was developed two decades before the term proteomics was coined [ , ] . the de entails the separation of complex protein mixtures by molecular charge in the first dimension and by mass in the second dimension. de analysis provides several types of information about the hundreds of proteins investigated simultaneously, including molecular weight, pi and quantity, as well as possible posttranslational modifications. de is extensively used but mostly for qualitative experiments and this method falls short in its reproducibility, inability to detect low abundant and hydrophobic proteins, low sensitivity in identifying proteins with ph values too low (ph < ) or too high (ph > ) and molecular masses too small (mr < kd) or too large (mr > kd) [ ] [ ] [ ] [ ] . poor separations of basic proteins due to "streaking" of spots and membrane proteins resolution [ ] are limiting factors in de. however, de is the only technique that can be routinely applied for parallel quantitative expression profiling of complex protein mixtures such as whole cell and tissue lysates [ ] and most widely used method for efficiently separating proteins, their variants and modifications (up to proteins). there are two ways to study posttranslational modifications by means of de. first, posttranslational modifications that alter the molecular weight and or pi of a protein are reflected in a shift in location of the corresponding protein spot on the proteomic pattern. second, in combination with western blotting, antibodies specific for posttranslational modifications can reveal spots on de patterns containing proteins with these modifications [ ] . protein extraction and solubilization are key steps for proteomic analysis using de, highly hydrophobic proteins tend to precipitate during isoelectro focusing (ief), low copy number and the insolubility of transmembrane proteins renders quantitative analysis of these peptides and polypeptides are very challenging [ ] . in order to enhance protein extraction and solubilization, different treatments and conditions are necessary to efficiently solubilise different types of protein extracts [ , ] . the major challenge for protein visualization in de is the compatibility of sensitive protein staining methods with mass spectrometric analysis. therefore, several fluorescent staining methods have been developed for the visualization of de patterns, including sypro stainings and cy-dyes [ ] . although sypro ruby [ ] and silver staining [ , ] have a comparable sensitivity, sypro ruby staining allows much higher reproducibility, a significantly wider dynamic range and less false-positive staining. in addition, sypro ruby allows for the detection of lipoproteins, glycoproteins, metalloproteins, calcium-binding proteins, fibrillar proteins, and low molecular weight proteins that are less "stainable" using other methods. finally, a large number of protein spots on de patterns contain several proteins with a similar pi. a ph gradient with a narrow range allows zooming into different proteins with the same molecular weight. increased separation distance × cm gels using ca-ief [ ] could increase the proteome coverage up to proteins. use of overlapping narrow range ipgs "zoom" gels and increase in separation area could yield better membrane protein separation [ ] . this technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. proteins can also be fluorescently labelled with cy , cy , or cy prior to de [ ] . cydyes are cyanine dyes carrying an n-hydroxysuccinimidyl ester reactive group that covalently binds the e-amino group of lysine residues in proteins. during dige [ ] , proteins in each of up to samples can be labelled with one of these fluorescent dyes, and the differentially labelled samples can be mixed and loaded together on one single gel, allowing the quantitative comparative analysis of three samples using a single gel ( figure ). the dige technique has exhibited higher sensitivity as well as linearity, eliminated postelectrophoretic processing (fixing and destaining) steps and enhanced reproducibility by directly comparing samples under similar electrophoretic conditions [ , ] . the resulting images are then analyzed by software such as de-cyder which are specifically designed for d-dige analysis [ ] . the major advantages of d-dige are the high sensitivity and linearity of its dyes, its straightforward protocol, as well as its significant reduction of intergel variability, increasing the possibility to unambiguously identify biological variability, and reducing bias from experimental variation. moreover, the use of a pooled internal standard, loaded together with the control and experimental samples, increases quantification accuracy and statistical confidence [ ] . the dige technique has dramatically improved the reproducibility, sensitivity, and accuracy of quantitation; however, its labeling chemistry has some limitations; proteins without lysine cannot be labeled, and they require special equipment for visualization, and fluorophores are very expensive [ , ] . tag (icat) . gel-free, or ms based, proteomics techniques are emerging as the methods of choice for quantitatively comparing proteins levels among biological proteomes, since they are more sensitive and reproducible than two-dimensional gel-based methods. icat is one of the most employed chemical isotope labeling methods and the first quantitative proteomic method to be based solely on using ms [ , ] . each icat reagent consists of three essential groups: a thiol-reactive group, an isotope-coded light or heavy linker, and a biotin segment to facilitate peptide enrichment. in an icat experiment, protein samples are first labeled with either light or heavy icat reagents on cysteine thiols. the mixtures of labeled proteins are then digested by trypsin and separated through a multistep chromatographic separation procedure. peptides are identified with tandem ms, and the relative quantifications of peptides are inferred from the integrated lc peak areas of the heavy and light versions of the icat-labeled peptides [ ] . the icat concept has been widely used after its introduction [ ] [ ] [ ] . different software programs were developed to analyze icat labeled ms data (e.g., proicat from applied biosystems, spectrum mill from agilent technologies, and sashimi from the institute of system biology [ ] ). icat is extremely helpful to detect peptides with low expression levels, which is one of the bottleneck issues in analytic protein techniques [ , ] . however, major limitations of this technique include selective detection of proteins with high cysteine content and difficulties in the detection of acidic proteins [ , ] . the methods for direct comparison of dige and icat for the identification and quantification of proteins in complex biological mixtures are also being considered [ ] . while the icat reagent only interacts with the free sulfhydryl of homocysteine and % protein is noncysteine, the silac has emerged as a valuable proteomic technique [ ] which becomes more common for cell types and have been applied in many fields [ ] [ ] [ ] . the silac technique can be effectively expanded to compare the differential expression levels of tissue proteome at different pathological states, which allows to identify new candidate biomarkers [ ] . compared with the icat, a popular in vitro labeling, silac as an example of in vivo coding requires no chemical manipulation, and there is very little chemical difference between the isotopically labeled amino acid and its naturally occurring counterpart [ ] . in addition, the amount of labeled proteins requires for analysis using silac technique is far less than that with icat. therefore, the silac-based method has broadly applied in many areas of cell biology and proteomics. except that the silac-based quantitative method is powerful in comparative/differential proteomics, it has been widely used in analyzing protein posttranslational modification, such as protein phosphorylation, detection of protein-protein or peptide-protein interactions and investigating signal transduction pathways [ , ] .though there are numerous advantages for using silac-based methods compared to chemical labeling, a major drawback of silac is that it cannot be applied to tissue protein analysis directly. to overcome this shortcoming, silac has been successfully applied to tissue proteome based on n isotope labeling [ ] . microorganisms such as malaria parasite can be labeled with isoleucine [ ] . latterly the culturederived isotope tags (cdits) method was developed as an alternative quantitative approach for studying the proteome of mammalian tissues based on the application of silac [ ] . o stable isotope labeling. differential o/ o coding relies on the o exchange that takes place at the cterminal carboxyl group of proteolytic fragments, where two o atoms are typically replaced by two o atoms by enzyme-catalyzed oxygen exchange in the presence of h o [ ] . the resulting mass shift between differentially labeled peptide ions permits identification, characterization, and quantitation of proteins from which the peptides are proteolytically generated. in contrast to icat, o labeling does not favor peptides containing certain amino acids (e.g., cysteine), nor does it require an additional affinity step to enrich for these peptides [ ] . unlike itraq, o/ o labeling does not require a specific ms platform nor does it depend on fragmentation spectra (ms ) for quantitative peptide measurements. it is amenable to the labeling of human specimens (e.g., plasma, serum, tissues), which represents a limitation of metabolic labeling approaches (e.g., silac). taken together, recent advancements in the homogeneity of o incorporation, improvements made on algorithms employed for calculating o/ o ratios and the inherent simplicity of this technique should result in increased use of o labeling [ ] . in general, o labeling suffers from two potential drawbacks, inhomogeneous o incorporation and inability to compare multiple samples within a single experiment. a dual o labeling using a non-gel-based platform has been developed to overcome the major problems of existing proteolytic o labeling methods [ ] . (itraq). the itraq reagent is well known for relative and absolute quantitation of proteins. the itraq technology offers several advantages, which include the ability to multiplex several samples, quantification, simplified analysis and increased analytical precision and accuracy [ ] [ ] [ ] . the interest of this multiplexing reagent is that or analysis samples [ ] can be quantified simultaneously. in this technique, the introduction of stable isotopes using itraq reagents occurs on the level of proteolytic peptides ( figure ). this technology uses an nhs ester derivative to modify primary amino groups by linking a mass balance group (carbonyl group) and a reporter group (based on n-methylpiperazine) to proteolytic peptides via the formation of an amide bond [ ] . due to the isobaric mass design of the itraq reagents, differentially labelled peptides appear as a single peak in ms scans, reducing the probability of peak overlapping. when itraq-tagged peptides are subjected to ms/ms analysis, the mass balancing carbonyl moiety is released as a neutral fragment, liberating the isotope-encoded reporter ions which provides relative quantitative information on proteins. an inherent drawback of the reported itraq technology is due to the enzymatic digestion of proteins prior to labelling, which artificially increases sample complexity and this approach needs a powerful multidimensional fractionation method of peptides before ms identification. prefractionation of proteins based on electrokinetic methodologies in free solution essentially relaying on the isoeletric focusing (ief) has gained wide acceptance. many commercial devices are now constructed to take the advantage of this principle ( table ). reproducible fractionation steps will break down the sample complexicity while concentrating low abundant species, resulting in more confident protein identifications and quantification by d gels, mass spectrometry, and protein arrays. a good example of a innovation is liquid-phase isoelectric focusing (ief) as a prefractionation tool before the first dimension of d gel electrophoresis [ , ] . for more consistent pi separation, the zoom ief fractionator [ ] and multicompartment electrolyser (mce) [ ] are being used to prefractionate the proteins. the fractionated samples can be directly applied on standard narrow range ipg strips for d electrophoresis. this allows at least to separate proteins to be analyzed, including proteins of very low abundance. ief, a highresolution electrophoresis technique, has been widely used in shotgun proteomic experiments [ ] . ief runs in a buffer-free solution containing carrier ampholytes or in immobilized ph gradient (ipg) gels. the use of ipg-ief for the separation of complex peptide mixtures has been applied to the analysis of plasma and amniotic fluid [ , ] as well as to bacterial material [ ] . the ipg gel strip is divided into small sections for extraction and cleaning up of the peptides. this technique recovers the sample from the liquid phase and was demonstrated to be of great interest in shotgun proteomics [ ] . ief is not only a high resolution and high capacity separation method for peptides, it also provides additional physicochemical information like their isoelectric point [ , ] . the pi value provided is used as an independent validating and filtering tool during database search for ms/ms peptide sequence identification [ ] . the recent introduction of commercially available offgel fractionator system by agilent technologies provides an efficient and reproducible separation technique [ ] . this separation is based on immobilized ph gradient (ipg) strips and permits to separate peptides and proteins according to their isoelectric point (pi) but is realized in solution [ ] . its micropreparative scale provides fraction volumes large enough to perform subsequent analyses as reverse phase (rp)-liquid chromatography (lc)-maldi ms/ms. the combined use of itraq labeling and offgel fractionation methods for the proteomic study of complex sample is also being considered [ , ] . in this procedure, a large well is used to separate the sample by page and lanes are created on the membrane containing immobilized protein with the use of a manifold [ ] . compatible combinations of primary antibodies are predetermined, with the criterion of being able to identify proteins that do not comigrate. different combinations of primary antibodies are added to each well, with appropriate dilutions of each primary antibody so that expressed proteins are detected in a single condition. the scalability of the system depends on defining suitable combinations of primary antibodies, with up to antibodies in lanes being used in the largest screens. detection software is used to identify proteins based on their expected and observed gel mobility. unlike d page and hplc-ms/ms, large-scale western blotting only identifies proteins for which antibodies are already available. while this is not an appropriate screen for identifying uncharacterized proteins, it greatly simplifies the verification and functional analyses of proteins that are detected. in addition, this approach is highly flexible, and can be focused to particular sets of proteins or protein function, such as cell signaling molecules. importantly, the foundation of this approach is the large amount of data on individual antibodies, which are already available and characterized in the literature [ ] . another approach to analyse proteomes without gels is "shotgun" analysis using mudpit [ ] . in the mudpit approach, protein samples are subject to sequencespecific enzymatic digestion, usually with trypsin and endoproteinase lysc, and the resultant peptide mixtures are separated by strong cation exchange (scx) and reversed phase (rp) high performance liquid chromatography (hplc) [ , ] . peptides from the rp column enter the mass spectrometer and ms data is used to search the protein databases [ ] . the mudpit technique generates an exhaustive list of proteins present in a particular protein sample, it is fast and sensitive with good reproducibility however, it lacks the ability to provide quantitative information [ ] [ ] [ ] . a combination of hplc, liquid phase isoelectric focusing, and capillary electrophoresis provides other multimodular options for the separation of complex protein mixtures [ ] . high throughput production of human proteins using different methods is being developed to make protein array approach more practical. recently simple and efficient production of human proteins using the versatile gateway vector system has been developed [ ] . in this approach, protein expression system is applied to the in vitro expression of human proteins and assessed their biological activity in two functional categories and developed "human protein factory" infrastructure which includes the resources and expression technology for in vitro proteome research. in another approach, dna array to protein array (dapa) is utilized, which allows the "printing" of replicate protein arrays directly from a dna array template using cellfree protein synthesis [ ] . based on the nucleic acid programmable protein array (nappa) concept, high-density self-assembling protein microarray is developed to display thousands of proteins that are produced and captured in situ from immobilized cdna templates [ ] . this method will enable various experimental approaches to study protein function in high throughput. the adventage of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, proteinnucleic acid, and protein-small molecule interactions, as well as posttranslational modifications [ , ] . the microarray format provides a robust and convenient platform for the simultaneous analysis of thousands of individual protein samples, facilitating the design of sophisticated and reproducible biochemical experiments under highly specific conditions [ ] . the principal challenges in protein array development are -fold: ( ) creation of a comprehensive expression clone library; ( ) high-throughput protein production, including expression, isolation, and purification; ( ) adaptation of dna microarray technology to accommodate protein substrates [ ] . functional protein microarrays differ from analytical arrays in that functional protein arrays are composed of arrays containing fulllength functional proteins or protein domains (figure ) . these protein chips are used to study the biochemical activities of an entire proteome in a single experiment. they are used to study numerous protein interactions, such as protein-protein, protein-dna, protein-rna, proteinphospholipid, and protein-small molecule interactions [ , ] . companies have introduced protein arrays aimed not only at proteomic analysis but also functional analyses of proteins (e.g., biacore ab, ciphergen biosystems inc., phylos inc.). affinity proteomics aim to produce antibodies to every protein expressed by the human genome and these will be characterized against purified antigens and tested on tissue arrays to collect information about their specificity for tissue antigens [ ] . companies are focused to produce various binding partners, for example, affibodies, monoclonal antibodies, and their fragments [ ] . protein chips will likely be the next major manifestation of the revolution in proteomics and offer another solution to analyze low abundant proteins and have the potential for high throughput applications to identify biomarkers [ ] . protein chips differ from previously described methods; whereas screening by de or lc ms/ms can potentially detect any protein, and protein chips can only provide data on set of proteins selected by the investigator [ ] . the development and application of high throughput, multiplex immunoassays that measure hundreds of known proteins in complex biological matrices, is becoming a significant tool for quantitative proteomics studies, diagnostic discovery, and biomarker-assisted drug development. two broad categories of antibody microarray experimental formats have been developed [ ] , direct labelling, single antibody experiments [ ] , dual antibody, sandwich immunoassays are described [ , ] . in the direct labelling method, all proteins in a complex mixture are tagged, providing a means for detecting bound proteins following incubation on an antibody microarray. in the sandwich immunoassay format, proteins captured on an antibody microarray are detected by a cocktail of detection antibodies, each antibody matched to one of the spotted antibodies. in addition, a variety of microarray substrates have been described, including nylon membranes, plastic microwells, planar glass slides, gel-based arrays and beads in suspension arrays. much effort has been expended in optimizing antibody attachment to the microarray substrate. finally, various signal generation and signal enhancement strategies have been employed in antibody arrays, including colorimetry, radioactivity, fluorescence, chemiluminescence, quantum dots and other nanoparticles, enzyme-linked assays, resonance light scattering, tyramide signal amplification, and rolling circle amplification. each of these formats and procedures has distinct advantages and disadvantages, relating broadly to sensitivity, specificity, dynamic range, multiplexing capability, precision, throughput, and ease of use. in general, multiplexed microarray immunoassays are ambient analyte assays [ ] . given the heterogeneity of antibody array formats and procedures currently in use in proteomics studies, and the absence of a "gold standard," there exists an urgent need for development and adoption of standards that permit platform comparisons and benchmarking. regardless of the choice of a given proteomic separation technique, gel-based or gel-free, a mass spectrometer is always the primary tool for protein identification. during the last decade, significant improvements have been made in the application of ms for the determination of protein sequences [ ] . mass spectrometers consist of an ion source, the mass analyzer, and an ion detection system. analysis of proteins by ms occurs in three major steps (a) protein ionization and generation of gas-phase ions, (b) separation of ions according to their mass to charge ratio, and (c) detection of ions [ ] . in gel-free approaches such as icat and mudpit, samples are directly analyzed by ms whereas, in gel-based proteomics ( de and d-dige), the protein spots are first excised from the gel and then digested with trypsin. the resulting peptides are then separated by lc or directly analyzed by ms. the experimentally derived peptide masses are correlated with the peptide fingerprints of known proteins in the databases using search engines (e.g., mascot, sequest). there are two main ionization sources which include matrix assisted laser desorption/ionization (maldi) and electrospray ionization (esi) and four major mass analyzers, which are time-of-flight (tof), ion trap, quadrupole, and fourier transform ion cyclotron (ftic) which are currently in use for protein identification and characterization [ ] . a combination of different mass analyzers in tandem such as quadrupole-tof and quadrupole-ion trap has combined the individual strengths of different types of mass analyzers and greatly improved their capabilities for proteome analysis [ ] . simple mass spectrometers such as maldi-tof are used for only measurement of mass, whereas tandem mass spectrometers are used for amino acid sequence determination [ ] . in maldi the sample of interest is crystallized with the matrix on a metal surface and a laser ion source causes excitation of matrix along with the analyte ions, which are then released into the gas phase. maldi measures the mass of peptides derived from a trypsinized parent protein and generates a list of experimental peptide masses, often referred to as "mass fingerprints" [ , ] . in esi, the analyte is ionized from a solution and transferred into the gas phase by generating a fine spray from a high voltage needle which results in multiple charging of the analyte and generation of multiple consecutive ions. tandem mass spectrometry or ms/ms is performed by combining two different ms separation principles. in tandem ms, individual trypsin-digested peptides are fragmented after a liquid phase separation. tandem ms instruments such as triple quadrupole, quadrupole ion trap, fourier transform ion-cyclotron resonance, or quadrupole time-of-flight are used in lc-ms/ms or nanospray experiments with electrospray ionization (esi) to generate peptide fragment ion spectra [ ] . ion mobility spectrometry (ims) has been utilized as a rapid gas-phase separations strategy for biomolecular ions [ , ] . the strategy provides high sensitivity because the gas-phase dispersion of peptide ions separates features corresponding to low abundance species from interfering chemical noise [ ] . reduced spectral congestion also allows for the use of shorter experimental run times (lc separations) without sacrificing throughput; short analysis time scales are key to measuring the large numbers of samples required to determine normal protein variability prior to realizing individual plasma profiling. additionally, mobility-dispersed ions can be fragmented and mobility linked to fragment ions without ion loss from precursor mass selection [ ] . these advantages have been demonstrated in head-to-head comparisons with conventional lc-ms/ms technology using rapid ( minutes) lc gradients [ ] . accurate mass and time (amt) tag approach [ ] addresses an analogous situation in lc-ms-based proteomics studies. in this approach, initial lc-ms/ms analyses are performed on prefractionated peptide samples in order to provide peptide sequence identifications. these experiments are relatively low throughput because the peptide prefractionation can be quite extensive and require separate lc-ms/ms analyses for each fraction. the high-throughput accurate mass and time (amt) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-negative bacterium rhodobacter sphaeroides . . . there has been a recent trend in proteomics toward the development and application of technologies for the targeted analysis of proteins within complex mixtures [ ] . selected reaction monitoring (srm) is a powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest [ , ] . the specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics. the use of tandem mass spectrometry data acquired on an ltq ion trap mass spectrometer can accurately predict which fragment ions will produce the greatest signal in an srm assay using a triple quadrupole mass spectrometer [ ] . one of the biggest benefits of a targeted assay on a triple quadrupole mass spectrometer is high throughput. using the selectivity of multiple stages of mass selection of a tandem mass spectrometer, these targeted srm assays are the mass spectrometry equivalent of a western blot [ ] . an advantage of using targeted mass spectrometry-based assay over a traditional western blot is that it does not rely on the creation of any immunoaffinity reagent. while its application is novel in the proteomics community, srm has been utilized for several decades in the toxicology and pharmacokinetics disciplines [ ] . peptidebased immunofractionation methods show potential for proteome wide screening approaches but are limited by the availability of antibodies [ , ] . the stable isotope standards with capture by antipeptide antibodies (siscapa) approach is based on the addition of stable isotope labeled standard peptides to the digested clinical sample followed by immunoaffinity enrichment of standard and analyte peptide by highly specific antipeptide antibodies [ , ] . this approach enables the absolute quantification of selected diagnostic peptides from digested clinical samples down to physiologically relevant analyte concentrations (ng/ml) at high precision ( % cv) and accuracy [ , ] . further improvement of mrm-based biomarker quantification should be possible if whole sets of analyte peptides can be enriched by immunofractionation. since this method relies on one specific antibody per target protein/peptide the generation of more than antibodies is necessary for proteome wide screening approaches. novel peptide affinity enrichment strategies enabling proteome wide analyses of signature peptides may provide an important addition to future proteome workflows. undoubtedly, the accuracy, high throughput, and robustness of ms technologies have made the characterization of entire proteomes a realistic goal [ , ] . the major bottlenecks in proteomics research today are related to data analysis to create an environment where computer scientists and biologists and the people who collect data can work closely together, so they can develop the necessary analytical tools that will help interpret the data [ ] [ ] [ ] . processing and analysis of proteomics data is indeed a very complex multistep process ( figure ). the meaningful comparison, sharing, and exchange of data or analysis results obtained on different platforms or by different laboratories remain cumbersome mainly due to the lack of standards for data formats, data processing parameters, and data quality assessment. accurate, consistent, and transparent data processing and analysis are integral and critical parts of proteomics workflows [ ] . we can now generate huge amounts of data, and currently there is an enormous challenge to figure out how to actually analyze this data and generate real biological insights. the necessity of an integrated pipeline for processing and analysis of complex proteomics data sets has therefore become critical. validation. this step consists of the assignment of ms/ms spectra to a database search using one of several engines available (e.g., sequest, mascot, comet, x!tandem, etc.). one of the difficulties related to the use of sequest for peptide identifications is the lack of methods to globally evaluate the quality of data and the lack of methods to access global changes created by filtering schemes and/or database changes [ ] . most approaches are matching and scoring large sets of experimental spectra with predicted masses of fragment ions of peptide sequences derived from a protein database. results are scored according to a scheme specific to each search engine that also depends on the database used for the search. usually tools are linked to one specific platform or were optimized for one instrument type. the various search engines do not yield identical results as they are based on different algorithms and scoring functions, making comparison and integration of results from different studies or experiments tedious [ , ] . peptide identification via database searches is very computationally intensive and time-demanding. high quality data allow more effective searches due to tighter constrains, that is, tolerance on precursor ion mass and charge state assignment, which will drastically reduce the search time in case of an indexed database. in addition, accurate mass measurements of fragment ions further simplify the database searches and add confidence to the results. the association of identified peptides with their precursor proteins is a very critical and difficult step in shotgun proteomics strategies as many peptides are common to several proteins, thus leading to ambiguous protein assignments. therefore it becomes critical to have an appropriate tool that is able to assess the validity of the protein inference and associate a probability to it. protein prophet database tool combines probabilities assigned to peptides identified by ms/ms to compute accurate probabilities for the proteins present [ ] . : - , . impossible. the lack of common standards and protocols has led to this situation and often resulted in duplication of efforts. results were usually reported as a set of identified proteins (i.e., list of peptides identified and associated proteins) with minimal supporting data. obviously the large volume of such data sets has made publication of detailed results using classical mechanisms very challenging. sharing and exchange of data and results requires the definition of standard formats for the data at all levels (including raw mass spectrometric data, processed data, and search results) as well as a better definition (and/or standardization) of the parameters used for the data processing or the database searches. organellar proteomics aims to describe the full complement of proteins of subcellular structures and organelles. identification of the proteins contained in subcellular organelles has become a popular proteomics endeavor [ ] . when compared with whole-cell or whole-tissue proteomes, the more focused results from subcellular proteomic studies have yielded relatively simpler datasets from which biologically relevant information can be more easily extracted [ ] . subcellular fractionation consists of two major steps, disruption of the cellular organization (homogenization) and fractionation of the homogenate to separate the different populations of organelles. such a homogenate can then be resolved by differential centrifugation into several fractions containing mainly ( ) nuclei, heavy mitochondria, cytoskeletal networks, and plasma membrane; ( ) light mitochondria, lysosomes, and peroxisomes; ( ) golgi apparatus, endosomes and microsomes, and endoplasmic reticulum; ( ) cytosol. each population of organelles is characterized by size, density, charge, and other properties on which the separation relies [ ] . analyzing subcellular fractions and organelles allows tracking proteins that shuttle between different compartments, for example, between the cytoplasm and nucleus. a high dynamic range of proteins can be partially achieved by fractionation of the proteome into subproteomes by applying affinity purification may allow proteomic analysis of low copy number proteins [ ] . the nuclear, chloroplast, amyloplast, plasma membrane, peroxisome, endoplasmic reticulum, cell wall, and mitochondrial proteomes were successfully characterized in arabidopsis [ ] . several groups have taken advantage of this approach to recover a higher percentage of membrane proteins from subcellular extracts using various nonionic and zwitterionic detergents or phase-partitioning methods. these efforts resulted in the successful determination of the protein complement of the thylakoid and envelope membrane systems of the chloroplast [ ] . by enriching for the protein class of interest based on a particular chemical/physical characteristic(s), offer the advantage of reducing sample complexity and access to lower abundance proteins in a discoverydriven experimental approach [ ] . free flow electrophoresis (ffe) utilizes differences in electrophoretic mobility rather than density to separate cells or subcellular organelles [ ] . ffe has previously been used in separating endosomes from hamster ovary cells [ ] , plasma membrane from human platelets [ ] , and insulin transporting vesicles in liver cells. the separation is based on the electrophoretic motility of cells or cell organelles suspended in a vertical free flowing buffer film on which an electric field is applied at a right angle to the flow direction. ffe has been a most valuable tool in the investigation of the composition of secretory vesicles and in addition, it has clarified how the membrane of plasma membrane vesicles is oriented after nitrogen disruption of human neutrophils [ ] . importantly, subcellular fractionation is a flexible and adjustable approach that may be efficiently combined not only with d gel electrophoresis but also with gelindependent techniques. however, they do have limitations of considerable cross-contamination with other subcellular organelles. ptms of proteins are considered to be one of the major determinants regarding organisms complexity [ ] . to date, at least more than different types of ptms have been identified of which only a few are reversible and important for the regulation of biological processes. specific functions are usually mediated through ptms, such as phosphorylations, acetylations, or glycosylations, which places additional demands on the sensitivity and precision of the method [ ] . one of the most studied ptms is protein phosphorylation, because it is vital for a large number of protein functions that are important to cellular processes spanning from signal transduction, cell differentiation, and development to cell cycle control and metabolism. enzymes and receptors can be switched "on" and "off " by phosphorylation and dephosphorylation. it was estimated that - % of proteins are phosphorylated. phosphorylation often occurs on serine, threonine, and tyrosine residues in eukaryotic proteins [ ] . analysis of the entire cellular phosphoproteome has been an attractive study subject since the discovery of phosphorylation as a key regulatory mechanism of cell life. unfortunately, phosphoproteins analysis is not straightforward for five main reasons. first, the stoichiometry of phosphorylation is generally relatively low, because only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time as a result of a stimulus. second, the phosphorylatation sites on proteins might vary, implying that any given phosphoprotein is heterogeneous (i.e., it exists in several different phosphorylated forms). third, many of the signaling molecules, which are major targets of phosphorylation events [ ] , are present at low abundance within cells and, in these cases; enrichment is a prerequisite before analysis. fourth, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, which means that although major phosphorylation sites might be located easily, and minor sites might be difficult to identify. finally, phosphatases could dephosphorylate residues unless precautions are taken to inhibit their activity during preparation and purification steps of cell lysates. in addition, various methods for protein phosphorylation site determination have been developed, yet this task remains a technical challenge [ ] . western blot has been widely used to determine the presence of ptms. however, this technique relies on the prior knowledge of the type and position of specific modifications and the availability of antibodies. it has low throughput and not ideal for studying highly complicated samples. specific chemical or affinity enrichment steps are usually incorporated into the sample preparation or fractionation stages of the general scheme of proteomic studies [ , ] . well established methods involving the analysis of p-labeled phosphoproteins by edman degradation and two-dimensional phosphopeptide mapping have proven to be powerful but not without limitations. consequently, mass spectrometry (ms) has emerged as a reliable and sensitive method for the characterization of protein phosphorylation sites [ ] and may therefore represent a method of choice for the analysis of protein phosphorylation [ ] . immobilized metal affinity chromatography (imac), metal oxide affinity chromatography (moac), and covalent methods are all capable of selectively enriching phosphopeptides [ ] . moac based on adsorption to tio is especially attractive, but as with all techniques, loading, rinsing, and elution solutions must be carefully selected to minimize nonspecific adsorption and to maximize the detection of both monophosphorylated and multiphosphorylated species. imac might not provide the selectivity available with tio enrichment, but with appropriate reagents, imac can be selective and sensitive for monophosphorylated and tetraphosphorylated peptides. however, some buffers and reagents such as edta are not compatible with imac, so hplc purification may be needed prior to this technique [ ] . when trying to isolate and identify as many phosphoproteins as possible in a cell lysate, chromatographic column-based methods are required. multiple elutions from imac or moac columns or even gradient elutions can help to simplify fractions of proteins and reveal more peptides [ , ] . a combination of techniques can reveal large numbers of phosphopeptides in complex samples, but comprehensive phosphoproteomics is still not possible. for the highest protein coverage, future phosphoproteomic techniques will likely employ multiple enrichment techniques along with two-dimensional separations, but such studies are time consuming. combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies, and mass spectrometry are particularly attractive for systematic investigation of posttranslationally modified proteins in proteomics [ ] . organisms. the application of proteomics and related technologies for the analysis of proteome is severely hampered by the lack of publicly available sequence information for most of the unsequenced organisms [ ] . despite the precision of the mass information yielded by the seldi technique, a significant number of proteins were found to have no similarity to known peptides, an aforementioned weakness of proteomics studies in nonmodel organisms [ ] . in order to circumvent this limitation, different strategies and tools were developed to make unsequenced organisms amenable to high-throughput proteomics [ ] (figure ). however, an evaluation of their performance in an integrated proteomics strategy using high-throughput shotgun ms data is currently missing. in principle, two different approaches can lead to an increase in protein identifications from unsequenced organisms. in the first approach, ms/ms data are searched against a protein database of an evolutionarily closely related organism. however, as a matter of principle of database-dependent searches, only proteins can be identified that contain at least one peptide with exactly the same sequence as the peptide from a protein in the database. with increasing evolutionary distance this will be an increasingly severe restriction [ ] . in the second approach, the amino acid sequence of a peptide is extracted from the ms/ms spectrum for de novo sequencing, that is, in a fully databaseindependent manner using exclusively the information contained in the ms/ms spectrum. several software tools for peptide de novo sequencing are now available and some of them provide sufficiently good results when applied to high-quality spectra [ ] . a basic limitation of ms de novo sequencing methods is the necessity for backbone cleavage between each pair of adjacent amino acids; a mass value representing a terminal fragment containing only one of the two residues is a first requirement for ordering of a specific pair [ , ] and this limitation urged the need for bioinformatics approaches that can help interpret the proteomics data [ ] . in the past several years there have been very important extremely useful advances in proteomics methods based on bottom-up display and bottom-up identification using peptides [ ] . these methods offer more sensitivity, greater rapidity and greater proteome coverage are often made with the explicit or implicit assertion that these methods are bound to replace more traditional methods based on topdown analysis, especially using d gels [ , ] . the combination of bottom-up display and bottom-up identification has achieved very important successes in detecting the presence of large numbers of different proteins in cells or subcellular organelles [ , ] . the use of specific fractionation schemes and prudent adoption of methods to increase the number of proteins able to be identified and quantified is enabling significant biological advances to be made. further technological developments that enable a larger proportion of the proteome to be visualized will further enhance our ability to characterize biological systems. as such, these advances in proteomics will impact not only academic pursuits but also pharmaceutical, biotechnology and diagnostic research and development [ ] . in the future gel-free techniques mudpit, itraq and o stable isotope labeling could be expected to gain more importance as they become more established. sample prefractionation system provides a highly valuable tool to fractionate proteins and peptides from complex eukaryotic samples like plasma. this approach has a positive influence on the number of proteins identified compared to scx method [ ] . itraq is a very powerful tool, recognised form its ability to relatively quantify proteins. itraq reagent improves maldi ionisation, especially for peptides containing lysine. although silac labelling is easy for any laboratory that uses cell culture, the ms technology that is required is still beyond the capabilities of most groups. one of the factors that contributed to the rapid acceptance of the silac technology was the availability of an open-source program, msquant, for interpreting results. protein microarrays offer the ability to simultaneously survey multiple protein markers in an effort to develop expression profile changes across multiple protein analytes for potential use in diagnosis, prognosis, and measurement of therapeutic efficacy [ ] . this technology is an excellent high-throughput method used to probe an entire collection of proteins for a specific function or biochemistry. it is an exceptional new way to discover previously unknown multifunctional proteins, and to discover new functionalities for well-studied proteins [ ] . a systematic and efficient analysis of vast genomic and proteomic data sets is a major challenge for researchers today. to overcome limitations of current proteomics strategies in regard to the dynamic range of peptides detected and alternative mass spectrometrybased approaches are being explored. targeted strategies exemplified by multiple reaction monitoring detect, quantify, and possibly collect a product ion spectrum to confirm the identity of a peptide with much greater sensitivity because the precursor ion is not detected in the full mass spectrum [ ] . a systematic and efficient evaluation of large-scale experimental results requires ( ) automatic retrieval of user defined information to construct a customized, queryable database; ( ) an intuitive graphical and query platform to display and analyze experimental data in the context of the customized database; ( ) efficient utilization of webbased bioinformatics software tools for data interpretation, prediction of function, and modeling; ( ) scalability and reconstruction of the database in response to changing user needs and an ever-expanding base of knowledge and bioinformatics tools [ ] . creating a software tool to encompass the four crucial features outlined above is a challenging and ongoing task, particularly with respect to the ever-expanding publicly available base of knowledge and bioinformatics tools. the data processing and analysis bottleneck can be overcome through integration of the entire suite of tools into one linear pipeline. the good news is that all of the various proteomics strategies are in phases of very rapid technological development and that important advances in sensitivity, throughput, and proteome coverage can be expected in the near future for all of them. post translational 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workflow to increase the detection rate of proteins from unsequenced organisms in high-throughput proteomics experiments improving gene annotation using peptide mass spectrometry proteomics studies confirm the presence of alternative protein isoforms on a large scale plips, an automatically collected database of protein lists reported by proteomics studies proteome analysis by mass spectroscopy detection technologies in proteome analysis two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains is proteomics heading in the wrong direction? proteomic mapping of brain plasma membrane proteins how much of the proteome do we see with discovery-based proteomics methods and how much do we need to see? peptides offgel electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative itraq labeling development and standardization of multiplexed antibody microarrays for use in quantitative proteomics protein microarray technology integrating biological databases new paradigms in cellular function and the need for topdown proteomics analysis key: cord- -p ikd ns authors: hansra, satyender; pujhari, sujit; zakhartchouk, alexander n title: exploration of new sites in adenovirus hexon for foreign peptides insertion date: - - journal: open virol j doi: . / sha: doc_id: cord_uid: p ikd ns adenoviral vectors are now being explored as vaccine carriers to prevent infectious diseases in humans and animals. there are two strategies aimed at the expression of a vaccine antigen by adenoviral vectors. the first includes an insertion of the foreign gene expression cassette into the e region. the second strategy is antigen incorporation into the viral capsid proteins. to extend this methodology, we have searched for new sites at the human adenovirus serotype major capsid protein hexon for a vaccine antigen insertion. to this end, we utilized sites in the hexon hypervariable region (hvr) , and to display a -mer peptide containing the main neutralizing epitope of porcine reproductive and respiratory syndrome virus. however, we could not rescue the viruses with the insertions of the peptide into hvr and , consistent with the viruses being unable to tolerate insertions at these sites. in contrast, the virus with the insertion of the peptide in hvr was viable - growing well in cell culture and the inserted peptide was exposed on the virion surface. human adenovirus type (had ) vector has been shown to be an excellent vaccine delivery system in humans and animals [ , ] . the replication-defective (e -deleted) and replication-competent (e -deleted) had have both been used as vaccine delivery vectors. for biosafety concerns, a replication-defective had is considered to be more suitable for vaccination. replication-defective vectors may also have deletion in the e region to increase the amount of foreign dna that can be inserted. conventional strategy for the expression of a vaccine antigen by adenoviral vector includes insertion of the foreign gene expression cassette into the e region. this antigen is expressed as transgene after the infection of permissive cells with recombinant adenovirus. apparent drawbacks of the conventional transgene expression of antigen include an inability of the had -based vectors to produce a potent humoral response against certain antigens and, in some cases, the inability of the vector to express a foreign gene [ ] . to overcome such hurdles, the "antigen capsid-incorporation" strategy was developed [ ] . incorporating an immunogenic peptide into the had capsid offers several potential advantages. importantly, the processing of capsid-incorporated antigen via the exogenous pathway could result in a strong humoral response, similar to that generated by native had capsid proteins, and this strategy may also be useful in boosting antigen-specific antibody immune responses. adenoviral capsid consists of the hexon, penton base and fiber [ ] . antigenic epitopes could be incorporated into these proteins as well as into the minor capsid protein ix [ ] . importantly, hexon is the most abundant of the capsid proteins, accounting for more than % of the capsid protein [ ] . furthermore, hexon is shown to be a vaccine adjuvant [ ] . early analysis of the hexon protein sequences revealed that, in addition to the conserved regions, there were discrete hypervariable regions (hvrs) [ ] . in a subsequent study, they are reclassified as regions to [ ] . these hvrs do not appear to be involved in the binding of any other viral proteins, and the loops at the top of the hvrs are the most amenable to modifications. it was also shown that short heterologous peptides can be incorporated within the hvrs of hexon without affecting the viability of the virus [ , ] . incorporating antigens into hexon is applicable towards vaccination for several pathogens including the poliovirus, pseudomonas, b. anthracis, influenza, malaria and hiv [ ] [ ] [ ] [ ] [ ] [ ] . in selected studies, protective immune responses in a mouse model are documented [ , ] . in had , hvr through , and have been used for the incorporation of foreign peptides [ , ] . in this study, we explored different sites in hvrs , and of had hexon for the insertion of a peptide corresponding to the porcine reproductive and respiratory syndrome virus (prrsv) main neutralizing epitope b [ ] of the gp protein. our results provide potentially applicable information for adenovirusvectored vaccine development involving such hexon modifications, particularly in hvr . in order to access new sites in had hexon, tailored for the incorporation of foreign peptide sequences, we genetically inserted a amino acid (aa) residue peptide into hvr , hvr or hvr region of the had hexon. the peptide consisted of embedding a aa residue sequence containing the prrsv major neutralizing epitope flanked by the lgs spacer sequences (fig. ) . fig. ( ) . sites in the hvrs that were modified with a peptide containing the prrsv major neutralizing epitope b. the arrows mark the position where the peptide was inserted. the numbers show the positions of the amino acid residues in had hexon. peptide sequence corresponding to the prrsv epitope is underlined. insertion of the dna encoding peptide into the had hexon gene was achieved by pcr followed by bacteriumbased homologous recombination. the linearized dna of the plasmid containing the right portion of the had genome with modified hexon and the bp deletion in the e region was co-transfected into human embryo kidney (hek) cells together with a plasmid containing the left portion of the had genome with an e -deletion. homologous recombination in hek cells led to generation of the recombinant virus, termed ad hvr epb. multiple attempts to rescue the other two recombinant viruses (ad hvr epb and ad hvr epb) were not successful. this inability to rescue recombinant viruses with the foreign peptide insertion into hvr and hvr of hexon suggested that the insertion interferes with the formation of viral particles. interestingly, in a previous study [ ] , hexon-modified virus with incorporation of the his peptide into hvr was reported to be viable. our result with hvr is contradictory, evidently due to the differences in the peptides sequences and their lengths. further, while the two studies are made with changes within hvr , the specific locations of the incorporations differ as well. notably our study uses a foreign sequence inserted between asn and gly ( fig. ) , while wu et al. [ ] placed his between pro and trp substituting for aa residues of hexon. to corroborate the insertion of the peptide encoding dna into the hexon gene of had , the viral genome fragment was amplified using pcr from the viral dna extracted from ad hvr epb-infected cells, and the pcr product was digested with avrii since the peptide encoding dna contained the single avrii site. predictably, a bp pcr fragment was amplified from ad hvr epb dna (fig. , lane ), and its site specific cleavage by avrii provided two fragments: bp and bp (fig. , lane ) . the results of this analysis suggested that recombinant ad hvr epb contained the epitope b encoding sequence in the hvr of hexon. the growth kinetics of the recombinant virus ad hvr epb was analyzed in hek cells. cultures of the cells were infected with ad hvr epb or ad -empty (e -deleted and partially e -deleted had with unmodified hexon), and the cells were harvested at , , , , and hr post infection intervals. virus in each sample was released by freeze-thawing and titered on monolayers of hek cells. fig. ( ) shows the insertion of the peptide in hvr , resulting in similar virus yields to unmodified virus with maximal titers ranging from x to tcid per ml in a culture of x cells. we also determined the viral particle/infectious particle (vp/ip) ratio for cscl gradient purified preparations of the control virus ad -empty, as well as the hexon-modified virus ad hvr epb. a . fold increase was observed in the vp/ip ratio of ad hvr epb preparations as compared to the unmodified control ad -empty ( table ) . a similar observation is reported for hexon-modified had with peptide incorporation into hvr or [ ] . a typical vp/ip ratio of unmodified adenoviruses ranges from to [ ] . we next examined whether prrsv epitope was presented on the surface of the virion, using an elisa binding assay. different amounts of cscl gradient purified viruses (ad hvr epb and ad -empty) were immobilized on the wells of -well plates and incubated with peptide antisera. the binding of antibodies to the virions was detected with alkaline phosphatase (ap)-conjugated secondary antibody, followed by ap color reaction hvr : ……. development. evidently, the anti-peptide antibodies bind well to adhvr epb virions and not to ad -empty (fig. ) . this suggests that the prrsv epitope inserted into hvr of hexon is exposed on the virion surface. while a previous study showed that epitopes were exposed on the virion surface when incorporated in hvr , hvr and hvr , and not exposed in hvr and hvr , as well as poorly exposed when a peptide was placed in hvr [ ] . in the current study, we find a new site in hvr for incorporation of foreign peptide into had hexon, and determine that the peptide was also exposed on the virion surface making it readily accessible for antibody binding and be potentially useful for vaccination. . ( ) . the prrsv epitope incorporated in hvr of hexon is accessible in the context of an intact virion. in the assay, varying amounts of purified viruses were immobilized in the wells of elisa plate and incubated with anti-peptide antibody. the binding was detected with an ap-conjugated secondary antibody. values are expressed as the mean and standard deviation. * indicates a p value of < . . herein, we report a novel adenovirus vector (ad hvr epb) with the insertion of the prrsv main neutralizing epitope b in different hvr regions of the major capsid protein hexon. while the insertion of the peptide into hvrs and were not tolerated by the adenovirus, the -mer epitope placed within hvr is determined to be exposed on the virion surface. plasmid puc-ad -hex was used as a template for pcr and as a cloning vector. this plasmid contained a . kb dna fragment of had genome encoding a portion of the hexon gene. to insert the dna sequence encoding the main neutralizing epitope b of prrsv in hvr and obtain the dna fragment for cloning, four consequent pcrs were performed using phusion high-fidelity dna polymerase (neb). first, a kb fragment was amplified using primers bl f and nhe-r, and puc-ad -hex as a template. second, a kb fragment was amplified using primers blf and nhe-r, and the product of pcr as a template. third, a bp fragment was amplified using primers bl r and nde-f, and puc-ad -hex as a template. fourth, a . kb fragment was amplified using primers nde-f and nhe-r, and the mixture of the products of pcr and from above as a template. the final . kb pcr product was digested with ndei and nhei and cloned into ndei and nhei sites of puc-ad -hex, creating puc-ad hex-hvr epb. similar strategy was used to construct puc-ad hex-hvr epb and puc-ad hex-hvr epb. specific sequences of the primers are presented in table . the plasmid ph r-hexhvr epb was generated by homologous dna recombination in e. coli bj [ ] between a . kb dna fragment excised by kpni and ndei from puc-ad hex-hvr epb and bamhi-linearized ph r [ ] . similar strategy was used to construct ph r-hexhvr epb and ph r-hexhvr epb. fig. ( ) shows the sites of the insertion of the peptide in the had hexon protein sequence. human embryonic kidney (hek) cells were transfected with paci-digested dnas of the plasmids ph r-hexhvr epb, ph r-hexhvr epb or ph r-hexhvr epb and paci-digested ph -l [ ] using lipofectamine- (invitrogen). homologous recombination in the transfected cells led to generation of the recombinant adenovirus, and the virus was detected by developing cytopathic effect (cpe) in the cell monolayer. if cpe was not detected by the th day after transfection, the cells and the cell culture media were collected and freeze-thawed. this material was used to infect a fresh monolayer of hek cells. the virus was amplified by passaging in hek cells and purified by cscl gradient centrifugation [ ] . the concentration of virus particles (vp) in purified virus preparations was determined by measuring the optical density at nm as previously described [ ] , whereas numbers of infectious particles (ip) were determined by the tissue culture infective dose (tcid ) method [ ] . hek cells grown in wells of -well plates were infected with ad -empty or ad hvr epb viruses at a multiplicity of infection (m.o.i.) of . infected cells were harvested at the indicated time intervals post-infection. cells were lysed in the growth medium by freezing-thawing three times. the titres of infectious viral progeny were determined by tcid method in monolayers of hek cells. hek cells grown in t flasks were infected with the virus ad hvr epb. after hours post infection, when full cpe was observed, the cells and cell culture media were harvested followed by cycles of freezing and thawing. cell debris was removed by centrifugation at , rpm for min, and the supernatant ( µl) was used for viral dna isolation. briefly, the supernatant was incubated with µl of dnase i ( mg/ml) at c for min, followed by addition of µl of . m ethylenediaminetetraacetic acid (edta) (ph . ), . µl of % sodium dodecyl sulfate (sds), . µl of proteinase k ( mg/ml) and incubation at c for h. total dna was further extracted using a geneclean spin kit (mp biomedicals) according to the manufacturer's instructions. the isolated dna was subjected to pcr using fermentas ( x) pcr master mix and primers ( '-atcatgcagctgggagagtc- ' and '-cattgcccagcaacattgag- ') that were designed to anneal in the sites flanking the hexon hvrs. amplification product was digested by avrii and size-separated by electrophoresis in % agarose gel. the peptide, containing amino acid residues of the prrsv neutralizing epitope b (shlqliynl), was synthesized by genscript inc. the peptide was conjugated to keyhole limpet hemocyanin (klh) or bovine serum albumin (bsa) as carrier molecules. two new zealand white rabbits were immunized with the conjugated peptide ( µg/rabbit) emulsified with freund's complete adjuvant (fca) followed by two injections of conjugated peptide ( µg/rabbit) in freund's incomplete adjuvant (fia) at four weeks apart. serum was collected ten days after the third injection and tested for ability to bind to the peptide by elisa. the enzyme-linked immunosorbent assay (elisa) binding assay was used to test if the peptide incorporated into the hexon protein is accessible to anti-peptide antibody at the virion level. different amounts of purified virions ranging from to viral particles were immobilized on the wells of a -well plate (immulon, thermo scientific) by overnight incubation in ul/well of mm carbonate buffer (ph . ) at c. after washing with pbs, containing . % tween , the immobilized virus was incubated with : diluted anti-peptide rabbit serum for h at c, followed by ap-conjugated goat anti-rabbit antibody (cedarlane) incubation. color reaction was performed with p-nitrophenyl phosphate (sigma-aldrich), and optical density at nm was measured with a microplate reader. new insights on adenovirus as vaccine vectors use of adenoviral vectors as veterinary vaccines alternative codon usage of prrs virus orf gene increases eucaryotic expression of gp( ) glycoprotein and improves immune response in challenged pigs capsid-incorporation of antigens into adenovirus capsid proteins for a vaccine approach adenovirus structure tropism-modification strategies for targeted gene delivery using adenoviral vectors structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response analysis of adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues identification of sites in adenovirus hexon for foreign peptide incorporation optimization of capsidincorporated antigens for a novel adenovirus vaccine approach protective immunity to pseudomonas aeruginosa induced with a capsid-modified adenovirus expressing p. aeruginosa oprf expression of a foreign epitope on the surface of the adenovirus hexon hiv antigen incorporation within adenovirus hexon hypervariable for a novel hiv vaccine approach epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity replacing adenoviral vector hvr with a malaria b cell epitope improves immunogenicity and circumvents preexisting immunity to adenovirus in mice characterization of a permissive epitope insertion site in adenovirus hexon using multivalent adenoviral vectors for hiv vaccination identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain production and release testing of ovine atadenovirus vectors generation of recombinant adenovirus using the escherichia coli bj recombination system severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice preparation and titration of cscl-banded adenovirus stocks evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy construction of capsidmodified recombinant bovine adenovirus type this work was supported by grants from the canadian swine health board and the saskatchewan ministry of agriculture. we thank the staff of the vido-intervac animal care unit for immunizations of rabbits. this paper was published with the permission of the director of vido-intervac, journal series no. . the authors confirm that this article content has no conflict of interest. key: cord- -psguw w authors: feng, jianyu; guo, hong; li, sen; lu, tun title: a study of the mechanism of the chaperone-like function of an scfv of human creatine kinase by computer simulation date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: psguw w a new application of antibodies is to use them as macromolecular chaperones. protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. however, not all antibodies that bind to one antigen could act as a chaperone. experiments show that some screened anti-human creatine kinase single chain antibodies (scfv) could assist in the folding and stabilizing of the enzyme, while others could not. we built the model of the single chain antibody (scfv-a ) that increased the stability of human creatine kinase (hck) by the homology modeling method. epitopes of human creatine kinase were predicted by computer and then the binding of scfv-a and hck was modeled with computer. the calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scfv-a -hck complex. based on the above study we gave an explanation about how scfv-a could act as a macromolecular chaperone assisting the folding of hck. this study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies' chaperone-like function. in recent years, a number of human diseases, such as alzheimer's, huntington's, parkinson's, and creutzfeldt-jakob's diseases, were reported to be related to the misfolding and aggregation of proteins [ , ] . molecular chaperones are a kind of protein that are capable of assisting nascent peptides in correctly folding to functional proteins by binding to the folding intermediate to avoid kinetic traps, suppressing aggregation of the substrate [ , ] . traditional molecular chaperones could be classified according to their molecular weights and sequences to families such as hsp , hsp , hsp and nucleoplasmin. they have low specificity and react with many kinds of proteins. the low specificity of conventional molecular chaperones enables them to help many house-keeping proteins simultaneously. but a protein-misfolding disease might be caused by only one specie of protein which carries point mutants while most other housekeeping proteins are normal [ ] . thus the use of traditional molecular chaperones as therapeutic molecules for misfolding diseases may have problems such as low efficiency and undesired side-effects. a new field in development is to design or screen specific macromolecules which could be used as chaperones for target proteins, inhibiting their misfolding or coagulation to cure the related protein-misfolding diseases [ ] [ ] [ ] . antibodies are the most common macromolecules that can bind specifically to target proteins. previous researches had shown that some antibodies could excert a chaperone-like function on their antigens [ ] . therefore antibodies with a chaperone-like function were considered as the therapeutic drug candidates for protein misfolding diseases because they only affect mutant proteins, leaving normal proteins intact. in addition, antibodies with a chaperone-like function were helpful research tools for protein folding researches. human creatine kinase (hck) is a protein of important physiological function, which is closely related to intracellular energy operation, muscle contraction and atp regeneration [ ] . according to existing researches in the folding of hck, the dysfunction of hck could be a highly possible pathogenic factor of many serious diseases [ , ] . our previous studies [ ] indicate that hck expressed in e. coli existed as inclusion bodies. antibodies produced by using hck expressed by e. coli as antigen could be used to study the renaturation of inclusion bodies, such as capturing the intermediates during the folding process of hck to study the structural characteristics of the intermediates. an scfv is a fragment of a conventional antibody which is constructed by connecting the variable domains of the antibody heavy chain and the light chain by a segment of linker peptide. scfvs with high affinity and specificity to their antigens had been isolated from phage display libraries by many groups [ ] [ ] [ ] [ ] . schlattner [ ] have successfully isolated several scfv clones from a human antibody phage display library that recognize cytosolic bb-ck. in our previous work [ ] several scfvs had been screened out from a phage library using recombinational hck as antigen. only one of the scfvs named scfv-a has a significant chaperonelike function, preventing the aggregational precipitation of hck during its folding and accelerating its recovery to nature conformation. in order to comprehend the unique chaperone property of scfv-a , the binding between scfv-a and hck must be analyzed. the top priority would be identifying the portion of hck bound by scfv-a . molecular docking by computer has been widely used in the study of the binding mechanism of protein-protein or proteinligand systems, including themes such as protein-folding mechanism [ , ] , protein-protein surface modification [ , ] , improving the binding between proteins and polypeptides or small molecules [ ] [ ] [ ] . the number of degrees of freedom of protein-protein interactions was so large that no docking program nowadays can search the conformational space thoroughly even at the classical mechanics level of theory. therefore, no current software is able to correctly dock two proteins ab initio. one approach to reduce the search space for protein-protein interactions is to predict protein-protein interaction sites first and then use the orientational docking method to test the possibility of the binding. this is the approach used in this work. as to antigen-antibody binding, the parts of the antibodies bound to antigens were defined as cdr regions (complementarity determining regions) which could be identified by sequence analysis, while the interaction sites on the antigens were called the epitopes. many bioinformatics software tools had been developed to predict epitopes. b-cell epitopes typically belong to one of the two classes: linear (continuous) epitopes or conformational (discontinuous) epitopes. at present the prediction methods for linear epitopes had been extensively studied and the prediction accuracy had been improved to be as high as . % [ ] . thus linear epitopes prediction programs could be used to predict the potential binding sites of hck. one technique recently developed to screen potential linear epitopes was the peptide array membrane technique [ ] . peptide fragments covering the whole sequence of a protein antigen were synthesized and spotted on the membrane, the membrane was then treated with antibodies. after washing away the unbound antibodies and labeling the bound antibodies with secondary chromogenic antibodies, the peptide segments of potential epitopes could be identified. such identified peptides were only potential epitopes because when they were fixed on the membrane their conformation might not be the same as when they were parts of a protein. but we expected more reliable results could be deduced by combining the two methods. after the potential interaction sites of both interacting proteins had been identified, molecule docking software packages were used to generate d models for the scfv-antigen complexes. the quality of the d complex models can be used to verify the validity of the previous prediction based on d sequence. the chaperone-like function of the antibody was explained by investigating their interactions. the steps of this research were summarized as below. first, the three-dimensional structure of scfv-a was constructed by the homology modeling method. then potential epitopes of hck were predicted by software tools and the peptide array membrane experiment. the d models of scfv-a -hck complex were built by orientational docking. finally, we analyzed the interaction between hck and scfv-a and speculated the mechanism underlying the chaperone-like function of the scfv-a . this research also provided a new approach for studying antibodyantigen interactions based on the peptide array membrane experiment and bioinformatics software tools. the cdrs and the linker of scfv-a were inferred from the alignment as shown in figure . the model of the antibody was constructed by the modeller v program as shown in figure a . the distributions of w-y dihedral angles of the backbone were checked by the procheck program [ ] . a ramachandran map was used to describe the dihedral angles of the low-energy conformation ( figure b ). it showed that . % of the residues fell into the best area, . % of the residues into the other license area, . % of the residues into the permitted area, . % of the residues into the not allowed area. therefore, dihedral angles of the structure of the model were reasonable. for . % residues the d- d compatibility score is greater than . by verify- d [ ] (a high score indicated the compatibility of the sequence and the tertiary structure is good). the amino acids with scores less than . were gly -lys which belonged to the linker segment ( figure c ). since no template was used for modeling the linker the poorer model result of this area was expected. the above evaluations verified that the three-dimensional model of scfv-a was reasonable. the peptide array membrane was shown in figure . each square contained a mer peptide. first set of peptides covered hck sequence was spotted in squares a to m . peptides at the spots n to t were the peptides picked from first set in which first amino acid sequence numbers were odd number; this just served as a repeat experiment. after secondary antibodies and chromogenic substrates were added, six sets of continuous spots were developed. each set of the continuous spots corresponds to a peptide segment of hck. spots a -a correspond to asn -tyr of hck; spots of e -e correspond to ser -lys of hck; spots of f -f correspond to lys -met of hck; spots of i -i correspond to glu -leu of hck; spots of i -i correspond to lys -asn of hck; spots of k -k correspond to thr -thr of hck. these six peptides were potential scfv-a binding sites on hck. there were linear epitopes predicted by ellipro [ ] and linear epitopes predicted by fbcpred [ ] . these predicted epitopes were potential binding sites for all antibodies. for example, some predicted peptides might be the epitopes for anti-hck antibodies with no chaperone-like functions. there were a total of eight overlapping epitopes in the two groups of epitopes predicted by both methods. these eight epitopes were shown in table . for these eight peptides, four peptides fell in the nterminal domain, one peptide fell in the linker area and three fell in the c-terminal domain. comparing to the peptide array membrane experiment results, there were two overlapping sequences, lys -met and lys -asn , which were positive in both methods. an orientational docking of scfv-a and hck epitopes was carried out by zdock [ ] docking server. if we combined the results from peptide array membrane experiment and bioinformatics predictions, we could reduce the workload by only docking the two overlapping peptides. for experimental completeness, we did the orientational docking for all of the six peptides screened out by the peptide array experiment. zdock did not generate any reasonable models for four of the six peptides (ser -lys , glu -leu , thr -thr , asn -tyr ). it was interesting because it agreed with the bioinformatics prediction that they were not epitopes. to look into the details of the six peptides, we investigated their structure feature. as shown in figure , the first three peptides were embedded inside the hck monomers, thus they were not accessible by scfv-a . for peptide asn -tyr , it was buried between the dimerization interfaces of the two hck monomers therefore it was also inaccessible for scfv-a . only the rest two peptides (lys -met and lys -asn could be docked to scfv-a by zdock server and two complex models were obtained. because zdock used rigid body approximation, to obtain a more accurate protein complex structure, complex models generated by zdock were redocked by rosettadock [ ] . which implemented the flexible docking method and came with a more accurate scoring function. rosettadock generated new complex models for each of the two epitopes respectively. the output results were ranked by docking scores. we discarded models in which the binding sites of the antibody were outside the cdr regions and picked the best complex models by the best rank scores. two final complex models for the two epitopes were named as complex-i and complex-ii respectively and shown in figure . antibodies and their functional derivatives such as scfv and fab are known for their specific binding and have already been used in protein folding researches [ ] . antibodies with a chaperone-like function had the potential to be used as therapeutic agents for misfolding diseases. more researches need to be done to elucidate the mechanism of antibodies' chaperone-like function. a protein antigen usually carries more than one epitope, therefore an antigen can interact with several different types of antibodies. obviously the different types of antibodies have different impacts on the antigen's properties. we had previously screened out several scfvs for hck and one of them named scfv-a showed strong chaperone-like activity. to elucidate why scfv-a had the chaperone-like function, we combined computer modeling and peptide array membrane technique to study the interactions between scfv-a and hck. firstly, bioinformatics software tools were used to predict the interacting sites between scfv-a and hck and then the results were combined with the peptide array membrane experiment results to build the d models of the binding complex. the prediction of protein-protein interactions by computer is still a challenging subject, and it is inappropriate to rely entirely on the energy scoring function to filter the results directly. thus, we used the orientational docking method followed by redocking, the cumulated results were then sorted by the scoring function to get reliable protein-protein binding models. first the binding sites of the antigen and the antibody were predicted respectively. after that the two proteins were orientationally docked according to the predicted binding sites. sequence based epitope prediction was still difficult especially for the conformational epitopes. we combined the prediction results of two different software tools, and only those epitopes reported by both methods were considered as potential epitopes. the accuracy of each method was % (fbcpred) and % (ellipro) respectively, so the accuracy of the prediction of the two methods combined could reach %. the number of predicted epitopes was further reduced by comparing them with the peptide array membrane experiment results. the epitopes we studied were linear epitopes, and for linear peptide epitopes, the binding solely depends on amino acid sequences. if an antibody could bind to a linear peptide epitope, it will bind to the peptide irrespective of whether the peptide was on the protein, on the folding intermediate or on the membrane. consequently only the epitopes which were positive in peptide array membrane experiment were selected for further modeling. two antigenantibody model complexes were screened out as the final result. there are two major entry points for scfv-a to exert its chaperone-like function on hck, on the hck folding intermediate or on the folded hck. yan et al found that the c-terminal domain of hck was more likely to be the binding site during rabbit muscle ck (rmck) aggregation [ ] and the linker played a crucial role in rmck folding [ , ] . after further investigate, they found that the linker act as a lid to protect the hydrophobic side chains of phe and val against exposure to solvent [ ] . in complex model-i, scfv-a bound to the opposite site of hck thus has not impact on these residues (figure a ). in complex model-ii, the binding position of scfv-a was close to the two hydrophobic residues and actually formed a bulky protrusion (figure b) , therefore for complex model-ii, scfv-a bound to hck would hinder the aggregation of hck by steric effects.webb [ ] found that the c-terminal portion of the chick ck (gly to lys ) was much more resistant to digestion than the n-terminal portion (pro to gly ), and the middle of the hck sequence (arg to arg ) was most sensitive to proteolysis. they proposed that the folding intermediate consisted of a folded c-terminal domain and a partially folded n-terminal domain separated by an unfolded central linker. gross et al.'s studies suggested that the c-terminal fragment ( - ) of mitochondrial ck represented an autonomous folding unit, and that the folding of the c-terminal domain might precede the conformational stabilization of the n-terminal moiety in vivo [ ] . the binding site of complex model-i was located between these two terminal domains, which indicated that in complex model-i, scfv-a would not significantly interfere with the major folding path ways of the two domains. it may stabilize the folded c-terminal domain and assist in the folding of the nterminal domain. there was a v endoproteinase sensitive fragment in the chick ck intermediate. when the chick ck had been resurrected, this fragment resisted to the proteinase, since it became less exposed in the folded kinase [ ] . therefore, this fragment can be regarded as a characteristic part of the creatine kinase intermediate. this fragment identified by v endoproteinase was ifkkag [ ] , the corresponding sequence of hck was ile -gly . it is interesting that in complex model-ii, scfv-a also bound to this segment which indicated that scfv-a could bind with the intermediate and participate in hck folding or resurrecting process. to summarize, in complex model-ii scfv-a can exhibit a chaperone-like function either by hindering the aggregation by the steric effects or by assisting the folding of hck and stabilizing the folded conformation. in complex model-i, scfv-a exhibits chaperone-like function by assisting the folding of hck and stabilizing the whole structure. in this study we combined computer modeling and the peptide array membrane method to investigate the interaction between scfv-a and hck. the mechanisms underlying scfv-a 's chaperone-like function were discussed. the traditional method of studying antigen-antibody interactions is by constructing many antigen mutants and observing the changes of the binding activity. the whole process is tedious and time consuming. epitopescanning by peptide array membrane method has been made cheap and fast nowadays, our work demonstrated that the combination of the peptide array membrane technique with bioinfomatics methods could be a more efficient approach for epitope mapping. for the two complex models we proposed, further study can be done to verify the models by engineering point mutants on the predicted epitopes of hck and investigating the change of the system's behaviors. homology modeling in our study was done in the following three steps: ( ) speculating the linker, the light and the heavy chain segments of the antibody. the sequence of scfv-a was used to do a blast search in the non-redundant protein sequences database of ncbi. a scfv sequence (id: acz . ) [ ] which had the highest score and the lowest e value was picked out to be used as reference. the sequences of acz . and scfv-a were aligned by clustalx . [ ] and each area of the scfv-a sequence was appointed by comparing the sequence with acz . . ( ) homology modeling templates preparation. since linker peptides of the single chain antibody were often absent in the pdb database [ ] , the sequence of the linker peptide would be excluded when aligned with the template antibody. the sequence of scfv-a without linker was used to search the pdb database. the light chain and the heavy chain were processed separately. we obtained a template ( dd [ ] ) with a higher identity which was a fab antibody fragment. the sequence identity of the light chain of the fab was %, while that of the heavy chain was %. both chains of the fab were used as templates for building the d model of scfv-a by modeller v [ ] . ( ) optimization of the model. since the linker was modeled without an appropriate template, optimization work was focused on the linker. the dope-based loop modeling protocol [ ] was used to optimize the linker structure. the cdrs are the major region of the antibodies that bind to antigens. since the cdrs of the scfv acz . was known, the cdrs of scfv-a was inferred from the alignment result of the two sequences ( figure ). the resulted cdr assignment was checked to be complied with the kabat numbering rules [ ] . the crystal structure of hck was available in pdb file i e [ ] . hck is a dimmer in nature but the i e only contains a each peptide epitope was noted by its sequence region on hck. there were total eight epitopes predicted by both software and their overlapping region was listed at the right column. doi: . /journal.pone. .t figure . positions of the peptides screened out by the peptide array membrane method on the hck d structure. the six experimental positive peptides were colored in salmon and represented by using the stick model. as shown in the picture, asn -tyr is at the position of dimerization interface of the two monomers; ser -lys is completely embedded inside the structure; glu -leu is in a dent of the surface; lys -asn which contains v endoproteinase fragment was also in a dent of the structure; lys -met is located at the bottom of the structure; thr -thr is located in the largest groove of the structure. doi: . /journal.pone. .g monomer and the structure of two peptide fragments, met -his and gly -gly , was missing. thus we use another ck structure, the u r for rabbit ck in pdb as the homology modeling template. the sequence identity between rabbit ck and hck is % and the u r file contains the structure of the two missing fragments peptides of the i e file. we used fbcpred [ ] based on the machine learning method and ellipro [ ] based on the d structure for epitope prediction. an epitope predicted by both methods would be regarded as a potential epitope. as for fbcpred, default parameters were adopted for linear epitope prediction, the length of the linear epitopes was set to , and the specificity was % as default. no parameter was needed for ellipro. mer peptides from the hck sequence were synthesized on nitrocellulose membrane as individual spots. peptide sequences were started from hck n-terminal and each subsequent peptides shift one position to the right. a set of such peptides completely sampled the whole hck sequence and formed an array on the membrane. the peptide array membrane was incubated with purified scfv-a antibodies in the mp buffer ( mm tris-hcl, ph . , mm nacl, . mm kcl, . % tween , % sucrose) overnight at uc with gentle shaking. unbound antibodies were removed with tbs ( uc) andthe antibodies bound were electro-transferred onto a pvdf membrane using a semi-dry blotter ( . ma/cm , three hours). the membrane was blocked with % non-fat dry milk for h and incubated with the primary antibody (anti-v antibody) overnight at uc. after being washed with tbs buffer for three times, the membrane was incubated with the alkaline phosphatase-conjugated secondary antibodies for hour and developed using the enhanced chemiluminescence method. the zodck server was used to dock scfv-a to hck. the zdock server allowed user to pick contact and blocking residues of the proteins for docking. the hck residues of the predicted epitopes were chosen as contact residues for docking. there were six linear peptides exhibiting positive reactivity with scfv-a in the peptide array membrane experiment and the corresponding portions of hck were designated as the orientational docking sites of hck. no blocking residues of hck were selected during docking, but for scfv-a , residues of the linker segment were selected as the blocking residues. rosettadock was used to optimize the models build by zdock because it implemented the flexible docking method and had a scoring function of better performance. the perturbation docking method of the rosetta-dock program was applied and the three default perturbation parameters were used. one thousand models were produced for each selected zdock model complex. conceived and designed the experiments: jyf tl. performed the experiments: hg sl. analyzed the data: jyf tl. contributed reagents/ materials/analysis tools: sl. wrote the paper: jyf tl. figure . the scfv-a -hck complexes. model i is shown at left. model ii is shown at right.scfv-a is colored in marine. hck is shown as dimmer. one hck monomer is colored in lime. the n-terminal of the other hck monomer is colored in slate and c-terminal in cyan, the linker is colored in orange. the v endoproteinase sensitive fragment is colored in magentas. the binding residues are represented by using the stick model. the phe , val are represented using the sphere model. doi: . /journal.pone. .g chaperone-like function of an scfv plos one | www.plosone.org protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions targeting misfolded proteins to fight neurodegenerative diseases molecular chaperones: proteins essential for the biogenesis of some macromolecular structures molecular chaperones: assisting assembly in addition to folding effects of the single point genetic mutation d g on muscle creatine kinase activity, structure and stability autoantibodies against chaperonin cct in human sera with rheumatic autoimmune diseases: comparison with antibodies against other hsp family proteins heat shock protein : the cancer chaperone. heat shock proteins in cancer therapeutic approaches to protein-misfolding diseases antibodies as specific chaperones delayed-onset muscle soreness does not reflect the magnitude of eccentric exercise-induced muscle damage. scandinavian journal of medicine the creatine kinase/creatine connection to alzheimer's disease: ck inactivation, app-ck complexes and focal creatine deposits mitochondrial creatine kinase in human health and disease a scfv antibody against human muscle creatine kinase as fully functional chaperone the selection of intracellular antibodies design and use of a phage display library the use of phage display for the development of tumour targeting agents isolation of high affinity human antibodies directly from large synthetic repertoires isoenzyme-directed selection and characterization of anti-creatine kinase single chain fv antibodies from a human phage display library design, structure and stability of a hyperthermophilic protein variant refolding the engrailed homeodomain: structural basis for the accumulation of a folding intermediate automated design of specificity in molecular recognition computational design of a new hydrogen bond network and at least a -fold specificity switch at a protein-protein interface computational design of second-site suppressor mutations at protein-protein interfaces computational design of affinity and specificity at protein-protein interfaces hsp / cdc chaperone/co-chaperone complex, a novel junction anticancer target elucidated by the mode of action of herbal drug withaferin a predicting flexible length linear b-cell epitopes peptide microarrays for the characterization of antigenic regions of human chromogranin a aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr a method to identify protein sequences that fold into a known three-dimensional structure ellipro: a new structure-based tool for the prediction of antibody epitopes zdock: an initial-stage protein docking algorithm protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations conformational change in the cterminal domain is responsible for the initiation of creatine kinase thermal aggregation role of the linker between the n-and c-terminal domains in the stability and folding of rabbit muscle creatine kinase mutation of the conserved asp in the linker impedes creatine kinase reactivation and refolding chaperone-like effect of the linker on the isolated c-terminal domain of rabbit muscle creatine kinase protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase the tryptophan residues of mitochondrial creatine kinase: roles of trp- , trp- , and trp- in active-site and quaternary structure formation monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase neutralizing antibodies of botulinum neurotoxin serotype a screened from a fully synthetic human antibody phage display library multiple sequence alignment using clustalw and clustalx the protein data bank structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody comparative protein structure modeling using modeller sequences of proteins of immunological interest: us dept. of health and human services structure of human muscle creatine kinase key: cord- -d iwkatn authors: henry, kevin a.; arbabi-ghahroudi, mehdi; scott, jamie k. title: beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: d iwkatn for the past years, phage display technology has been an invaluable tool for studies of protein–protein interactions. however, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. this review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. the filamentous bacteriophage (genera inovirus and plectrovirus) are non-enveloped, rod-shaped viruses of escherichia coli whose long helical capsids encapsulate a single-stranded circular dna genome. subsequent to the independent discovery of bacteriophage by twort ( ) and d 'hérelle ( ) , the first filamentous phage, f , was isolated in loeb ( ) and later characterized as a member of a larger group of phage (ff, including f , m , and fd phage) specific for the e. coli conjugative f pilus (hofschneider and mueller-jensen, ; marvin and hoffmann-berling, ; zinder et al., ; salivar et al., ) . soon thereafter, filamentous phage were discovered that do not use f-pili for entry (if and ike; meynell and lawn, ; khatoon et al., ) , and over time the list of known filamentous phage has expanded to over members (fauquet et al., ) , including temperate and gram-positivetropic species. work by multiple groups over the past years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in marvin, ; rakonjac et al., ; rakonjac, ) . in the mid- s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by smith and colleagues (smith, ; parmley and smith, ) . based on the ideas described in parmley and smith ( ) , groups in california, germany, and the uk developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (bass et al., ; devlin et al., ; mccafferty et al., ; scott and smith, ; breitling et al., ; kang et al., ) . this technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. many excellent reviews are available on phage-display libraries and their applications (kehoe and kay, ; bratkovic, ; pande et al., ) . however, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. these tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. a final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( table ) . the display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. display may be achieved by fusing dna encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type display on pviii, type display on piii, etc.), resulting in fully recombinant phage. much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type display if both recombinant and wild-type pviii genes are on the phage genome, type + display if the parmley and smith ( ), mcconnell et al. ( ) , rondot et al. ( ) hybrid (type and + systems) type + system < smith and scott ( ) , smith and petrenko ( ) pvi hybrid (type + system) yes < > kda hufton et al. ( ) pvii fully recombinant (type system) no ∼ > kda kwasnikowski et al. ( ) hybrid (type + system) yes < gao et al. ( ) pviii fully recombinant (landscape phage; type system) no ∼ - residues kishchenko et al. ( ) , petrenko et al. ( ) hybrid (type and + systems) type + system ∼ - > kda scott and smith ( ) , greenwood et al. ( ) , smith and fernandez ( ) pix fully recombinant (type + * system) yes ∼ > kda gao et al. ( ) hybrid (type + system) no < gao et al. ( ) , shi et al. ( ) , tornetta et al. ( ) asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. the copy number depends on polypeptide size; typically < copy per phage particle but for pviii peptide display can be up to ∼ % of pviii molecules in hybrid virions. the total number of pviii molecules depends on the phage genome size; one pviii molecule is added for every . nucleotides in the viral genome. recombinant gene is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type * + display). by far the most commonly used coat proteins for display are the major coat protein, pviii, and the minor coat protein, piii, with the major advantage of the former being higher copy number display (up to ∼ % of recombinant pviii molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number ( - per phage particle). while pviii display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than per virion (sidhu et al., ) . for the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in e. coli cells. early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (meynell and lawn, ) and that only the major coat protein, pviii, and the minor coat protein, piii, are targeted by antibodies (pratt et al., ; woolford et al., ) . thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la cruz et al. ( ) . the phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pviii display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pviii or piii (de la cruz et al., ) . as library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; geysen et al., ; knittelfelder et al., ) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; table ) , including malaria and human immunodeficiency virus type (hiv- ). when displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein were immunogenic in mice and rabbits (de la cruz et al., ; greenwood et al., ; willis et al., ; demangel et al., ) , and antibodies raised against the latter cross-reacted with the full-length protein. various peptide determinants (or mimics thereof) of hiv- gp , gp , gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (minenkova et al., ; di marzo veronese et al., ; de berardinis et al., ; scala et al., ; chen et al., ; van houten et al., van houten et al., , , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di marzo veronese et al., ; scala et al., ) . the list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( table ) . while in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (irving et al., ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (van regenmortel, ); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. more recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. immunization with phage displaying alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (frenkel et al., (frenkel et al., , esposito et al., ; tanaka et al., ) , possibly reduced amyloid plaque formation in mice (frenkel et al., ; solomon, ; esposito et al., ) , and may have helped maintain cognitive abilities in a transgenic mouse model of alzheimer's disease (lavie et al., ) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. yip et al. ( ) found that antibodies raised in mice against an erbb /her peptide could inhibit breast-cancer cell proliferation. phage displaying peptide ligands of an anti-ige antibody elicited antibodies that bound purified ige molecules (rudolf et al., ) , which may be useful in allergy immunotherapy. several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. for example, immunization with phage displaying follicle-stimulating hormone peptides on pviii elicited antibodies that impaired the fertility of mice and ewes (abdennebi et al., ) . phage displaying or chemically rubinchik and chow ( ) conjugated to sperm antigen peptides or peptide mimics (samoylova et al., a,b) and gonadotropin-releasing hormone (samoylov et al., ) are also in development. for the most part, peptides displayed on phage elicit antibodies in experimental animals ( table ) , although this depends on characteristics of the peptide and the method of its display: piii fusions tend toward lower immunogenicity than pviii fusions (greenwood et al., ) possibly due to copy number differences (piii: - copies vs. pviii: estimated at several hundred copies; malik et al., ) . in fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (bsa) and keyhole limpet hemocyanin (klh; melzer et al., ; su et al., ) , and has comparatively few endogenous b-cell epitopes to divert the antibody response from its intended target (henry et al., ) . excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. the overall picture is considerably bleaker than that painted by table , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. irving et al. ( ) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. while the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (henry et al., ) . this may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see immunological mechanisms of vaccination with filamentous phage below). the filamentous phage has been used to a lesser extent as a carrier for t-cell peptide epitopes, primarily as fusion proteins with pviii ( table ) . early work, showing that immunization with phage elicited t-cell help (kölsch et al., ; willis et al., ) , was confirmed by several subsequent studies (de berardinis et al., ; ulivieri et al., ) . from the perspective of vaccination against infectious disease, de berardinis et al. ( ) showed that a cytotoxic t-cell (ctl) epitope from hiv- reverse transcriptase could elicit antigen-specific ctls in vitro and in vivo without addition of exogenous helper t-cell epitopes, presumably since these are already present in the phage coat proteins (mascolo et al., ) . similarly, efficient priming of ctls was observed against phage-displayed t-cell epitopes from hepatitis b virus (wan et al., ) and candida albicans (yang et al., a; wang et al., wang et al., , d , which, together with other types of immune responses, protected mice against systemic candidiasis. vaccination with a combination of phagedisplayed peptides elicited antigen-specific ctls that proved effective in reducing porcine cysticercosis in a randomized controlled trial (manoutcharian et al., ; morales et al., ) . while the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (plotkin, ) , in certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (gram et al., ) . e. coli f a-g adhesin (van gerven et al., ) , hepatitis b core antigen (bahadir et al., ) , and hepatitis b surface antigen (balcioglu et al., ) all elicited antibody responses when displayed on piii, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. phage displaying schistosoma mansoni glutathione s-transferase on piii elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (rao et al., ) . two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. immunization with phage displaying the e idiotype scfv (mimicking a vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from vibrio anguillarum challenge (xia et al., ) . a chemically linked phage-bcl tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a b-cell lymphoma model (roehnisch et al., ) , and was welltolerated and immunogenic in patients with multiple myeloma (roehnisch et al., ) . one study of dna vaccination with an anti-laminarin scfv found that dna encoding a piii-scfv fusion protein elicited stronger humoral and cell-mediated immune responses than dna encoding the scfv alone (cuesta et al., ) , suggesting that under some circumstances, endogenous phage t-cell epitopes can enhance the immunogenicity of associated proteins. taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional t-cell help to displayed or conjugated proteins. however, the low copy number of piii-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (figure ) . the phage particle is immunogenic without adjuvant in all species tested to date, including mice (willis et al., ) , rats (dente et al., ) , rabbits (de la cruz et al., ) , guinea pigs (frenkel et al., ; kim et al., ) , fish (coull et al., ; xia et al., ) , non-human primates (chen et al., ) , and humans (roehnisch et al., ) . various routes of immunization have been employed, including oral administration (delmastro et al., ) as well as subcutaneous (grabowska et al., ) , intraperitoneal (van houten et al., ) , intramuscular (samoylova et al., a) , intravenous (vaks and benhar, ) , and intradermal injection (roehnisch et al., ) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. antibodies are generated against only three major sites on the virion: (i) the surface-exposed n-terminal ∼ residues of the pviii monomer lattice (terry et al., ; kneissel et al., ) ; (ii) the n-terminal n and n domains of piii (van houten et al., ) ; and (iii) bacterial lipopolysaccharide (lps) embedded in the phage coat (henry et al., ) . in mice, serum antibody titers against the phage typically reach : - : after - immunizations, and are maintained for at least year postimmunization (frenkel et al., ) . primary antibody responses against the phage appear to be composed of a mixture of igm and igg b isotypes in c bl/ mice, while secondary antibody responses are composed primarily of igg and igg b isotypes, with a lesser contribution of igg c and igg isotypes (hashiguchi et al., ) . deletion of the surface-exposed n and n domains of piii produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van houten et al., ) . figure | types of immune responses elicited in response to immunization with filamentous bacteriophage. as a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. the phage likely activates t-cell independent antibody responses, either via phage-associated tlr ligands or cross-linking by the pviii lattice. after processing by antigen-presenting cells, phage-derived peptides are presented on mhc class ii and cross-presented on mhc class i, resulting in activation of short-lived ctls and an array of helper t-cell types, which help prime memory ctl and high-affinity b-cell responses. frontiers in microbiology | www.frontiersin.org although serum anti-phage antibody titers appear to be at least partially t-cell dependent (kölsch et al., ; willis et al., ; de berardinis et al., ; van houten et al., ) , many circulating pviii-specific b cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates t-cell-independent b-cell responses in addition to highaffinity t-cell-dependent responses (murira, ) . filamentous phage particles can be processed by antigen-presenting cells and presented on mhc class ii molecules (gaubin et al., ; ulivieri et al., ) and can activate t h , t h , and t h helper t cells (yang et al., a; wang et al., d) . anti-phage t h responses were enhanced through display of ctla- peptides fused to piii (kajihara et al., ) . phage proteins can also be cross-presented on mhc class i molecules (wan et al., ) and can prime two waves of ctl responses, consisting first of short-lived ctls and later of long-lived memory ctls that require cd + t-cell help (del pozzo et al., ) . the latter ctls mediate a delayed-type hypersensitivity reaction (fang et al., ; del pozzo et al., ) . the phage particle is self-adjuvanting through multiple mechanisms. host cell wall-derived lps enhances the virion's immunogenicity, and its removal by polymyxin b chromatography reduces antibody titers against phage coat proteins (grabowska et al., ) . the phage's singlestranded dna genome contains cpg motifs and may also have an adjuvant effect. the antibody response against the phage is entirely dependent on myd signaling and is modulated by stimulation of several toll-like receptors (hashiguchi et al., ) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (molenaar et al., ) , particularly of the liver and spleen, where it is retained for days (zou et al., ) , potentially activating marginal-zone b-cell responses. thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former soviet union and eastern europe (reviewed in sulakvelidze et al., ) . the filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of inovirus and plectrovirus includes many pathogens of medical importance, including salmonella, e. coli, shigella, pseudomonas, clostridium, and mycoplasma species. in an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "trojan horse" to introduce various antibacterial agents into cells. m and pf phage engineered to express either bglii restriction endonuclease (hagens and blasi, ; hagens et al., ) , lambda phage s holin (hagens and blasi, ) or a lethal catabolite gene activator protein (moradpour et al., ) effectively killed e. coli and pseudomonas aeruginosa cells, respectively, with no concomitant release of lps (hagens and blasi, ; hagens et al., ) . unfortunately, the rapid emergence of resistant bacteria with modified f pili represents a major and possibly insurmountable obstacle to this approach. however, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (hagens et al., ) or phage engineered to repress the cellular sos response (lu and collins, ) . filamentous phage f infection inhibited early stage, but not mature, biofilm formation in e. coli (may et al., ) . thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. more advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (figure ) . the first work in this area showed as proof-of-concept that phage encoding a gfp expression cassette and displaying a her specific scfv on all copies of piii were internalized into breast tumor cells, resulting in gfp expression (poul and marks, ) . m or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of staphylococcus aureus growth than high-concentration free chloramphenicol (yacoby et al., ; vaks and benhar, ) . m phage loaded with doxorubicin and displaying a targeting peptide on piii specifically killed prostate cancer cells in vitro (ghosh et al., a) . tumorspecific peptide:pviii fusion proteins selected from "landscape" phage (romanov et al., ; abbineni et al., ; fagbohun et al., fagbohun et al., , lang et al., ; wang et al., a) were able to target and deliver sirna-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (jayanna et al., a; wang et al., a wang et al., ,b,c, b bedi et al., bedi et al., , bedi et al., , ; they were non-toxic and increased tumor remission rates in mouse models (jayanna et al., b; wang et al., b,c) . using the b -ova tumor model, eriksson et al. ( ) showed that phage displaying peptides and/or fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (eriksson et al., ) . phage displaying an scfv against β-amyloid fibrils showed promise as a diagnostic (frenkel and solomon, ) and therapeutic (solomon, ) reagent for alzheimer's disease and parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (ksendzovsky et al., ) . similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (rakover et al., ) . the advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. first and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating lps, in the range of ∼ - endotoxin units (eu)/ml (boratynski et al., ; branston et al., ) , which have the potential to cause severe adverse reactions. lps is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (smith and gingrich, ; branston et al., ) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (boratynski et al., ; zakharova et al., ) , polymyxin b chromatography (grabowska et al., ) , and treatment with detergents such as triton x- or triton x- (roehnisch et al., ; branston et al., ) . these strategies routinely achieve endotoxin levels of < eu/ml as measured by the limulus amebocyte lysate (lal) assay, well below the fda limit for parenteral administration of eu/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated lps which may be undetectable. a second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in dalmasso et al., ) or for detection of foodborne pathogens post-production (reviewed in schmelcher and loessner, ) . filamentous phage displaying a tetracysteine tag on piii were used to detect e. coli cells through staining with biarsenical dye . m phage functionalized with metallic silver were highly bactericidal against e. coli and staphylococcus epidermidis . biosensors based on surface plasmon resonance (nanduri et al., ) , piezoelectric transducers (olsen et al., ) , linear dichroism (pacheco-gomez et al., ) , and magnetoelastic sensor technology (lakshmanan et al., ; huang et al., ) were devised using filamentous phage displaying scfv or conjugated to whole igg against e. coli, listeria monocytogenes, salmonella typhimurium, and bacillus anthracis with limits of detection on the order of - bacterial cells/ml. proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (li et al., b) and eggs (chai et al., ) . the filamentous phage particle is enclosed by a rod-like protein capsid, ∼ nm long and nm wide, made up almost entirely of overlapping pviii monomers, each of which lies ∼ angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (henry et al., ) . the regularity of the phage pviii lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (figure ) . the most commonly used approach is functionalization of amine groups with nhs esters (van houten et al., (van houten et al., , yacoby et al., ) , although this can result in unwanted acylation of piii and any displayed biomolecules. carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (li et al., a) . carrico et al. ( ) developed methods to specifically label pviii n-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (ng et al., ) or enzymes (chen et al., ; hess et al., ) , but this can be cumbersome and is less general in application. for more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. it has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (welsh et al., ) . lee et al. ( ) engineered m phage to display a zns-binding peptide on piii and showed that, in the presence of zns nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament hess et al., ) , this pioneering figure | chemically addressable groups of the filamentous bacteriophage major coat protein lattice. the filamentous phage virion is made up of ∼ , - , overlapping copies of the -residue major coat protein, pviii, arranged in a shingle-type lattice. each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). the n-terminal residues generally exposed to the immune system for antibody binding are in bold underline. figure adapted from structural data of marvin, , freely available in pdb and scope databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (mao et al., (mao et al., , , nanoparticles , and nanocomposites (oh et al., ; chen et al., ) . using hybrid m phage displaying co o -and gold-binding peptides on pviii as a scaffold to assemble nanowires on polyelectrolyte multilayers, nam et al. ( ) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (nam et al., ) . the electrochemical properties of such batteries were further improved through piii-display of single-walled carbon nanotube-binding peptides (lee et al., ) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. phagebased nanomaterials have found applications in cancer imaging (ghosh et al., b; yi et al., ) , photocatalytic water splitting (nam et al., a; neltner et al., ) , light harvesting (nam et al., b; chen et al., ) , photoresponsive technologies (murugesan et al., ) , neural electrodes (kim et al., ) , and piezoelectric energy generation (murugesan et al., ) . thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. it is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. magnetic alignment of high-concentration filamentous phage in solution can partially order dna, rna, proteins, and other biomolecules for measurement of dipolar coupling interactions (hansen et al., (hansen et al., , dahlke ojennus et al., ) in nmr spectroscopy. because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in husimi, ; wichman and brown, ; kawecki et al., ; hall et al., ) . the filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. first and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on piii. libraries of variant fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (marks et al., ; bradbury et al., ) . however, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; koide and koide, ) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. iterative methods have been developed to combine computationally designed mutations (lippow et al., ) and circumvent the screening of combinatorial libraries, but these have had limited success to date. recently, esvelt et al. ( ) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (pace), which allows multiple rounds of evolution per day with little experimental intervention. the authors engineered m phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene iii expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone dna polymerases. by supplying limiting amounts of receptive e. coli cells to the engineered phage variants, esvelt et al. ( ) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. carlson et al. ( ) later showed that pace selection stringency could be modulated by providing small amounts of piii independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated piii variant that impairs infectivity in a dominant negative fashion. pace is currently limited to protein functions that can be linked in some way to the expression of a gene iii reporter, such as protein-protein interaction, recombination, dna or rna binding, and enzymatic catalysis (meyer and ellington, ) . this approach represents a promising avenue for both basic research in molecular evolution (dickinson et al., ) and synthetic biology, including antibody engineering. filamentous bacteriophage have been recovered from diverse environmental sources, including soil (murugaiyan et al., ) , coastal fresh water (xue et al., ) , alpine lakes (hofer and sommaruga, ) and deep sea bacteria (jian et al., ) , but not, perhaps surprisingly, the human gut (kim et al., ) . the environmental "phageome" in soil and water represent the largest source of replicating dna on the planet, and is estimated to contain upward of viral particles (ashelford et al., ; chibani-chennoufi et al., ; suttle, ) . the few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from to % of all viral particles (demuth et al., ; pina et al., ; hofer and sommaruga, ) . there was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (hofer and sommaruga, ) . environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. the existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (roux et al., ) and reclaimed and potable water (rosario et al., ) but have much higher frequencies in wastewater and sewage (cantalupo et al., ; alhamlan et al., ) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. there are no data describing the population dynamics of filamentous phage and their host species in the natural environment. at the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. this can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. temperate filamentous phage may also play a role in genome evolution (reviewed in canchaya et al., ) . perhaps the best-studied example of virulence modulation by filamentous phage is that of vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding ctxφ phage (waldor and mekalanos, ) . integration of ctxφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs φ, and a satellite filamentous phage, tlc-knφ (hassan et al., ) . thus, filamentous phage species interact and coevolve with each other in addition to their hosts. infection by filamentous phage has been implicated in the virulence of yersinia pestis (derbise et al., ) , neisseria meningitidis (bille et al., (bille et al., , , vibrio parahaemolyticus (iida et al., ) , e. coli :k :h (gonzalez et al., ) , xanthomonas campestris (kamiunten and wakimoto, ) , and p. aeruginosa (webb et al., ) , although in most of these cases, the specific mechanisms modulating virulence are unclear. phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen ralstonia solanacearum (yamada, ) . since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (lin et al., ) . finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (petrenko and makowski, ) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (curtis et al., (curtis et al., , . engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (curtis et al., ). the filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. while novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. kh and js conceived and wrote the manuscript. ma-g read the manuscript and commented on the text. evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization generating fsh antagonists and agonists through immunization against fsh receptor n-terminal decapeptides metagenomics-based analysis of viral communities in 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antigen mimotopes displayed on filamentous phage molecular evolution picks up the pace filamentous phages specific for the i sex factor design of specific immunogens using filamentous phage as the carrier uptake and processing of modified bacteriophage m in mice: implications for phage display genetically engineered phage harbouring the lethal catabolite gene activator protein gene with an inducer-independent promoter for biocontrol of escherichia coli inexpensive anti-cysticercosis vaccine: s pvac expressed in heat inactivated m filamentous phage proves effective against naturally acquired taenia solium porcine cysticercosis recognition by human sera and immunogenicity of hbsag mimotopes selected from an m phage display library characterization of molecular correlates of the chronic humoral immune response: clues towards eliciting broadly neutralizing antibodies against hiv characterization of filamentous bacteriophage pe infecting ralstonia solanacearum strains virusbased photo-responsive nanowires formed by linking site-directed mutagenesis and chemical reaction virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes stamped microbattery electrodes based on self-assembled m viruses biologically templated photocatalytic nanostructures for sustained light-driven water oxidation virus-templated assembly of porphyrins into light-harvesting nanoantennae spr biosensor for the detection of l. monocytogenes using phage-displayed antibody production of hydrogen using nanocrystalline protein-templated catalysts on m phage quantitative synthesis of genetically encoded glycopeptide libraries displayed on m phage graphene sheets stabilized on genetically engineered m viral templates as conducting frameworks for hybrid energy-storage materials affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of salmonella typhimurium detection of pathogenic bacteria using a homogeneous immunoassay based on shear alignment of virus particles and linear dichroism phage display: concept, innovations, applications and future antibody-selectable filamentous fd phage vectors: affinity purification of target genes potential applications of phage display to bioremediation a library of organic landscapes on filamentous phage abundance, morphology and distribution of planktonic viruslike particles in two high-mountain lakes correlates of protection induced by vaccination targeted gene delivery to mammalian cells by filamentous bacteriophage conditional lethal mutants of the small filamentous coliphage m . ii. two genes for coat proteins selection of antigenic and immunogenic mimics of hepatitis c virus using sera from patients towards a solution for hepatitis c virus hypervariability: mimotopes of the hypervariable region can induce antibodies crossreacting with a large number of viral variants filamentous bacteriophages: biology and applications, " in els (the encyclopaedia of life sciences) filamentous bacteriophage: biology, phage display and nanotechnology applications antigen-specific therapy of eae via intranasal delivery of filamentous phage displaying a myelin immunodominant epitope expression of a -kilodalton glutathione s-transferase antigen of schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential structural requirements for the activity of the mirb ferrisiderophore transporter of aspergillus fumigatus chemically linked phage idiotype vaccination in the murine b cell lymphoma model phage idiotype vaccination: first phase i/ii clinical trial in patients with multiple myeloma phage display selection of peptides that affect prostate carcinoma cells attachment and invasion a helper phage to improve single-chain antibody presentation in phage display metagenomic analysis of viruses in reclaimed water assessing the diversity and specificity of two freshwater viral communities through metagenomics recombinant expression and neutralizing activity of an mhc class ii binding epitope of toxic shock syndrome toxin- epitope-specific antibody response to ige by mimotope immunization some physical-chemical and biological properties of the rod-shaped coliphage m generation and characterization of phage-gnrh chemical conjugates for potential use in cat and dog immunocontraception phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife infective and inactivated filamentous phage as carriers for immunogenic peptides vaccination with filamentous bacteriophages targeting dec- induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants the use of filamentous bacteriophage fd to deliver hla-a -restricted peptides and to induce strong antitumor ctl responses selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv- -positive sera application of bacteriophages for detection of foodborne pathogens searching for peptide ligands with an epitope library de novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pix fusion proteins high copy display of large proteins on phage for functional selections filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface effect of dna copy number on genetic stability of phage-displayed peptides hydroxyapatite chromatography of phage-display virions phage display libraries of peptides and proteins displayed on filamentous phage generation of anti-β-amyloid antibodies via phage display technology towards alzheimer's disease vaccination filamentous bacteriophage as a novel therapeutic tool for alzheimer's disease treatment comparison of phage pviii and klh as vector in inducing the production of cytokines in c bl/ j mice bacteriophage therapy viruses in the sea a mimotope peptide of aβ fibril-specific antibodies with aβ fibrillation inhibitory activity induces anti-aβ conformer antibody response by a displayed form on an m phage in mice accessibility of peptides displayed on filamentous bacteriophage virions: susceptibility to proteinases antibody fab display and selection through fusion to the pix coat protein of filamentous phage an investigation on the nature of ultra-microscopic viruses antigenic properties of hcmv peptides displayed by filamentous bacteriophages vs. synthetic peptides in vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines presentation of the functional receptor-binding domain of the bacterial adhesin f a-g on bacteriophage m engineering filamentous phage carriers to improve focusing of antibody responses against peptides filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide basic research in hiv vaccinology is hampered by reductionist thinking lysogenic conversion by a filamentous phage encoding cholera toxin induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine crosspresentation of phage particle antigen in mhc class ii and endoplasmic reticulum marker-positive compartments bio-mimetic nanostructure self-assembled from au@ag heterogeneous nanorods and phage fusion proteins for targeted tumor optical detection and photothermal therapy enhanced tumor delivery and antitumor activity in vivo of liposomal doxorubicin modified with mcf- -specific phage fusion protein paclitaxel-loaded peg-pe-based micellar nanopreparations targeted with tumor specific landscape phage fusion protein enhance apoptosis and efficiently reduce tumors hybrid phage displaying slaqvkytsassi induces protection against candida albicans challenge in balb/c mice protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of candida albicans heat shock protein in c bl/ j mice enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein paclitaxel-loaded polymeric micelles modified with mcf- cell-specific phage protein: enhanced binding to target cancer cells and increased cytotoxicity cytoplasmic delivery of liposomes into mcf- breast cancer cells mediated by cell-specific phage fusion coat protein bacteriophage and phenotypic variation in pseudomonas aeruginosa biofilm development evidence for tilted smectic liquid crystalline packing of fd inovirus from x-ray fiber diffraction experimental evolution of viruses: microviridae as a model system immunological properties of foreign peptides in multiple display on a filamentous bacteriophage adsorption protein of bacteriophage fl: solubilization in deoxycholate and localization in the fl virion sensitive and selective bacterial detection using tetracysteine-tagged phages in conjunction with biarsenical dye phage display particles expressing tumor-specific antigens induce preventive and therapeutic anti-tumor immunity in murine p model development of a phage displayed disulfide-stabilized fv fragment vaccine against vibrio anguillarum high frequency of a novel filamentous phage, vcyφ, within an environmental vibrio cholerae population targeting antibacterial agents by using drug-carrying filamentous bacteriophages filamentous phages of ralstonia solanacearum: doubleedged swords for pathogenic bacteria prophylactic vaccination with phage-displayed epitope of c. albicans elicits protective immune responses against systemic candidiasis in c bl/ mice epitope mapping of mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes m phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors comparison of phage piii, pviii and gst as carrier proteins for peptide immunisation in balb/c mice spontaneous assembly of viruses on multilayered polymer surfaces characterization of murine coronavirus neutralization epitopes with phage-displayed peptides purification of filamentous bacteriophage for phage display using size-exclusion chromatography conformational mimicry of a chlamydial neutralization epitope on filamentous phage f , a rodshaped male-specific bacteriophage that contains dna biodistribution of filamentous phage peptide libraries in mice this work was supported by funding from the national research council of canada (kh, ma-g) and the canada research chair program (js). we thank jyothi kumaran and roger mackenzie for critical appraisal of the manuscript, and jasna rakonjac for inviting us to contribute it. this is national research council canada publication number . key: cord- - mxi dzi authors: memczak, henry; lauster, daniel; kar, parimal; di lella, santiago; volkmer, rudolf; knecht, volker; herrmann, andreas; ehrentreich-förster, eva; bier, frank f.; stöcklein, walter f. m. title: anti-hemagglutinin antibody derived lead peptides for inhibitors of influenza virus binding date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: mxi dzi antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. however, development, production and quality control of antibodies is expensive and time consuming. to circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza a spike glycoprotein. their binding properties were studied experimentally, and by molecular dynamics simulations. two peptide candidates showed binding to influenza a/aichi/ / h n . one of them, termed peb, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. peb matches best the conserved receptor binding site of hemagglutinin. peb bound also to other medical relevant influenza strains, such as human-pathogenic a/california/ / h n , and avian-pathogenic a/mute swan/rostock/r / h n . strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. the peptides and their derivatives are of great potential for drug development as well as biosensing. influenza a virus is an enveloped virus belonging to the orthomyxoviridae family. it can cause annual epidemics and infrequent pandemics [ ] . the spanish flu pandemic of as well as the asian flu of and the hongkong flu in pandemics caused the death of millions of people [ ] . in the pandemic swine origin influenza a h n virus as well as the outbreak of h n in china in has reminded the world of the threat of pandemic influenza [ ] [ ] [ ] [ ] . the genome of influenza virus consists of eight segmented negative rna strands. the envelope bilayer harbors the two spike glycoproteins hemagglutinin (ha) and neuraminidase (na), and the m proton channel. the homotrimeric ha is the most abundant protein on the viral surface. it mediates attachment to the host cell surface via binding to sialic acid (sa) residues of cellular receptors, and upon endocytic virus uptake it triggers fusion of the envelope with the endosomal membrane releasing the viral genome into the cytoplasm. na cleaves glycosidic bonds with terminal sa facilitating the release of budding virions from the cell. in diagnostics, antibodies against spike proteins are the preferred tool for identification and serotyping of viruses. development of therapeutic antibodies against influenza is a challenge, as the high viral mutation rate (antigenic drift) and genetic reassortment of the virus genome (antigenic shift) continuously lead to new strains escaping from neutralization by antibodies [ , ] . this goes along with adaptation to small molecule inhibitors (e.g. oseltamivir) [ ] . vaccines can only temporarily control the recurring epidemics of influenza, because antigenic changes are typical for ha and na. avian and bat serotypes of influenza a virus ha (h -h ) are known, but only three (h , h , and h ) have been adapted to humans. antibodies binding to regions of hemagglutinin conserved among serotypes have been developed which demonstrated broad specificity and neutralization potency [ ] [ ] [ ] [ ] [ ] [ ] . however, development, production and quality control of antibodies is expensive and time consuming. as an alternative, short peptides binding specifically to the spike proteins can be produced in automated high-throughput synthesis at low costs. ha-binding peptides have been recently obtained by phage display, lead structure optimization of natural products and specific toxins, bioinformatics tools and discovery from side effects of known anti-inflammatory peptides [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . some of them showed antiviral activity [ , [ ] [ ] [ ] [ ] [ ] . a more epitope-oriented accession to binding peptides is the search for paratope-derived peptides from variable regions of specific antibodies [ ] . antibodies against ha have been described, and at least antigenic sites (a-f) on the ha-trimer have been identified, localized either at the receptor binding site, the interface of the three ha-monomers, or at other sites like the stalk [ , , ] . several structures of ha-antibody complexes have been published deposited in the protein data bank (pdb) [ ] [ ] [ ] [ ] . indeed, an antibody was described, whose ha binding is mediated mainly by one cdr, namely hcdr [ ] . inspired by this finding, we chose linear peptides corresponding to the cdrs of vh of monoclonal antibody hc , having the majority of contacts with the ha domain of the strain a/aichi/ / [ , ] . the antibody and the derived peptides bind to ha at the sa binding site, in particular to the -loop and the -helix, which belong to the antigenic sites a and b, respectively. this binding site is conserved among several ha serotypes providing a basis for a peptide with broader specificity [ ] . we used complementary experimental and theoretical approaches to select ha binding vh-cdr peptides and to improve their potential to inhibit binding, and finally, infection of cells by influenza a virus. the inhibitory potential of the most efficient cdr-peptide was improved by microarray-based site-directed substitutions of amino acids. we could demonstrate a broader specificity of the selected peptides as they bound to ha of human and avian pathogenic influenza strains. influenza strain a/aichi/ / h n x (aichi h n ), reassorted with a/puertorico/ / h n , and low pathogenic a/mute swan/rostock/r / h n k (rostock h n ) were harvested from allantoic fluid of hen eggs. virus isolates were clarified upon low speed centrifugation ( x g, min) and concentrated by ultracentrifugation ( x g, h). for safety reasons, viruses prepared for spr experiments were inactivated by min irradiation with uv-light on ice. infectivity of inactivated virus was precluded using mdck ii based cellassays, remaining binding ability of ha was proven using standard hemagglutination assay (ha) with human red blood cells [ ] . for for infection and cytotoxicity assays madin-darby canine kidney epithelial cells (mdck ii, nbl- , ccl- ) were used (atcc). cells were cultivated under standard cell culture conditions with dmem (supplemented with % fetal calf serum (fcs), mm l-glutamine) in humid atmosphere at °c and % co . hemagglutination inhibition assays (hai) were performed using either human erythrocytes (α- , '-sialosugars) for aichi h n , or turkey erythrocytes (α- , '-sialosugars) for rostock h n [ ] . peptides were synthesized with a linker for immobilization or without linker for in vitro inhibition assays. for spr based binding experiments, antibody derived peptides were extended with an n-terminal lysine linker: kkkk-sgfllisn-amide (pea-lys), kkkk-fydydvfy-amide (peb-lys), kk-ßaßa-lgviwaggntny-amide (pec-lys). in the spr based screen for virus binding, single and double mutated variants of peb-lys were used. all lysine variants were purchased from biosyntan or genecust with > % purity. these peptides were dissolved in water and diluted in appropriate buffers for immobilization on spr sensor chips. spr based binding inhibition assays were performed with the full length peptides sgfllisngvhwv-amide (pea), ardfydydvfyyamd-amide (peb), and its double mutant ardfygydvffyamd-amide (peb gf ). for the biological assays peb, peb gf and the control peptide ardfydpdvfyyamdamide (peb p ) were applied. these peptides, and the peptides eb, s ( - ), phage (p ) h n and phage (l-p ) h n , mucroporin m- were synthesized by rudolf volkmer (charité, berlin). hplc purification and analysis were achieved using a linear solvent gradient (a: . % tfa in water; b: . % tfa in acetonitrile; gradient: - % b over min; uv detector at nm; rp- column). the identities of the peptides were validated by mass spectrometry using mal di-tof (microflex lt, bruker daltonik), and esi (q-tof micro, micromass). lyophilized peptides could be stored for at least one year at - °c as controlled by hplc/ms analysis. stock solutions of peb, peb gf and peb p were prepared by initial dissolving in dmso, followed by dilution in pbs ( mm peptide with % dmso v/v). solvation was improved by sonication. stock solutions could be stored for several weeks at °c. circular dichroism (cd) spectroscopy for determination of secondary structure and melting temperature was performed (s fig) using a jasco j- cd spectrometer. cd spectra of μg ml - peptide solutions were recorded at nm wavelength steps. the primary signal in millidegrees (mdeg) was recorded over s, and then the molar ellipticity (θmrw, [deg cm dmol - − ]) was calculated. measurements were performed on a biacore™ t (ge-healthcare bio-sciences ab) using cm chips, and running buffer hbsp ( mm hepes, mm nacl, . % tween , ph . ) at °c. peptides and proteins were immobilized via amine coupling with edc/nhs at a flow rate of μl min - , according to the manufacturer's instructions (ge healthcare). optimum preconcentration was evaluated before, using acetate and phosphate buffers ( mm) of ph to , and ligand concentrations of μg ml - . for the immobilization of biotinylated sialyllactose (lectinity), neutravidin (thermo fisher scientific) ( μg ml - ) was immobilized by amine coupling at ph . enabling high ligand density. biotinylated sialyllactose was then injected at a concentration of μg ml - in hbsp for min. several concentrations of analyte (ha, virus) were applied at least onto two channels with generally s association and a s dissociation time, followed by a s regeneration step with mm naoh. a flow rate of μl min - was used. quantitative data were obtained by extracting the amount of bound material s after injection was terminated (mean value over s). reverse peptide binding experiments were run, with immobilized ha or aichi h n , under the same conditions. double referencing of binding curves was performed, using a control flow cell without ligand, and using control injections of buffer, in order to eliminate bulk refractive index effects and unspecific binding to the sensor surface. the dissociation constant (k d ) was calculated from a plot of steady state binding levels against analyte concentration, using a biacore t evaluation software. a constant concentration of or μg ml - aichi h n virus was used as analyte, while the concentration of the inhibitors was varied. the suspensions of virus and inhibitor were mixed and subsequently incubated on a thriller thermoshaker at rpm and °c for at least min to reach equilibrium before injection on the spr surface. the inhibitors used were peptides or α- , 'and α- , '-sialyllactose (carbosynth). the suspensions of virus and inhibitor were mixed and subsequently incubated for at least min to reach equilibrium before injection on the spr surface. all data were referenced against buffer or inhibitor injection of identical concentration. the decrease of binding capacity of the chip over time was considered by referencing against multiple virus injections at various time points during the experiment. inhibition was calculated against the mean value of at least five binding events with the virus alone, using the sigmoidal four parameter logistic fit (origin). microarray-based substitutional analysis of peptide peb was performed using a pepstar peptide library spotted on glass slides by jpt peptide technologies. the slides were used without additional treatment. for the labeling of proteins with a fluorescent dye, dyomics dy- (λ ex = nm, λ em = nm, fluorospin kit (emp biotech) was used, according to the manufacturer´s instructions. the following materials were labeled: newyork h n , victoria h n , aichi h n , and california h n . labeled analytes were incubated several hours or overnight at indicated concentrations using femtotip buffer (ftp) ( mm tris, % glycerol, % polyvinylpyrrolidon , . % tween , ph . ) for dilution. the slides were washed twice in ftp and twice in ultrapure water and subsequently dried under a stream of nitrogen. fluorescence measurements were performed using an axon a laser scanner (molecular devices). fluorescence intensity was evaluated using genepix pro . software. for evaluation of binding efficiency the contrast (c) was calculated from the assay was performed according to potier et al. [ ] . aichi h n or rostock h n viruses were incubated with human or turkey erythrocytes, respectively, to yield agglutination (in the absence of an inhibitor) and concentration-dependent inhibition of agglutination (in the presence of an inhibitor). inhibitors were twofold serially diluted in pbs. then, hemagglutination units (hau) containing Á virus particles were added to all wells. viral particle concentration was estimated as described by desselberger et al. [ ] . after min incubation at room temperature, μl of a % erythrocyte solution (~ Á cells μl - ) was added, gently mixed and incubated for min at room temperature. for aichi h n human erythrocytes (α- , '-sialosugars), for rostock h n turkey erythrocytes (α- , '-sialosugars) have been used. the inhibitor constant k i (hai), reflects the lowest inhibitor concentration, which is necessary to achieve complete inhibition of hemagglutination caused by the influenza virus. to check for full hemagglutination inhibition, the microtiter plate was tilted by °to cause droplet formation from the red blood cell pellet [ ] . the experiment has been assessed using an mts reagent (promega) according to the manufacturer's protocol: , mdck ii cells were seeded the day before infection. aichi h n or rostock h n were pretreated with peptides in a twofold dilution series for min at room temperature under slight agitation. cells were washed once with pbs (supplemented with . mm mg + , . mm ca + ), before pretreated virus (moi . ) was added to the cells and maintained for h at room temperature to allow binding. unbound virus was removed by washing once with infection medium (dmem, mm l-glutamine, . % fcs, . % bovine serum albumin (bsa), . μg ml - l-(tosylamido- -phenyl) ethyl chloromethyl ketone (tpck) treated trypsin, u ml - penicillin and μg ml - streptomycin), and subsequently incubated for h at °c. then, μl mts solution was added to each well followed by hours incubation at °c. finally, absorbance at nm was recorded and data were normalized as follows: experiments have been performed for both virus strains in at least triplicate experiments. the microneutralization assay was performed according to the protocol of klimov et al. [ ] with minor modifications. briefly, peptide dilutions were mixed with either tcid aichi h n or tcid rostock h n in infection medium for min at room temperature. subsequently, , mdck ii cells were added to each well, followed by incubation for h at °c. next, the medium was removed, washed with pbs and fixed with ice cold acetone ( % v/v). after removal of the fixative, plates were dried and undergone immunostaining. cells were stained with igg primary antibody against the influenza a nucleoprotein (millipore, cat. # mab , mouse monoclonal, : in antibody diluent: pbs, . % tween (v/v) and % (w/v) nonfat, dry milk) for h at room temperature. following, cells were washed with washing buffer (pbs, . % tween ), before a secondary antibody ( : goat anti-mouse igg, kpl, cat. # - in antibody diluent) conjugated to horseradish peroxidase (hrp) was added for another hour at room temperature. next, the antibody was removed and the cells were washed with washing buffer. finally, σ-phenylenediamine dihydrochloride (opd) in citrate ( . m phosphate-citrate, . % sodium perborate, ph . at °c), was added for approximately minutes until the supernatant turned yellow. the reaction was stopped with . m sulfuric acid and absorbance of the reaction product in the microwell plate was recorded at nm. after subtraction of the background of non-infected cells, neutralization was calculated in ratio to untreated, infected cells. one day before treatment , mdck ii cells were seeded. on the following day media was removed and replaced with a twofold serial dilution of either peb, peb gf or peb p in supplemented dmem (see above). next, cells have been incubated in the presence of inhibitor for h at °c. then, μl mts solution was added to each well. finally, absorbance at nm was measured and data were normalized to untreated cells as follows: cell viability % ð Þ ¼ ðtreated cells À medium backgroundÞ ðuntreated cells À medium backgroundÞ x an ensemble of configurations for the complexes ha-pea, ha-peb, ha-pec and ha-peb gf was built using the amber package of programs [ ] . original coordinates for all atoms were taken from crystal structure pdbid = vir. in each case, the antibody moiety excepting the sequence of the peptide under study was erased. in the case of peb gf , in silico d g and y f mutations were performed. systems were then solvated using the tip p water molecule model [ ] , extending at least Å from the complex. nearly water molecules were added to solvate the complex and the resulting truncated octahedral box size was nearly Å x Å x Å. an appropriate number of chloride ions were added to keep the total system charge neutral. all bond lengths involving hydrogen atoms were constrained using the shake algorithm allowing the usage of a fs time-step [ ] . the temperature was fixed at k using a langevin thermostat with a collision frequency of ps - . the electrostatic interactions were treated using the particle-mesh ewald (pme) scheme with a fourth-order b-spline interpolation and a tolerance of - [ ] . the non-bonded cut-off was Å and the non-bonded pair list was updated every fs. simulations were carried out according to the same protocol that has been used in our previous studies [ ] [ ] [ ] [ ] [ ] , with the amber ff sb force field [ ] . briefly, each complex configuration was first optimized by steps of steepest descent followed by another steps of conjugate gradient minimization, keeping all atoms of the complex restrained to their initial position with a weak harmonic potential. each system was then simulated for ps at constant volume with a kcal mol - Å - restraint on the complex, in order to equilibrate the solvent at k without undesirable drifts of the structure. subsequently, a ps md simulation with a kcal mol - Å - restraint on each complex at a pressure of , kpa was conducted to relax the density using berendsen's barostat. after ns without restraint equilibration phase, a ns simulation at constant pressure was carried out and the coordinates were stored every ps, resulting in configurations for each simulation. subsequently, molecular mechanics-poisson-boltzmann surface area (mm-pbsa) calculations were performed. in the mm-pbsa method, the binding free energy of the receptor-ligand complex, Δg bind , is determined from where g com , g rec , and g lig denote the absolute free energies of the complex, receptor and the ligand, respectively. this method has been discussed elsewhere [ ] [ ] [ ] [ ] [ ] . the free energy g for each species is estimated from here, e mm is the molecular mechanical energy in the gas phase, g solv the solvation free energy, and -ts mm the contribution from the conformational entropy. the term e mm is comprised of the internal (bond, angle, dihedral) (e int ), electrostatic (e elec ), and van der waals energies (e vdw ), according to to incorporate all possible nonbonded interactions, the term e mm was estimated for each snapshot with no cut-offs. the solvation free energy, g solv , is approximated as the sum of the polar (g pol ) and the nonpolar contribution (g np ) using a continuum representation of the solvent according to here, γ = . kcal mol - Å - and b = - . kcal mol - . the popular linear poisson-boltzmann method was used to estimate the polar component of the solvation free energy. here, sasa is the solvent accessible surface area estimated by the linear combination of pairwise overlap (lcpo) algorithm using a probe radius of . Å [ ] . the electrostatic contribution to the solvation free energy was estimated from the poisson-boltzmann (pb) approach using the pbsa solver implemented in amber. in order to solve the pb equation the grid spacing was set to . Å in all dimensions and the relative dielectric constants in the protein and in the water were chosen to be and , respectively. the ionic strength was set to . m. the ratio between the longest dimension of the rectangular finitedifference grid and that of the solute was chosen to be . . the linear pb equation was solved with a maximum of iterations. in order to understand the inhibitor-residue interaction in more detail, the interaction energy was further decomposed into the contributions from each residue of the peptide by using the theory of free energy decomposition [ ] . the peptide-residue interaction is approximated by where Δe vdw and Δe elec are the contributions from the van der waals and electrostatic interactions between the inhibitor and each residue in the gas phase. the polar solvation free energy, Δg pb , was estimated using the pbsa module of amber. the d structure of the fab-hemagglutinin complex of the antibody hc (pdb: vir; complex of immunoglobulin igg with ha of aichi h n ) was analyzed to identify peptide sequences of the fab fragment at the ha contact area [ ] . essentially, three hypervariable complementarity determining regions (cdr) of the heavy chain variable domain (vh) of fab interact with ha ( fig a, b and c ). from these three loop-like organized sequences interacting with the sa binding pocket of ha, peptides pea, peb and pec ( fig d) were derived containing as a core the cdr sequences (table ) . in order to predict and compare binding affinities to ha of the three peptides, corresponding peptide-ha complexes were simulated in explicit water model for ns using md. as deduced from the root-mean-square deviation for peb already after three ns a steady state for binding was observed (fig a) . in contrast, peptide pea dissociated from ha within the simulation time of ns. gibbs free energies of peptide binding to ha were - . and - . kcal mol - for peb and pec, respectively (table ) . even though absolute values for free energy changes derived from md simulation have been proven not to correlate exactly with experimental values, a comparison between md derived values implicates a stronger binding of peb to ha. also, as shown in fig b and c , peb fits best the conserved sa binding site of aichi h n ha. based on this and experimental results (see below) we selected peb as a potential inhibitor of virus binding to host cells. as expected for its short primary sequence, and revealed experimentally by cd spectroscopy, peb displayed no secondary structure elements in solution at physiological ph (s a and s b fig) . to unravel the molecular basis of peb binding to ha, an analysis of energetic contribution was conducted by a combined md/mm-pbsa approach. molecular configurations obtained from md simulations of the complexes in explicit water were used for calculation of binding free energies using an implicit solvation scheme. the binding free energy was decomposed into contributions of the individual peptide residues (fig b, peb) . residues within and close to the loop (y to y ) provide the largest contributions to the binding free energy. significant attractive contributions to the binding free energy change arise from f (- . kcal mol - ), y (- . kcal mol - ), and f (- . kcal mol - ). however, d and d contribute repulsively by . kcal mol - and . kcal mol - , respectively, to the binding free energy. hence, exchanging d and d to other amino acids would predict to reduce the repulsive interactions of these residues and, thus, to increase the affinity of the modified peb to ha. the detailed analysis of electrostatic, van der waals and polar contributions to the free energy change of each residue is presented in s fig. we have also analyzed the contributions of the individual residues of ha to this complex. attractive contributions mainly originate from residues s (- . kcal mol - ), n (- . kcal mol - ), and l (- . kcal mol - ), whereas the most significant repulsive contribution is caused by e ( . kcal mol - ) (numbering according to pdb vir) (s fig). the attractive contributions from the polar residues s and n arise mainly from the intermolecular electrostatic interaction energy, whereas the hydrophobic residue l derives most of its contribution from van der waals interactions. binding of aichi h n virus to surface immobilized peptides was measured using spr. for this purpose, peptides pea-lys, peb-lys, and pec-lys, containing lysine residues at the amino terminus, acting as spacer were used. as deduced from md simulations, the n-terminus of peb does not significantly contribute to binding of the peptide to ha, as the contribution of these residues to the total free energy change is close to kcal mol - (fig b, peb) . binding significant virus binding was observed on peb-and pec-, but not on pea-modified surfaces consistent with our md simulations. the minimum detection level, corresponding to the lowest virus concentration, and showing binding times above the spr response of a buffer control, was . μg and . μg aichi h n virus protein ml - for immobilized peb and pec, respectively. the higher sensitivity of peb in the viral binding assay confirms the selection of this cdr derived peptide for further experiments, as mentioned above. to confirm specificity three peb variants containing single amino acid residue exchanges, which were immobilized with the same ligand density, were investigated. no binding was observed with the three variants, suggesting that the binding of virus is indeed specific to the cdr sequence ( fig a) . the results also exclude the possibility that binding is mediated by the oligolysine terminus. furthermore, several well-known proteins were tested for unspecific binding to the peptide modified chip surface ( fig b) . all proteins were injected at the same mass concentration. only for core-streptavidin and lysozyme minor binding to peb was found showing that surface inertness is not impaired. very likely, viruses bind to the peptide-covered surfaces in a multivalent manner (see discussion). as it is not possible to calculate reliable binding constants from the multivalent binding, the inverse experiment using surface immobilized ha from aichi h n was performed. from steady-state affinity calculation for peb a binding constant k d of . μm (r max = . ru; chi = . ), was obtained (fig a and b ). to assess whether peb binds to and thus blocks the sialic binding pocket of aichi h n neuraminidase, an enzyme activity assay with the substrate munana was performed (for details see material and methods). while the neuraminidase inhibitor dana (n-acetyl- , -dehydro- -deoxyneuraminic acid), serving as a positive control, was able to inhibit neuraminidase activity (ic to further support specificity of binding, inhibition of binding of aichi h n to surface immobilized fetuin by peb was studied. fetuin contains α-linked , '-and , '-sialic acids, table) . all proteins were diluted to a concentration of μg ml - . columns represent the mean value of duplicate experiments. the error bars show the sem. provide evidence that peb competes successfully for the sa binding site and, thus, may serve as a virus binding inhibitor. our md simulations have shown that the main contributions of peb binding to ha originate from residues to of the peptide. in search for peptides with higher binding affinity, a full substitutional analysis using microarrays was performed. thus, peb mutant variants were generated. in each of them, the full-length sequence was preserved except for only one amino acid from the eight loop-forming amino acids of the original peb peptide (ardfydydv-fyyamd) which was substituted with the remaining natural amino acids (fig b) . to assess binding, aichi h n was labeled with a protein reacting fluorophore. most notably, substitution of d generally leads to an increased binding signal. this is in agreement with our md simulations, predicting that this residue causes repulsive forces (+ . kcal mol - ) by interacting with ha. furthermore, most substitutions (out of d, g, t) of f , which was predicted to provide high attractive contribution (- . kcal mol - ), reduced the obtained fluorescence signal. although the results are essentially consistent with the theoretical predictions, we noticed a deviation: following the microarray results, aspartic acid d seems to be essential for binding while the md simulation assigned it to be repulsive. notably, for some substitutions of y we observed also an enhanced binding of virus. the microarray approach was applied to other influenza virus strains which are of higher medical relevance (s fig): new york h n , victoria h n and california h n . similar as for aichi h n , most substitutions of d increased binding of california h n and-although rather marginal-of new york h n . however, for the latter as well as for victoria h n substitutions of other residues caused significantly enhanced binding. for both strains all substitutions of y lead to enhanced affinity. virus binding of selected peptide variants was evaluated by spr to identify variants with improved binding. particularly, we were interested in variants showing binding to various influenza virus strains. to this end, several doubly substituted variants were probed. based on the results for monosubstituted variants (see above), we have chosen d and y for substitution. fig c shows the results for the binding of three virus strains to peb-lys and monoand double-substituted variants. the best variant was peb gf with the sequence ardfygydvffyamd. the repulsive amino acid d (+ . kcal mol - ) and the only slightly attractive residue y (- . kcal mol - ) (see md simulation above) were replaced by g and f, respectively. contribution to the total binding free energy change of every amino acid of peb gf binding to ha of aichi h n obtained by md-simulations is shown in fig b. d g and y f mutations decreased the contribution of free energy changes of these residues (s fig and discussion) . y f alters only moderately its interaction with ha, but it induces a favorable contribution change in the interaction of its neighbor residue phenylalanine with ha. the side chain of residue is oriented towards the solvent in peb, due to its polar property. on the other hand, the absence of a -oh group in the residue side chain in peb gf induces a slight change in the orientation of the loop, tilting now the phenylalanine side chain towards a hydrophobic region of the ha αhelix, located between residues - . the amount of hemagglutinin bound to peb gf compared to peb was -, -and -fold for aichi h n , new york h n and california h n , respectively. specific binding of peb gf to the receptor binding site of ha was evidenced by spr ( fig a, table ). compared to peb, peb gf showed a -fold lower ic ( μm vs μm) for inhibition of aichi h n binding to a fetuin-immobilized spr surface. for peb we observed a minor tendency to bind to immobilized fetuin, which becomes obvious from calculated inhibition values higher than %. to characterize the potential of antibody derived peptides to prevent virus replication, we studied virus binding and infection of host cells in the presence of peb, peb gf and peb p . the latter is assumed to be a structurally distinct variant due to the presence of a proline residue, used as a negative control. both peb and peb gf efficiently prevented aichi h n mediated aggregation of red blood cells (rbc) as assessed by hai (fig a) . however, peb gf was much more efficient in comparison to peb. the inhibitory constant k i (hai) was μm and . μm for peb and peb gf , respectively. furthermore, we could show binding inhibition of rostock h n table . mean values of ic [μm] from inhibition experiments using spr (left column), together with its sem (n! ). spr binding of virus aichi h n to fetuin is shown in fig a. inhibitor constant ki(hai) [μm] from hemagglutination inhibition experiments with its sem (n! ). data is plotted in fig . mean values of ic [μm] from infection inhibition (fig b and c ) and microneutralization assays (fig d and e ) are presented together with its sem (n! ). (table ) . hemagglutination by both viral strains was not inhibited by peb p . both peptides also protected efficiently mdck ii cell from infection (moi . ) by aichi h n (fig b) and rostock h n (fig c) . peb p did not compromise the infection at all. importantly, all three peptides did not impair cell viability within h treatment with peptides up to the maximum concentration ( μm) studied (s fig). similar results were obtained from a microneutralization assay, in which the peptide inhibitors are present in solution throughout the whole h incubation (fig d and e and table ). here, viral nucleoprotein expression was detected by immunostaining of infected cells, and the results were compared to untreated, infected cells. the ic values of rostock h n were similar to that obtained by the mts based infection inhibition assay. however, the ic values for aichi h n were somewhat lower ( - fold) with respect to the mts assay. although micromolar ic values seem to be rather high concentrations from the point of view of medical applications, the results are very promising for developing multivalent inhibitors (see discussion). to evaluate the inhibitory potential of the antibody derived peptides in relation to that of already published ha binding peptides, we compared these peptides in the hai assay (fig ) [ - , , ] . we normalized k i (hai) values to that of , -sialyllactose as the natural receptor for h . with reduction of k i (hai) by and fold, peb respectively peb gf showed superior potential compared to other published peptides, with the exception of the "entry blocker" eb. as in previous experiments, peptide peb gf showed an higher inhibitory potency than peb as observed by its -fold lower k i (hai). additionally both peptides showed lower k i (hai)-values than peptides obtained by phage display against h n (s ( - )), h n and h n . only the entry blocker eb showed superior hemagglutination inhibition, revealed by its -fold lower k i (hai) as compared to peb gf . however, due to its amphiphilic character, eb forms micelles and thus may act in a multivalent manner [ ] . therefore, it inhibits aichi h n hemagglutination more efficiently than other monovalent peptides. for the sake of completeness we mention that mucroporin m- showed strong hemolytic effects and strong interference with gold surfaces, which made it impossible to obtain reliable data [ ] . our aim was to obtain peptide inhibitors that recognize the conserved region of the sialic acid binding pocket of ha with a broader specificity to cover several influenza virus strains. the three cdrs of the vh-chain of antibody hc against hemagglutinin of influenza virus aichi h n were used as templates to design peptides being a potential inhibitor of virus binding to host cells. we used complementary experimental approaches as spr and peptide microarrays, and md to select appropriate peptides and to enhance their affinity to ha by site directed substitutions of amino acids. this was validated by assessing their potential to prevent virus binding to cell surfaces and, eventually, infection of host cells. three peptides-pea, peb and pec-have been derived from the three cdr sequences of the hc antibody's heavy chain. in agreement with predictions from our md calculations of peptide-ha complexes, we found by spr experiments that binding of surface immobilized peptide peb to ha from aichi h n was superior to pec (fig ) . therefore, and as peb matches the conserved sialic acid binding site better than pec (pdb vir) [ ] , subsequent studies were performed with peb and its derivatives. inhibition of virus binding to surface immobilized fetuin and , '-sialyllactose by peb confirmed the specific binding of the peptides to the sialic acid binding pocket of ha. the ic values are in the range of - μm, depending on the assay, which is about one order of magnitude lower than the ic of α- , -sialyllactose. an important observation which may be also of interest for upcoming studies is that we found specific binding of viruses to surface immobilized peptides consisting only of amino acids representing the peb core sequence, and a four lysine long linker (fig ) . nevertheless, peb and its derivatives harboring only those amino acids which are essential for the recognition of sa are presumably not efficient binders in their monomeric form. the affinities of the peptides are in the micromolar range, whereas the reported k d value of antibody hc binding to hemagglutinin from aichi h n is ± nm [ ] . however, weak binding interactions for such shorter monomeric peptides could be enhanced by a multivalent interaction as it occurs very likely in the spr based experiments, where binding of even . Á viruses ml - to immobilized peb could be detected (fig ) . extensions of the c-and n-termini of the cdr sequences could be important for (i) the formation of a stable peptide structure, which exposes the cdr loop region in its native arrangement or (ii) direct involvement in binding and foster interaction with ha. according to the md simulations peb adopts a strand-loop-strand conformation with the loop consisting of residues d to y . as shown in fig b, residues within and adjacent to the loop (y to y ) include the residues yielding the largest contributions to the binding free energy. most of these contributions are attractive (negative free energies changes) but some are also repulsive (positive free energies changes). the contributions of each amino acid to the free energy calculated by mm-pbsa were largely consistent with the results of microarray-based substitutional analysis, despite the described discrepancy for d . possibly, substitution of d changes the secondary structure leading to a lower affinity. in the light of improving the affinity of peb and to enable a broader specificity we also probed the binding of the peptide to ha of other virus strains by microarray-based substitutional analysis. we identified that y is also an important residue for improving binding. based on that, we investigated the affinity of selected variants of peb having two substitutions. in this screen, we identified peb gf as the best binder. the two substitutions, d g and y f, altered the ligand moiety and its binding contacts, resulting in favorable changes to the contribution to the free energy change of residues , and ( fig b) . the mutation of residue (d g), replacing a charged amino acid by a neutral one, resulted in two major changes. firstly, it induces a decrease of its own electrostatic contribution to the binding free energy, but such a change is largely overcome by an increase in the contribution to the polar solvation free energy change (s fig). on the other hand, one should notice that residues and are located in close contact to each other in the loop region. as a consequence for peb, this involves repulsive interaction between two negatively charged residues (d and d ). the replacement of d by an uncharged residue in peb gf probably induces a slight reorganization in the loop structure, favoring van der waals and polar solvation interaction contributions between this residue and the ha moieties (s fig). due to its orientation towards the sa binding region of ha, cdr sequences of antibody hc [ , ] against ha of aichi h n are promising candidates for getting variants with broad influenza strain specificities. indeed, both peptides peb and peb gf were found to bind to ha of other strains (rostock h n , new york h n , and california h n ). it is worth to note that all has of different influenza subtypes, which could be bound and inhibited by peptides peb or peb gf reveal strong structural similarities, especially for those amino acids involved in peptide binding (s table) . this close relation supports the wide binding ability and inhibitory potency of peb for a variety of influenza a strains. such broader specificity is also supported by the similarity between the vh-cdr of the mouse antibodies against aichi h n hemagglutinin in pdb ken (antibody hc , amino acids - : aafyydydfffdy) and pdb vir, antibody hc , amino acids - : rdfydydvfyyam), and also a human antibody against h : (egdydiltgyyyyfdy), respectively [ , , ] . notably, there is enrichment of aromatic amino acids tyr and phe, and of asp. in line with these findings, a recently published human antibody against h shows a higher proportion of aromatic amino acids in the hcdr domain, too [ ] . the crucial role of an asp residue and hydrophobic amino acids in the ha binding of many receptor mimicking antibodies is highlighted by lee et al [ ] . as shown exemplarily for aichi h n and rostock h n inhibition of virus binding by peptides leads to a strong protection from virus infection of host cells. in the mts based infection inhibition assay peb gf was found to be superior with respect to peb, too. a similar observation was made for rostock h n when using the microneutralization assay, which provides a direct measure for viral replication. however, for aichi h n we did not find a preference for peb gf indicating strain dependent differences of the antiviral potential of the peptides. the inhibitory potential of our antibody derived peptides demonstrated a remarkable performance compared to other published peptides binding to ha (fig ) . as summarized in table , peb and peb gf provides better inhibition against aichi h n compared to rostock h n , with the exception of the results from the hai assay. however, we should take into consideration that the performance of a peptide as inhibitor depends on many factors. as a consequence, the hemagglutination inhibition efficiency may not necessarily be stronger against aichi h n , even though the peptide was derived from an antibody binding to this influenza strain. one can speculate that rostock h n is more prone to neutralization by blocking this binding site, while for aichi h n peb and peb gf can be displaced more easily in a binding competition experiment with erythrocytes. this trend could also be explained by the use of erythrocytes derived from different species (see material and methods). similar strain-dependent efficiencies have been obtained by lópez-martinez et al. [ ] . although a peptide was designed against broadly conserved epitopes, the antiviral efficiency was not the same against all tested serotypes (three different h n strains and h n ). short oligomeric biomolecules with sufficient affinity are of high interest for diagnostics and drug design, as development, synthesis and further chemical modifications are easy to implement. linear short cdr derived peptides as used here can be expected to differ from the loop structure they adopt within the antibody, leading to lower affinity. however, there are promising ways to largely improve them. cyclisation of cdr peptides using a d-pro-l-pro template has been shown to force them into canonical conformations [ ] . in this report, the l loop of the hc antibody adopted a hairpin structure and an aromatic t-stacking as in the antibody crystal structure. this and other strategies to generate peptidomimetics by stabilizing or mimicking turns, ß-sheets and helices, have been recently reviewed [ ] . whereas the gain in affinity by cyclization of a peptide is limited by the affinity of the native structure, larger improvements can be achieved by multivalency, especially when a virus with a high target protein density on its surface has to be bound [ ] [ ] [ ] [ ] . for example, sialyl-containing oligosaccharides constituting the natural receptors of hemagglutinin on mammalian epithelial cells, exhibit a millimolar : affinity, but due to multivalent virus-cell attachment the binding is strong enough to enable infection. effective inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles and other glycoarchitectures has been described [ , ] . more recently, a trivalent glycopeptide mimetic containing three sialyl residues matching the three sialyl binding sites in a ha trimer, was reported to bind to h hemagglutinin fold better than the monomer [ ] . recently, we demonstrated that the antiviral effectivity of acylated peb gf can be increased, upon presentation in a multivalent fashion by -fold against rostock h n and by -fold against aichi h n [ ] . further, multivalent presentation of the short s peptide (arlpr) with a dendrimer resulted in sub-micromolar inhibitory activities [ ] . the antibody derived peptide peb and its mutant variant peb gf are promising hemagglutinin binding peptides, which both could have potential for application in viral diagnostics and therapeutics. infection inhibition could be achieved at micromolar concentrations. strategies to improve the peptide structure and to apply the peptides for developing multivalent binders with high affinities are discussed. finally, the workflow presented involving complementary experimental and theoretical approaches could be a template for the development of antiviral agents, which could be also applied for influenza detecting biochips. in a coevolutionary approach the peptide's primary sequence may also be adapted to seasonal upcoming mutations of the influenza virus with minor additional efforts, costs and in short time without the additional development of novel monoclonal antibodies. contributions of individual residues of peb (black) and peb gf (grey) to the binding free energies of the corresponding peb-ha or peb gf -ha complexes. a) electrostatic, b) van der waals and c) polar contributions to the total binding free energy change. the mutation of residue (d g) replacing a charged amino acid by a neutral one, decreases the electrostatic contribution, but such a change is largely overcome by an increase in the contribution to the solvation free energy change, as calculated. mutation of amino acid (y f) appears not to alter directly the contribution of the interaction between this residue and the ha, but it induces a favorable free energy change contribution in the interaction between residue and the ha, essentially by a more favorable van der waals interaction energy change between uncharged residues. (tif) labeled influenza california h n (left), new york h n (middle) and victoria h n (right) were used as analytes. numbers represent mean value of the contrast relative to contrast for positive control fetuin. false-colors are used to illuminate fluorescence intensities, color changes from blue (lower) to yellow (higher intensity than peb). data just represent qualitative relation between peb mutants within single influenza strains. quantitative comparison between different strains is not valid due to the varying types of samples, while precise quantitation fails due to unknown ligand density (most likely varying per peptide). table. alignment of ha sequences of used influenza viruses. amino acids involved in binding of peb as obtained from md-simulations are highlighted in gray. pdb vir: sequence obtained from protein database; a/mute/swan/r / -h n : sequence obtained from prof. harder (friedrich-loeffler-institut, riems, germany); all other sequences were obtained from influenza virus resource (ivr) as indicated by their accession numbers [ ] . while l is identical for all subtypes, s and e are replaced in some cases by functional closely related t or d, respectively. the sequence differ mostly in n , which is replaced by s, a or v. the n substitutions could be the main reason for differences in the observed binding ability. (docx) influenza neuraminidase: a druggable target for natural products influenza: the mother of all pandemics unraveling the mystery of swine influenza virus human infection with a novel avian-origin influenza a (h n ) virus avian-origin influenza a(h n ) infection in influenza a(h n )-affected areas of china: a serological study emergence of a novel swine-origin influenza a (h n ) virus in humans variation and infectivity neutralization in influenza structural basis of immune recognition of influenza virus hemagglutinin influenza virus resistance to antiviral therapy a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins a highly conserved neutralizing epitope on group influenza a viruses cross-neutralization of influenza a viruses mediated by a single antibody loop heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity structural insights into key sites of vulnerability on hiv- env and influenza ha a common solution to group influenza virus neutralization identification and characterisation of a novel anti-viral peptide against avian influenza virus h n sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy phage displayed peptides to avian h n virus distinguished the virus from other viruses bovine lactoferrin-derived peptides as novel broad-spectrum inhibitors of influenza virus virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses a novel family of peptides with potent activity against influenza a viruses inhibition of influenza a virus infection in vitro by peptides designed in silico influenza a virus entry inhibitors targeting the hemagglutinin systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins structural identification of the antibody-binding sites of hong kong influenza haemagglutinin and their involvement in antigenic variation antigen distortion allows influenza virus to escape neutralization refined three-dimensional structure of the fab fragment of a murine iggl,lambda antibody structurally conserved binding sites of hemagglutinin as targets for influenza drug and vaccine development hemagglutination inhibition assays. seminars in avian and exotic pet medicine fluorometric assay of neuraminidase with a sodium ( -methylumbelliferyl-alpha-d-n-acetylneuraminate) substrate relation of virus particle counts to the hemagglutinating activity of influenza virus suspensions measured by the ha pattern test and by use of the photometric hcu method influenza virus titration, antigenic characterization, and serological methods for antibody detection comparison of simple potential functions for simulating liquid water numerical-integration of cartesian equations of motion of a system with constraints-molecular-dynamics of n-alkanes particle mesh ewald-an n.log(n) method for ewald sums in large systems importance of polar solvation for cross-reactivity of antibody and its variants with steroids energetic basis for drug resistance of hiv- protease mutants against amprenavir origin of decrease in potency of darunavir and two related antiviral inhibitors against hiv- compared to hiv- protease energetics of mutation-induced changes in potency of lersivirine against hiv- reverse transcriptase mutation-induced loop opening and energetics for binding of tamiflu to influenza n neuraminidase comparison of multiple amber force fields and development of improved protein backbone parameters approximate solvent-accessible surface areas from tetrahedrally directed neighbor densities insights into protein-protein binding by binding free energy calculation and free energy decomposition for the ras-raf and ras-raigds complexes identification of the minimal active sequence of an anti-influenza virus peptide receptor binding and membrane fusion in virus entry: the influenza hemagglutinin a complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site an antibody that prevents the hemagglutinin low ph fusogenic transition broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin structural mimicry of canonical conformations in antibody hypervariable loops using cyclic peptides containing a heterochiral diproline template structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes monomeric inhibitors of influenza neuraminidase enhance the hemagglutination inhibition activities of polyacrylamides presenting multiple c-sialoside groups polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. insight into mechanism of inhibition multivalency as a chemical organization and action principle inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles inhibition of influenza virus activity by multivalent glycoarchitectures with matched sizes a nanomolar multivalent ligand as entry inhibitor of the hemagglutinin of avian influenza potential of acylated peptides to target the influenza a virus synthesis and influenza virus inhibitory activities of carbosilane dendrimers peripherally functionalized with hemagglutinin-binding peptide influenza virus resource the authors thank ines kretzschmar (charité, berlin) for help with peptide synthesis, prof. harder (friedrich-loeffler-institut, riems, germany) for providing virus rostock h n , dr. zierenberg (gsk dresden) for providing the influenza monobulks a/victoria/ / h n and a/newyork/ / h n , and dr. thaa (fu berlin) for providing the turkey erythrocytes. for support with cd spectroscopy measurements we thank dr. jahnke (university of potsdam). conceived and designed the experiments: ffb ah eef ws. performed the experiments: hm dl pk sdl. analyzed the data: hm dl pk sdl vk ws. contributed reagents/materials/analysis tools: rv. wrote the paper: ws ah dl. key: cord- -q f el authors: farhadi, tayebeh; hashemian, seyed mohammadreza title: computer-aided design of amino acid-based therapeutics: a review date: - - journal: drug des devel ther doi: . /dddt.s sha: doc_id: cord_uid: q f el during the last two decades, the pharmaceutical industry has progressed from detecting small molecules to designing biologic-based therapeutics. amino acid-based drugs are a group of biologic-based therapeutics that can effectively combat the diseases caused by drug resistance or molecular deficiency. computational techniques play a key role to design and develop the amino acid-based therapeutics such as proteins, peptides and peptidomimetics. in this study, it was attempted to discuss the various elements for computational design of amino acid-based therapeutics. protein design seeks to identify the properties of amino acid sequences that fold to predetermined structures with desirable structural and functional characteristics. peptide drugs occupy a middle space between proteins and small molecules and it is hoped that they can target “undruggable” intracellular protein–protein interactions. peptidomimetics, the compounds that mimic the biologic characteristics of peptides, present refined pharmacokinetic properties compared to the original peptides. here, the elaborated techniques that are developed to characterize the amino acid sequences consistent with a specific structure and allow protein design are discussed. moreover, the key principles and recent advances in currently introduced computational techniques for rational peptide design are spotlighted. the most advanced computational techniques developed to design novel peptidomimetics are also summarized. different diseases may be caused by pathogens or malfunctioning organs, and using therapeutic agents to heal them has an old recorded history. small molecules are conventional therapeutic candidates that can be easily synthesized and administered. however, many of these small molecules are not specific to their targets and may lead to side effects. moreover, a number of diseases are caused due to deficiency in a specific protein or enzyme. thus, they can be treated using biologically based therapies that are able to recognize a specific target within crowded cells. under the biologic conditions, some macromolecules such as proteins and peptides are optimized to recognize specific targets. therefore, they can override the shortcomings of small molecules. recently, pharmaceutical scientists have shown interest in engineering amino acid-based therapeutics such as proteins, peptides and peptidomimetics. [ ] [ ] [ ] theoretical and experimental techniques can predict the structure and folding of amino acid sequences and provide an insight into how structure and function are encoded in the sequence. such predictions may be valuable to interpret genomic information and many life processes. moreover, engineering of novel proteins or redesigning the existing proteins has opened the ways to achieve novel biologic macromolecules with desirable therapeutic functions. protein sequences comprise tens to thousands of amino acids. besides, the backbone and side chain degrees of freedom lead to a large number of configurations for a single amino acid sequence. protein design techniques give minimal frustration through precise identification of sequences and their characteristics. [ ] [ ] [ ] [ ] considering energy landscape theory, the adequately minimal frustration in natural proteins occurs when their native state is adequately low in energy. the de novo design of a sequence is difficult because there are huge numbers of possible sequences: n for n-residue proteins with only natural amino acids. peptide design should incorporate computational approaches. it can benefit from searching the more advanced fields used for small molecules and protein design. however, the straightforward adoption of computational approaches employed to small-molecule and protein design has not be accepted as a reasonable solution to the peptide design problem. [ ] [ ] [ ] in the peptide drug design, the conformational space accessible to peptides challenges the small-molecule computational approaches. besides, the necessity for nonstandard amino acids and various cyclization chemistries challenges the available tools for protein modeling. furthermore, the aggregation of peptide drugs during production or storage can be an unavoidable problem in the peptide design procedure. rational design of a peptide ligand is also challenging because of the elusive affinity and intrinsic flexibility of peptides. peptide-focused in silico methods have been increasingly developed to make testable predictions and refine design hypotheses. consequently, the peptide-focused approaches decrease the chemical spaces of theoretical peptides to more acceptable focused "drug-like" spaces and reduce the problems associated with aggregation and flexibility. , for the discussions that follow, peptides can be defined as relatively small ( - residues) polymers of amino acids. in physiological conditions, several problems such as degradation by specific or nonspecific peptidases may limit the clinical application of natural peptides. moreover, the promiscuity of peptides for their receptors emerges from high degrees of conformational flexibility that can cause undesirable side effects. besides, some properties of therapeutic peptides, such as high molecular mass and low chemical stability, can result in a weak pharmacokinetic profile. therefore, peptidomimetic design can be a valuable solution to circumvent some of undesirable properties of therapeutic peptides. , in the biologic environment, peptidomimetics can mimic the biologic activity of parent peptides with the advantages of improving both pharmacokinetic and pharmacodynamic properties including bioavailability, selectivity, efficacy and stability. a wide range of peptidomimetics have been introduced, such as those isolated as natural products, synthesized from novel scaffolds, designed based on x-ray crystallographic data and predicted to mimic the biologic manner of natural peptides. using hierarchical strategies, it is possible to change a peptide into mimic derivatives with lower undesirable properties of the origin peptide. over the past years, computational methods have been developed to discover peptidomimetics. in a part of this review, novel computational methods introduced for peptidomimetic design have been summarized. peptidomimetics can be categorized as follows: peptide backbone mimetics (type ), functional mimetics (type ) and topographical mimetics (type ). the first generation of peptidomimetics (type ) mimics the local topography of amide bond. it includes amide bond isosteres, pyrrolinones or short fragments of secondary structure, such as beta-turns. such mimetics generally match the peptide backbone atom-for-atom, and comprise chemical groups that also mimic the functionality of the natural side chains of amino acids. a number of prosperous instances of type peptidomimetics have been reported. the second type of peptidomimetics is described as functional mimetics or type mimetics, which include small, non-peptide compounds that are able to identify the biologic targets of their parent peptide. at first, they were assumed to be conservative structural analogs of parent peptides. however, using site-directed mutagenesis, their binding sites to biologic targets were investigated. the results indicated that type peptidomimetics routinely bind to protein sites that are different from those selected by the original peptide. therefore, type mimetics maintain the ability to interfere with the peptide-protein interaction process without the necessity to mimic the structure of the natural peptide. type peptidomimetics reveal the best conception of peptidomimetics. they consist of the necessary chemical groups that act as topographical mimetics and contain novel chemical scaffolds that are unrelated to natural peptides. here, theoretical and computational techniques to design proteins, peptides and peptidomimetics are reviewed. however, the current review does not deeply highlight the computational aspects of amino acid-based therapeutic design, but only discusses the methods used to design the mentioned therapeutics. figure summarizes the key concepts presented in this study. as some examples, the structures of aldesleukin, leuprolide and spaglumic acid, important amino acid-based therapeutics approved by the us food and drug administration (fda), are shown in figure a computer-aided design of amino acid-based therapeutics figure a ) and leuprolide (pdb id: yy ; figure b ) were obtained from the protein data bank (pdb; http://www. rcsb.org/) and visualized by pymol tool. the structure of spaglumic acid was retrieved (in mol format) from pub-chem database (https://pubchem.ncbi.nlm.nih.gov/) with the pubchem id ( figure c ) and visualized using pymol. aldesleukin, a lymphokine, is a recombinant protein used to treat adults with metastatic renal cell carcinoma (https://www.drugbank.ca/drugs/db ). leuprolide, a synthetic nine-residue peptide analog of gonadotropin releasing hormone, is used to treat advanced prostate cancer (https://www.drugbank.ca/drugs/db ). spaglumic acid is used in allergic conditions such as allergic conjunctivitis. the drug belongs to a class of peptidomimetics known as hybrid peptides. hybrid peptides contain at least two dissimilar types of amino acids (alpha, beta, gamma or delta) linked to each other via a peptide bond (https://www.drugbank.ca/ drugs/db ). in the current study, all fda-approved therapeutics (in ) were retrieved from drugbank (https://www.drugbank. ca/biotech_drugs) and an analysis was conducted to compare their percentages. protein-based therapies, gene or nucleic acid-based therapies, vaccines, allergenics and cell transplant therapies made up . %, . %, . %, . % and . % of total approved therapeutics, respectively. small-molecule drugs made up . % of the approved therapeutics ( figure ). computational designing of proteins can be classified as follows: ) template-based designing in which three-dimensional ( d) farhadi and hashemian structure of a predefined template is adapted to design a sequence and ) de novo designing in which the amino acids' arrangement is changed to generate both sequence and d structure of a completely novel protein. the problem of predicting the fold of an unknown sequence could be solved by utilizing templates. since the fold is unaltered, the backbone atoms are directly located on this framework. moreover, to generate a functional protein, the side chains that can effectively stabilize the structure are added to the backbone. , routine concerns and methods for template-based protein design are reviewed below. selecting the template (scaffold) protein the template (also named as scaffold protein) contains a group of backbone atom coordinates. the coordinates can be retrieved from an available x-ray crystal structure or cautiously from a nuclear magnetic resonance (nmr) structure. computer-aided design of amino acid-based therapeutics fixing the backbone decreases the computational complication, but it may inhibit the main chain modifications to adjust sequence alternation. backbone flexibility can generate designed functionalities over the protein's normal function. the backbone flexibility is introduced through incorporating other closely associated conformations to an existing structure. [ ] [ ] [ ] recently, new functionalities were effectively introduced into the tim-barrel topology. this fold has been detected as one of the most shared structures in distinct protein superfamilies. sequence search and characterization in a design procedure, a protein sequence is selected such that it meets the energetic and geometric constraints established by the chosen fold. sequence search techniques sample different sequences and estimate their energies to gain the one owing the minimum energy. in order to identify the sequences subject to an objective function or a specific energy, a diverse strategies including optimization and probabilistic approaches have been developed. optimization processes may recognize candidate sequences using stochastic or deterministic methods. probabilistic approaches focus on characterizing the sequence space probabilistically. deterministic methods: to achieve a sequence folded into a global minimum energy conformation, deterministic methods search the whole sequence space and identify the global optima. , these methods include dead-end elimination (dee), self-consistent mean field, graph decomposition and linear programming. stochastic algorithms search the sequence space in an exploratory manner. these algorithms include monte carlo algorithms (simulated annealing), graph search methods and genetic algorithms. some of the most commonly used methods are discussed below. dee has been considered as a thorough search algorithm. to find and remove sequence-rotameric positions that are not portions of the global minimum energy conformation, dee compares two amino acid rotamers and removes the one with greater interaction energy. interaction energies are computed for each rotamer of the test amino acid, along with all rotamers of every other amino acid. the situation is repetitively examined for total amino acid states as well as their rotamers until it no longer holds true. , expanding the sequence length increases the combinatorial complication of dee exponentially. therefore, to design sequences of amino acids or larger, application of dee may be restricted. details of the theorem are explained elsewhere. , stochastic search algorithms: as mentioned before, deterministic approaches are perfect to design proteins with small sizes, but show the applied disadvantages with extension of sequence size. stochastic or heuristic methods are valuable to design large proteins. the most widely used method for protein design includes monte carlo sampling. , monte carlo method samples positions of complicated proteins in a way related to a selected probability distribution such as boltzmann distribution. boltzmann distribution specially weighs low-energy configurations. the monte carlo algorithm performs iterative series of calculations. at the primary step of each search, a partially accidental test sequence is generated, and its energy is calculated via a physical potential. during the primary step, both rotamer state and amino acid identity are adjusted and an efficient temperature controls the probable energy alterations. in the next step, named simulated annealing, the temperature gradually decreases and permits favorable sampling of lowerenergy configurations. multiple independent calculations are carried out to converge the system to a global minimum. , for more explanation about the theorems and details of the formulation of the probability distribution and weights, readers are referred to study previous reports. , probabilistic approach: probabilistic approaches are frequently employed when thorough information is not accessible for protein design. in a probabilistic approach, sitespecific amino acid probabilities may be utilized, rather than particular sequences. the procedure is partially motivated by the uncertainties to find sequences consistent with a specific structure. briefly, the backbone atoms are fixed or greatly constrained, side chain conformations are discretely handled, energy functions are estimated and solvation is handled by simple models. however, in order to offer valuable sequence information for design experiments and to find structurally significant amino acids, probabilistic techniques leverage structural characteristics of interatomic interactions. generally, monte carlo methods give a probabilistic sampling of sequences. , in addition, an entropy-based formalism has been defined to predict amino acid probabilities for a certain backbone structure. , the method employs concepts from statistical thermodynamics to assess the sitespecific probabilities. to address the whole space of existing compositions, the theory is not restricted by the computational enumeration and sampling. large protein structures with . variable residues can be supplied simply. sampling sequence space to generate conformations the chemical variability of a sequence and the number of various amino acids permitted at each position are defined as "degrees of freedom for each amino acid". moreover, each of the natural residues search the whole sequence space. drug design, development and therapy : submit your manuscript | www.dovepress.com to decrease the degrees of freedom for each amino acid and searching the sequence space, diverse approaches such as hydrophobic patterning have been proposed. monomers can be used to probe a protein structure and improve its function, other than the naturally occurring amino acids. sampling of side chain conformational space to form conformations side chain conformations are typically consistent with the energy minima of molecular potentials and can be obtained from a structural database. rotamer statuses are related to the repeatedly detected values of dihedral angles in the side chain of each amino acid. for example, the simplest amino acids including alanine and glycine have only one rotamer status, while the bigger amino acids have . diverse rotamer statuses. a variety of rotamer libraries including backbonedependent, secondary structure-dependent and backboneindependent libraries have been developed for protein design. , by using a rotamer library, one can discretize a meaningful state space to decrease the computational difficulty. rotamer libraries can be extended beyond the natural amino acids. the effective rotamers can model cofactors, ligands, water and posttranslational modifications. for example, to improve the modeling of protein-protein interactions and model water within proteins interiors, the structurally definite water molecules can be inserted as a solvated rotamer library. energy functions have been employed to quantify sequencestructure compatibilities. they include linear associations of hydrogen bonds made by backbone atoms, repulsion among atoms, hydrophobic attraction among non-polar groups and electrostatic interactions among sequential neighbors. the sequence of a protein is selected so that it can adjust the energetic and geometric constraints enforced by the favorite fold. constraints typically contain several intramolecular interactions such as van der waals, hydrophobic, polar and electrostatic interactions, as well as hydrogen bonds. generally, by using a scoring function, it is possible that energetic contributions of the mentioned parameters are taken into account. , , de novo design: designing the sequence and d structure through assembly of proteins fragments , or secondarystructure elements, , novel structures can be modeled de novo. in the design procedures, the backbone coordinates are generally constrained. summary and important findings of some proteins designed using computational approach including a retroaldol enzyme, the kemp elimination enzyme, a novel βαβ protein, a redesigned procarboxypeptidase, a novel α/β protein structure and the top are shown in table . peptide design methods have been categorized as ligand-and target-based design methods. in the ligand-based designing procedure, information derived from peptides is used to design novel therapeutic peptides. in the target-based method, information derived from target proteins is specifically utilized. typically, a hybrid approach including both ligand-and target-based design is utilized. ligand-based peptide design the ligand-based design has been classified as follows: ) sequence-based, ) property-based and ) conformationbased design. sequence-based approach uses the information of conserved regions and analyzes the multiple sequence alignments. this method is directed by the hypothesis that conserved regions are functionally and structurally significant. computational tools allow the ligand-based peptide design, although they lag behind bioinformatics strategies developed for protein designing. recently, using a method based on a pam matrix, the relationship between a series of collagen peptides and antiangiogenic activity including proliferation, migration and adhesion was analyzed. the pam matrix captured information of mutation rates among all pairs of amino acids. based on the results, regions at the c and n termini of the peptides were detected to be significant for an ideal activity and suggested as two distinct binding sites. the approach showed the potential worth of the sequence-based peptide design. in another report, a computational platform called sarvision was developed to support sequence-based design. sarvision signifies an important step for peptide sequence/activity relationship (sar) analysis. moreover, it pools the improved visualization abilities with advanced sequence/activity analysis. compared to small molecules, property-based design methods for peptides are in the early stages of development. in a recent study, the Δg decomposition per residue and the physicochemical characteristics of amino acids, such as hydrophilicity, hydrophobicity and volume, were used computer-aided design of amino acid-based therapeutics to model peptide binding to targets of interest. , finally, a model was built to estimate peptide Δg values for binding to the class i major histocompatibility complex (mhc) protein hla-a* . furthermore, in a wide range of studies, antimicrobial peptides were successfully analyzed by using the property-based approach. for example, a machinelearning method was employed to design novel antimicrobial peptides. the victory of the property-based methods with antimicrobial peptides may be explained by the fact that the desired biologic activity of membrane disruption is relatively nonspecific. in the case of conformation-based peptide design, computational techniques were developed to predict the conformational ensembles or structure of peptides and analyze the sars. , pep-fold is an online tool used to predict the d structures of peptides of length - residues. a remarkable suggestion from the data is that pep-fold seems to solve the conformational sampling problem. , in order to search conformational spaces of a peptide, long timescale molecular dynamic simulations have been employed. , besides, quantum mechanical calculations are promising to address the scoring deficiency in the peptide conformational examination. apparently, to affect the peptide design processes positively, improving the major theoretical and technical issues is necessary before such computationally sophisticated and costly procedures. conformation of a peptide may be modeled to generate a d pharmacophore hypothesis. a certain pharmacophore hypothesis is useful to determine the adme/tox activities or particular potencies of a peptide. for example, screening of a peptide library was jointed to generate a pharmacophore hypothesis to identify potent agonists of melanocortin- receptor isoforms. a combinatorial tetrapeptide library was screened, and sar and ligand-derived pharmacophore templates were generated. the pharmacophore hypothesis was proposed to allow continuous attempts in the rational design of melanocortin receptor molecules. target-based peptide design compared to ligand-based peptide design, target-based design appears to be in a more improved level. targetbased design is initiated with the computer-aided survey of a ligand-bound or unbound protein target to recognize its potential binding sites, prospective specificity surfaces and other pharmacologic activity elements. the phase is generally followed by an in silico design phase where computational methods perform, refine and evaluate peptide design ideas. some recently developed computational methods for targetbased peptide design are reviewed below. recently, an increase in the number of protein-peptide d structures deposited in the pdb has assisted to search the molecular mechanism and structural basis of peptide recognition and binding. information of crystal structures of protein-peptide complexes can improve our knowledge of the farhadi and hashemian chemical forces involved in the binding and special modes of binding. dynamic data of the complexes can be partially extracted from the solution nmr structures deposited in the pdb. to record the structures and functions of various protein-peptide complexes, the experimentally resolved structure data were gathered, annotated and analyzed, and several distinctive databases such as pepx, pepbind and peptiddb were generated. the pepx database, derived from the pdb, comprises unique protein-peptide interface collections. the pepbind database contains , proteinpeptide complex structures from the pdb. peptiddb is a curated database of protein-peptide complexes. the abundance of the structural information specifically on monomeric proteins could be gathered to design proteinpeptide interactions with no requirement for their sequence homology. protein-peptide docking precise docking of a highly flexible peptide is a major challenge. traditional docking protocols, such as autodock, vina , and moe-dock, developed for docking of small molecules, were also used to dock a peptide to a protein receptor. however, comparative studies revealed that these techniques would face failure if the docked peptides were . residues long. therefore, development of peptide-focused docking protocols is very important. other protein-protein docking tools such as z-dock and hex have been used for the computational peptide design in some studies. below, details of recently developed peptide-focused docking approaches are discussed. first, heuristic evolution procedures were applied to search the large conformational space of linear peptides before the binding. however, these procedures were not efficient and their use was limited. then, a scheme based on conformational sampling became common in the peptide docking. besides, several illustrative approaches were proposed to balance between the accuracy and efficacy of the flexible peptide docking. in this aspect, docscheme, dynadock and pepspec were integrated to online userfriendly interfaces and introduced. recently, pepcrawler and flexpepdock were developed as the peptide docking tools. it is reported that flexpepdock has sub-angstrom accuracy in reproducing the crystal structures of protein-peptide complexes. all of the flexpepdock-based methods assume previous information about the peptide-binding site. anchordock, a recently described algorithm, allows powerful blind docking calculations through relaxing the constraint. the program predicts anchoring origins on a protein surface. following recognition of the anchoring origins, an assumed peptide conformation is refined using an anchor-constrained molecular dynamic process. haddock, a well-known protein-protein docking tool, has been recently expanded to run the flexible peptide-protein docking. to handle a docking procedure, haddock uses ambiguous interaction restraints based on the experimental information about intermolecular interactions. this rigid body peptide docking is followed through a flexiblesimulated annealing process. the novel haddock strategy initiates docking computations from an ensemble of three dissimilar peptide conformations (eg, α-helix, extended and polyproline-ii) that are high informative inputs. cabs-dock is a recently introduced protein-peptide docking tool and runs a primary docking procedure whose outcomes can be refined by other tools such as flexpepdock. in the primary phase of the procedure, random conformations of a peptide are predicted and located around the protein target of interest. the process is followed by replica exchange monte carlo dynamics. subsequently, models are selected for the last optimization using the modeller tool to gain accurate scoring and ranking poses. , galaxypepdock was developed to use experimentally resolved protein-peptide structures for running the template-based docking pooled by flexible energy-based optimization. atomistic simulation atomistic monte carlo and molecular dynamics simulations are accurate, but they are meticulous techniques to investigate peptide-protein binding interactions. these techniques can also detect the thermodynamic profile and trajectory included in protein-peptide identification. these methods predict the association among conformations of a peptide in solution or protein. in a study, in order to describe the binding of a decapeptide to the cognate sh receptor, a long-term molecular dynamic simulation was used and a two-state model was built. in the first step, a relatively quick diffusion phase, nonspecific encounter complexes were generated and stabilized by using electrostatic energy. the secondary step was a slow modification phase, in which the water molecules were emptied out from the space between the peptide ligand and the receptor. in another report, by using monte carlo method, the mentioned two-state model was verified to trace some oligopeptide routes for binding to various pdz (post synaptic density protein, drosophila disc large tumor suppressor, and zonula occludens- protein) domains. drug design, development and therapy : submit your manuscript | www.dovepress.com computer-aided design of amino acid-based therapeutics the affinity of bh peptides to bcl- protein was investigated, and results showed the higher affinity of bound peptides occurred when the corresponding peptides were in a lower degree of disorder in unbound states and vice versa. these results showed that the highly structured peptides could increase their affinity through reducing the entropic loss associated with the binding. overall, in addition to the electrostatic and hydrophobic forces, protein-peptide interactions can be affected by the entropic effect and conformational flexibility that could be willingly examined with atomistic simulations. very recently, using a fast molecular dynamics simulation, the energetic and dynamic features of protein-peptide interactions were studied. in most cases, the native binding sites and native-like postures of protein-peptide complexes were recapitulated. additional investigation showed that insertion of motility and flexibility in the simulation could meaningfully advance the correctness of protein-peptide binding prediction. peptide affinity prediction most features of computational peptide design are based on the accuracy and efficacy of affinity prediction. hence, the fast and reliable prediction of peptide-protein affinity is significant for rational peptide design. in this aspect, two categories of prediction algorithms including sequence-and structure-based approaches were developed. the sequencebased method uses the information derived from primary polypeptide sequences to approximate and evaluate the standards of the binding affinity. the structure-based process takes the information derived from d structures of proteinpeptide complexes to predict the binding affinity. at the sequence level, the quantitative structure-activity relationships (qsars) have been widely utilized to forecast the binding affinity of peptides and conclude the biologic function. to model the statistical correlation between sequence patterns and biologic activities of experimentally assessed peptides, machine-learning methods such as partial least squares (pls), artificial neural networks (ann) and support vector machine (svm) have been used. the obtained correlations have been used to infer experimentally undetermined peptides. the relationship between the biologic activity and molecular structure is an important issue in biology and biochemistry. qsar is a well-established method employed in pharmaceutical chemistry and has become a standard tool for drug discovery. however, the predictive capacity of qsar techniques is generally weaker than statistics-based approaches. therefore, a combination of the qsar method with a statistic-based technique may bring out the best in each other and can be a trend in future developments of drug discovery. at the structural level, numerous reports on affinity prediction have addressed the mhc-binding peptides. plentiful mhc-peptide complex structure records have been deposited in the pdb. the significance of domain-peptide recognition has been recently illustrated in the metabolic pathway and cell signaling. to predict the protein-peptide binding potency, a number of strict theories were suggested based on the potential free energy perturbation. the theories computed the alteration of free energies upon the interaction between phosphor-tyrosine-tetra-peptide (pyeei) and human lck sh domain. furthermore, to obtain a deep insight into the structural and energetic aspects of peptide recognition by the sh domain, a number of molecular modeling experiments such as homology modeling, molecular docking and mechanism dynamics were used. peptide array strategies confirmed that some peptide candidates may be potent binders of the abl sh domain. very recently, an approach including quantum mechanics/molecular mechanics, semiempirical poisson-boltzmann/surface area and empirical conformational free energy analysis was developed to quantitatively illustrate the energetic contributions involved in the affinity losing of pdz domain and oppa protein to their peptide ligands. , de novo peptide design recently, in order to de novo target-based peptide design, two remarkable methodologies including the vital method and an approach developed by bhattacherjee and wallin were introduced. the vital method pools verterbi algorithm with autodock to design peptides for the binding sites of a target. the "bhattacherjee and wallin" approach explores both peptide sequence and conformational space around a protein target at the same time. this approach was tested on three dissimilar peptide-protein domains to assess its ability. a brief list of the existing computational resources employed in peptide design is presented in table . in recent years, some computational methods have been proposed to design peptidomimetics. these methods can be classified based on their specificity to translate peptides to peptidomimetics. to select the best method, drug design, development and therapy : submit your manuscript | www.dovepress.com farhadi and hashemian awareness about the structure of peptide-protein complexes is important. , herein, recently introduced methods for computer-aided design of peptidomimetics are presented. growmol is a combinatorial algorithm employed in the peptidomimetics design. growmol searches a variety of probable ligands for the binding sites of a target protein and produces molecules with the chemical and steric complementarity for the d structure of binding sites. this method was used to generate peptidomimetic inhibitors of thermolysin, hiv protease and pepsin. by using the x-ray crystal structures of pepstatin-pepsin complexes, growmol predicted therapeutic peptidomimetics against the aspartic proteases. the algorithm created some cyclic inhibitors bridging the side chains of cysteine residues in the pl and p inhibitor subsites. the binding modes were checked using x-ray crystallography. , ludi is another interesting software referring to the de novo methodology. by using natural and non-natural amino acids as building blocks, the software designed peptidomimetics against renin, thermolysin and elastase. conformational flexibility of each novel peptidomimetic was searched through sampling the multiple conformers of each amino acid. peptide-driven pharmacophoric hypothesis is the most perceptive computational technique discovered in the peptidomimetics design. the method is especially useful when the x-ray structures of protein-protein complexes exist. the main idea is to adapt the hot spot concept into the associated pharmacophoric feature concept. with a pharmacophorebased virtual screening process, this strategy can determine novel type mimetics. in fact, the side chains of each amino acid can be simply categorized based on the conventional pharmacophoric characteristics, such as hydrogen bond donors and acceptors, aromatic ring and charged and hydrophobic centers. for example, in a report, pharmacophore model directed synthesis of the non-peptide analogs of a cationic antimicrobial peptide identified an anti-staphylococcal activity. to make a pharmacophore hypothesis, a model of rna iii-inhibiting peptide (rip), a well-known heptapeptide inhibitor of the staphylococcal pathogenesis, was utilized. through the virtual screening of , commercially available small molecules based on the rip-based pharmacophore, hamamelitannin was discovered as a non-peptide mimetic of rip. hamamelitannin is a tannin derivate extracted from hamamelis virginiana. , in another study, two rounds of in silico screening were performed to discover potential peptidomimetics able to mimic a cyclic peptide (cyclo- [cpfvktqlc] ) that is known to bind the anb integrin receptor. at the end of the process, the most potent representatives were at least , times better than the original cyclopeptide (around mm). in a prosperous instance, virtual screening was done by using multi-conformational forms of a large commercial library. a target-based pharmacophoric model mapped the cd -binding site on hiv- gp . the pharmacophore hypothesis was made based on a homology model of the protein cavity. in a cell-based assay, two of the top scoring molecules were detected as micromolar inhibitors of hiv- replication. computer-aided design of amino acid-based therapeutics the pharmacophore-based screening was used to find the novel alzheimer's therapeutics as mimetics of neurotrophins. the therapeutic utilization of neurotrophins might be restricted because of several deficiencies such as its reduced central nervous system penetration, decreased stability and potency to enhance neuronal death through interaction with the p ntr receptor. the mimetism of particular nerve growth factor domains could inhibit neuronal death. peptidomimetics of the loop and loop domains of nerve growth factor can prevent neuronal death induced by p ntr-dependent and trk-related signaling. in another study, a full-computational pharmacophorebased approach assessed the fda-approved drugs as valuable candidates to inhibit protein-protein interactions. peptide structures were designated in terms of pharmacophores and searched against the fda-approved drugs to detect same molecules. the top ranking drug matches contained several nuclear receptor ligands and matched allosterically to the binding site on the target protein. the top ranking drug matches were docked to the peptide-binding site. the majority of the top-ranking matches presented a negative free energy change upon binding that was comparable to the standard peptide. geometry similarity method geometry similarity methods create a geometric similarity between non-peptide templates and peptide patches. in a study, the supermimic tool was developed to recognize peptide mimetics. in the program, a complex library of peptidomimetics composed of several protein structure libraries has been deposited. moreover, supermimic includes the d-peptides, synthetic components (reported as betaturn or gamma-turn mimetics) and peptidomimetic ligands obtained from the pdb. in the program, the searching process allows scanning a library of small molecules that mimic the tertiary structure of a query peptide followed by scanning of a protein library where a query for small molecule can adopt into the backbone. , sequence-based method recently, a method has been developed to rank peptide compound matches that are limited to short linear motifs in proteins and compounds with amino acid substituents. the algorithm allows mapping the side chain-like substituents on every compound of a large chemical library. the complete molecule can be signified by a short sequence, and each fragment in the molecule can be represented as a distinct letter abbreviation. a cross-search between the pubchem database (about . million molecules) and a non-redundant collection of , peptides obtained from pdb demonstrated that the algorithm can be useful for high-throughput measurements. to recognize a true positive, the method explored identified protein motifs against the national cancer institute developmental therapeutic program compound database. in another study, the similarity of amino acid motifs to compounds web server was developed to ease screening of identified motif structures against bioactive compound databases. the methodology was reported to be efficient since the compound databases were preprocessed to maximize the accessible data, and the necessary input data was minimal. in similarity of amino acid motifs to compounds, motif matching can be full or partial that may decrease or enhance the number of potential mimetics, respectively. using a novel search algorithm, the web service can perform a fast screening of known or putative motifs against ready compound libraries. the classified results can be examined by linking to appropriate databases. , fragment-based method replacement with partial ligand alternatives through computational enrichment is a fragment-based approach. by using structures of peptide-bound proteins as design anchors, the program can computationally find a non-peptide mimetic for specific determinants of known peptide ligands. hybrid peptide-driven shape and pharmacophoric method development and application of strategies for pharmacophore modeling indicate that the medicinal chemistry community has broadly accepted the intuitive nature of the pharmacophore concept. besides, shape complementarity has been identified as a significant element in the molecular identification between ligands and their targets. in virtual screening efforts, using the pharmacophore-and shape-based techniques distinctly may increase the rate of false-positive results. therefore, incorporating both pharmacophore-and shape-matching techniques into one program can potentially diminish the rate of false positives. recently, to discover novel peptidomimetics, a weboriented virtual screening tool named pepmmsmimic was developed to pool the conventional pharmacophore matching with shape complementarity. a library of million conformers were extracted from . million commercially available chemicals and gathered in the mmsinc database. the database was used as a skeleton to develop farhadi and hashemian pepmmsmimic. in the pepmmsmimic interface, the d structure of a protein-bound peptide is used as an input. then, chemical structures able to mimic the pharmacophore and shape similarity of the original peptide are proposed to involve in the protein-protein recognition. a list of in silico methods used to design potential peptidomimetics along with their strengths and weaknesses is presented in table . overall, design and development of therapeutics are tedious, expensive and time-consuming procedures. therefore, using modern approaches including computer-aided design methods can lessen the examination phase, price and failure of therapeutics discovery. computational methods used to design amino acid-based therapeutics can increase the range of available biotherapeutics. benefiting from the dramatic advance in bioinformatics, computational tools can be used to find and develop therapeutic proteins, peptides and peptidomimetics. , moreover, using the computational tools decrease the cost of therapeutics development, from concept to market, by up to %. however, in the computational protein designing, there are some challenges such as our inadequate knowledge of folding and physical forces that stabilize protein structures. moreover, sequences and local structures have many degrees of freedom that can complicate the sequence search. therefore, there is a requirement for effective methods to find sequences related to a particular structure and measure essential protein folding criteria. overall, in silico design of amino acid-based therapeutics includes many challenges that should be removed to improve the overall performance of the design processes. for example, although structure determination of all disease-related proteins through crystallography and nmr is a laborious task, it is necessary to gather much structural information of peptide-protein interactions. besides, development of vigorous algorithms to calculate protein-protein binding energies is essential. the estimation of binding constant between two macromolecules with an appropriate speedaccuracy tradeoff needs millisecond scale molecular dynamics. moreover, understanding of both protein-protein and protein-peptidomimetics recognition processes in a molecular level can be improved using higher accurate force fields such as quantum mechanical polarizable force. in recent years, there are growing examples on the approval of monoclonal antibodies (therapeutic antibodies) by the fda for treatment of various diseases. this important area of amino acid-based therapeutics has been covered in more depth elsewhere. , for more explanation about the theorems and details of antibody informatics for 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protein-peptide complexes rosetta flexpepdock web server -high resolution modeling of peptide-protein interactions pdock: a new technique for rapid and accurate docking of peptide ligands to major histocompatibility complexes predicting peptide binding sites on protein surfaces by clustering chemical interactions protein-peptide complex prediction through fragment interaction patterns pep-sitefinder: a tool for the blind identification of peptide binding sites on protein surfaces vital: viterbi algorithm for de novo peptide design drug design, development and therapy : submit your manuscript | www.dovepress.com submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are the features of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord- -n ptr p authors: reddy, vijayalakshmi; desai, anita; krishna, shankar susarla; vasanthapuram, ravi title: molecular mimicry between chikungunya virus and host components: a possible mechanism for the arthritic manifestations date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: n ptr p background: chikungunya virus (chikv), a reemerging pathogen causes a self limited illness characterized by fever, headache, myalgia and arthralgia. however, – % affected individuals develop persistent arthralgia which contributes to considerable morbidity. the exact molecular mechanisms underlying these manifestations are not well understood. the present study investigated the possible occurrence of molecular mimicry between chikv e glycoprotein and host human components. methodology: bioinformatic tools were used to identify peptides of chikv e exhibiting similarity to host components. two peptides (a&b) were identified using several bioinformatic tools, synthesised and used to validate the results obtained in silico. an elisa was designed to assess the immunoreactivity of serum samples from chikv patients to these peptides. further, experiments were conducted in a c bl/ j experimental mouse model to investigate if peptide a and peptide b were indeed capable of inducing pathology. findings: the serum samples showed reactivity of varying degrees, indicating that these peptides are indeed being recognized by the host immune system during chikv infection. further, these peptides when injected into c bl/ j mice were able to induce significant inflammation in the muscles of c bl/ j mice, similar to that observed in animals that were injected with chikv alone. additionally, animals that were primed initially with chikv followed by a subsequent injection of the chikv peptides exhibited enhanced inflammatory pathology in the skeletal muscles as compared to animals that were injected with peptides or virus alone. collectively these observations validate the hypothesis that molecular mimicry between chikv e protein and host proteins does contribute to pathology in chikv infection. introduction chikungunya fever is caused by a arbovirus belonging to family togaviridae and genus alphavirus. chikv is positive sense rna virus with about . kb long genome. the prevalence of chikv has increased globally. it caused massive outbreaks when it re-emerged in in french reunion islands where it affected about % of the total population. chikv outbreaks also occurred in india during the same period, southern states in india recorded a total of . million cases [ , ] . chikungunya fever is characterized by fever, headache, myalgia and arthralgia. though chikungunya is a self limiting illness [ ] , a small proportion of - % of affected individuals develop persistent arthralgia. the risk factors associated with the development of persistent arthralgia include older age of patients (> years) and pre existing rheumatic problems. however, the precise molecular mechanisms of pathogenesis that lead to the development of these complications are poorly understood. experimental evidence of chikv persistence in macrophages of macaca species has been demonstrated [ ] and it has been suggested as one of the factors contributing to residual arthralgia. although, molecular mimicry as the cause of prolonged joint manifestations had not been proved conclusively in chikungunya infection, there are reports which suggest that such a phenomenon might be operational. therefore, in this study we investigated the possible occurrence of molecular mimicry between chikv e and host components using a three pronged strategy: (i) identification of homologous regions between chikv proteins and host tissue components using bioinformatics tools, (ii) establishing cross reactivity between serum samples obtained from chikv infected patients and peptides exhibiting molecular mimicry and (iii) validating the ability of the cross reactive peptides in inducing joint and muscle pathology in a mouse model. we demonstrate the occurrence of molecular mimicry between chikv envelope glycoprotein (e ) and the host components. a clinical isolate of chikv (chikungunya virus strain drde- ; genbank accession number: ef . ) was used for all the in vivo experiments in this study. the bioinformatics related work was carried out using the chikv e protein sequence from the prototype strain chikv s available in the swiss prot (id:q jux ). further, a multiple sequence alignment of the e glycoprotein of drde- sequences and chikv s revealed a % homology between the two strains. peptides chikv peptides were custom synthesised from commercial sources (hysel pvt ltd., india) and obtained as a lyophilised powder. the non-specific peptide was a gift from xcyton diagnostics private ltd, bangalore, india. rabbit anti-human polyclonal-hrp conjugate was procured from dako, denmark, while goat anti-mouse igg-hrp was obtained from genei, bangalore. all work related to animals was conducted with good animal practice defined by committee for the purpose of control and supervision of experiments of animals. the use of animals was approved by the institutional animal ethics committee (iaec) of nimhans (approval reference no: aec/ / (b)/nv dated . . ). the animals were housed in cages maintained in hygienic conditions with good ventilation, in a room maintaining the usual day and night cycle. the animals used for the experiments were euthanized by cervical dislocation and animal ethics were strictly adhered to at all times, while bleeding and sacrificing the animals. the use of human samples for the study was approved by was approved by institute ethics committee at nimhans (approval reference no: nimhans/ th iec/ ) which adheres to the ethical guidelines for biomedical research on human participants developed by the indian council for medical research (icmr). written informed consent was obtained from all the subjects themselves in the study. c bl/ j strain of mice were obtained from nimhans central animal research facility and used in the study. eight day old mouse pups were procured from the animal facility along with the mother and the mouse pups were used for the experiments. the human samples used in this study were received at the department of neurovirology, national institute of mental health and neurosciences (nimhans), which is one of the twelve designated national apex laboratories for the diagnosis of chikungunya in india. all the subjects enrolled in the study presented to the hospital/clinics with fever, joint pain, rash, myalgia, conjunctival redness, and headache. additionally, the prevalence and local outbreaks in the region aided in making a clinical diagnosis of chikungunya fever. blood samples ( - ml clotted blood) were collected from thirty six subjects, serum separated and stored in aliquots at - ˚c until all the tests were performed. the chikv infection was confirmed by detection of chikv specific igm antibodies using an elisa (national institute virology, pune) and/or chikv rna by taqman real time pcr targeting the nsp region [ ] . serum samples collected from healthy individuals served as controls. chikv was grown in c / cell line and infectious fluid was harvested. chikv infected c / fluid was centrifuged at , rpm for minutes to remove debris and nacl was added to the supernatant to obtain a final concentration of . molar. subsequently, polyethylene glycol was added to the mixture to obtain a final concentration of % (w/v) and the suspension stirred on ice bath for minutes. the mixture was incubated overnight at ˚c, and centrifuged at rpm for minutes to obtain the virus rich precipitate. the precipitate was dissolved in / th of original infected cell culture fluid volume using gtne buffer. chikv was purified using a discontinuous sucrose gradient method. briefly, ml of % sucrose (w/v) in gtne buffer was carefully overlaid onto . ml of % (w/v) sucrose. subsequently, . ml of chikv obtained after peg concentration was overlaid onto the discontinuous sucrose gradient and centrifuged at , rpm for hours at ˚c using a ultracentrifuge (beckman coulter, usa). the band at the inter-phase was collected and resuspended in - volumes of pbs (ph . ) and centrifuged at , rpm for hours to obtain a purified virus pellet. the pellet was dissolved in ml of fresh pbs and frozen in small aliquots at - ˚c. the complete genome sequence of a prototype chikv s belonging to african genotype was obtained from the gen bank. the sequence of chikv e glycoprotein was obtained from swissprot (q jux ). this sequence was uploaded into immune epitope database and analysis resource (iedb) server available at http://www.immuneepitope.org/. the server predicts the antigenic determinants using five different algorithms-chou and fasman beta turn prediction, emini surface accessibility prediction, karplus and schulz flexibility prediction, kolaskar and tongoankar antigenicity prediction, parker hydrophilicity prediction. the antigenic peptides from e region were deduced after considering hydrophilicity, surface probability, chain flexibility and secondary structure antigenic index both as text and graphs. the results obtained from the server were combined to construct a graph using ms-excel, which in turn yielded putative epitopic regions of chikv e glycoprotein. the peaks with antigenic propensity, surface accessibility, flexibility, hydrophilicity and beta turns were considered. the results obtained through iedb were further confirmed using additional server-european molecular biology open software suite (emboss) available at http://liv.bmc.uu.se/cgi-bin/emboss/antigenic. the results obtained using the chou and fasman criteria for beta turn prediction was also verified using coudes server. the existence of sequence similarity between chikv e glycoprotein and human hla-b was investigated using blast. the existence of structural similarity between the chikv e glycoprotein and human host components were determined by using bioxgem server and number of hits obtained in the non-redundant protein database (nrpdb) were limited to first in the output. multiple sequence alignment of e glycoprotein sequences of alphaviruses-chikv, onnv, rrv, sfv, and eeev was done using clustalw available at http://www.ebi.ac.uk/tools/clustalw /index.html. the optimal concentration of the peptide to be coated onto the elisa plate was predetermined in an initial experiment and was found to be μg/well. the peptides were coated onto the elisa microwells using carbonate buffer and incubated overnight at ˚c. the plate was washed three times with phosphate buffered saline with tween (pbst) and quenched using % skimmed milk powder in pbst for one hour at ˚c. the plates were washed with pbst and reacted with μl of serum samples ( : dilution in pbs containing . % triton-x ) obtained from patients infected with chikv (n = ) as well as serum samples obtained from control subjects (n = ). the samples were incubated for minutes at ˚c, followed by five washes with x pbst. subsequently, rabbit polyclonal anti-human antibodies tagged with hrp (dako, denmark) was diluted : and μl was added to each well and the plate was incubated at room temperature for minutes. the plate was washed five times with pbst and μl of the tmb solution was added and incubated in the dark for minutes. the reaction was stopped by the addition n sulphuric acid and the od values were read at nm using elisa reader (thermo scientific, usa). mouse experiments to evaluate the role of chikv peptides in causing tissue damage by molecular mimicry eight day old c bl/ j pups were procured along with the mother and the pups were used for the in vivo experiments. they were assigned to nine different groups with each group comprising of pups as shown in table . prior to determining the role of peptides in the possible enhancement of pathology related to chikv infection, the pathological changes induced by the chikv in c bl/ j mice were studied (group ). for this purpose, chikv ( pfu/ μl) was inoculated into the foot pad of day old mice. the control group of mice (group ) received equal volume ( μl) of eagles minimum essential medium (emem). the mice were kept under observation for days only post infection. at the end of the observation period, blood was collected from the mice through retro orbital plexus bleeding, and the mice euthanized to obtain the following organsbrain, thymus, heart, lungs, spleen, liver, kidneys, upper limbs and lower limbs. for histopathological studies the tissues were fixed in % paraformaldehyde, while for pcr the tissues were collected in emem. chikv infection was confirmed by two methods-presence of chikv specific igg antibodies in the serum and detection of chikv nucleic acid in the serum and harvested tissues using taqman real-time pcr [ ] . eight day old pups in group , and were injected with two doses ( μg /dose) of chikv peptide a, chikv peptide b and non-specific peptide respectively on day and day . the peptides were reconstituted in sterile x pbs (ph . ). equal volumes of peptide solution and freund's incomplete adjuvant were mixed and emulsified to obtain water in oil emulsion. the emulsion was stored at - ˚c until use. in all these three groups blood was collected th day post inoculation by retro orbital bleeding and fresh tissues were harvested and processed for pcr and paraformaldehyde fixed tissues for histopathology. mice in group were injected with emem alone followed by pbs emulsified in freund's incomplete adjuvant days post infection with chikv. c bl/ j mice in group , and were injected with chikv ( pfu/ μl) followed by μg of the chikv peptide a, chikv peptide b and non-specific peptide respectively, on th day post chikv inoculation through the same route at the same site. in these groups of mice the blood was collected and tissues harvested on th day post infection, and processed for pcr and histopathological examination. detection of anti chikv igg antibodies in the serum of c bl/ j mice serum was separated from the blood and stored at - ˚c. polystyrene elisa microwells (nunc, denmark) were coated with purified chikv in carbonate buffer at a concentration of μg/well diluted in carbonate buffer (appendix i). the plate was incubated overnight at ˚c, washed thrice with pbst and quenched using x pbst containing % skimmed milk powder. serum samples were diluted : in pbst and μl added to the wells and incubated at ˚c for hour followed by washing for five times with pbst. subsequently, μl of a : dilution of goat anti-mouse igg conjugated with hrp (genei, bangalore) was added to the wells, incubated at ˚c for hour followed by washing for times with pbst. finally, μl of the substrate solution (tmb) was added to all the wells and incubated in the dark for minutes. the reaction was stopped by the addition n sulphuric acid. the od values were read at nm using an elisa reader (thermo scientific, china). the tissues collected in emem were homogenised using a motorised hand held homogeniser. the homogenates were spun at , rpm for minutes at ˚c. the supernatants were collected, μl of the supernatant was used for rna extraction using qiamp viral rna extraction kit (qiagen, germany). the eluted rna was converted to cdna using high capacity reverse transcription kit (abi, usa). the cdna was stored at - ˚c until tested. similarly whole blood rna extraction kit was used to extract rna from blood and converted to cdna which was used in the taqman real time pcr as described earlier [ ] . all tissues were fixed in paraformaldehyde and embedded in paraffin for processing and μm thick sections obtained were mounted on selin coated glass slides. subsequently, the tissue sections were de-paraffinised with two changes of xylene and rehydrated in absolute alcohol followed by washing briefly in tap water. staining of the sections was carried out using harris haemotoxylin for - minutes, followed by washing under running tap water for minutes and differentiated in % acid alcohol for seconds. the sections were subsequently washed under running tap water for minute, treated with bluing saturated lithium carbonate solution for - seconds and washed under tap water for minute. counter staining of sections was carried out using eosin-phloxine solution for minute followed by dehydration in % alcohol and absolute alcohol for minutes each. the sections were finally immersed in xylene twice for minutes each for clearing and then mounted with dpx. bioinformatics approach to deducing molecular mimicry as described in the materials and methods section, the sequence of the african prototype of chikv s ei glycoprotein (q jux ) was obtained from the swissprot database and subjected to immune epitope analysis using five algorithms in iedb and emboss programs. the results are depicted in fig . the scores obtained for the five algorithms were uploaded into an ms excel sheet to generate a combined graph which yielded the putative epitopic regions of e glycoprotein of chikv. analysis of the peaks in the graph with respect to antigenic propensity, surface accessibility, flexibility, hydrophilicity and b turns enabled prediction of the following epitopic regions: these regions of chikv e glycoprotein satisfy all the criteria necessary for a given peptide to be considered immunogenic and capable of eliciting an immune response in the host system. subsequently multiple sequence alignment of e glycoprotein of alphaviruses-chikv, onyong onyong virus (onnv), ross river virus (rrv), semiliki forest virus (sfv), and eastern equine encephalitis virus (eeev) was done through clustalw. the results of the alignment are depicted in fig . as evident from the figure, the alignment revealed two motifs skd and kca present only in arthritogenic alphaviruses (chikv,onnv, rrv) and not in encephalitogenic alphaviruses (sfv, eeev). furthermore, the amino acid sequences skd and kca were present in the immunodominant peptides and deduced from e glycoprotein respectively. all further experiments using human serum samples and mouse models were therefore restricted only to these two peptides. the sequence similarity between the chikv e glycoprotein and hla-b was determined using blast. the alpha chain of hla-b molecule shared a partial homology from the stretch ranging from - of chikv e glycoprotein as well as the immunodominant region of peptide a (fig ) . the output obtained through the bioxgem d blast performed on the chikv e was limited to first hits. the output of the blast was further analyzed and limited only to human proteins ( table ). further analysis of these human proteins was limited to those that are known to contribute to the inflammation and arthritic pathology. amongst these, six human proteins-human complement component , complement component [ ] , fibronectin [ ] , kelch like protein [ ] , mast/stem cell growth receptor [ ] and beta arrestin [ ] which exhibited maximum similarity to chikv ei protein were only considered for further analysis. prominent among these six were complement proteins c and c . when the structural similarity between the e glycoprotein and complement component c was analyzed, the sequence of amino acids in the region of chikv e glycoprotein which shared homology with complement component c were also present in peptide a and peptide b (fig ) . all the patients in the study were from the state of karnataka, south india. among the patients with confirmed chikv infection, were females and were males. the mean age of the patients was . years. all the patients whose samples were used in the study presented with fever, while ( %) had arthralgia, ( %) had myalgia, ( %) had complained of headache. rash and gastrointestinal symptoms were seen in ( %) and ( %) of the patients each, and conjunctival redness was seen in ( %) of the patients. amongst / patients ( %) persistent arthralgia was reported at weeks after onset of initial symptoms. hence follow up samples blood samples could be collected from these three patients at weeks. in all other patients no follow up samples could be collected. a sample was considered to be positive in the peptide elisa if it had an od value equal to or greater than that of the cut-off value. the cut off value for each of the peptides was calculated by using od values obtained with sera of healthy control subjects (n = ) using the formula mean od of control samples + sd. the cut off od value for peptide a was . and for peptide b it was . . amongst the samples obtained from confirmed chikv patients, ( . %) showed reactivity to the peptide a and ( %) to peptide b (fig ) . the od values of chikv positive samples towards these peptides ranged from . to . for peptide a, while it varied from . to . for peptide b. these experiments were carried out to determine the possible synergistic role of peptides in the enhancement of pathology in chikv infection: all the mice were confirmed to have chikv infection by the detection of anti chikv antibodies in the serum by elisa. the cutoff in the elisa was determined using the mean + sd od values of serum samples obtained from uninfected control mice and it was . . the od values of serum samples from all the infected animals were found to be > . and hence considered positive for chikv-specific antibodies. in addition, the presence of chikv nucleic acids was demonstrable in the muscle tissue by taqman real time pcr while, it was not detected in the blood and other tissues of the infected group of mice. all the control group of animals were negative for chikv nucleic acids. in order to have an objective assessment of the pathological features noted in all groups of animals, a semi quantitative scale for scoring the degree of inflammation centred mainly around the muscles was evolved and the slides were evaluated by a pathologist blinded to the groups and the same is depicted in fig . the inflammation was graded as minimal ( +), mild ( +), moderate ( +) and severe ( +). the salient histopathological features noted in chikv infected mice were as follows: (i) the soft tissue around the elbow and knee joints were relatively normal with no evidence of synovitis or arthritis, while tissues close to the shoulder and the hip joint revealed moderate degree of lymphohistiocytic infiltration virus, (ii) the major muscles of the hip and the shoulder had multifocal lymphocytic infiltrate in the endomysium with myonecrosis, (iii) random and occasion muscle fibres close to inflammation revealed cytoplasmic basophilia and central nucleation with prominent nucleolus indicative of regenerative activity, similar to polymyositis noted in human subjects (iv) the synovium and periarticular soft tissue had sparse inflammation while the articular cartilage and the articular cavity were free of inflammation, (v) the epimysium around the muscle and tendinous portion close to the insertion had variable lympho-histiocytic inflammation indicating tenosynovitis, (vi) the striking pathology was mineralization of the necrosed muscle belly especially the lateral group of muscles close to the hip and shoulder joints similar to some cases of chronic polymyositis in human subjects. other than these features noted in the limbs and joints all the other organs did not reveal any significant pathological changes. some of the salient features noted in chikv infected mice (group ) are depicted in fig . the degree of pathological damage centred on the muscles in the various groups of mice was graded and a comparative chart was prepared ( table ) . as evident from the table, the group of mice that were mock infected (group ) and subsequently did not receive any peptides revealed sparse ( +) inflammation in the muscles probably related to the procedure. similar findings were also noted in the mock infected animals that received a single dose of freund's incomplete adjuvant (group ). in mice that received two doses of non-specific peptide but no virus (group ) hyperplasia of the bone marrow was noted with sparse inflammation ( +). on the other hand, mice that received two doses of chikv specific peptides but no virus (groups & ) exhibited myositis, muscle necrosis, vasculitis and hyperplasia of the marrow (immune mediated inflammatory muscle and marrow reactive changes) and an overall inflammation score of + (fig ) . in the three groups of mice that received an initial inoculum of virus followed by a single dose of either chikv specific peptides (groups & ) or non-specific peptide (group ) the pathological features were more florid (fig ) . amongst these three groups of animals, the mice that received virus followed by non-specific peptide exhibited features similar to those observed in mice that received virus alone (group ). the animals in groups and had the highest overall inflammatory score ( +) and showed multiple features including myositis, muscle necrosis, focal regeneration, mineralization and calcification of the necrotic muscles, hyperplasia of marrow in the long bones. molecular mimicry represents shared immunologic epitopes between a microbe and the host. in a viral system, viruses have been shown to have cross reactive epitopes with host self proteins [ ] . molecular mimicry can be either in the form of sequence homology wherein the host and the infectious agent share the sequence of similar or identical amino acids or it might be due to the conformational similarity between the host and the infectious agent in question [ ] . molecular mimicry is one of the major mechanisms for the induction of autoimmune diseases through the activation of autoreactive t cells in the host immune system. several elegant examples of molecular mimicry leading to autoimmune manifestations have been described following bacterial and viral infections [ ] . chikungunya fever is a self limiting illness, however in - % of the patients arthralgia persists for a period of two years and above [ ] . more than half of all chikv infected patients in la reunion island during the - epidemics had complaints of persistent joint pain / recurring stiffness [ ] . the arthritis attributed to chikv infection indeed mimics rheumatoid arthritis, as discussed by bouquillard et al [ ] wherein patients infected with chikv in reunion islands developed ra. further, malvy et al [ ] suggested that molecular mimicry may be responsible for chronic manifestations as symptoms continue to persist despite chikv not being detectable in the synovial tissue. we investigated molecular mimicry in this study by using a combined approach of identifying homologous regions between chikv glycoprotein e protein and host tissue components using bioinformatics tools, the ability of these designed peptides to cross react with serum samples from chikv infected patients and inducing immune mediated joint and muscle pathology in a mouse model. in order to determine if there are any "arthritogenic" motifs within the e protein, a multiple sequence alignment of amino acid sequences of e glycoprotein of chikv was carried out with other alphaviruses such as onnv, rrv, sfv and eeev using clustalw (fig ) . the alignment revealed that two common motif(s) skd and kca were present only in arthritogenic alphaviruses such as chikv, rrv and onnv but not in the 'encephalitogenic' alphaviruses such as sfv and veev (fig ) . the presence of these two motifs seen only in arthritogenic alphaviruses lead us to postulate that these two motifs may have a role in the development of arthralgia which is a hallmark in the disease produced by these group of viruses. simultaneously, the immunodominant epitopes in the chikv e glycoprotein were deduced using iedb and emboss programs which use criteria like presence of beta turn, surface accessibility, flexibility, antigenicity and hydrophilicity of the regions to be studied. on combining the common results obtained with these two programs, four immunodominant regions were obtained in chikv e glycoprotein (peptide a, b, c and d). interestingly, it was observed that the "arthritogenic" motifs skd and kca were also present in the immunodominant peptides a and b respectively. sequence homology comparisons between chikv e glycoprotein and various human proteins using blast revealed that a homology of four consecutive amino acids tqlv/telv exist between the chikv e glycoprotein and hla-b molecule (fig ) . it is a well established that hla b has been implicated in the pathogenesis of autoimmune arthritis and ankylosing spondylitis [ ] . interestingly this consecutive sequence of amino acids was also present in one of the immunodominant peptides (peptide a) of chikv e glycoprotein ( fig ) . having ascertained that there is amino acid homology between chikv e glycoprotein and hla b molecule, the occurrence of structural homology was also explored using the bioxgem program. this program searches for the longest common substructures existing between the query structure and every structure in the database. the output of the query while executing the program was however limited to the first hits. amongst these proteins, further analysis was restricted only to human proteins present in the output. amongst the human proteins in the list (table ) , six proteins were shortlisted as they are known to contribute to either inflammation or arthritic pathology. the most prominent amongst them was complement components c and c . indeed, c complement component has been implicated in inflammation and tissue injury in other related alphaviral infections [ ] and therefore further analysis was confined to the homology between the chikv e glycoprotein and the c complement component. the analysis showed that the homology occurred between von willebrand factor (vwf) domain of c and chikv e glycoprotein (panel a, fig ) . interestingly, the amino acid sequences in the region of chikv e glycoprotein exhibiting homology were also present in the immunodominant peptides a and b (panel b, fig ) . in summary, combining the data from the various bioinformatics approaches and employing a logical algorithm relevant to the pathogenesis of arthritis the choice narrowed down to two immunodominant peptides of chikv e protein (peptides a & b) .therefore all further experiments were carried out using only these two peptides.the peptides a and b were used in an elisa to assess the immunoreactivity of serum samples obtained from chikv confirmed patients as well as healthy control subjects. the serum samples obtained from chikv confirmed patients (n = ) showed reactivity to both the peptides to varying degrees. antibodies to peptide a was noted in / ( . %) of samples while / ( %) of serum samples showed reactivity to peptide b (fig ) . these results indicate that the two peptides are indeed being recognized by the host immune system during chikv infection. as evident form fig , it was interesting to note that the sera from three patients who had persistent arthralgia weeks after the onset of initial symptoms indeed exhibited much higher od values ( . to . for peptide a and . for peptide b) to the two peptides a & b as compared to other patients (od values were close to cut off and between . and . ). although, a quantitative elisa would have delineated these differences better our elisa was designed only as qualitative assay. further, experiments were conducted to investigate if peptide a and peptide b were capable of inducing pathology in an experimental mouse model. the results indicated that these two peptides on their own were able to induce significant inflammation in the muscles of c bl/ j mice (fig and table ) similar to that observed in animals ( +) that were injected with chikv. further, animals that were primed initially with chikv followed by a subsequent injection of the two chikv peptides exhibited enhanced inflammatory pathology ( +) as compared to animals that were injected with peptides or virus alone (fig and table ). on the contrary, animals that received an unrelated peptide (i.e. not containing the arthritogenic motifs or exhibiting homology to host proteins) either before or after priming with chikv exhibited minimal muscle inflammation ( +). collectively these observations validate the hypothesis that molecular mimicry between chikv e protein and host proteins does contribute to pathology in chikv infection. such observations have not been reported hitherto in chikv infection although molecular mimicry as a mechanism leading to autoimmune phenomena has been demonstrated in several microbial infections including viruses and bacteria [ ] . among the viruses, molecular mimicry has been noted in theiler murine encephalitis virus (tmev), hepatitis b virus (hbv), and sfv and coxsackie viral infections [ ] . in sfv infection of c bl/ j mice a similar approach using bioinformatic tools derived peptides and validation of these peptides invivo for their ability to induce autoimmune demyelination was undertaken [ ] . an algorithmic approach was used to demonstrate amino acid homology between immunogenic epitopes of sfv and various myelin proteins. the criterion used for the occurrence of molecular mimicry was the presence of three similar consecutive amino acids. it was observed that myelin oligodendrocyte protein (mog) shared homology with sfv e glycoprotein. the injection of a peptide containing the sequence of shared amino acids into mice caused demyelination with presence of multifocal vacuolation in the cns white matter [ ] . the phenomenon of molecular mimicry has also been studied in sars infection [ ] . eleven peptides derived from sars spike(s) protein which shared homology with various human proteins were synthesised and their reactivity was assessed using serum samples obtained from sars patients. serum samples recognised only / peptides and the authors concluded that these two peptides may contribute to viral pathogenesis through the phenomena of molecular mimicry. the limitations of the present study are twofold. firstly, hla-b typing was not performed in the chikv confirmed patients and controls of this study. however, serum reactivity of peptide a which shared homology with hla b molecule suggests that this molecule may have an important role to play in persistent arthralgia as / patients exhibited high reactivity to it. the geographical and ethnic variation in the prevalence of hla-b globally is well documented [ ] and further its occurrence in chikv related complications is also reported to be very low [ , ] . therefore it may be argued that the observance of molecular mimicry between chiv e glycoprotein and hla-b may not be the major factor contributing to complications that ensue following acute infection. secondly, the time interval between the injection of chikv and the peptides into animal the model (groups & ) is rather short rendering it difficult to conclude whether the inflammatory response noted was due to the immunopathologic process of molecular mimicry or a direct inflammatory effect of both the virus and the peptide. however, the muscle fibrosis and calcification in the muscles noted in the experimental animals recapitulates immune mediated polymyositis in humans during the evolution and progression. the other limitation with all studies conducted using animal models to demonstrate molecular mimicry as a mechanism of pathogenesis is that an adjuvant such as cfa/fica is invariably required to elicit immune mediated damage. this suggests that in addition to having cross reacting disease inducing epitopes, sufficient activation of antigen presenting cells is required. consequently, the most difficult part in the studies investigating molecular mimicry is to correlate/extrapolate the findings obtained in vitro and/or animal studies to the disease occurring in the natural host. despite this limitation, the concept of molecular mimicry remains a viable hypothesis for framing questions and approaches to understanding the pathogenetic mechanisms involved during the disease process. therefore, it would be definitely worthwhile to pursue future studies with the two chikv peptides described in this study using transgenic animal models and human t and b cell responses to the peptides. for their help with histopathology and mr. kumar from department of neurovirology for help with the animal experiments. we would like to thank late. dr. v. kumaraswami for being inspiration for the study. re-emergence of chikungunya virus in india chikungunya virus infection-a resurgent scourge imported chikungunya infection chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages utility of igm elisa, taqman real-time pcr, reverse transcription pcr, and rt-lamp assay for the diagnosis of chikungunya fever the role of complement in inflammatory diseases from behind the scenes into the spotlight citrullination of fibronectin in rheumatoid arthritis synovial tissue identification of specific autoantigens in sjögren's syndrome by serex the critical role of mast cells in allergy and inflammation increased expression of beta-arrestin and in murine models of rheumatoid arthritis: isoform specific regulation of inflammation molecular mimicry and immune-mediated diseases chikungunya virus infection. a retrospective study of cases post-epidemic chikungunya disease on reunion island: course of rheumatic manifestations and associated factors over a -month period rheumatoid arthritis after chikungunya fever: a prospective follow-up study of cases destructive arthritis in a patient with chikungunya virus infection with persistent specific igm antibodies hla-b : natural function and pathogenic role in spondyloarthritis complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease molecular mimicry as a mechanism of autoimmune disease molecular mimicry between a viral peptide and a myelin oligodendrocyte glycoprotein peptide induces autoimmune demyelinating disease in mice peptide mimicrying between sars coronavirus spike protein and human proteins reacts with sars patient serum what's new? specific management of post chikungunya rheumatic disorders: a retrospective study of cases in reunion island from we thank ms. priyanka s and ms. pavithra s for help with bio-informatics analysis. we thank mr. prasanna and ms. rajashakti from human brain tissue repository (hbtr) from nimhans conceived and designed the experiments: vr rv ad ssk. analyzed the data: vr rv ssk ad. wrote the paper: rv vr ssk ad. key: cord- -o r ktie authors: kokoszka, malgorzata e.; kay, brian k. title: mapping protein–protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning date: - - journal: peptide libraries doi: . / - - - - _ sha: doc_id: cord_uid: o r ktie one avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. with the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of months. to illustrate this approach, we use a library of bacteriophage m particles, which display -mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the sh domain of the human lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase cbk . the binding properties of the selected peptide ligands are then dissected by sequence alignment, kunkel mutagenesis, and alanine scanning. finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. very often in research projects, there is interest in mapping the protein-protein interactions of a protein of interest as a way of understanding its function in the cell or virus. while a variety of techniques exist for this purpose, such as yeast two-hybrid screening, mass spectrometry, and protein complementation assays, another approach is the use of phage-displayed combinatorial peptide libraries. in such an approach, one takes a purifi ed, recombinant protein and isolates peptide ligands to it through affi nity selection (fig. ) . interestingly, the phage-displayed peptides bind at "hot spots" for molecular interaction and very often share the same primary structure as short regions within cellular interacting proteins. several examples where this approach has proven useful include such targets as protein interacting domains [ , ] . to demonstrate the utility of this approach, we describe its application to two targets, human protein tyrosine kinase, lyn, and the yeast serine/threonine-protein kinase cbk . lyn was fi rst discovered as a viral oncogene [ ] and later appreciated to be a proto-oncogene in humans. it is a non-receptor protein tyrosine fig. a general workfl ow diagram for isolating and characterizing the peptide ligands to protein domains or fragments using phage display methods. in principle, coding regions of any protein domain of interest can be converted into recombinant dna and expressed as a fusion protein to a partner such as glutathione s-transferase (gst). with a soluble protein in hand, one can perform affi nity selections with phage-displayed combinatorial peptide libraries to identify its peptide ligands. the binding properties of those ligands can be then further characterized through affi nity and specifi city measurements. to assess the importance of each residue in the peptide ligand, a mutagenic analysis known as alanine scanning can be performed on the recombinant dna. with that knowledge, potential interacting partners of the protein of interest can be predicted [ ] . finally, improving affi nity and specifi city of selected ligands through directed evolution may lead to the development of antagonists, which could be used as inhibitors of specifi c cellular interaction for proof-of-principle experiments of drug development kinase, which is a member of the src family of proteins. it has a modular architecture: a src homology (sh ) domain, a src homology (sh ) domain, several linker regions, and a catalytic domain that phosphorylate tyrosines in proteins [ ] . the sh domain plays a role in mediating protein-protein interactions and has been described to bind proline-rich motifs in proteins. the cbk belongs to a large family of ndr/lats protein kinases, which is conserved across eukaryotes and includes members such as myotonic dystrophy kinase [ ] . cbk plays a role in controlling cell separation after cytokinesis, cell integrity, and polarized growth in saccharomyces cerevisiae [ ] . to date, only a few substrates of cbk have been reported. one of them, ace , a transcription factor that is activated by cbk via three phosphorylation sites [ ] , is responsible for transcriptional control of enzymes required for septum degradation after cytokinesis [ ] . the other, ssd , is an rnabinding protein that suppresses translation of certain mrnas and which loses activity when phosphorylated by cbk [ ] . we chose these two proteins as targets in parallel affi nity selection experiments to illustrate that the same phage-displayed combinatorial peptide library can yield very different peptide ligands to different targets. the lyn sh domain has previously been used in affi nity selection experiments, and so it served as a positive control. the cbk protein had not been used previously in affi nity selection of combinatorial peptide libraries. we also show that the kunkel mutagenesis in combination with alanine scanning and elisa are very simple and expedient methods for evaluating the contribution of certain amino acids in the peptide ligands for binding to their targets. prepare using deionized water (dih o). autoclave or fi lter sterilize and store at room temperature, unless indicated otherwise. single-stranded dna samples were isolated with the qiaprep spin m purifi cation kit (qiagen). . covalently closed, circular double-stranded m dna, synthesized via kunkel mutagenesis [ , ] , was purifi ed with qiaquick pcr purifi cation kit (qiagen). . concentrations of dna samples were determined using nanodrop nd- uv spectrophotometer (thermo fisher scientifi c, inc.) and measuring the optical absorbance at nm. . the following steps: phosphorylation of oligonucleotides, annealing to the template, and synthesis of covalently closed circular double-stranded dna (cccdna) were performed using dna engine dyad ® thermal cycler (bio-rad). in described methods, affi nity selections with phage-displayed combinatorial peptide libraries (figs. and ) were employed to identify peptide ligands that bind to human lyn sh domain and yeast cbk protein kinase. in general, once peptide ligands have been identifi ed for a protein target, an interacting motif can be deduced via sequence alignment (logo, fig. ) of the primary structures of the displayed binding peptides. however, to assess the functionality of this motif, amino acid replacements of the critical residues are generally necessary. while this is possible through the chemical synthesis of variant peptides, it is more expedient to use mutagenesis techniques, such as alanine scanning (fig. , [ ] ). for the purpose of this selection protocol, the target domain was overexpressed in escherichia coli in the form of a glutathione s-transferase (gst) fusion protein. recombinant protein can be prepared via commercially available pgex vectors (ge healthcare). figure presents a schematic diagram of the selection protocol utilizing glutathione-conjugated magnetic beads. to maintain optimum sterility and eliminate carryover, only fi ltered tips should be used throughout the selection procedure. . add μl of glutathione magnetic bead slurry ( μl of settled beads) into ml eppendorf tube, and wash twice with μl of ice-cold wash buffer # (pbs) on magnetic separator. remove the supernatant. . remove the supernatant. resuspend in μl of blocking buffer # . add μg of gst protein to remove potential gst-binding phage clones and library equivalents (e.g., if the library size is clones and the titer pfu/ml use μl). bring the volume to μl with ice-cold wash buffer # . incubate at °c for h on the rotating shaker. . remove the supernatant. wash three times with μl of icecold wash buffer # (pbst). remove supernatant, add μl of ice-cold wash buffer # , and transfer into a new ml eppendorf tube to eliminate any plastic-bound phage. . repeat the washing step two more times with wash buffer # . remove the supernatant. . elute the phage with μl of elution buffer. incubate for min at room temperature, and gently mix the content every couple minutes. . place the tube on magnetic separator, and transfer the eluted phage supernatant into a new . ml eppendorf tube containing μl of neutralization buffer, and mix well. fig. c ) revealed a known y/fxfp docking motif to cbk [ ] . also, it should be noted that sequence (fig. c ) contains the fkfp motif, which is present in ssd , a known cbk substrate [ , ] . our fi ndings clearly indicate that potential binding partners of certain targets can be deduced via selections with phage-displayed peptide libraries. as only three sequences were used for alignment, any additional amino acid preference analysis could be biased. ( c ) further characterization of the isolated y/fxfp motif showed preference to positively charged residues, r and k, at the "x" position. other allowed residues at that position included v, m, t, q, when at least one positively charged residue was observed outside the motif, or c, when a second cysteine was present following the motif. all sequences incorporated in the logo ( isolates, data not shown) were isolated by screening phage-displayed focused library. the library was synthesized via kunkel mutagenesis (as described in subheading . . if blue-white screening can be performed, add μl of mm iptg and μl of % x-gal. . immediately before plating, add ml of × yt top agar ( . %), gently swirl, and pour over prewarmed × yt agar plates. incubate overnight at °c. . add anti-m hrp-conjugated antibody diluted : , in pbst (same per well volume as the target protein), and incubate min to h at room temperature. . wash the plates with μl of pbst three more times. . add chromogenic substrate solution (subheading . , item ) (same per well volume as the target protein), and incubate for - min. . quantify the results by measuring the optical absorbance at nm, using a microtiter plate spectrophotometer. all binding phage clones isolated via elisa are amplifi ed and their binding regions subsequently identifi ed by dna sequencing ( see note ). if the selection generates enough unique isolates, the binding motif of the target can be predicted by sequence alignment known as logo plot (fig. ) . with that knowledge, one can attempt to better defi ne the known interactions and to predict new substrates of the target. to further assess the importance of each residue in the motif, mutagenic analysis known as alanine scanning can be performed (fig. , [ ] ). in this method, each residue is replaced, one by one, by alanine (or by glycine if originally occupied by alanine). one way to generate all the variants is to incorporate them into m phage genome via kunkel mutagenesis (described in subheading . . ) [ , , ] . once the phage-displayed mutant pool is generated, the effect of the substitutions on their binding to the target is evaluated by phage elisa (described in subheading . ). for the purpose of this protocol, a modifi ed m phage vector containing an amber stop codon has to be fi rst obtained [ ] . in the vector constructed by scholle et al., an amber stop codon, tag, has been placed at the n-terminus of the coding region of gene iii, following the signal sequence of protein iii (piii). that modifi cation eliminates the need for generation of uracil-containing single-stranded dna (u-ssdna) template, using e. coli strain cj ( dut − ung − ). a mutagenic oligonucleotide, containing both complementary and exogenous dna fragments, is then annealed to the ssdna template, with the exogenous fragment looping over the tag-containing vector region. once the double-stranded dna (dsdna) is synthesized from the ssdna template, it is electroporated into non-suppressor e. coli strain (e.g., ss ). if the tag triplet-containing region has not been replaced by the exogenous fragment, the phage will not be propagated as the minor coat protein, piii, cannot be translated. the wild-type phage can be propagated via the suppressor e. coli strain such as tg cells. the amber stop codon is then translated into glutamine. the use of tag-containing template facilitates nearly % recombination effi ciency [ ] . . if low quantities of target are available, a small amount can be used to coat the beads (e.g., μg of protein and μl of slurry, respectively). also, decreasing the amount of used target can facilitate isolation of higher affi nity clones. . for convenience, target-bound beads can be prepared for the entire selection procedure and stored at °c in the blocking buffer # (pbs containing % bsa (w/v)) for up to a week. . as long as no contamination is introduced, phage particles can be stored in a culture medium at °c for many years. . since the phage titer, recovered after fi nal round of selection, depends on many controllable and uncontrollable factors such as stringency of the selections (i.e., number of washes, amount of target used) or type and structure of the target, from our experience, we recommend to cover a wide dilution range from at least − - − . . if fi ltered tips are used for phage transfer, it is convenient to briefl y rinse and remove the tips with multichannel pipette. . in order to identify dna sequences of potential "binders," the phage is fi rst amplifi ed by infecting μl of tg cells (from a mid-log phase culture) with μl of phage supernatant (or single plaque) and incubated overnight in a shaking incubator ( rpm), in ml fi nal volume. the pelleted cells are then used for isolation of dsdna that consists of replicative form of phage dna. subsequently, the samples are analyzed via sequencing. remaining phage supernatant can be stored at °c ( see note ). . in order to isolate high quantity of ssdna template, large amount of phage particles has to be fi rst collected. amplifi cation of phage by infecting cells with just a single plaque may result in low quantity of template. thus, we suggest a two-step preparation process, where the peg-precipitated concentrated virions are used to infect the fresh mid-log cells. . to remove any polyethylene glycol (peg) residues from the surface of the tube prior to phage reconstitution, it is recommended to fi rst briefl y rinse the side of the tube with pbs, avoiding the phage pellet. usually - ml of room temperature pbs is used to resuspend the pelleted virions. one of the major advantages of affi nity selection of phage-displayed combinatorial peptide libraries is the potential for rapid discovery of peptide ligands to a target protein. it is relatively straightforward to use homemade or commercially purchased libraries to isolate peptide ligands to a given target and then deduce a binding motif. a peptide with a consensus motif can then be used to study the biology of the target (i.e., predict cellular interacting partner, solve the three-dimensional structure of the peptide and target in a complex, and to inhibit the target in cells). however, when only one or a few peptide ligands are isolated to a target, it is diffi cult to know a priori what residues in the phage-displayed peptide ligand contribute to binding. while one can explore the binding parameters of the peptide sequence through chemical synthesis of peptides with systematic amino acid replacements across the primary structure, we present an alternative, faster, and easier approach involving kunkel mutagenesis, alanine scanning, and elisa. we fi nd that by coupling these three techniques, one can readily determine at fi rst pass many important aspects of the binding interaction. exploring protein -protein interactions using peptide libraries displayed on phage mapping intracellular protein networks the yesrelated cellular gene lyn encodes a possible tyrosine kinase similar to p lck src family kinases: regulation of their activities, levels and identifi cation of new pathways cbk p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in saccharomyces cerevisiae the saccharomyces cerevisiae mob p-cbk p kinase complex promotes polarized growth and acts with the mitotic exit network to facilitate daughter cell-specifi c localization of ace p transcription factor the ndr/lats family kinase cbk directly controls transcriptional asymmetry cbk regulation of the rna-binding protein ssd integrates cell fate with translational control effi cient construction of a large collection of phage-displayed combinatorial peptide libraries effi cient site-directed mutagenesis using uracilcontaining dna highresolution epitope mapping of hgh-receptor interactions by alanine-scanning mutagenesis improvements to the kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries rapid and effi cient sitespecifi c mutagenesis without phenotypic selection convergent evolution with combinatorial peptides can we infer peptide recognition specifi city mediated by sh domains? distinct ligand preferences of src homology domains from src, yes, abl, cortactin, p bp , plcy, crk, and grb proteome-wide discovery of evolutionary conserved sequences in disordered regions this work was funded by the chicago biomedical consortium, with support from the searle funds at the chicago community trust. we would like to thank mr. kyle schneider, dr. brian yeh, and dr. eric weiss (nu) for providing the gst-cbk dna constructs and purifi ed gst-cbk fusion protein. we are grateful to dr. michael kierny and dr. renhua huang (uic) for their helpful comments on this manuscript. key: cord- -nlaiurau authors: ansari, junaid; kaur, gaganpreet; gavins, felicity n. e. title: therapeutic potential of annexin a in ischemia reperfusion injury date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: nlaiurau cardiovascular disease (cvd) continues to be the leading cause of death in the world. increased inflammation and an enhanced thrombotic milieu represent two major complications of cvd, which can culminate into an ischemic event. treatment for these life-threatening complications remains reperfusion and restoration of blood flow. however, reperfusion strategies may result in ischemia–reperfusion injury (i/ri) secondary to various cardiovascular pathologies, including myocardial infarction and stroke, by furthering the inflammatory and thrombotic responses and delivering inflammatory mediators to the affected tissue. annexin a (anxa ) and its mimetic peptides are endogenous anti-inflammatory and pro-resolving mediators, known to have significant effects in resolving inflammation in a variety of disease models. mounting evidence suggests that anxa , which interacts with the formyl peptide receptor (fpr) family, may have a significant role in mitigating i/ri associated complications. in this review article, we focus on how anxa plays a protective role in the i/r based vascular pathologies. cardiovascular diseases (cvd) are the main cause of morbidity and mortality worldwide and were solely responsible for the death of about . million people in , with the vast majority being due to either ischemic heart disease (ihd) or cerebrovascular disease [ ] . this has led to the exponential increase in healthcare costs with estimated increase in total medical care costs in united states (us) alone rising from $ billion to $ billion between the years and . additionally, the burden of cvds has increased tremendously in developing countries and are predicted to overtake infectious disease as the leading cause of mortality by the year [ ] . many acute cardiovascular events, such as acute ischemic stroke (ais) and myocardial infarction (mi), are due to, e.g., unhealthy vascular states, resulting from prolonged atherosclerosis, coronary artery disease, diabetes, uncontrolled hypertension, and aging [ ] . additional studies have suggested behavioral, psychosocial including bereavement [ ] and environmental factors can also trigger and contribute to acute cardiovascular events [ ] . both ais and mi are life-threatening conditions which require prompt treatment consisting of rapid restoration of blood flow followed by subsequent management consisting of anti-inflammatory strategies and neuro-and cardio-protection for secondary prevention [ , ] . however, restoration of blood supply and re-oxygenation paradoxically lead to further exaggeration of tissue injuries and tissue destruction (termed ischemia-reperfusion injury (i/ri)). to restore homeostasis, it is essential mechanisms of activation where t cells accumulate at sites of ischemia and reperfusion [ , ] . multiple studies have shown that enhanced generation of oxidants results in the activation and deposition of complement and phospholipase a (pla )-mediated production of ltb and platelet activating factor (paf). furthermore, there is abundant evidence suggesting the role of platelets in i/ri [ ] by mediating microvascular thrombosis as well as ensuing inflammation through platelet-platelet as well as platelet-leukocyte interactions. these communications lead to the release of a broad range of pro-inflammatory molecules including high-mobility group box protein (hmgb ), cell differentiation ligand (cd l), polyp, and interleukin- alpha/beta (il- α/β) furthering the thrombo-inflammatory environment and contributing in both innate and adaptive immune responses [ ] . annexin a (anxa ) is a -kda member of superfamily of thirteen annexin proteins, comprising amino acids with at least twelve distinct ca + and phospholipid binding proteins [ ] . this member of the superfamily was aptly named anxa due to its capability to "annex" phospholipid membranes [ ] . the protein itself was discovered in the s and s by its ability to inhibit pla activity and eicosanoid synthesis [ ] . further anti-inflammatory actions were seen in guinea pig lung models, glucocorticoid (gc)-treated rat models and rat medulla cell experiments [ ] . anxa has a unique n-terminal region (active biological portion) embedded within its core, which is only exposed under elevated ca + levels (≥ mm). the n-terminal region of each member of the annexin superfamily is believed to be the "fingerprint", endowing its biological activity, however there are other regions of interest in the core protein which are known to also possess anti-inflammatory properties, for instance, antifammin- [ , ] . anxa is abundantly present in cells of myeloid origin such as neutrophils, monocytes and macrophages. under resting conditions, anxa is present within the cytoplasm and is only extruded upon cell activation [ ] . the molecular mechanisms responsible for secretion vary from cell to cell, e.g., in neutrophils, anxa resides in the gelatinase granules and is released upon neutrophil activation (e.g., neutrophil-endothelial interaction), attaching itself to the outer leaflet of the plasma membrane and undergoing further conformational change to expose the n-terminal region [ ] . in the case of macrophages, anxa is released via the atp-binding cassette (abc) transporter system [ ] . anxa is regulated by gcs, with gcs increasing both the anxa gene and also its secretion from existing intracellular pools by stimulating protein kinase c (pkc) activity [ ] . the anti-inflammatory and pro-resolving effects of this kda protein are mediated by the shedding of l-selectin resulting in decreased neutrophil adhesion to the endothelium and restricting transmigration [ ] . pharmacological intervention of anxa has been shown to decrease neutrophil rolling and adhesion to endothelium while increasing detachment of adherent and inducing neutrophil apoptosis [ ] . perretti and flower ( ) demonstrated that anxa attenuated il- and il- induced neutrophil migration into the murine air pouch [ ] . additionally, getting et al. showed that both endogenous and exogenous anxa were able to inhibit the neutrophil and monocyte recruitment in murine peritoneal cavity [ ] . these findings suggest that anxa retains its anti-inflammatory action irrespective of stimuli. furthermore, anxa may further undergo changes such as cleavage, induced by neutrophil specific proteases (e.g., cathepsin g (cg) [ ] , neutrophil elastase (ne) and proteinase- [ ] ), although it is unknown as to whether the cleavage process occurs to inactivate anxa and produce homeostasis, or to produce bioactive fragments which can act as a pro-drug [ ] . anxa and its mimetic peptides, such as the n-terminal derived ac - , bind to the formyl peptide receptor (fpr) family of seven transmembrane g-protein-coupled receptors (gpcrs) [ ] . various cell types express fprs, especially myeloid cells, e.g., neutrophils and monocytes. three fpr members exist in humans and they are termed: fpr , fpr /alxr (also known as the lxa receptor), and fpr . fpr /alxr shares % amino acid sequence homology with fpr , and fpr shares % amino acid homology to fpr and % to fpr /alx [ , ] . in mice, the fpr family is more complex, consisting of at least eight members. mouse fpr shares % sequence homology with human fpr , and fpr has % homology to fpr /alxr [ ] . the fprs are primarily coupled to g proteins (g iα , g iα ). upon the binding of a ligand, such as formyl-met-leu-phe (fmlp), g proteins are activated and trigger the release of several second messengers such as ca + from intracellular stores, through activation of phospholipase c (plc), pld and pla [ ] . neutrophils predominantly sense inflammatory stimuli via fprs to perform both pro-as well as anti-inflammatory functions, depending upon the pathophysiological status and ligand binding. in inflammatory states, neutrophil fprs participate in various biological functions including chemotaxis, degranulation, ros production, promoting neutrophil-platelet interactions [ ] , and enabling apoptosis and phagocytosis [ ] . the ability of fprs to perform such wide range of biological functions is due to their ability to interact with multitude of agonists and antagonists, ranging from formylated and non-formylated proteins/peptides to small molecular weight compounds, e.g., fmlp, his-phe-tyr-leu-pro-met (hfylpm) (chemoattractants for monocytes and neutrophils), anxa , and hiv envelope proteins gp and gp [ ] . a detailed description of the fprs, their ligands and biological functions is given in tables and . among the fpr members, fpr /alx is the most versatile and can interact with a multitude of ligands resulting in both anti-inflammatory and pro-resolving (anxa and lxa ) as well as pro-inflammatory (serum amyloid a (saa) and cathelicidin) functions (table ) . these diverse effects also seem to be partly due to the ability of fpr /alx to facilitate biased agonism and enable different dimerization states after ligand binding [ , ] . binding of saa and/or the cathelicidin-associated antimicrobial peptide leucine- (ll ) to fpr /alx results in neutrophil nf-κb activation, cytokine release, increased neutrophil infiltration and lifespan [ ] . however, binding of anti-neuronal nuclear antibody (anna ) inhibits neutrophil infiltration, promotes neutrophil apoptosis, and pushes macrophages towards a less pro-inflammatory phenotype, increasing the rate of phagocytosis by macrophages [ ] . ongoing studies are showing that fpr signaling is associated with neutrophil oxidative burst by inducing a rapid and reversible increase in the mitochondrial membrane potential within neutrophils [ ] . in addition, mitochondrial-derived fpr ligands also function as chemotactic damage-associated molecular pattern molecules (damps also known as alarmins or danger signals), activating the innate immune system [ ] . the function of fpr is less well characterized, although it is expressed on eosinophils, monocytes, macrophages, and dendritic cells. evidence suggests that fpr plays role in allergic reaction and dendritic cell maturation [ ] . cerebrovascular disease is the second most common cause of death after ihd, the third leading cause of disability-adjusted life-years (dalys) and accounted for eight million deaths worldwide in [ , ] . ais is the most common manifestation of cerebrovascular disease with limited therapeutic options to date [ ] , mainly due to complex pathophysiology. ais evolves locally due to underlying atherogenesis and vasculopathy (thrombotic occlusion), or from a distant embolus arising from the heart (embolic occlusion) [ ] . these events result in activation of an ischemic cascade, culminating in sudden tissue hypoxia, cellular dysfunction, cell death and subsequent disruption of blood-brain barrier [ ] . the main goal in ais management is rapid recanalization of the occluded vessel to restore blood supply and limit focal ischemic insult and global hypoxic injury. however, various animal models have demonstrated that reperfusion can actually result in tissue injury at both molecular and cellular levels [ ] as translated into recurrent strokes seen in many patients [ ] . as such, there are several strategies currently under development to generate neuroprotective agents to prevent i/ri and irreversible brain injury in ais [ ] . i/ri plays a significant detrimental role in ais resulting in widespread inflammation due to immune responses. ischemia results in the complex accumulation of various cellular mediators including activated leukocytes (neutrophils, monocytes and lymphocytes), activated platelets, alongside activation of resident cells (e.g., microglia and astrocytes) superoxide generation and cytokines (tumor necrosis factor-α (tnf-α) and interleukin- beta (il- β)) production [ ] . under hypoxia, the deficiency of vasodilator in particular endothelial nitric oxide (no) has been shown to enlarge stroke size [ ] . tlrs have also been shown to play a significant role in cerebral i/ri especially mediating responses through microglial cells, and tlr has been demonstrated to upregulate the expression of cellular adhesion molecules (cams and selectins), facilitating neuronal injury [ ] . the activation of microglia and macrophages increases the expression of, e.g., icam- , p and e-selectin, which enhance leukocyte rolling and sticking to the vessel surfaces [ ] . additionally, there is a crosstalk between thrombosis and inflammation in cerebral ischemia resulting in a thrombo-inflammatory state [ ] . subsequently, restoration of blood flow results in cellular and molecular responses involving innate as well as adaptive arms of immunity, activation of the complement system, and the coagulation system. these responses lead to a further damage of the affected tissue. additional injury is caused by the production of ros, and the involvement of other inflammatory cells such as dendritic cells [ , ] . neutrophils play a huge role as these are first immune cells to infiltrate the ischemic region of brain [ ] and accumulate within six hours of reperfusion and increase the tissue infarct size between six and twenty-four hours (the time of maximal infarct related closely with the time course of neutrophil infiltration) [ ] . during reperfusion, in addition to interacting with other cells, e.g., endothelial cells and platelets, neutrophils also produce many pro-thrombotic mediators such as neutrophils extracellular traps (nets) and serine proteases, e.g., cg and ne, which promote thrombus formation. furthermore, matrix metalloproteinase- (mmp- ) and the surface integrin receptor mac- (cd b/cd or macrophage- ag), expressed by neutrophils play significant roles in compromising blood-brain barrier (bbb) and accelerating thrombus formation by binding to fibrinogen, von willebrand factor (vwf), and glycoprotein ib (gpib), respectively [ , ] . the role of annexin a in ais anxa has been shown to act in various ways to mitigate cerebral i/ri. our work has shown a significant effect of anxa (and its peptide mimetics) on diminishing leukocyte adhesion and trafficking (mainly by acting in an in an autocrine/paracrine fashion to decrease leukocyte-endothelial interactions) [ ] , inhibiting the release of pro-inflammatory and pro-thrombotic cytokines, and regulating neutrophil-platelet interactions [ , , ] . human recombinant anxa and its peptide mimetics have shown to markedly reduce the lesion size, clinical score and markers of leukocyte infiltration in murine middle cerebral artery occlusion (mcao) model [ , , ] . recently, we showed that exogenous administration of anxa n-terminal peptide ac - attenuated neutrophil and platelet activation and neutrophil-platelet aggregation in the murine cerebral microvasculature after induction of cerebral i/ri in mice [ ] . ac - has also been shown to be protective in rats, as demonstrated by intracerebroventricular administration of the peptide reducing infarct size and cerebral edema two hours post cerebral ischemia [ ] . mcarthur et al. demonstrated that animals lacking the anxa receptor fpr /alx (i.e., fpr /alx knock-out or fpr /alx −/− mice) showed markedly greater bbb leakage post-ischemia than their wild-type (wt) counterparts [ ] . additionally, anxa plays a role in mediating the permeability of the bbb. cristante et al. showed that anxa knock-out (anxa −/− ) mice possess a disorganized actin cytoskeleton due to increase in activity of rhoa small gtpase and a disruption of occludin and ve-cadherin [ ] . furthermore, it is not just blood cells that express fpr /alx, but also brain resident cells such as microglia [ ] and as such are potential targets for the anti-inflammatory and pro-resolving effects of ac - and its parent compound [ ] . ihd is the leading cause of mortality with an estimated . million deaths worldwide in [ ] . despite significant improvement over the years in survival rates (due to advances in reperfusion therapy), secondary heart failure, due to ensuing inflammation, is a major cause of disease burden after myocardial infarction. overproduction of ros [ ] , cardiac tissue remodeling and ensuing pro-inflammatory immune response are the main causes of mi/ri [ ] . the pharmacological administration of anti-oxidants and calcium channel blockers has shown the promising results under experimental settings, however both interventions failed to achieve success in clinical studies [ ] . if mi lasts more than twenty minutes, cardiomyocyte death is initiated in the subendocardium, expanding towards epicardium [ ] . the depletion of atp under ischemia due to halt in oxidative phosphorylation can inhibit myocardial contractile function [ ] . ischemia leads to ca + overload, which can cause damage to ultrastructure of myocardial [ ] . initially, it was thought that reperfusion can limit infarct size and preserve left ventricular (lv) systolic function, however, with growing scientific evidence, it was proven that reperfusion can itself induce cardiomyocyte death [ ] . reperfusion in ihd results in a massive outpouring into the ischemic tissue of activated leukocytes (especially neutrophils) and other mediators e.g., complement fragments, ros and pro-inflammatory cytokines, all of which promote further injury and cardiomyocyte death [ ] . several interventions have been shown to reduce infarct size up to % under experimental mi/r conditions. these interventions include administration of biologics to inhibit p-selectin, cd , cd and icam- and pharmacologic inhibition of complement activation and leukocyte depletion [ ] . neutrophils play a central role in the mi/ri. they are the first cells to be summoned, resulting in accelerated pro-inflammatory responses including production of inflammatory mediators (e.g., cg, ne and mmp- [ ] ) and ros, leading to microvascular injury, cardiomyocyte death and extracellular matrix (ecm) degradation and remodeling [ ] . neutrophil activation in i/ri promotes the expression of adhesion molecules, leading to adherence of neutrophils to the endothelium followed by transmigration, and direct interaction with the cardiac myocytes [ ] . the involvement of anxa in ihd anxa and its n-terminal peptides have both been shown to be cardioprotective in various i/ri models [ , ] . d'amico et al. demonstrated a decrease in infarct size ( %) upon infusion of recombinant anxa and the protective effects of anxa were further confirmed by la et al. as evidenced by the administration of the anxa n-terminal peptide, ac - , to decrease infarct size and reduce mpo and il- β content in infarcted hearts [ ] . treatment with anxa also attenuated loss of fiber organization, decreased mpo activity, reduced tnf-α and macrophage inflammatory protein (mip- α) levels and leukocyte extravagation in cardiac tissues [ ] . peptide ac - has been shown to preserve cardiomyocyte contractile function and reduce cardiac myocyte injury, with protection, at least in part, being associated with activation of pkc, p -mapk and k atp channels [ ] . additionally, qin et al. demonstrated that exogenous administration of ac - at the onset of reperfusion increased cardiomyocyte viability and recovery of lv function, which was associated with fpr /akt signaling [ ] . hematopoietic progenitor stem cell (hpsc) mobilization and differentiation has been shown to be regulated by anxa [ ] , e.g., anxa deficiency results in increased cardiac necrosis, inflammation, hypertrophy and fibrosis resulting in exaggerated cardiac infarct size after eight days of myocardial infarction [ ] . these effects were linked to a greater expansion and altered mobilization of hpsc, with increased circulating neutrophils and platelets and altered macrophage inflammatory phenotype and function [ ] . more recently, there has been a large focus on the pharmacological concept of biased-agonism (multiple active conformations of a receptor exist and elicit distinct signals yielding to multiple functional outcomes). the small-molecule fpr /fpr /alx agonist, compound b (cmpd b), has been shown to have biased agonism, by enabling fpr based pro-resolving biology without changing fpr / -mediated calcium mobilization, hence providing superior cardio-protection. cmpd b, similar to anxa , attenuates both early and late inflammatory responses after reperfusion in mi [ ] , with erk / -akt kinases seeming to play a significant role [ , ] . kidney transplantation is the leading cause of kidney i/ri. at the end of , there were approximately , people waiting for a kidney transplant. due to greater availability, the vast majority of kidney transplants which take place use organ(s) from a cadaveric donor. for instance, out of , kidney transplants that took place in , , organs came from cadaveric donors and only came from living donors [ ] . however, cadaveric donor kidneys are more susceptible to i/ri due to prolonged ischemia under cold conditions [ ] . in the case of living donors, although ischemic period is shorter, the organ is still subjected to warm ischemia during harvesting which may also result in i/ri [ ] . finally, a more severe injury occurs during the reperfusion phase in the recipient due to the activation of innate and adaptive immune responses. indeed, many renal transplant recipients are under immunosuppressant therapy to prevent graft dysfunction. figures show that % of kidney transplants will fail within a month of transplantation, % within a year and % within three years. furthermore, each year, more than % of total kidney transplants are re-transplanted due to failure of the transplanted kidney [ ] . therefore, there is an urgent unmet clinical need to find better therapies to prevent graft rejection. renal i/ri is characterized by a decrease in glomerular filtration rate (gfr, which is the volume of the fluid filtered from the kidney each minute). ischemia leads to the depletion of atp which in turn inhibits the activity of tubular na + k + atpase and the redistribution of the na +/ h + exchanger. both conditions cause a decrease in the reabsorption of na + by tubular cells. due to increased na + filtration, gfr is limited via tubuloglomerular feedback mechanism to avoid na + and water loss [ ] . additionally, ros production is increased during i/ri causing renal tubule injury via oxidation of proteins and lipids on membrane, dna damage and thereby triggering apoptosis of tubular epithelial cells which can shed into tubular lumen causing tubular obstruction [ , ] . the paracellular diffusion of water, ions and macromolecules becomes unregulated upon the loss of tubular cells and this increases the back-leak into the interstitium and blood vessels, which also decreases gfr. thus, it can be said that gfr can also be decreased by the synergetic effect of cell surface area and tubular obstruction. additionally, apoptosis leads to the decrease in cell-cell interactions increasing the vascular permeability [ , ] . the depletion of atp also inhibits the activity of ca + atpase causing an increase in intracellular calcium levels which can induce cytoskeletal damage and increase in vascular permeability [ ] . inflammation plays a major role in renal i/ri due to enhanced production of pro-inflammatory cytokines, e.g., il- and tnf-α, which have been shown to work mainly through jak/stat signaling pathways such that inhibition of the jak/stat signaling pathway has been shown to improve kidney i/ri outcomes. other cytokines, e.g., mcp- , il- β, il- , and il- , are released upon renal tubular cell activation and further the damage due to i/ri [ ] . as with other i/ris, in renal i/ri, neutrophils are the first cells to respond to the injury site, which results in the production of multiple mediators including ne, tissue type plasminogen activator, hepatocyte growth factor and cd expression. neutrophils also cause renal microvasculature obstruction as they bind to endothelial cells and platelets. additionally, adhesion molecules, e.g., icam- and p-selectin, which are major regulators of neutrophil recruitment have been shown to be involved in kidney i/ri. more recently, kelly et al. showed that kidneys from icam- −/− mice displayed protection against i/ri, which was further confirmed by blocking icam- with an icam- specific antibody [ , ] . takada et al. showed that soluble p-selectin ligand decreased the neutrophil transmigration in rat ischemic kidneys [ ] . the role of anxa in kidney i/ri is in its nascent stage. however, some studies have shown that anxa may exert a protective role, e.g., in a rat model of renal i/ri, exogenous administration of peptide ac - reduced: (a) tubular necrosis; (b) the influx of phagocytic cells (neutrophil and monocytes); (c) sodium and potassium excretion; and (d) gfr, thereby improving functional recovery and outcome [ ] . cyclosporine is an immunosuppressant and is used to avoid organ transplant rejection. the major side effect of cyclosporine treatment is nephrotoxicity, as the compound decreases renal blood flow and increases renal vascular resistance (both common conditions in renal i/ri). however, exogenous administration of ac - has been shown to reverse these side effects [ ] . diabetes is metabolic syndrome which affects many organs including the kidneys, with - % of diabetic patients developing nephropathy [ ] . anxa has been shown to afford protection in murine diabetic nephropathy by decreasing p , erk and jnk, and activating akt signaling, suggesting that anxa could be a potential therapeutic strategy for treating renal dysfunction caused by diseases such as diabetes [ ] . i/ri in the gi system is mainly seen with abdominal and thoracic vascular surgery, but it can also be associated with small bowel transplantation and hemorrhagic shock [ ] . most of these gi complications lead to mesenteric ischemia and i/ri. the mortality rate associated with mesenteric i/ri is in the range of - % [ ] . enterocytes are the main intestinal cells and are resistant to short term hypoxia, however prolonged hypoxia can cause irreversible cell death resulting in the production of various damps, e.g., nucleic acids and heat shock proteins. ischemia causes inactivation of enzymes such as cytochrome oxidase and superoxide dismutase, so upon re-oxygenation, oxidative phosphorylation does not occur, resulting in ros production which can further trigger apoptosis of enterocytes further causing intestinal barrier disruption. there is also increase in intracellular calcium concentration leading to changes in cytoskeleton, which further damages cell-cell junctions, thereby increasing vascular permeability [ ] . systemic inflammation is a significant component of intestinal i/ri. the loss of barrier function facilitates movement of microbiota and their products from the gut to the blood stream. the molecular patterns associated with these microbes and their products, known as pathogen-associated molecules pattern (pamps). pamps and damps can bind tlrs on immune cells, activating local inflammatory response such as production of cytokines, cell adhesion molecules, and chemokine receptors via induction of nf-κb. these steps lead to recruitment and infiltration of leukocytes causing tissue damage [ ] . the inflamed intestinal mucosa also produces as whole plethora of different inflammatory cytokines and chemokines, e.g., tnf-α, il- , il- and il- , which can significantly increase leukocyte infiltration and exacerbate inflammation and disease [ ] . the protective role of anxa in gi-associated i/ri anxa has been shown to play a protective role in gastrointestinal i/ri. we have shown that intravenous administration of ac - inhibits leukocyte adhesion and emigration in a mouse mesenteric i/ri model, as induced by clamping of the superior mesenteric artery, followed by reperfusion [ ] . the loss of epithelial cell barrier is major characteristic of intestinal i/ri. the effect of anxa or its peptides on epithelial cell barrier has not been studied in intestinal i/ri models but they have afforded protection in other gastrointestinal inflammatory murine models including colitis and gastric ulcers. leoni et al. demonstrated that ac - , when delivered locally in the intestine using polymeric nanoparticles, enhanced the epithelial cell restoration in colitis [ ] . martin et al. showed the positive effect of peptide ac - in enhancing the healing process of gastric mucosal damage and reducing the ulcer areas. furthermore, this same group also demonstrated that hemorrhagic lesions were greater in anxa −/− mice upon induction of gastric ulcers [ ] . leoni et al. suggested that anxa exhibits an fpr /nox -dependent positive role in intestinal wound healing: upon binding to fpr , anxa activates src and downstream nox- [ ] . ros produced by nox- causes rapid and reversible oxidation and subsequent inactivation of phosphatase pten and ptp-pest. this inactivation of phosphatases leads to activation of focal adhesion protein involved in cell movement, fax and paxillin, thereby increasing the intestinal epithelial cell movement and wound repair [ ] . intestinal i/ri is also a major cause of acute lung injury (ali). treatment with exogenous ac - has been shown to reduce pulmonary vascular leakage, decrease neutrophil infiltration and mpo content in lung tissues following intestinal i/ri, possibly by inducing release of anti-inflammatory cytokine il- and decreasing the release of pro-inflammatory cytokine tnf-α [ ] . babbin et al. also showed in a dextran-sulfate sodium (dss)-induced colitis model that anxa was able to regulate intestinal mucosal injury and inflammation via engagement with fpr /alx. in addition, anxa deficient mice failed to regain weight and showed no improvement in disease activity index and mucosal injury upon withdrawal of disease in the dss-induced colitis model [ ] , advocating the crucial and beneficial role of anxa . ac - (fpr , fpr /alx) decreases neutrophil-endothelium interactions in flow chamber regulates leukocyte-platelet response in the cerebral microvasculature decreases pulmonary arterial pressure decreases inflammatory cytokine production decreases lung tissue damage following i/r decreases infarct size post myocardial i/r reduces myeloperoxidase activity prevents ifn-γ and endotoxin induced inotropic and cyclooxygenase gene expression decreases adhesion and transmigration in inflammatory mesentery in vivo ais [ ] i/r induced lung injury [ ] mi [ , ] mesenteric ischemia [ ] [ , , , , ] ac - (fpr ) activates neutrophil nadph oxidase inflammation [ ] annexin a (fpr /alx) cyclosporine h (fpr ) decreased neutrophil activation inflammation [ ] dca (fpr ) inhibits fmlp-induced monocyte and neutrophil chemotaxis and calcium mobilization inflammation [ ] ebola peptides (fpr ) inhibits fmlp interaction in cho cells viral pathogenesis [ ] flipr (fpr /alx) fpr /alx inhibitory protein (flipr) exerts anti-inflammatory activity by inhibiting calcium mobilization and cell migration toward chemoattractants. inflammation [ ] hiv- peptides (fpr ) inhibits fmlp interaction in cho cells viral pathogenesis [ ] isopropylureido-flflf (fpr ) inhibits chemotaxis inflammation [ ] spinorphin (fpr ) inhibits calcium mobilization and fmlp induced neutrophil chemotaxis inflammation [ ] wrw (fpr /alx) inhibits chemotaxis, calcium flux, superoxide generation and erk phosphorylation neurodegenerative diseases, ais [ ] mi, myocardial infarction; mi/r, myocardial ischemia reperfusion; ais, acute ischemic stroke; ifn-γ, interferon gamma; mapk, mitogen-activated protein kinase; erk, extracellular signal-regulated kinase; akt, serine/threonine-protein kinase; pmn, polymorphonuclear leukocytes; cho cells, chinese hamster ovary; atl, aspirin triggered lipoxin; asa, aspirin ; igg, immunoglobulin g; fmlp, formyl-met-leu-phe (fmlp), g proteins; flipr, fpr /alx inhibitory protein in summary, cvds are the leading cause of morbidity and mortality worldwide, with mi and is being the primary causes of death. the majority of cvd related complications are due to i/ri. hence, it has become imperative to develop preventative as well as therapeutic strategies to mitigate these life-threatening complications. in this review, we focus on anxa and its peptide mimetics as possible therapeutic strategies for the treatment of i/ri, based on their ability to alleviate thrombosis and inflammation and promote resolution ( figure ) . indeed, drug development programs focused on endogenous anti-inflammatory and pro-resolving agents, such as anxa -based pharmacologic strategies, offer great therapeutic potential as they are devoid of metabolic side effects because they mimic the way inflammation naturally subsides in the body. however, the development of novel therapeutics should also be mindful of ascertaining not only if the drug is anti-inflammatory, but also if it is resolution-toxic (i.e., deranges or impairs timely and/or complete resolution), which could ultimately outweigh the overall therapeutic efficacy in treating i/ri and other inflammatory disorders. figure . schematic representation of the protective effects of anxa in i/ri. by binding to members of the fpr family, anxa and its peptide mimetics can (a) decrease rolling, adhesion and emigration of leukocytes (monocytes and neutrophils) [ , , , , , , ] . in addition, platelet aggregates are diminished [ , , ] and pro-inflammatory mediators (e.g., myeloperoxidase (mpo), reactive oxygen species (ros) and cytokines) are moderated [ , ] . (b) anxa promotes the balance between pro-inflammatory [ , , , , , , ] and anti-inflammatory cytokines, thereby fostering homeostasis in the host. 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whereas an inhibitory signal is generated independently of the fpr family receptors enhancing the interaction between annexin- and formyl peptide receptors regulates microglial activation to protect neurons from ischemia-like injury cathepsin g-dependent modulation of platelet thrombus formation in vivo by blood neutrophils identification of neutrophil granule protein cathepsin g as a novel chemotactic agonist for the g protein-coupled formyl peptide receptor mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like /mouse formyl peptide receptor-like as the receptor and acts as an immune adjuvant the soluble d d ( - ) fragment of the urokinase receptor inhibits monocyte chemotaxis and integrin-dependent cell adhesion defective polymorphonuclear leukocyte formyl peptide receptor(s) in juvenile periodontitis n-formyl-methionyl-leucyl-phenylalanine (fmlp) promotes osteoblast differentiation via the n-formyl peptide receptor -mediated signaling pathway in human mesenchymal stem cells from bone marrow formylated humanin activates both formyl peptide receptor-like and t /dp , an ectodomain peptide of human immunodeficiency virus type gp , is an activator of human phagocyte n-formyl peptide receptor the hiv- cell entry inhibitor t- potently chemoattracts neutrophils by specifically activating the n-formylpeptide receptor t /dp , a synthetic leucine zipper-like domain of the hiv- envelope gp , attracts and activates human phagocytes by using g-protein-coupled formyl peptide receptors n , a synthetic n-terminal heptad repeat domain of the hiv- envelope protein gp , is an activator of human phagocytes lipoxins and novel -epi-lipoxin analogs display potent anti-inflammatory actions after oral administration ll- , the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like (fprl ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells synthetic peptide mmk- is a highly specific chemotactic agonist for leukocyte fprl human mitochondria-derived n-formylated peptides are novel agonists equally active on fpr and fprl , while listeria monocytogenes-derived peptides preferentially activate fpr pituitary adenylate cyclase-activating polypeptide is a functional ligand for formyl peptide receptor-like internalization of prp - by the formyl-peptide-receptor-like- in glial cells a novel nonpeptide ligand for formyl peptide receptor-like regulatory effects of deoxycholic acid, a component of the anti-inflammatory traditional chinese medicine niuhuang, on human leukocyte response to chemoattractants cross-talk between fmlp and vitronectin receptors triggered by urokinase receptor-derived srsry peptide a seven-transmembrane, g protein-coupled receptor, fprl , mediates the chemotactic activity of serum amyloid a for human phagocytic cells activation of the chemotactic peptide receptor fprl in monocytes phosphorylates the chemokine receptor ccr and attenuates cell responses to selected chemokines utilization of two seven-transmembrane, g protein-coupled receptors, formyl peptide receptor-like and formyl peptide receptor, by the synthetic hexapeptide wkymvm for human phagocyte activation boc-mlf and boc-flflf are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor characterization of chenodeoxycholic acid as an endogenous antagonist of the g-coupled formyl peptide receptors n-terminal residues of the chemotaxis inhibitory protein of staphylococcus aureus are essential for blocking formylated peptide receptor but not c a receptor peptides derived from hiv- , hiv- , ebola virus, sars coronavirus and coronavirus e exhibit high affinity binding to the formyl peptide receptor the immunosuppressant cyclosporin a antagonizes human formyl peptide receptor through inhibition of cognate ligand binding a new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like n-terminus urea-substituted chemotactic peptides: new potent agonists and antagonists toward the neutrophil fmlf receptor the endogenous opioid spinorphin blocks fmet-leu-phe-induced neutrophil chemotaxis by acting as a specific antagonist at the n-formylpeptide receptor subtype fpr identification of peptides that antagonize formyl peptide receptor-like -mediated signaling key: cord- -y w plro authors: mcclorey, graham; banerjee, subhashis title: cell-penetrating peptides to enhance delivery of oligonucleotide-based therapeutics date: - - journal: biomedicines doi: . /biomedicines sha: doc_id: cord_uid: y w plro the promise of nucleic acid based oligonucleotides as effective genetic therapies has been held back by their low bioavailability and poor cellular uptake to target tissues upon systemic administration. one such strategy to improve upon delivery is the use of short cell-penetrating peptides (cpps) that can be either directly attached to their cargo through covalent linkages or through the formation of noncovalent nanoparticle complexes that can facilitate cellular uptake. in this review, we will highlight recent proof-of-principle studies that have utilized both of these strategies to improve nucleic acid delivery and discuss the prospects for translation of this approach for clinical application. this century is being heralded as the era of genomic medicine. with the mapping of the human genome sequence, advancement in the field of genetic engineering has transformed our understanding of how genes underpin biological processes in health and disease states and has principally brought the whole genome amenable to sequence-specific therapeutic intervention via nucleic acid based molecules. the concept of "personalized medicine" has thus become a reality and various gene-manipulative technologies such as antisense methodologies have demonstrated "proof-of-principle" for treatment of rare genetic diseases. however, these antisense biomolecules-namely, small interfering rnas (sirnas), classical antisense oligonucleotides (asos), splice-switching asos (ssos), micrornas (mirnas), and anti-mirnas (antagomirs)-are generally high molecular weight, highly charged entities and thus cannot easily traverse the cell membrane. this is especially the case for neurodegenerative disorders where the tightly-regulated blood-brain barrier (bbb) prevents uptake of most pharmaceuticals. this hurdle of low bioavailability and poor cellular uptake of biologically active nucleic acids means there is an urgent need to address this in the field of drug delivery. cell-penetrating peptides (cpps, also known as protein transduction domains) could be one of the solutions to this problem. in comparison to the extensively studied traditional delivery systems like liposomes, viral vectors, or polymeric cation-based systems (polyplexes) [ , ] , cpps are unique in that they are short (fewer than amino acids) cationic and/or amphipathic peptides that translocate small drugs/cargo across cell membranes [ , ] . many of the early identified cpps derive from peptide sequences found in naturally occurring protein elements that exhibited inherent translocating properties. some of the most important of these for subsequent cpp iterations include: the transactivator of transcription from hiv (hiv-tat) [ ] ; penetratin- , which is derived from the homeodomain of antennapedia, a drosophila transcription factor [ ] ; transportan, a chimeric peptide derived from galanin and the wasp-venom peptide toxin mastoparan [ ] ; and cationic polyarginine and polylysine sequences such as arg [ ] . several types of cargoes, for example, proteins, nucleic acid based macromolecules such as sirna, plasmid dna, asos, and small drug molecules, can be transported by cpps to overcome the natural cellular biological barriers. this has led to increased interest in the utilization of cpps as delivery tools of genetic modifiers, with some of the greatest promise demonstrated for nucleic acid delivery to organs of the body with inherently poor uptake by naked delivery. examples of some of these cpps and their applications are listed in table and this review intends to highlight the progress of cpp development for oligonucleotide delivery that could expand the repertoire of novel "druggable" targets. lgaqsnf sso ( ome) for dmd [ ] (rxr) rxrrxrrxrrxr anti-viral anti-bacterial [ , ] nanoparticle within the context of cpp-mediated delivery, effector nucleic acids can either be directly conjugated to the cpp or noncovalently complexed, typically forming nanoparticle structures ( figure ). covalent conjugation occurs with a defined structure and stoichiometry between predominantly charge-neutral oligonucleotide chemistries, such as peptide nucleic acids (pna) and phosphorodiamidate morpholino (pmo), and cationic cpps. cpps ( - residues long) join covalently to the cargo antisense nucleic acid and then traverse the biological membranes through various peptide-mediated uptake mechanisms. neutral-charge backbone chemistries are an ideal cargo for charged cpps as they avoid problems of electrostatic interaction and subsequent aggregation that can occur with anionic chemistries. one of the earliest studies to demonstrate the potential of direct cpp conjugation was the addition of four lysines to a pna sequence that had enhanced splice-switching activity in a gfp reporter mouse model compared to naked pmo [ ] . the ability of cpps to enhance pna activity was further investigated across a range of cpp classes, including transportan, oligo-arginine, ptat, penetratin, kff, synb , and nls, in luciferase-based splice-switching assays, with transportan having the highest efficacy [ ] . a key finding of this study was that presence of serum led to inhibition of activity of a subset of cpps and this may partly explain the relatively few successful in vivo studies with cpp-pnas. nevertheless, cpp-pnas have been utilized in preclinical studies targeting c-myc for severe combined immunodeficiency [ ] and as a proof of concept as potent in vivo inhibitors of mirna upregulation following lipopolysaccharide induction [ ] . one of the most promising neutral-charge chemistries are pmos that consist of a morpholine ring in place of ribose and phosphorodiamidate linkages in place of phosphodiester [ ] . these compounds act primarily through steric blockage of complementary rna sequences to either inhibit protein translation or to modify pre-mrna splicing. as pmos do not efficiently enter cells on their own, the strategy of cpp conjugation has been utilized to improve cellular uptake and enhance their antisense activity [ ] . conjugation of cpp to pmo uses several methods, including maleimide linkage, disulfide linkage, click chemistry, or amide linkage [ ] and enhances the pmo pharmacokinetic profile, biodistribution, and stability [ ] . the most promising results to date have been with arginine-rich peptides that contain aminohexanoic acid and beta-alanine spacers to improve delivery of splice-switching oligonucleotides (ssos) [ ] . for therapeutic purposes, this approach has been explored most extensively for duchenne muscular dystrophy (dmd), which affects in newborn boys and is caused primarily by out-of-frame deletions in the dmd gene that results in the loss of the structural muscle protein dystrophin. absence of dystrophin causes progressive muscle degeneration, resulting in loss of ambulation and premature death due to respiratory and cardiac failure. targeting of pre-mrna splicing through an "exon-skipping" approach restores the mrna reading frame around the deletion, producing an internally deleted yet still functional protein. this approach has been used successfully with naked pmo that resulted in the conditional approval of eteplirsen, a pmo targeting exon of the dmd gene [ ] . however, despite the safe profile of this drug and evidence of dystrophin production, a limitation was the relatively low treatment efficacy, with restoration of dystrophin an average of . % of normal levels following weeks treatment. a need therefore remains for a more effective compound to improve dystrophin levels and thus functional benefit from this approach. the use of cpps to improve pmo delivery is one such approach and was first demonstrated with arginine-rich b-peptide (b-pmo) that demonstrated approximately % wild-type dystrophin levels following a single mg/kg dose [ ] concurrent with partial restoration in cardiac function measures following dobutamine-mediated stress induction, and improved muscle function [ ] following intravenous systemic administration into the mdx mouse model of dmd. this compares extremely favorably to naked pmo, as weekly administration of pmo at mg/kg for weeks could only achieve % wild-type dystrophin levels [ ] . subsequent utilization of a fusion peptide of b-peptide with a muscle-targeting peptide that was identified through a phage display (msp) [ ] was able to improve activity -to -fold after multiple mg/kg dosing, although interestingly, in a further study, the orientation of the peptide fusion in relation to pmo conjugation was shown to be an important factor in activity [ ] . b-pmo has also been utilized for studies in canine models of dmd that greater recapitulate the human disease pathology and thus represent a sterner test of the ability of cpp-pmos to effectively restore dystrophin expression. intravenous administration of repeat low dose ( mg/kg per aso) b-pmo induced body-wide dystrophin restoration at approximately % of wild-type levels, including in the heart where amelioration of cardiac conduction abnormalities was observed after treatment [ ] . this was an important proof-of-principle study to demonstrate that cpp-pmo could have success in dystrophic muscle environments that can contain additional barriers to uptake, such as fibrosis and fat deposition. b-pmo has also been utilized as a therapeutic strategy for targeting myotonic dystrophy type i, where a ctg expansion in the untranslated region of the dystrophia myotonica-protein kinase (dmpk) gene results in a pathogenic transcript that complexes with rna-binding proteins such as muscleblind-like (mbnl ), resulting in widespread aberrant splicing abnormalities. systemic administration of b-pmo targeting this repeat element blocked mbnl sequestration, resulting in normal nuclear distribution and subsequent correction of abnormal rna splicing, including for chloride channel gene, which is a primary contributor to myotonia [ ] . our group has more recently developed a cpp class called pna/pmo internalization peptides (pip) that comprises a hydrophobic core region flanked on each side by an arg-rich domain containing aminohexanoic (x) and β-alanine (b) spacers [ ] . a single mg/kg dose induced - % wild-type dystrophin levels in body-wide skeletal muscle similar to b-pmo, but most significantly, introduction of this core sequence conferred approximately % dystrophin expression in the heart, which was absent for b-pmo at the same dosing level [ ] . further iterations of core design, namely pip series of peptides, demonstrated variable levels of activity that suggested a minimal core sequence length is required to maintain activity, although precise sequence was less important [ ] . furthermore, a pip -pmo was utilized to investigate the pharmacodynamics of this approach, including the observation that approximately % wild-type dystrophin protein levels are required to protect against eccentric contraction-induced muscle damage, as well as parameters such as minimal efficacious dosing levels and maintenance of effect [ ] [. other novel cpps have been developed for muscle targeting including m identified through phage display performed on c c myoblasts [ ] . conjugation to pmo resulted in - % wild-type dystrophin levels following a single systemic administration although at dosing levels -to -fold higher than that for comparable efficacy with b and pip cpps. our group has more recently developed a cpp class called pna/pmo internalization peptides (pip) that comprises a hydrophobic core region flanked on each side by an arg-rich domain containing aminohexanoic (x) and β-alanine (b) spacers [ ] . a single mg/kg dose induced - % wild-type dystrophin levels in body-wide skeletal muscle similar to b-pmo, but most significantly, introduction of this core sequence conferred approximately % dystrophin expression in the heart, which was absent for b-pmo at the same dosing level [ ] . further iterations of core design, namely pip series of peptides, demonstrated variable levels of activity that suggested a minimal core sequence length is required to maintain activity, although precise sequence was less important [ ] . furthermore, a pip -pmo was utilized to investigate the pharmacodynamics of this approach, including the observation that approximately % wild-type dystrophin protein levels are required to protect against eccentric contraction-induced muscle damage, as well as parameters such as minimal efficacious dosing levels and maintenance of effect [ ] [. other novel cpps have been developed for muscle targeting including m identified through phage display performed on c c myoblasts [ ] . conjugation to pmo resulted in - % wild-type dystrophin levels following a single systemic administration although at dosing levels -to -fold higher than that for comparable efficacy with b and pip cpps. one of the biggest challenges of the nucleic acid therapeutic field has been systemic delivery to the central nervous system (cns), where the blood-brain barrier represents a formidable barrier even for small-molecule drugs. the potential for cpps to deliver nucleic acid cargo was demonstrated in a study whereby systemic administration of an arginine-rich cpp conjugated to a fitc-labelled pmo cargo resulted in widely detected uptake into all areas of the brain, most notably to the cerebellum and purkinje cells, although the functional benefit of this uptake was not reported [ ] . more recently, cpp-pmos have been explored in preclinical models of spinal muscular atrophy (sma) whereby mutations in the smn gene results in the loss of survival motor neuron (smn) protein and subsequent severe motor developmental delay and premature death. a paralogous gene, smn , also encodes smn protein but only produces low levels due to a sequence variant that results in exclusion of exon from ~ % of mature transcript which in turn produces a truncated, nonfunctional protein. a strategy of employing ′-o'methoxyethyl ssos (nusinersen) to target an intronic silencing element to promote inclusion of exon in the smn gene has recently been clinically approved, having demonstrated significant improvement in motor function and survival rates in infants [ , ] . one of the biggest challenges of the nucleic acid therapeutic field has been systemic delivery to the central nervous system (cns), where the blood-brain barrier represents a formidable barrier even for small-molecule drugs. the potential for cpps to deliver nucleic acid cargo was demonstrated in a study whereby systemic administration of an arginine-rich cpp conjugated to a fitc-labelled pmo cargo resulted in widely detected uptake into all areas of the brain, most notably to the cerebellum and purkinje cells, although the functional benefit of this uptake was not reported [ ] . more recently, cpp-pmos have been explored in preclinical models of spinal muscular atrophy (sma) whereby mutations in the smn gene results in the loss of survival motor neuron (smn) protein and subsequent severe motor developmental delay and premature death. a paralogous gene, smn , also encodes smn protein but only produces low levels due to a sequence variant that results in exclusion of exon from~ % of mature transcript which in turn produces a truncated, nonfunctional protein. a strategy of employing -o'methoxyethyl ssos (nusinersen) to target an intronic silencing element to promote inclusion of exon in the smn gene has recently been clinically approved, having demonstrated significant improvement in motor function and survival rates in infants [ , ] . delivery to the cns is essential for this approach and is achieved clinically through intrathecal administration which, whilst tolerated, places a considerable burden on patients and clinicians. systemic administration would likely be a preferable route and may also have benefit in restoring smn levels in tissues outside the cns. to address this, intravenous administration of pip a-pmo into a severe mouse model of sma increased mean survival drastically (~ -fold), improved neuromuscular junction morphology and rescued levels of circulating insulin like growth factor at -fold lower doses than comparable efficacy observed for naked pmos [ ] . notably, the severity of this mouse model necessitates delivery before postnatal day to show functional benefit, where it is likely that the bbb may not be fully formed, and as such does not reflect the clinical situation for therapeutic intervention. to address this, systemic administration of pip a-pmo to a phenotypically unaffected adult mouse model of sma was performed in the same study with demonstration of a . -to . -fold increase in corrected smn transcript in brain and spinal cord, and an approximately -fold increase in liver and skeletal muscles. a subsequent study using the same adult mouse model explored putative brain-targeting motifs to enhance pmo delivery [ ] . a branched chain peptide designed to target the apolipoprotein receptor br-apoe(k→a) induced a . -fold increase in exon inclusion in the spinal cord and to a lesser extent in the brain. one of the limitations of positively charged cpps is that conjugation to negatively-charged nucleic acids such as sirnas and -o-me asos results in electrostatic interactions that can self-aggregate and potentially interfere with oligonucleotide target binding. in an attempt to address this, a -mer phage display was performed to identify noncharged homing peptides that would enhance delivery of omeps sso in the mdx mouse model [ ] . conjugation of a candidate peptide (lgaqsnf) increased exon skipping activity approximately - % in skeletal and cardiac tissues following a -week subcutaneous administration protocol, although notably an increase in protein restoration was not observed. as well as modulating splicing to correct a genetic defect, ssos can be utilized for destructive exon skipping whereby induction of an out-of-frame transcript results in nonsense-mediated decay and subsequent loss of protein [ , ] . as significant knockdown of gene expression, and hence protein levels, would be required for a functional effect, current naked sso delivery technologies are likely to be too inefficient in vivo. thus, pmos conjugated to b peptide were utilized in an exemplar study for targeting myostatin, a negative regulator of skeletal muscle growth and differentiation, in combination with dmd exon skipping as a complementary strategy for dmd [ ] . combinatorial treatment improved some, but not all, functional measures of muscle pathology in the mdx model to a greater extent than dystrophin expression alone, demonstrating the potential of destructive exon skipping as an alternative to more widely used sirna and rnaseh gene knockdown strategies [ ] . this combinatorial approach has also been utilized to simultaneously target two genes using a single cpp-pmo construct. addition of a second pmo through either "click" chemistry or a disulfide linker allowed two pmos to be delivered by a single cpp with similar splice-switching activity as having two separate cpp-pmos, potentially offering the advantage of reduced cpp toxicity burden [ ] . cpp-aso approaches have also been developed as antibacterial agents, (reviewed in [ ] ), as asos alone have poor capability to penetrate the bacterial cell membrane. in an exemplar study, intranasal administration of (rxr) -pmo targeting acpp in a mouse acinetobacter pneumonia model showed increased survival time and reduced pulmonary bacterial levels compared to saline controls [ ] . noncovalent cpp strategies depend on the formation of nanoparticle complexes due to electrostatic and/or hydrophobic interactions between anionic oligonucleotides and predominantly amphipathic peptides that consist of a hydrophilic (polar) domain and a hydrophobic (nonpolar) domain. amphipathicity of these peptides can be conferred either due to sequence-specific ordering along the peptide chain of hydrophobic and hydrophilic residues (primary peptides) or through the conformational structure of the peptide that confers hydrophilic and hydrophobic sides, such as an α-helical or β-sheet structure (secondary peptides). one of the earliest reported primary amphipathic cpps for aso delivery was the mpg peptide that contains a hydrophobic domain derived from the fusion sequence of hiv gp and a hydrophilic domain derived from the nuclear localization sequence of sv t-antigen [ ] . a modified version of this peptide, mpg- , was used to deliver sirna targeting the cell cycle regulator cyclin b in a xenograft tumor mouse model [ ] . the molar ratio of cargo to peptide was found to be a crucial determinant of efficacy, with optimal efficacy achieved at an mpg- /sirna molar ratio of / , which produced stable particles with diameters of nm. whilst intratumoral administration of mpg- /sirna complexes induced complete inhibition of tumor growth at . mg/kg, intravenous systemic injection of . mg/kg induced only a % decrease in tumor growth. to resolve this, mpg- /sirna particles were functionalized with cholesterol moieties, which are suggested to improve potency and stability through longer circulation times [ ] . addition of this moiety induced significantly increased mouse survival from % to % by day compared to cholesterol-free formulation and % reduction in tumor growth at only . mg/kg. the same group also reported development of a secondary amphipathic peptide (cady) that consists of aromatic tryptophan (trp) and cationic arginine residues that adopt a helical conformation within membranes that expose trp groups that favor cellular uptake [ ] . stability of sirna-to-serum exposure improved as cady molar ratio increased, indicating that cady protects the sirna within nanoparticle complexes. potency of these complexes was confirmed in vitro in difficult to transfect cell lines, however no in vivo data has been reported in the literature to date. one of the potential limitations of in vivo development of cpps is the potential for degradation by intracellular and extracellular proteases. to address this, cady-k peptide was modified into a retro-inverso peptide, whereby peptides consist of d-amino acids in the reverse sequence of naturally occurring l-isoforms [ ] . this peptide, termed rick (retro-inverso cady-k) had similar sirna-mediated knockdown efficacy to parent cady-k nanoparticles but with the advantage of improved resistance to enzymatic and serum degradation of sirna cargo [ ] . another approach to cpp-mediated sirna delivery has been the development of branched histidine-lysine (hk) polymers [ ] , whereby the histidine component is required for buffering and lysing of endosomes for cellular release of cargo and the lysine component important for electrostatic binding to nucleic acid. utilizing this noncovalent approach, hk polymers containing sirna directed against raf- , which has an important role in tumor angiogenesis, was able to effectively reduce xenograft tumor size by approximately % following multiple systemic dosing of µg of hk/raf- sirna complexes during tumor growth, with no overt toxicity detected [ ] . hydrophobicity can also be conferred to cpps through the addition of stearic acid modifications. one of the more widely published and successful examples is the pepfect peptide family. these peptides were initially developed through addition of a n-terminal stearic acid modification to transportan (tp ), termed pepfect , which enhanced splice-switching activity of -ome asos approximately -fold compared to unmodified peptide in a luciferase based cell assay [ ] . surprisingly, this peptide was inactive for rnai-mediated silencing and so to address this, an endosomotropic modification with a chemical analogue of chloroquine was introduced to enhance endosomal release (pepfect ) [ ] . as well as exhibiting enhanced sirna potency over commercially available reagents in vitro, pepfect sirna complexes induced potent target gene knockdown of - % in kidney, lung, and liver organs following a single dose of mg/kg with no apparent acute toxicity. further iterations of this peptide through modification of the tp sequence (pepfect ) were also effective for multiple nucleic acid cargoes in vitro [ , ] and most interestingly could demonstrate that it could retain activity after being dried as a solid formulation for several months [ ] . perhaps surprisingly, these lipid-functionalized cpps (pepfect and others) could be noncovalently formulated with otherwise difficult to transfect charge-neutral pmos so as to induce effective splice-switching activity in vitro, although this could not be replicated for in vivo delivery [ ] . a number of approaches to cpp design have been made to improve the selectivity of cell-type specific delivery of nucleic acid cargoes, especially for sirna. monoclonal antibodies or their fragments, aptamers, homing peptides, and small molecules can be employed to improve cell or tissue specific targeting and are generally conjugated or fused with cationic cpps to improve interaction with anionic sirna. conjugation of cd -specific single chain antibody to oligo- -arginine cpp was used for highly efficacious sirna delivery into primary human cd + t cells, with proof of targeting demonstrated in mice protected from hiv challenge following systemic administration of ccr -, vif -, and tat-targeting sirna nanoparticles to prevent viral spread and replication [ ] . a short peptide derived from the rabies virus glycoprotein (rvg), which is known to specifically bind to acetylcholine receptors in neuronal cells, was functionalized for sirna complexation through addition of nine arginine residues (rvg- r) [ ] . intravenous administration of µg of rvg- r sirna complexes into wild-type mice was able to induce approximately % reduction in cu-zn superoxide dismutase (sod ) levels in the brain but not in spleen and liver, with efficacy peaking at hours before returning to normal levels at around days. additionally, this approach was used in a mouse model of fatal flaviviral encephalitis with % survival in rvg- r antiviral sirna treated mice compared to control peptide-sirna groups who all died within days [ ] . an alternate to cell-specific targeting is to design activatable cpps (acpp) that improve uptake under specific environmental conditions. these cpps are generally hairpin structures that consist of a cationic and neutralizing anionic domain connected by a cleavable peptide loop that have limited cellular uptake due to neutralization of electrostatic interactions with the cell. however, in the presence of a disease-associated protease, the linker loop is cleaved, thus activating the cationic domain of the cpp [ ] . this approach has been demonstrated for targeting of cargo-free cpps to xenograft tumor models from different cancer sites where uptake is mediated by matrix metalloproteinase cleaveage of the cpp linker at the tumor-stromal interface [ ] . another reported acpp strategy is to create a ph-sensitive linkage, such as hydrazine, that will be catalyzed under decreased ph conditions such as that observed in tumor tissues. addition of a ph-sensitive acpp to a liposomal sirna carrier improved uptake and target-gene silencing in vitro under reduced ph conditions [ ] , although more optimization is required as the potency was not as high the original cpp and this approach has yet to be demonstrated in vivo to our knowledge. sensitivity to ph can also be exploited by fusogenic peptides that can be functionally activated through conformational changes at low ph such as in acidic endosome and lysosomes that aid in cargo release from these compartments. an early example of this is the ha peptide derived from hemagglutinin which has a random coil structure at physiological ph, but adopts a α-helical conformation within the acidic endosome that fuses and destabilizes the endosomal membrane [ ] . more recently, a fusion peptide, termed , consisting of a synthetic influenza virus-derived fusogenic domain and a cationic arginine domain, could induce significant target-gene silencing and subsequent decrease in cell invasiveness in an oral cancer cell model [ ] . tissue-specific targeting capabilities of this peptide was further enhanced through combining with an epidermal growth factor receptor targeting peptide and utilized for in vivo administration to xenograft oral cancer tumors, where % gene knockdown could be detected with the dual peptide nanoparticles but only % with the -sirna alone [ ] . the mechanism of cellular internalization of cpp, with or without cargo, can be broadly defined as either through energy-independent direct penetration or through energy-dependent endocytosis (graphically represented in figure ). the observation in early cpp experiments that uptake was similar at • c and • c, or under energy depletion conditions, suggested that direct translocation of the negatively-charged plasma membrane occurred via an energy-independent mechanism [ , ] . direct translocation is thought to occur primarily through membrane destabilization or transient pore formation. in the "carpet-like" model, accumulation of lytic cpps at the cell surface, primarily through electrostatic interaction, forms a localized "carpet", leading to reorganization of the lipid membrane such that transient holes are formed that allow additional cpps to enter [ ] . in contrast, the "barrel-pore" model describes the formation of transmembrane channels that are formed due to insertion of amphipathic α-helices peptides whereby the hydrophobic surface interacts with the lipid membrane, whilst the hydrophilic surface faces inwards, thus forming an aqueous pore [ ] . an alternate proposed model for direct uptake is the "inverted micelle model", whereby cpp interaction with the lipid bilayer would result in the formation of inverted micelles that would trap the peptides in the hydrophilic core until further interaction with the membrane would cause the inverse process and release cpps into the intracellular compartment [ ] . however, a key subsequent study suggested that some of these early observations of direct penetration may indeed have been an artifact of cell fixation methods [ ] , and as a consequence, the majority of microscopic studies on cpp localization are conducted in live cells. nevertheless, direct translocation cannot be entirely ruled out as, for example, cady cpps complexed with sirna do not colocalize with endosomal markers and were active in the presence in of endocytic inhibitors [ ] . since these early studies, a number of energy dependent endocytic pathways including micropinocytosis [ ] , clathrin- [ ] , and caveolae-mediated [ ] endocytosis have since been identified to be the primary driver of some cpps, with and without nucleic acid cargoes (reviewed here [ ] ). in a comparison of several cationic and amphipathic cpps conjugated to pna, results suggested that cationic conjugates relied on micropinocytosis, whilst amphipathic conjugates utilized clathrin-mediated endocytosis [ ] . however, endocytic pathways for each cpp class may not be very clearly defined as a large body of information suggests that the specific endocytic pathways utilized by cpps is highly dependent on properties of the cargo, relative concentration, and the cell line/tissues being targeted [ ] . a clear example of this is for pip a-pmo, whereby caveolae-mediated endocytosis is primarily responsible for uptake in skeletal muscle cells, but clathrin-mediated endocytosis was important for cardiomyocyte uptake [ ] . more recently, cell-surface proteoglycan (glycosaminoglycans like heparan sulphates) [ ] and other cofactors such as scavenger receptors (sr) have also been implied in the uptake mechanism of cpp nanoparticles [ ] . srs are a large family of pattern recognition receptors that are highly expressed in immune cells and play important roles in innate immunity and homeostasis [ ] . negatively charged pepfect -sso complexes were taken up into hela cells through a class a sr-dependent endocytosis pathway [ ] and in subsequent studies, it was also demonstrated that some amphipathic and cationic cpps could also trigger recruitment of sr-a and sr-a to the plasma membrane [ ] . srs have also been implicated in the uptake of spontaneously forming amphipathic cpp-pmo micelles, with sr knockout models having significantly reduced splice-switching activity in diaphragm and heart tissues compared to wild-type controls [ ] . in an elegant study to further understand and identify uptake mechanisms of cpps, rna expression profiling of the early cellular response was performed in hela cells transfected with pepfect with or without an oligonucleotide cargo [ ] . ipa analysis suggested that the autophagy pathway was being induced upon transfection and to validate this, ligands that modulate autophagy were coadministered with pf -sso in a hela luciferase splice-switching reporter system. modulation of autophagy-related intracellular pathways showed concentration dependent effects on splice correction activity, and furthermore, autophagy induction and colocalization with autophagosomes was confirmed by confocal microscopy and transmission electron microscopy. biomedicines , , x for peer review of caveolae-mediated [ ] endocytosis have since been identified to be the primary driver of some cpps, with and without nucleic acid cargoes (reviewed here [ ] ). in a comparison of several cationic and amphipathic cpps conjugated to pna, results suggested that cationic conjugates relied on micropinocytosis, whilst amphipathic conjugates utilized clathrin-mediated endocytosis [ ] . however, endocytic pathways for each cpp class may not be very clearly defined as a large body of information suggests that the specific endocytic pathways utilized by cpps is highly dependent on properties of the cargo, relative concentration, and the cell line/tissues being targeted [ ] . a clear example of this is for pip a-pmo, whereby caveolae-mediated endocytosis is primarily responsible for uptake in skeletal muscle cells, but clathrin-mediated endocytosis was important for cardiomyocyte uptake [ ] . more recently, cell-surface proteoglycan (glycosaminoglycans like heparan sulphates) [ ] and other cofactors such as scavenger receptors (sr) have also been implied in the uptake mechanism of cpp nanoparticles [ ] . srs are a large family of pattern recognition receptors that are highly expressed in immune cells and play important roles in innate immunity and homeostasis [ ] . negatively charged pepfect -sso complexes were taken up into hela cells through a class a sr-dependent endocytosis pathway [ ] and in subsequent studies, it was also demonstrated that some amphipathic and cationic cpps could also trigger recruitment of sr-a and sr-a to the plasma membrane [ ] . srs have also been implicated in the uptake of spontaneously forming amphipathic cpp-pmo micelles, with sr knockout models having significantly reduced splice-switching activity in diaphragm and heart tissues compared to wild-type controls [ ] . in an elegant study to further understand and identify uptake mechanisms of cpps, rna expression profiling of the early cellular response was performed in hela cells transfected with pepfect with or without an oligonucleotide cargo [ ] . ipa analysis suggested that the autophagy pathway was being induced upon transfection and to validate this, ligands that modulate autophagy were coadministered with pf -sso in a hela luciferase splice-switching reporter system. modulation of autophagy-related intracellular pathways showed concentration dependent effects on splice correction activity, and furthermore, autophagy induction and colocalization with autophagosomes was confirmed by confocal microscopy and transmission electron microscopy. figure . cell-penetrating peptide internalization occurs through direct penetration (left) or endocytic pathways (right). direct translocation of cpps into the cellular space can occur through energyindependent mechanisms that cause membrane destabilization such as the "carpet-like" model or formation of inverted micelles, or through direct pore formation such as the "barrel-stave" model. the majority of cpps are thought to be taken up through energy-dependent endocytic internalization either through clathrin-dependent, caveolin-mediated, and clathrin/caveolae independent endocytosis or micropinocytosis before escape from endosome compartments into the cellular environment. cell-penetrating peptide internalization occurs through direct penetration (left) or endocytic pathways (right). direct translocation of cpps into the cellular space can occur through energy-independent mechanisms that cause membrane destabilization such as the "carpet-like" model or formation of inverted micelles, or through direct pore formation such as the "barrel-stave" model. the majority of cpps are thought to be taken up through energy-dependent endocytic internalization either through clathrin-dependent, caveolin-mediated, and clathrin/caveolae independent endocytosis or micropinocytosis before escape from endosome compartments into the cellular environment. endocytic uptake of cpps and their cargo is followed by complex intracellular trafficking towards early endosomes, maturation in late endosomes/multivesicular bodies (mvbs), lysomes, or the golgi network [ ] . as one of the key limiting factors for the bioavailability of cpp cargo that enter the cell through these pathways is endosomal entrapment, researchers are actively pursuing bioengineering methods such as fusogenic lipids [ ] for early escape from endosomes to the cytosol to prevent destruction and thus aid in the delivery to active sites in the cytoplasm [ ] . a number of approaches have focused on taking advantage of the low ph environment of endosomes. the incorporation of ph-sensitive domains into the cpp has been used to destabilize lipid membranes under the acidic ph conditions in the endosome to aid escape [ ] . similarly, histidine moieties, which act as a proton sponge at endosomal ph, can increase osmotic pressure leading to endosomal rupture and cargo release [ ] . incorporation of lysomotropic moieties have also been used to successfully improve endosomal escape, as exemplified by pepfect , that incorporated four chloroquine analogs to improve efficiency of cargo delivery compared to the parent stearyl-tp peptide [ ] . whilst the field of cpp-mediated delivery of oligonucleotides has expanded rapidly within the last years, there have been no successful clinical trials to date that have utilized these approaches. to a large extent, this is a reflection as to where the field is at the moment, with studies focused on optimization of cpp design and generation of proofs of principle in early preclinical studies. improvement in cpp design for covalent and noncovalent applications of cpp are focused predominantly on improving cellular uptake, although tissue-specific targeting is an obvious goal. there are approximately unique cpps currently listed on the cppsite . database [ ] that have been experimentally validated, and as such, represent expensive and time-consuming studies. to attempt to improve upon cpp prediction, differences between true and non-cpps have formed the basis for various machine learning models to predict whether novel peptide sequences are cell-penetrating or not. a recent example of this includes a web-based server that also predicts uptake efficiency of candidate cpp sequences [ ] . although experimental validation will always be necessary, these algorithms may improve the hit-rate and broaden the design landscape further outside of the major cpp families. there are a number of advantages and disadvantages to developing covalent and noncovalent approaches for clinical application of cpp-aso delivery. for covalent conjugations, the advantages are that a clearly defined entity is produced that has high reproducibility and generally has a low molar ratio of cpp-to-aso cargo (typically : ), which is important in consideration of cpp toxicity. however, production of these compounds is quite laborious and limited predominantly to charge-neutral oligonucleotide backbones, which in turn have more limited biological application, being used primarily for steric-blocking approaches such as exon-skipping. the process of noncovalent complexation due to electrostatic and hydrophobic interactions between cpps and asos is more simplified, usually by coincubation at defined concentrations. there is also the advantage that this approach can be used for a wider range of nucleic acid chemistries as well as types of effector molecules and biological applications such as rna interference and plasmid dna delivery. however, with this simplified complexation process comes the drawback of heterogeneity of the nanoparticle-like complexes that are formed and the potential for aggregation. as size, shape, and charge of the nanoparticle are crucial for uptake efficacy, uniform production is desirable to be considered for clinical application. assessment of size and morphology of pepfect and nickfect nucleic acid nanoparticles by transmission electron microscopy demonstrated that a relatively homogenous population of nanoparticles ranging in size from to nm were produced in the absence of aggregation, suggesting that, at least for these peptides, relative homogeneity could be achieved [ ] . cpp mediated toxicity is also a concern for both approaches and for noncovalent approaches, especially where typically a higher molar ratio of cpp:aso from : to : is required for optimal efficacy [ , , , ] . for clinical development, safety, tolerability, pharmacokinetics, pharmacodynamics, and therapeutic index are major factors to be considered for nucleic acid delivery. unfortunately, in vivo assessment of cpp-aso toxicology has not been robustly reported, although assessment of serum markers of liver damage at efficacious dosing for cpp-pmo has not indicated any hepatic toxicity, at least for arginine-based cpps [ ] . one of the potential advantages of cpp-based approaches over viral-based delivery is the opportunity for readministration which is not possible for viral vectors which elicit a strong immune response upon subsequent administration or if patients have pre-existing viral exposure. in multiple preclinical studies, cpp-asos toleration of multiple-dosing regimens is reported, and for pepfect mediated delivery of sirna, no acute inflammatory cytokine response could be detected and no increases in serum markers of kidney or liver damage were observed following intravenous delivery [ ] . furthermore, addition of tat, antennapedia, and transportan cpps to epithelial cells at concentrations effective for cargo uptake, did not elicit toll-like receptor signaling or inflammatory cytokines [ ] . in the clearest insight of cpp-aso toxicity, administration of high dose b-peptide-pmo into rats demonstrated a decrease in body weight and elevated serum blood urea nitrogen and creatinine in a dose-dependent manner that suggested reduced renal output [ ] . administration at very high doses (> mg/kg) were also accompanied with mild lethargy immediately post-administration. however, it should be noted that these responses were observed at doses significantly higher than minimal effective doses. in a clinical development program by avi-biopharma (now sarepta therapeutics), nonhuman primates were administered once-weekly iv administration for weeks of mg/kg of (rxrrbr) peptide conjugated pmo that demonstrated an average of %, %, and % exon skipping in diaphragm, quadriceps, and heart, respectively [ ] . however, at this dose, mild tubular degeneration was detected in the kidneys and development of this compound has been dropped. however, recent investor presentations from sarepta have shown data from nonhuman primate studies of a novel cpp-pmo (srp- ) targeting exon of dmd that is well tolerated and shows efficacious exon-skipping activity. this has led to acceptance of an investigational new drug application and a phase / a trial is currently underway and estimated to be completed early (nct clinicaltrials.gov) the field of cpp delivery of oligonucleotides clearly shows promise as evidenced by the number of successful preclinical studies in a wide range of disease indications that have arisen over the last decade. the greatest success is likely to come for targeting organs that are most refractory to systemic naked oligonucleotide delivery such as the central nervous system, heart, and skeletal muscle where the need for effective delivery is paramount to realize the power of specific gene and disease mutation targeting that oligonucleotides offer. with advancement of design algorithms and better understanding of uptake mechanisms and intracellular trafficking, the therapeutic potential of this field will improve and likely we will see more cpp-aso candidate's move towards clinical drug development. viral and nonviral delivery systems for gene delivery non viral vectors in gene therapy-an overview peptides for nucleic acid delivery applications and challenges for use of cell-penetrating peptides as delivery vectors for peptide and protein cargos cellular uptake of the tat protein from human immunodeficiency virus the third helix of the antennapedia homeodomain translocates through biological membranes cell penetration by transportan arginine-rich peptides. an abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery cell-penetrating peptideconjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer systemic delivery of a peptide-linked morpholino oligonucleotide neutralizes mutant rna toxicity in a mouse model of myotonic dystrophy a fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice prevention of exercised induced cardiomyopathy following pip-pmo treatment in dystrophic mdx mice how much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse pip -pmo, a new generation of peptide-oligonucleotide conjugates with improved cardiac exon skipping activity for dmd treatment systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy effective dystrophin restoration by a novel muscle-homing peptide-morpholino conjugate in dystrophin-deficient mdx mice identification of a peptide for systemic brain delivery of a morpholino oligonucleotide in mouse models of spinal muscular atrophy peptide conjugation of -o-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice antiviral effects of antisense morpholino oligomers in murine coronavirus infection models gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo targeting cyclin b through peptide-based delivery of sirna prevents tumour growth a new potent secondary amphipathic cell-penetrating peptide for sirna delivery into mammalian cells a retro-inverso cell-penetrating peptide for sirna delivery a stearylated cpp for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy design of a peptide-based vector, pepfect , for efficient delivery of sirna in cell culture and systemically in vivo pepfect peptide vector for efficient gene delivery in cell cultures pepfect , a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation transvascular delivery of small interfering rna to the central nervous system fusogenic-oligoarginine peptide-mediated delivery of sirnas targeting the cip a oncogene into oral cancer cells dual peptide-mediated targeted delivery of bioactive sirnas to oral cancer cells in vivo systemic delivery of hk raf- sirna polyplexes inhibits mda-mb- xenografts systemically delivered antisense oligomers upregulate gene expression in mouse tissues evaluation of cell-penetrating peptides (cpps) as vehicles for intracellular delivery of antisense peptide nucleic acid (pna) inhibition of burkitt's lymphoma cells growth in scid mice by a pna specific for a regulatory sequence of the translocated c-myc efficient inhibition of mir- function in vivo by peptide nucleic acids uncharged stereoregular nucleic acid analogues. . synthesis of a cytosine-containing oligomer with carbamate internucleoside linkages cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides delivery of therapeutic oligonucleotides with cell penetrating peptides pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate vectorization of morpholino oligomers by the (r-ahx-r) peptide allows efficient splicing correction in the absence of endosomolytic agents eteplirsen in the treatment of duchenne muscular dystrophy. drug des functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-dna oligomers elucidation of muscle-binding peptides by phage display screening effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of duchenne muscular dystrophy improved cell-penetrating peptide-pna conjugates for splicing redirection in hela cells and exon skipping in mdx mouse muscle pip transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice arginine-rich cell-penetrating peptide dramatically enhances amo-mediated atm aberrant splicing correction and enables delivery to brain and cerebellum nusinersen versus sham control in later-onset spinal muscular atrophy nusinersen versus sham control in infantile-onset spinal muscular atrophy dystrophin isoform induction in vivo by antisense-mediated alternative splicing antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following octa guanidine morpholino oligomer treatment dual myostatin and dystrophin exon skipping by morpholino nucleic acid oligomers conjugated to a cell-penetrating peptide is a promising therapeutic strategy for the treatment of duchenne muscular dystrophy systemic antisense therapeutics for dystrophin and myostatin exon splice modulation improve muscle pathology of adult mdx mice bi-specific splice-switching pmo oligonucleotides conjugated via a single peptide active in a mouse model of duchenne muscular dystrophy advances in the delivery of antisense oligonucleotides for combating bacterial infectious diseases a new peptide vector for efficient delivery of oligonucleotides into mammalian cells mechanisms and optimization of in vivo delivery of lipophilic sirnas recent developments in retro peptides and proteins-an ongoing topochemical exploration highly branched hk peptides are effective carriers of sirna peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides cell-specific sirna delivery suppresses hiv- infection in humanized mice building cell selectivity into cpp-mediated strategies in vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer enhancing sirna-based cancer therapy using a new ph-responsive activatable cell-penetrating peptide-modified liposomal system structure and topology of the influenza virus fusion peptide in lipid bilayers a truncated hiv- tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides cell internalization of the third helix of the antennapedia homeodomain is receptor-independent cell-penetrating peptides: a reevaluation of the mechanism of cellular uptake direct translocation as major cellular uptake for cady self-assembling peptide-based nanoparticles interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis protein cargo delivery properties of cell-penetrating peptides. a comparative study cell membrane lipid rafts mediate caveolar endocytosis of hiv- tat fusion proteins uptake mechanism of cell-penetrating peptides deletion analogues of transportan cellular trafficking determines the exon skipping activity of pip a-pmo in mdx skeletal and cardiac muscle cells neuropilin- and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides scara involvement in the uptake of nanoparticles formed by cell-penetrating peptides scavenger receptors in homeostasis and immunity scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides cell-penetrating peptides recruit type a scavenger receptors to the plasma membrane for cellular delivery of nucleic acids self-assembly into nanoparticles is essential for receptor mediated uptake of therapeutic antisense oligonucleotides role of autophagy in cell-penetrating peptide transfection model delivery of endocytosed membrane proteins to the lysosome delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges transducible tat-ha fusogenic peptide enhances escape of tat-fusion proteins after lipid raft macropinocytosis an endosomolytic tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for dna transfection cppsite . : a repository of experimentally validated cell-penetrating peptides cppred-rf: a sequence-based predictor for identifying cell-penetrating peptides and their uptake efficiency characteristics of cell-penetrating peptide/nucleic acid nanoparticles cell penetrating peptides fail to induce an innate immune response in epithelial cells in vitro: implications for continued therapeutic use morpholinos and their peptide conjugates: therapeutic promise and challenge for duchenne muscular dystrophy the authors declare no conflict of interest. key: cord- -lhwhkada authors: kašička, václav title: recent developments in ce and cec of peptides date: - - journal: electrophoresis doi: . /elps. sha: doc_id: cord_uid: lhwhkada the article brings a comprehensive survey of recent developments and applications of high‐performance capillary electromigration methods, zone electrophoresis, itp, ief, affinity electrophoresis, ekc, and electrochromatography, to analysis, preparation, and physicochemical characterization of peptides. new approaches to the theoretical description and experimental verification of electromigration behavior of peptides and to methodology of their separations, such as sample preparation, adsorption suppression, and detection, are presented. novel developments in individual ce and cec modes are shown and several types of their applications to peptide analysis are presented: conventional qualitative and quantitative analysis, purity control, determination in biomatrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid and sequence analysis, and peptide mapping of proteins. some examples of micropreparative peptide separations are given and capabilities of ce and cec techniques to provide important physicochemical characteristics of peptides are demonstrated. peptides are an extremely abundant and prominent family of biologically active compounds. they play a significant role in control and regulation of many vitally important processes in all living organisms; their diverse biological functions include operating as hormones, neurotransmitters, immunomodulators, coenzymes, enzyme substrates and inhibitors, receptor ligands, drugs, toxins, and antibiotics. in the current era of proteomics, the comprehensive analysis of proteome, representing now the main approach to understand the molecular bases of biological processes and diseases, and to discover new biomarkers and drug targets, the importance of peptides even increasing. the reason for this fact is that both the structure and function of many proteins are identified via their peptide fragments generated by their enzymatic hydrolysis [ ] [ ] [ ] . this "bottom-up" or "shotgun" approach is one of the main directions in the proteome research [ ] . in addition, for more detailed understanding of both normal and pathological processes of the living cells, a comprehensive investigation of the whole peptide set of a cell (peptidome), peptidomics, a logical sequel to proteomics, has emerged [ ] [ ] [ ] . hence, no wonder that separation, analysis, purification, and characterization of peptides by capillary electromigration methods belong to the most challenging tasks of these high-performance separation techniques. this article presents a comprehensive survey of the recent advances in ce and cec of peptides, particularly in the years - with some overlap to the first half of . it is an update of the previous reviews on ce and cec of peptides [ ] [ ] [ ] [ ] , which cover the period - . fast and vigorous developments of both pure electrophoretic modes, zone electrophoresis (ze), itp, ief, affinity electrophoresis, and mixed, electrokinetic and chromatographic modes, ekc and electrochromatography, in the capillary and microchip format, have gone ahead also in the recent period. applications of these methods to analysis and characterization of peptides have been further broadened, and ce and where a is a constant, and k, the exponent of m r , is related to the shape of a peptide molecule [ ] . three basic semiempirical models for peptide mobility differ in the value of exponent k. it approaches / for the stokes model in which peptides are modeled as spherical particles with high charge density [ ] , k is equal to / for peptides considered as classical polymer (random coil) with a lower charge density [ ] , and k is close to / for offord's model [ ] for larger and more rigid structures, the frictional forces for which during the electrophoretic motion are proportional to the surface area of peptide molecules. in addition, some other semiempirical models have been suggested with different specific k values for particular sets of peptides or with logarithmic dependence of effective mobility on charge or on the number of amino acid residues of the (poly)peptide chain [ ] [ ] [ ] . the above three basic models were examined by cze of peptide hormones (oxytocin, eledoisin, bradykinin, met-and leu-enkephalins, triptorelin, and buserelin) in a broad ph range, - [ ] . the classical polymer model with k = / resulted in slightly better correlation within the whole investigated ph range. the plots of q/m r / against ph of the bge were used to predict ce separations of the above hormones and also of the opioid peptides (endomorphin , dynorphin a, and enkephalin derivatives) [ ] using the accurate acidity constants (pk a ) of the present ionogenic groups (obtained in previous work from the cze measurement of ph dependence of effective mobilities of these or similar peptides) for calculation of peptide charge. the agreement between the predicted and experimental mobilities was very good and the optimum ph for the separation of the analyzed peptides could be predicted from a limited amount of experimental data. combination of modeling of peptide mobility and ce-ms was shown to be useful also for a fast and simple characterization of the cleavage specificity of new recombinant enzymes as demonstrated by ce-ms separation of peptide fragments obtained from hydrolysis of cytochrome c by recombinant and natural pepsin a [ ] . modeling of peptide mobility using the calculated pk a values of ionogenic groups [ ] helped to elucidate the most probable amino acid sequence of peptide fragments generated by pepsin hydrolysis of cytochrome c and in this way to characterize the pepsin cleavage site. the above three semiempirical models of peptide mobility and logarithmic form of the eq. ( ) introduced by cross et al. [ , ] log m ep q ¼ Àk log m r ( ) have been used to investigate the structure-mobility relationship of gonadotropin-releasing hormones (gnrhs) and their analogs and fragments [ ] . none of the models was found to be quite definitively applicable for the whole set of ten gnrhs differing in size (tetrapeptide to decapeptide) and charge ( . to . elementary charges) which were analyzed in five conventional and isoelectric acidic bges, ph . - . . nevertheless, for the dependence of m ep on q/m r k , the highest coefficient of correlation, r = . - . , was obtained for k close to / for all five acidic bges. this indicates that the electrophoretic migration of the set of gnrhs can be best described by the classical polymer model and the most probable structure of gnrhs in the acidic bges will be a random coil. the advantage of the application of eq. ( ) is that the value of the exponent k of m r in the nonlogarithmic form of this relationship can be easily determined as a slope of the logarithmic dependence. graphs of the model of cross and the most suitable classical polymer model are shown in fig. . the quantitative structure-mobility relationship for prediction of peptide mobilities was developed using the offord's model and artificial neural networks [ , ] . in this model, the offord's charge-over-mass term (q/m r / ) as one descriptor is combined with the corrected steric substituent constant and molar reactivity descriptors to account for the steric effects and bulkiness of the amino acid side chains. the multilinear regression of the data set showed an improved predictive ability of the model over the simple offord's relationship. back propagation artificial neural network models resulted in a further significant improvement of predicted mobilities, especially for larger peptides and for peptides with higher charges containing the basic amino acids, his, lys, and arg. linear and nonlinear quantitative structure-property relationship models for prediction of peptide mobilities in cze were suggested using the linear heuristic method and a nonlinear radial basis function . adapted with permission from [ ] . neural network, respectively [ ] . the results of the nonlinear model were closer to the experimental data than those of the linear model. a new approach, bead model for modeling electrophoretic mobility and diffusion of peptides and proteins, is based on an approximate structural model of peptides and it also takes into account the electrohydrodynamic theory [ , ] . a peptide formed by x amino acids is modeled as n = x beads with two beads representing each amino acid in the chain. peptide conformations are randomly generated and the mobility and diffusion coefficients are computed for at least independent conformations. in general, the mobility is found to be dependent only weakly on peptide conformation, which is in agreement with the small differences of correlation coefficients when different values of exponent k of m r are used in the above eq. ( ) of the dependence of peptide mobilities on the shape of their molecules. the precedence of this model is that the input parameters are independent of experimental mobilities. hence, it is hoped that the model will be useful in the prediction of peptide mobility as well as in using peptide mobilities to extract information about peptide structure, conformation, and charge. in the other approach, the nmr measured translational diffusion constants of amino acids are used for modeling of diffusion and electrophoretic mobilities of peptides composed of these amino acids [ ] . piaggio et al. [ ] have shown that the important characteristics of short oligopeptides, such as effective (net) charge, hydrodynamic spherical radius, and hydration, can be explored through the cze determined experimental electrophoretic mobilites using the linderstrøm-lang electrophoresis model and its perturbed version. the other new approaches include the application of the machine learning techniques, such as back propagation artificial neural network, radial basis function artificial neural network and support vector regression for the prediction of peptide mobility in ce [ ] , and the use of a novel learning machine, support vector machine, for the development of the nonlinear quantitative structure-mobility relationship model of peptides based on the calculated molecular descriptors representing the structural features of the modeled compounds [ ] . a model reflecting the effect of ph, ionic strength, and temperature on electrophoretic behavior for a series of oligopeptides, oligoglycines, oligo-l-alanines, and oligo-lvalines, with a number of residues up to ten, has been developed on the basis of cze measurement of effective mobilities of these oligopeptides in bges within a broad ph scale, . - . , at two ionic strengths ( and mm) and at temperatures from to c [ ] . using this broad set of experimental data a semiempirical model was developed allowing to predict pk a values for any oligopeptide composed of amino acids with neutral lateral chains. the input parameters of this model are only the number of residues and the pk a of terminal amino acids in their free form. the model can predict the peptide pk a values at a given ionic strength and temperature and can also be used for selection of the optimum ph for the separation of mixtures of peptides the pk a of which are known or could be estimated. the proposed relationship allows a significant reduction of the experimental data needed for the development of suitable separation conditions. the mobilities and charges of the large polypeptides can be approximated by the models applied for modeling charge and mobilities of proteins [ ] . based on the basic electromigration properties of peptides, i.e., their charge, size, conformation, hydrophobicity, binding capabilities, and considering also their other properties, such as solubility, amphoteric character, chemical stability, and biological activity, the experimental conditions of their ce separations are selected, of course taking into account general rules for bge selection [ , ] . different aspects of the selection of the bge for the analysis of peptides and proteins by cze, such as concentration and types of the buffering constituents of the bge with respect to their buffering capacity, mobility and electric conductivity, ph, additives suppressing the adsorption or influencing the eof and selectivity, organic modifiers, temperature, and joule's heating effects were thoroughly discussed in the earlier published but still relevant review [ ] . preconcentration and preseparation (or "sample cleanup") are the necessary operations for sample preparations, when separation power and/or sensitivity of ce and cec are not sufficient for direct analysis of peptides present at low concentration levels and/or in complex mixtures of different (bio)matrices, such as body fluids, cell lysates, and tissue extracts. the reason for that is that ce techniques generally suffer from relatively low concentration sensitivity due to the minute (nano-to picoliter) applied sample volume and the short optical pathlength (typically - mm) of the most frequently used uv-absorption detector. the problems associated with sample preparation for peptides and proteins in biological matrices prior to their ce and lc analyses are thoroughly discussed in the recent reviews [ , ] , where the application of various sample-preparation techniques, such as homogenization, centrifugation, precipitation, solvent extraction, liquid-liquid extraction (lle), solid-phase (micro)extraction (spe or spme), affinity (micro)extraction, (micro)dialysis, and ultrafiltration, is described. the earlier used off-line classical sample cleanup and preconcentration methods, such as spe, spme, and lle, are recently replaced by on-line modes of these techniques. both preconcentration and preseparation are frequently solved by using a solid-phase packed sorbent at the inlet end of the capillary, a microvariation of spme cartridge, or more effectively and with smaller disturbance effect for the following ce separation using a microcartridge containing a membrane impregnated with different chromatographic stationary phases. the advances in on-line coupling of spme and ce were reviewed by the inventor of spme, liu and pawliszyn [ ] . one such system, on-column sample enrichment for the high sensitivity ce-ms analysis of peptides was developed by sandra et al. [ ] . peptides or glycopeptides are first retained and concentrated on a short ( - mm) c rp packed-bed situated in the fused-silica (fs) separation capillary, and they are subsequently liberated for ce separation by injection of a plug of organic solvent ( % v/v acn or % v/v -propanol in water). the concentration lods for standard peptides and for fetuin tryptic peptides were in the high picomolar range with a coaxial sheath-liquid flow ce-ms interface. the improved cruciform configuration of the analyte concentrator-microreactor device, designed by guzman and phillips [ ] for use in on-line immunoaffinity ce, enables to specifically trap, enrich, and elute an analyte from any biological fluid or tissue extract without any sample pretreatment except filtration, centrifugation, and/or dilution, allowing the separation and characterization of the target analytes faster, with higher sensitivity and lower cost than existing techniques. using fab' fragments of ig g raised against angiotensin ii and neurotensin, these neuropeptides present in low concentration in a complex matrix (urine) were captured by and eluted from the analyte concentrator/ microreactor. the following on-line ce analysis confirmed the presence of both peptides in the eluate. with this device -to -fold concentration was achieved, permitting quantitation of peptide analytes at the ca. ng/ml level using uv-absorption detection. recently also monolithic capillary columns have been employed for spme preconcentration and preseparation various analytes, including peptides [ ] . iron-protoporphyrin-modified monolithic fs capillary columns ( mm id mm long) have been prepared for on-line preconcentration of decapaptide hormone, angiotensin i, prior to its cze analysis [ ] . angiotensin was released from the sorbent by mm sodium phosphate, ph . /acn, / v/v, and then analyzed by cze in mm sodium phosphate, ph . . the monolithic column exhibited a high retention capacity for angiotensin i and showed as much as a -fold increase of concentration sensitivity achieving values up to . ng/ml with uv-absorption detection at nm. similar monolithic fs capillary columns modified with iminodiacetic acid and copper(ii) ions were employed for on-line affinity capturing of histidine-containing peptides from the model peptide mixture. his-containing peptides were released from the sorbent by a mm imidazole solution and separated by cze with uv-absorption detection [ ] . the same type of sorbent, monolithic column with cu(ii) iminodiacetic acid functional groups has been developed for preconcentration of low-abundance peptides and proteins from complex biological samples [ ] . the practical considerations that permit coupling or integration of sample treatment devices into commercial ce apparatuses are treated in the recent review [ ] where the off-line and on-line strategies are critically compared and the integrated in-capillary, in-vial, or in-replenishment-system methodologies for spme and lle are described. in addition to the chromatographic lle and spme principles, sample concentration is achieved also by electrophoretic approaches [ ] . electric field-amplified sample injection (fasi) also called field-enhanced sample injection, based on the increased intensity of the electric field in the injected zone of a sample dissolved in water or diluted bge and possessing higher electric resistance than the surrounding bge, has been used for high-sensitivity analysis of bioactive peptides by ce with esi-ms detection [ ] . after optimization the injection conditions, such as type and concentration of the acid and organic solvent in the sample solution, length of the water plug, sample injection time, and separation voltage, more than -fold improvement in detection signal was obtained, permitting analysis of bioactive peptides and tryptic protein digests in low nanomolar (fmol/ml) levels. using the fasi technique, carnosine and its related peptides, anserine, and homocarnosine, could be separated and determined by ce at mol/l level, with -to -fold enlargement of sensitivity without loss of separation efficiency as compared to conventional sample injection [ ] . the dynamic ph junction technique for peptide concentration [ ] is based on the presence of a dynamic ph junction within the capillary, which induces narrowing of the originally broad sample zone due to a sharp reduction in an analyte's migration velocity following a reversal of its electrophoretic direction from the acidic sample zone to the alkaline zone of the bge, see fig. . the method allowed injection of broad sample zones in relatively high-conductivity matrices, without deteriorating peak shape, resolution, and separation efficiency, which makes the method suitable for analyses of real samples. the size of the original sample zone was reduced almost times and the peak height of analyzed peptides has increased more than times. in the next study, the technique was further improved and about -to fold sensitivity enhancement was achieved in ce-esi-it-ms analysis of peptide hormones and tryptic peptides of cytochrome c, demonstrating the suitability of this approach for pmf in protein analysis and proteomics [ ] . the capturing of a protein or polypeptide in its pi, within an applied electric field in the capillary using a ph junction created by discontinuous buffer system is the base of over -fold concentration for microliter volumes of peptide and protein solutions [ ] . further investigation of the discontinuous buffer junctions using ph indicators and their optimization allowed up to -fold preconcentration of myoglobin [ ] . a ph-mediated base stacking has been developed for determination of oxidized and reduced forms of glutathione in liver microdialysates [ ] . a microfluidic device has been developed for sample cleanup of peptides and proteins via their electroimmobilization in a microflow stream [ , ] . after their capture, the electric field is decreased in a stepwise manner, causing sequential release of the captured peptides according to their electrophoretic mobility. tryptic peptides were sepa-rated and analyzed by maldi-ms. the concentrating and separation power was sufficient to increase the ionization yield of several peptides originated from the shotgun tryptic digestion of membrane protein extracts, which were not detected in the unprocessed sample. an extremely high, up to million-fold preconcentration of peptides and proteins has been achieved by a nanofluidic filter based on electrokinetic trapping mechanism [ ] . the device, fabricated by photolithography and etching techniques, generates an extended space charge region within a microchannel, in which peptides and proteins are very efficiently collected and trapped. this electrokinetic trapping and collection can proceed for several hours and concentration factors as high as - have been demonstrated. due to its simplicity, performance, and robustness the devices can be integrated in various analytical microsystems, including chip electrophoresis. preconcentration and preseparation of bulk components can be achieved also by application of itp as a preceding step of cze analysis realized either in column coupling system [ ] or as a transient itp (t-itp) process which is continuously converted into cze mode [ ] [ ] [ ] . selective enrichment and ultrasensitive identification of trace peptides in proteome analysis was achieved using t-itp-cze coupled with nano-esi-ms [ ] . a new simple and robust itp system with high concentration of leading ion and eof suppression can be integrated into microchip ce devices to achieve up to million-fold sample stacking [ , ] . peptides are mostly derivatized with a fluorophore to be detected with (laser-induced) fluorescence (lif) detection, the sensitivity of which is about two to three orders higher figure . dynamic ph junction model for acidic peptides and proteins. (a) long plug of sample in a low-ph matrix is injected into an uncoated capillary filled with a high ph bgs. (b) steep ph boundary develops at the front end of the plug and sweeps throughout the sample zone during electrophoresis, converting the cationic analyte into anionic, and significantly retarding its migration velocity. (c) focused peak migrates to the detector. reprinted with permission from [ ] . than that of the most frequently used and no derivatization requiring, uv-absorption detection. the list of selected pre-, on-, and postcolumn derivatization reactions to label peptides and other analytes in the nanomolar and subnanomolar range for ce with lif detection can be found in the review [ ] . the on-line coupling of sequential injection analysis (sia) and ce via an in-line injection microvalve with the port to port volume nl has been developed for on-line derivatization and analysis of peptides and amino acids [ ] . the sia system was used for automated derivatization of amino acids and peptides by dichlorotriazinylaminofluorescein enabling sensitive detection with lod ng/ml using argon ion lif detection at excitation/emission wavelengths / nm. in addition to more conventional procedures of preand postcapillary derivatization, new approach based on incapillary derivatization, adopted from the concept of capillary electrophoresis-mediated microanalysis (emma) [ ] , has started to be used for peptide and protein labeling. incapillary derivatization of amino acids and peptide buccaline with -fluoro- -nitro- , , -benzoxadiazole (nbd-f) was developed for their subsequent ce analysis with lif detection (argon ion laser, excitation/emission at / nm wavelength) [ ] . the in-capillary derivatization was achieved via zone-passing mode by introducing successive plugs of sample and nbd-f into an fused-silica (fs) capillary previously equilibrated with an alkaline borate buffer. to prevent nbd-f hydrolysis and to achieve a reliable derivatization, nbd-f was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and nbd-f reagent plugs. nbd-f/sample molar ratio above , sample/nbd-f plug lengths ratio equal to and increased temperature of c for amino acids and c for peptides were found to be optimal for high degree of derivatization. a good linearity between the peak heights and analyte concentrations was achieved allowing quantitative analysis of amino acids and buccaline peptide at the submicromolar level. in-capillary noncovalent labeling of insulin and a gastrointestinal peptide (arg-arg-gastrin) by commercially available dye (sypro orange) has been tested for analysis of these biopeptides by ce with lif detection [ ] . the peptide labeling and the fluorescent emission intensity (excitation at nm, emission at nm) were found to be sufficient only in the presence of cationic detergent, tetradecyltrimethylammonium bromide (ttab), in submicellar concentration in the borate bge. although the sensitivity attained was not as high as expected with lif detection, the advantage of this approach was that a rapid on-line labeling of peptides could be performed without the complications and limitations of multiple labeled species frequently encountered with covalent labeling. the integrated rapid on-chip sample derivatization employing naphthalene , -dicarboxyaldehyde (nda) and -mercaptoethanol ( -me) with an easily assembled microdialysis-chip electrophoresis was developed and tested as a prototype of micrototal analytical system for high-temporal resolution monitoring of amino acids and peptides in biological systems [ ] . the capability of ce and cec to analyze extremely small sample volumes (nano-to picoliter scale) put special demands on the techniques and microdevices to manipulate such low-volume samples. sample micromanipulations are integrated in the so-called micrototal analytical systems (mtas) [ ] where all operations, i.e., sample manipulation, potential preconcentration, clean-up and derivatization, separation, and detection, are performed on the microstructures fabricated on the chips (see section ) . different means for sample manipulations in the mtas, such as sample introduction to the chip, preseparation, derivatization, microdispensing, microinjection (electrokinetic, pressurized, piezoelectric, optical gating, microrotary valves), mixing by static mixers, centrifugal force, capillary force, pneumatic propulsion, and electromanipulation, are discussed in the recent review [ ] . these techniques are particularly useful for handling of small sample volumes, such as in single cell analysis [ ] and in the analysis of microbiopsy and microdialysis samples [ ] . several integrated systems for protein digestion and cebased separation have been developed also on the chips, e.g., the chip-based nanovials with immobilized proteolytic enzymes [ , ] . for in vivo monitoring of biomolecules in body fluids and tissues, e.g., neurotransmitters in the brain, microdialysis cells composed of a semipermeable membrane, typically - mm long and . - . mm in diameter, are fashioned into a probe, which is implanted in the brain or other tissues [ ] . adsorption of peptides, particularly longer polypeptides and peptides with increased number of hydrophobic amino acid residues, represents a serious disturbance factor in their ce analysis performed both in the most frequently used fs capillaries and plastic chips. thus, suppression of adsorption belongs to the challenging issues in ce of peptides. various strategies used to suppress the peptide and protein adsorption to the fs capillary or microchip wall have been surveyed in the recent review [ ] . in addition to the separations performed in extreme low or high ph and in high ionic strength bges, different ways of the fs capillary coatings are employed. dynamic coatings are based on reversible (dynamic) adsorption of small ions or hydrophilic polymers such as cellulose derivatives or synthetic polysaccharides added into the bge. static (permanent) modifications of the inner capillary wall involve the formation of a covalent bond between a suitable derivatized coating agent and the silanol groups of the silica, giving rise to an immobilized polymeric coating, or the bond may be noncovalent but yet "permanent" resulting from the irreversible physical adsorption of the polymer, e.g., polyvinyl alcohol (pva) or peg from the aqueous solution to the capillary wall. a new adsorptive polyamine coating for ce of basic peptides and proteins in fs capillaries was prepared using polymer poly-la , synthesized from , -diamino-n-methyldipropylamine and , -butanediol diglycidyl ether [ ] . the polymeric coating is formed by multisite electrostatic interaction between silanol groups and the positively charged nitrogen atoms, and hydrogen bonding due to the hydroxyl groups of the polymer. the physically adsorbed polymer resulted in stable coating within a broad ph range, - , even in the presence of organic modifiers (acn, methanol) and complex biological samples (plasma and cerebrospinal fluid (csf)). the coating procedure was found to be highly reproducible and provided fast (few minutes) and highly efficient separations of basic peptide hormones, e.g., enkephalins, oxytocin, angiotensin, bradykinin, and proteins, such as lysozym, ribonuclease a, a-chymotrypsinogen a, cytochrome c, and myoglobin. in addition to suppression of peptide sorption, the polycationic coating reversed the eof and generated strong anionic eof, which ensured relatively fast migration of all peptides, negatively charged, neutral and positively charged to anode. moreover, the coating is well compatible with esi-ms detection. semipermanent noncovalent coatings of the fs capillary by the double-chained surfactants, such as didodecyl-to dioctadecyldimethylammonium bromide and triple-chained tridodecylmethylammonium bromide, which have been successfully applied for cze separation of proteins in mm ammonium formate bge, ph . [ ] , have the potential to be applied also for separation of peptides. using this noncovalent coating concept, bilayers, triple layers, or even multiple layers can be formed by polymers of opposite charge being alternatively adsorbed on each other. noncovalently bilayer coated fs capillaries, prepared by successive flushing the capillary with cationic polybase (polybrene) and anionic polyacid, poly(vinyl sulfonate), have been shown to be suitable for separation proteins, such as interferon-a- b, myoglobin and carbonic anhydrase, and peptide fragments of insulin [ ] . with mm tris-phosphate bge, ph , a high efficiency ( - ) and favorable repeatability of migration times (rsd , . %) were obtained, even at high concentration of hsa in the sample, up to mg/ ml. commercially available dual layers-based dynamic coatings, e.g., ceofix kits (see www.microsolvtech.com/ceofix. asp), can be also used for ce separations of peptides. from different strategies tested for noncovalent fs capillary coating in nace of synthetic polypeptides poly(n e -trifluoroacetyl-l-lysine), such as (i) addition of viscous additives, ethyleneglycol, or glycerol, to the bge, (ii) adsorption of peg of different m r via hydrogen bonding, (iii) coating with binary mixtures containing ethylene glycol and peg, and (iv) adsorption of cationic polyelectrolyte (deae-dextran) layer, the last one, polycationic coating was found to be the most effective in suppressing the polypeptide adsorption [ ] . the problem of peptide and protein adsorption is serious also in plastic microchips. due to their hydrophobicity, the inner surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, including polypeptides and proteins, resulting in their significant adsorption to the microchannel walls. cationic quaternary ammonium starch derivatives used as dynamic coating agents were found to suppress the adsorption of fluorescently labeled oligopeptides and amino acids in their cze separation in poly(methyl methacrylate) (pmma) microfluidic devices. the effect was valid over the ph range . - . good solubility and low viscosity were the other advantages of this dynamic polymeric coating agent. effective capillary coating can be achieved also by low molecular mass compounds. adsorption of cationic diethylentriamine results in masking the silanol groups and other active adsorption sites on the fs capillary wall which are responsible for peptide and protein adsorption to the capillary [ ] . adsorption of barium(ii) cations originating from barium tetraborate bge to ionized silanols and barium silicate precipitation seem to be able to shield the silica surface from separands and to reverse the direction of eof as shown by ce separations of tryptic digests of peptides [ ] . dynamic or permanent capillary coatings have been used also for control of the eof, which influences separation efficiency, resolution, and speed of ce analyses of all types of analytes, including peptides and proteins. a high and reproducible anodic or cathodic eof for ce-ms separation of glycopeptides was achieved by noncovalent coating of the fs capillary with polycationic polybrene or polycationic polybrene/polyanionic dextrane sulfate single or multiple layers [ ] . the latter type of bilayer coating, produced by subsequently rinsing the fs capillary with a solution of polybrene and poly(vinyl sulfonate), was shown to be suitable for ce separation of enkephalins and tryptic peptides of cytochrome c in acidic mm formic acidbased bge, ph . , and compatible with esi-ms detection, causing no ionization suppression or background signals [ ] . dynamic coatings and precoatings with the commercially available agents eotrol tm and ultratrol tm have been reported to provide ph and bge composition independent cathodic or anodic eof at different flow rates [ ] . in addition to chemical ways, eof can be regulated also by physical tool, external radial electric field applied to the outer low-conductive capillary coating as shown by ce separation of oligoglycines [ ] . cze is the simplest, universal, and most frequently used ce mode applied to separation of peptides. ze mode is usually assumed when ce is spoken about without further specification of the separation mode [ ] . for that reason only some special new aspects of peptide cze separations are presented in this section, the others are mentioned also in the other sections. a new cze approach, the so-called ion-interaction cze (ii-cze), has been employed to the separation of synthetic cationic proteomic peptide standards comprising of three groups of nine decapeptides with net charges of , , and for all nine peptides within a group and with subtle changes of hydrophobicity within each group of nine peptides [ ] . this bidimensional ce approach provided an excellent resolution of the peptides with high peak capacity by combining the powerful inherent ze separation mechanism (different electrophoretic mobilities due to different charge/size ratios) with an hydrophobicity-based mechanism consisting in differences of peptide interactions with bge constituents, perfluorinated carboxylic acids (trifluoroacetic, pentafluoropropionic, heptafluorobutyric) present in high concentrations (up to mm) and adjusted by lioh to ph . . thus, simultaneously with a ze-based separation of the three differently charged groups of peptides, there is a hydrophobically mediated separation of the peptides within these groups, see fig. . this methology is quite different from other ce modes using complexing agents, such micelles or cds in ekc. the principle of ii-cze has been successfully applied also for the separation of peptides differing by a single amino acid substitution in deca-and dodecapeptides with model amino acid sequences [ ] . substitutions differed by a wide range of properties, e.g., alkyl side chains, polar side chains, and charged side chains. the hydrophobic pentafluoropropionic acid anion provided best separation of peptides that differ in hydrophobicity of their hydrophobic side chains but high concentration of the hydrophilic phosphate anion was the best for separation of peptides differing in their polar side chains. differential hydration/dehydration properties of side chains in the peptide and the hydration/ dehydration properties of the hydrophilic/hydrophobic anions as well as the electrostatic attraction between the peptide and the anions in solution play an important role in these solution-based effects. new types of bge constituents, "narrow ph cuts" of carrier ampholytes employed for ph gradient formation in ief, obtained by ief fractionation in agarose gel or in the rotofor device, have been investigated in common studies by peltre's and righetti's groups [ ] [ ] [ ] . they found them promising bges for the cze of proteins and peptides with respect to their low conductivity and good buffering capacity, permitting application of high intensity of electric field and thus achieving short separation times. these properties are provided also by single component isoelectric buffers, such as - mm iminodiacetic acid, which was used for cze separation of gnrhs and their analogs and fragments [ ] . extremely fast, about only s separations of jak tyrosine phoshorylated peptide fragment and its complex with sh -bb protein were achieved by cze in mm id, cm/ cm total/effective length capillary using a very high electric field strength of v/cm and a bge composed of mm tris, mm glycine, ph . [ ] . nace performed in pure organic solvents (e.g., acn or methanol) or cze in hydro-organic solvents, i.e., in the mixtures of organic solvents with water buffers also found their applications in the analysis of peptides and peptide derivatives. nace with methanol or methanol/acn mixture ( . : . v/v) as a solvent of bges composed of acetic acid, ammonium acetate, sodium acetate, and tetramethylammonium hydroxide has been used for separation of living (with free n-terminal amino group) and dead (with formylated n-terminus) water-insoluble synthetic homopolypeptides, poly(n e -trifluoroacetyl-l-lysine) [ ] . nace permitted the separation of the oligo-and polymers both according to their size (with the addition of tfa up to a dp of , ) and according to the nature of the end groups. interestingly, nace has provided better separation selectivities for a-helical polypeptides containing - amino acids than the aqueous ce under similar conditions [ ] . this result was attributed to the significantly increased helical secondary structure of analyzed peptides in methanolic bges than in aqueous bges as found by circular dichroism method. nace in different bges with electrochemical detection was tested for separation of enkephalin peptides [ ] . the best separation was achieved in bge consisted of mm ammonium acetate in mixed acn/ methanol ( : v/v) solvent. the recent applications of itp both in the capillary [ ] and in the microchip [ ] formats are more frequently dealing with the determination of low-molecular-mass ions than with the analysis of peptides and other biopolymers. one of the few exceptions is the application of capillary itp (citp) to the determination of the purity of lecirelin, synthetic analog of luteinizing hormone-releasing hormone (lhrh) [ ] . in addition to the determination of the purity of this peptide, citp has been used also for quantitation of anionic counterions of this strongly basic peptide. more frequently, itp or t-itp modes are employed as a preconcentration and/ or preseparation step [ , , ] prior to ce analysis of peptides and proteins present at low concentrations and/or in complex mixtures, such as, e.g., for selective enrichment and ultrasensitive identification of trace peptides in proteome analysis using t-itp/ze mode coupled to nano-esi-ms detection [ ] and in combination with carrier ampholyte-based ce (cabce) mode [ ] . cief is more suitable for evaluation of microheterogeneity of proteins, glycoproteins, and longer polypeptides than for peptides, since the effective charge of short peptides, similarly as that of amino acids and unlike that of proteins, may approach zero value at rather broad ph zone than at a sharp ph value, pi. different ways of pi determination of proteins and peptides by cief are presented and critically evaluated in the review [ ] . since the direct measurement of ph inside the capillary is unavailable, the estimation of pi values has to be based on the use of different types of pi markers, dyes, fluorescently labeled peptides, sets of proteins with known pi values. denaturing cief, i.e., ief in the presence of amphoteric detergents and/or urea, and cief with the whole-column imaging uv-absorption detection and with the liquid-core waveguide lif whole-column detection have been employed for investigation of protein/polypeptide conformational and chemical microheterogeneity, for characterization of proteins with identical pi values and for analysis of proteins and polypeptides of different origins, such as naturally fluorescent phycobiliproteins and noncovalently (nanoorange) labeled proteins, antibodies, and viruses [ , ] . due to its concentrating and focusing effect cief is frequently used as the first concentrating step in two-or multidimensional separations of complex mixtures of peptides and proteins. -d capillary format was realized by on-line hyphenation of cief and nongel sieving ce via a hollow-fiber membrane interface [ ] . -d separation, analogous to the classical slab gel ief/sds-page is realized by on-line coupling of cief with esi-ms detection [ ] , which provides both pi and m r values, similarly as classical -de, however, with much higher precision of m r determination. this system was applied to determination of peptide and protein concentrations using angiotensin ii and human tetrasialo-transferrin as the model analytes [ ] . the lod was . mm, i.e., about ten times lower than that with uv-detection. human transferrin could be detected with acceptable accuracy and repeatability at physiological concentration levels ( . - . mg/ml). a -d microcolumn platform consisting of online hyphenated cief in hydroxypropylcellulose coated capillaries and neutral stearyl-acrylate (c ) monolithic cec has been used for separation of model mixtures of peptides, proteins, and albumin-depleted human serum [ ] . in addition to cief, also noncapillary solution ief systems are worth to be mentioned due to their efficient application in the important areas of proteomics and peptidomics. a six-chamber device with isoelectric membranes as a preseparation step prior to hplc-ms analysis significantly improved peptide detection and identification in the investigation of insoluble nuclear protein fraction [ ] . an efficient fractionation and improved peptide and protein identification of human plasma and of an escherichia coli tryptic digest was attained also by peptide-offgel electrofocusing followed by hplc-ms analysis [ , ] . affinity electrophoresis performed in a capillary format, capillary affinity electrophoresis (cae), is mostly used as a mild and sensitive tool for the investigation of the (bio)molecular interactions and recognition, and for estimation of binding (association) or dissociation constants of the formed complexes [ , ] . cae involves several modes based either on the separation of the interacting species, such as in the hummel-dreyer method, the vacancy peak method and frontal analysis, or on the detection of a specific physicochemical property of the complexed ligand or the binding partner. all these methods utilize the differences in the migration velocities of the interacting species. both theoretical and experimental considerations for these methods and their applications for estimation of binding constants have been outlined in the recent review [ ] , particular recent applications to peptide interaction studies are presented in section . . methodologically new approach for estimation of binding constants of peptide ligands (n-acetyl and n-succinyl derivatives of a dipeptide d-ala-d-ala) to glycopeptide antibiotic receptor (vancomycin from streptomyces orientalis), flowthrough partial-filling cae (ftpf-cae), has been developed by gomez and coworkers [ ] . in this technique, the capillary is first partially filled with a zone of ligand followed by a sample plug containing receptor and noninteracting standards. after application of a voltage, the receptor and standards flow into the zone of ligand where a dynamic equilibrium is achieved between receptor and ligand. due to the continued electrophoretic movement, the receptor and standards migrate through the zone of the ligand plug prior to detection. evaluation of the change in the relative migration time ratio (rmtr) of the receptor, relative to the noninteracting standards, as a function of the concentration of ligand, yields a value for the binding constant, k b . in the technique of multiple-injection cae (micae) [ , ] , separate plugs of sample containing noninteracting standards, peptide i, buffer, and peptide ii, are injected into the capillary and electrophoresed. peptides i and ii migrate through the capillary at similar electrophoretic velocities and remain as distinct zones due to the buffer plug between peptides. the electrophoresis is then carried out in increasing concentrations of receptors, macrocyclic antibiotics, vancomycin, ristocetin, and teicoplanin, in the bge. continued electrophoresis results in a shift of migration times of the peptides upon interaction with antibiotic. analysis of the change in the rmtr of the resultant complexes relative to the noninteracting standards, as a function of the antibiotic concentration gives the k b . the advantage of micae technique is that it is able to measure k b of interaction of ligands of similar mobilities with the receptor without the need of individual binding experiments for each ligand. other new modification of micae is the partial filling micae [ ] , which has been also applied to investigation of the interactions between macrocyclic antibiotics, vancomycin, teicoplanin, and ristocetin, with fluoren- -ylmethoxycarbonyl (fmoc)-derivatized d-ala-d-ala terminus peptides. a special type of cae is the so called kinetic ce [ ] , which is defined as ce of species, which interact during electrophoretic movement inside the capillary. it represents a platform for development of homogeneous kinetic affinity methods for quantitative affinity measurements (binding and kinetic constants), affinity purification, and search for molecules with affinity to given ligands. affinity electrophoresis was carried out also in a microchip format [ ] and it was found suitable for highthroughput screening of biomolecular interactions, capable to provide quantitative data on the strength of the interaction with minimum sample and reagent consumption and within a very short analysis time ( - s). the latest developments and applications of cekc, namely its micellar mode (capillary mekc, cmekc) are summarized in the recent reviews [ , ] . cmekc with ionogenic detergents, anionic sds, cationic ctab, or zwitterionic chaps, is suitable for separation of electroneutral peptides, i.e., peptides with blocked or derivatized n-and c-terminus and other ionogenic groups of peptide chain, and/or for separation of peptides having the same or very similar charge to mass ratio but differing in their hydrophobicity. a partial filling mekc (pf-mekc) with a mixed micelle system composed of a zwitterionic surfactant -(n,n-dimethylhexadecylammonium)propanesulfonate (paps) and a nonionic surfactant peg dodecyl ether (brij ) has been applied to peptide mapping of bsa [ ] . the highly selective separation of tryptic bsa peptides was achieved in an optimized bge, composed of mm ammonium formate buffer, ph . , and containing mm paps and . % m/v brij . cekc with two polymeric pseudostationary phases, acrylamide-and siloxane-based polymers, provided very high efficiency and good selectivity for separation of ndaderivatized amino acids with selectivities different from those observed by sds micelles but these phases were found to be generally unsuitable for separation of ndalabeled peptides [ ] . being a hybrid electrokinetic and chromatographic technique, cec of peptides profits from the high selectivity of numerous stationary phases developed for peptide separation by hplc and from the low dispersion of electroosmotically driven motion of a mobile phase. another advantage of cec is that the composition of its mobile phase is more compatible with on-line coupling with ms detection than that of mekc. recent progress in cec is described in the reviews [ , ] and in the special issue devoted to advances in cec and ekc [ ] . cec separations of peptides have been performed in different separation modes, with rp, cationic or anionic stationary phases. mixed-mode stationary phase bearing an embedded quaternary ammonium group in a c alkyl chain has been used both for cec and hplc investigation of the chromatographic behavior of synthetic oligopeptides, eledoisins, [ile- ]-angiotensin iii, arg-arg-gastrin, rennin substrate, and epidermal growth factor [ ] . in hplc experiments, the variation of acn content in the mobile phase indicated that peptides are mainly separated by rp mechanism. the weak or negative retention factors observed as compared to c silica stationary phase suggested the involment of electrostatic repulsion in acidic conditions. comparison of hplc and cec separations showed that (i) ion-exclusion is more pronounced in hplc, and (ii) a high acn content in the mobile phase induced for some peptides an increase of retention in cec, pointing out the existence of a retention mechanism other than partitioning mainly involved in a chromatographic process. these facts demonstrate that the electric field has an important effect on peptide retention in cec and that chromatographic retention in cec cannot be simply predicted from the retention observed, even under identical experimental conditions, in hplc. performance of mixed-mode, rp (c )/strong cation exchanger (scx), sorbents for cec separation of peptides was tested by thrombin receptor antagonistic peptide and its alanine-scan analogs [ ] . by modulating the mobile phase composition, the hydrophobic or ion exchange interactions could be made to dominate the chromatographic retention of peptides in addition to their electrophoretic migration. using this strategy, a high resolution of six closely related synthetic peptides with very small differences both in their charge and hydrophobicity was achieved, however only within a narrow ph range around . of the mobile phase. cec on silica-based rp (c ) at acidic ph and hydrophobic (c )/strong-anion-exchange (sax) mixed mode phases at weakly alkaline ph showed that the former was more suitable than the latter for separation of model mixtures of short peptides ( - amino acids) [ ] . simple peptide mixtures are preferably separated under isocratic conditions, where significant changes in the separation are achieved by small changes of the individual parameters, such as ionic strength, organic modifier content, and temperature. on the other hand, complex peptide mixtures, such as protein digests often require gradient elution. the advantage of application of stepwise gradient of buffer concentration in cec with the mixed-mode stationary phase, -( -sulfo- , -naphthalimido)propyl-modified silylsilica, was demonstrated by improved and shortened separation of tryptic digests of cytochrome c [ ] . pressurized cec (pcec) with the possibility to variate a flow rate and to allow continuous gradient elution, where the mobile phase is driven by both eof and pressurized flow, facilitating fine tuning in selectivity of neutral and charged species, has been shown to be more efficient for the separation of model oligopeptides compared to the isocratic cec using mm id capillary column packed with mm size silica particles with c rp [ ] . monolithic materials are becoming a well-established stationary phase format for cec. both the simplicity of their in situ preparation and the large number of readily available chemistries make the monolithic separation columns an attractive alternative to the capillary columns packed with particulate materials. the current state-of-the-art in this rapidly growing area of cec including its application for peptide separations is summarized in the recent reviews [ , ] . a novel monolithic column for cec separation of oligopeptides was prepared using l-phenylalanine as a template and a covalent approach through the formation of schiff base with orthophthaldialdehyde (opa). the reaction mixture was introduced into the capillary and after the thermal polymerization the template was extracted with methanolic hcl solution. the capillary column ( ) cm mm id with a mobile phase of mm phosphate buffer (ph . )/methanol % v/v, applied voltage kv, and detection at nm, could separate several oligopeptides, such as angiotensin i and ii, oxytocin, vasopressin, tocinoic acid, b-casomorphin, within min, see fig. . the cec separation of this set of peptides was meadiated by a combination of chromatographic retention involving hydrophobic, hydrogen bonding, electrostatic interactions as well as schiff base formation with opa in the cavity of the templated polymer and also by electrophoretic migration of charged peptides. a neutral, nonpolar stearyl-acrylate monolithic column providing a relatively strong eof but being free of electrostatic interactions with charged solutes was developed for the rp-cec of neutral and charged species including peptides and proteins [ ] . another mixed-mode n-alkyl methacrylate-based monolith has been used for separation of therapeutic peptides [ ] . while the sulfonic acid (scx) moiety supported the generation of a stable eof at both acidic and basic ph, the butyl ligands provided the nonpolar sites for chromatographic resolution. another cec mode, open tubular cec (ot-cec), which is based on the attachment of thin layer stationary phase to the inner capillary wall [ ] , is also used for separation of peptides and proteins. novel type of capillary columns for ot-cec are based on etched chemically modified fs capillaries. fabrication, properties, and applications of these columns have been recently reviewed by pioneers of ot-cec, pesek et al. [ ] . etching of the inner capillary wall is performed by heating the capillary at temperature of - c in the presence of ammonium hydrogen difluoride. etching process substantially (ca. -fold) increases the surface area, which alleviates the relatively low capacity of the bare fs capillary, and creates a surface that is fundamentally different from the bare fs. after the chemical modification of the surface, the bonded organic moiety forms the stationary phase for ot-cec. etched fs capillaries chemically modified with n-butylphenyl and cholesterol- -undecanaoate [ ] have been used for ot-cec separation of two sets of peptides, each having structurally similar amino acid sequences, and the resolving power of the ot-cec was found to be superior in comparison with gradient rp-hplc. ot-cec in fs capillaries with covalently attached gquartet-forming dna oligonucleotides provided better resolution of fibrinogen peptides when g-quartet structure was destabilized by increase of temperature from to - c [ ] . the applications of (metallo)porphyrins-based physically adsorbed or chemically bonded stationary phases for ot-cec separations of peptides, amino acids, and other analytes have been reviewed by deyl et al. [ ] . in spite of the high separation power of individual ce and cec modes, for complete separation of complex peptide and protein mixtures present in body fluids, tissue extracts and enzymatic digests of large proteins, apart from super-complex mixtures of proteome or peptidome of different organisms, cells and organelles, a combination of two or more complementary separation principles, such as ief and sds-page in the -de [ ] , is necessary. the increasingly important role of multidimensional peptide separations in proteomics and peptidomics, i.e., in comprehensive analysis of all or part of protein and peptide content in the cell, organ or biofluid, has been emphasized in recent reviews on this topic [ , ] . the ultimate goal is to have a rapid separation system that can provide identification and comprehensive monitoring of the changes in concentration, interactions, and structures of proteins and peptides in the proteome and peptidome. two-or multidimensional peptide separations are based on utilization of two or more independent physical properties of peptides to separate their mixtures into individual components. when the properties are truly independent, the separation methods can be considered as "orthogonal" and the peak capacity of this multidimensional separation is approximately equal to the product of the individual peak capacities of each dimension. different combinations of separation principles are used for -d separations of peptides and proteins, such as lc-ce, ce-ce, ce-lc, where lc and ce represent different modes, so that many different combinations are available, such as, e.g., rplc-cze, ion-exchange chromatography (iec)-cze, sec-cze, rplc-cief, affinity lc-cekc, cze-cief, cze-cekc, cekc-cze, citp-cze, cze-cge, cief-cge, cze-capillary lc (clc), and cief-clc. when these -d systems are on-line connected with ms or ms/ms detection, then really powerful multidimensional systems are obtained capable to identify hundreds up to two thousands of peptides/proteins in complex mixtures such as body fluids, tissue extracts, and cell lysates. for more details see the above mentioned reviews [ , ] , here only few examples will be presented. a high-speed -d ce system with a compact fluorescence detector for high-sensitivity peptide and protein analysis has been developed by dovichi's group [ ] . fluorescently labeled proteins/polypeptides are separated according to their size by cze in a replaceable % dextran sieving matrix in bge composed of mm tris/ches, . mm sds, ph . , in the first dimension. the second-dimensional separation is performed by mekc in an on-line connected second capillary containing mm tris/ches bge with mm sds micellar pseudophase. as soon as the first peptide/protein zone approaches the capillary outlet, the fractions from the first capillary are successively transferred to a second capillary, where they are separated according to their hydrophobicity to generate, in serial fashion, a -d electrophoregram. the comprehensive -d separation involving analysis of fractions lasted min. the system has been applied to -d protein/polypeptide fingerprinting of barrett's esophagus tissues. the same combination of sieving ce and mekc modes has been used also in the multiplexed -d ce system which allows separation of five samples in parallel [ ] . samples are injected into five first-dimensional capillaries, the fractions are transferred across an interface to five second-dimensional capillaries and analytes are detected by lif in a five-capillary sheath-flow cuvette. the instrument produced lods at the zeptomole level for -( -furoyl)quinoline- -carboxyaldehyde-labeled trypsin inhibitor. another ce-based -d orthogonal separation system, separating proteins/polypeptides according to pi by cief in the first dimension and according to the m r in the second dimension by a non-gel sieving ce, employes a hollow-fiber membrane interface for coupling the two capillaries [ ] . -d microcolumn platform consisting of on-line coupled cief in hydroxypropylcellulose-coated fs capillaries and neutral stearyl-acrylate (c ) monolithic cec has been developed by zhang and el rassi [ ] and applied to separations of model mixtures of peptides, proteins, and albumin-depleted human serum. the theoretical peak capacity of this orthogonal cief-cec platform was estimated as more than . sec and ce have been on-line coupled via spme and tsplit interface, see fig. . this -d system was applied to peptide analysis in biological fluids [ ] . the sec column fractionated the sample by m r ; the low-m r fraction containing peptides is directed to a c spme column, where the peptides are captured and concentrated. the peptides are desorbed from spme column by nl acn, and the effluent is introduced into the t-split interface, which hydrodynamically splits ( : ) the flow and in this way it ensures appropriate injection of analytes into the ce system. the system enabled determination of enkephalins in csf with lods of about ng/ml and good linearity over . - mg/ml range for injection of ml spiked csf samples. since recently there are attempts to integrate multidimensinal separations on chips. an integrated protein/ peptide concentration/separation system combining nonnative ief with sds gel electrophoresis has been constructed on a polymer microfluidic chip with planar dimensions as small as cm cm [ ] . instead of time-consuming sequential sampling in classical capillary systems, the zones of concentrated and focused proteins are electro- kinetically transferred into an array of orthogonal microchannels and further resolved by sds gel electrophoresis in a parallel and high-throughput format. resolved proteins are monitored by noncovalent, environment-sensitive fluorescent probes such as sypro red. a comprehensive -d separation is accomplished within min with an overall capacity of about spots. combination of affinity selection of specific peptides, e.g., histidine-containing peptides by immobilized metal affinity chromatography, cysteine-containing peptides by covalent chromatography, glycopeptides by lectin chromatography with subsequent ce or ce-ms separation and detection of these specific peptides represent other examples of off-line multidimensional separations applicable in proteomics studies [ ] . ms and ms/ms can be considered as the second and third separation dimensions when used as detection mode in ce or cec; these set-ups represent a special and numerous class of multidimensional separations, for review see, e.g. [ , , ] , and section . . different aspects of ce-ms analysis of peptides and proteins have been recently thoroughly discussed and reviewed [ , ] . from the different ms ionization modes, involving atmospheric pressure chemical ionization (apci), inductively coupled plasma (icp), fab, esi, and maldi, the last two modes, esi, and maldi are most suitable and mostly used for peptides and proteins. combination of advanced ms technique, fourier transform ion cyclotron resonance (ft-icr), with ce or cief is suitable for high-throughput analysis of complex peptide/protein mixtures in proteomics [ ] . on-line cief-esi-ms was applied to quantitative analysis of peptides and proteins [ ] . cief-ms using % v/v pharmalyte - and a sheath liquid containing water/ methanol/acetic acid ( / / ) was capable to resolve angiotensin i and ii (differing by dpi . ) with resolution of . and to determine human tetrasialo-transferrin in the . mg/ml concentration. [ ] . the special on-line interface for coupling lc and ce is a flexible device made of pdms, which can be used with conventional capillaries, allows coupling to any kind of ms detector and provides high injection repeatability (rsd , . %). the applied ft-icr-ms detector provides ultrahigh resolution of detected ions, mass accuracy at ppm level, and high sensitivity. sequence coverage for bsa of % showed a high recovery of sample in the different transfer steps through the system. the lod for peptide identification is in the low picomole range. the system seems to be the most qualified and promising for analysis of complex biological samples, such as those in proteomics and peptidomics. another -d system, an on-line spme-ce-ms setup, was developed by tempels et al. [ ] . analytes are preconcentrated using a c microcolumn ( mm . mm) and then introduced into the ce system via a valve interface. the ce is connected with esi-it-ms using a coaxial sheathliquid sprayer. the potential of the system was demonstrated by analysis of csf samples spiked with enkephalins and by separation of tryptic peptides of cytochrome c. immunoassays combined with ce can be also considered as multidimensional separation systems providing both selectivity and sensitivity, which is competitive with any method currently available for molecular analysis. immunoassays are mostly coupled with ce in the precapillary offline mode. in the competitive immunoassays sample antigen is incubated with labeled antigen or antibody and the amount of free or bound antigen or of free or bound antibody is determined by ce. for recent advances of ce-immunoassays see [ ] . the relatively strong absorption of a short-wave uv radiation ( - nm) by peptide bond allows application of uvabsorption detection at this wavelength range as a universal detection principle for monitoring peptide separations in ce and cec with lods in the micromolar range in a ca. nanoliter detection cell volume with millisecond time resolution. the earlier developed special detection cell constructions with increased optical path and thus enhanced sensitivity (zshaped cell, buble cell, or sleeve cell), and some new designs of the uv-absorption detector for capillary and chip separations are described in the recent reviews on optical detection schemes [ , ] . applications of a new type of optical detector, based on the whole-column imaging detection system working alternatively in absorption, refraction index, and fluorescence mode can be found in refs. [ , , ] . the cm section of the fs capillary with removed polyimide coating is illumi-nated by the light guided from the light source (deuterium lamp, xenon lamp, he-ne laser, diode laser) by an optical fiber bundle and focused by cylindrical lens to the capillary. the intensity of light after passage across the capillary is measured by a ccd camera, placed in the direction of illuminating light for absorption or refractive index gradient mode, or placed perpendicularly to the direction of the illuminating light for the fluorescence mode. absorption mode was found to be the most practical due to its quantitative ability and universal characteristics not only for the cief of proteins and peptides, for which it was originally developed in order to eliminate the disturbing mobilization step of cief required for single point detection after focusing process, but also for cze separation of these and other analytes allowing to study the dynamics of electroseparation processes. a new type of uv-absorption detector, utilizing a complementary metal oxide semiconductor (cmos) active pixel sensor, has been constructed for real time visualization of electrophoretically mediated in-capillary reactions [ ] . imaging of analyte peaks absorbing at nm and migrating over the length of mm in the capillary dimensions enabled measurement of velocities and lengths of reactant and product zones. the system was used for monitoring glutathione oxidation by hydrogen peroxide. cmos imaging allowed the whole process of reaction, separation, and quantification to be perfomed in nanoliter volumes on-capillary in single run within ca. min. enhanced sensitivity of peptide detection at ultralow uv region below nm has been utilized, e.g., in cze determination of zinc-bacitracin in feedstuff when this peptide antibiotic was detected at nm at low mg/ml level [ ] . a universal optical detector based on dual capillary backscatter interferometry with density gradient compensation has been constructed for refractive index measurements in nanoliter volume for microchip ce with sensitivity at the femtomole level [ ] . lif is the most sensitive detection scheme in ce, cec, and other microanalytical systems as documented in the recent reviews on advances in optical detection methods [ , ] . with special designs of detection cells, such as liquid sheathflow cuvettes and integrated separation and detection devices, lif detection has a potential to achieve the detection limit of few or even of a single molecule [ ] . the latest developments and applications of lif detector in ce and cec of peptides are summarized in comprehensive reviews [ , ] . the disadvantage of the lif detection of peptides is the necessity of their derivatization by fluorogenic labels. the native fluorescence can be utilized only for detection of peptides containing aromatic amino acid residues of trp, tyr, and phe. however, for their excitation, deep uv-laser systems, such as nd/yag laser operating at nm, or multiphoton excitation are necessary. the latter approach has been utilized in a compact lif detector based on two-photon excitation of the native fluorescence of proteins and peptides containing the above mentioned aromatic amino acids [ ] . the fluorescence is excited by a solid-state diode pumped microchip laser operating at nm, with an average power . mw, a pulse width ps, repetition rate . khz, and beam diameter . mm. concentration lods for free amino acids, phe, tyr, and trp, were mm, . mm, and nm, respectively, in a volume of fl, for bsa the limit was nm. a multichannel native fluorescence detection system has been constructed for analysis of amino acid and peptide neurotransmitters in single neurons [ ] . a hollow-cathode metal vapor heag laser emitting at nm is used as excitation source. fluorescence is collected orthogonally to the excitation optics with an mgf -coated, all-reflective microscope objective with a . numerical aperture and spectrally distributed by a series of dichroic beam-splitters into three wavelength channels: - nm, - nm, and . nm. a separate photomultiplier tube is used for detection of the fluorescence in each of the three wavelength ranges. with this detector, analytes can be separated and identified not only by their electrophoretic mobilities but also via their multichannel signature consisting of the ratios of relative fluorescence intensities in each wavelength channel. the lods for neurotransmitters are in the low nanomolar (attomole) range. more frequently, fluorescence detection of peptides in ce and cec is based on precolumn or postcolumn derivatization of these analytes with a fluorescent label (see section . ). the disadvantage of this approach is that due to the usual presence of multiple derivatization sites in peptide and protein molecules, several derivatives with different electrophoretic mobilities and consequently multiple peaks may be obtained for originally single peptide or protein species. cief with the liquid-core waveguide lif whole-column detection described in more detail in the above section . on uv-absorption detection has been applied for separation and analysis of several different proteins and polypeptides, for microheterogeneity characterization, and pi determination of antibodies, glycoproteins, and viruses [ , , ] . a high-brightness blue light-emitting diode (led) serving as the excitation source at nm and an optical fiber collecting and guiding the emitting fluorescence to photomultiplier tube were used in the combined led-induced fluorescence and contactless conductivity detection applied for analysis of fitc-labeled amino acids and petides [ ] . a home-made instrument with a frequency-doubled nd/yag laser operating at nm was used as excitation source for lif detection of rhodamine b isothiocyanate-derivatized amino acids and peptides (bradykinin, angiotensin, and insulin) [ ] . ms represents an extremely powerful detection principle for ce, cec, and other separation techniques because of its universality, sensitivity, and selectivity, as evidenced by impressive recent developments of ms, ce-ms, and cec-ms methodologies in general [ , ] and in analysis of peptides and proteins especially [ , ] . importance of ms as analytical and structure elucidation method has significantly increased in the last years due to its key role in the proteomics-associated analysis of complex peptide and protein mixtures [ ] [ ] [ ] . consequently, the importance of both on-and off-line coupling (hyphenation) of ms with ce and cec separations of peptides and proteins, especially in proteomics, peptidomics, and peptide mapping, is also growing, as shown in some reviews [ , , ] . especially introduction of esi and maldi ionization techniques brought tremendous progress in on-line and offline characterization of electrophoretically separated peptides and proteins by ms [ ] . combination of ce with esi-ms and maldi-ms allows not only high-accuracy determination of m r of ce separated peptides, proteins, and other biomolecules, but also provides important structural data on amino acid sequence, the sites of post-translational modifications, peptide mapping, and noncovalent interactions of peptides and proteins. esi is the preferred mode for on-line coupling ce with ms, namely because of its capability to generate the ions with multiple charges so that the ion m/z (mass/charge) values for even very large species as polypeptides and proteins may fall within the limited m/z detection range of most mass spectrometers. a novel, rugged sheathless ceand cec-esi interface, in which an open tubular separation capillary and an electrospray tip are integrated with a nafion tubing junction, is coupled to ms detector has been developed for ce and cec separations of amino acids and peptides [ ] . a stable electrospray was generated at nanoflow rates by applying a positive electric potential at the nafion membrane junction. to sustain the stable spray, an eof to the spray was supported by coating the fs capillary with lupamin, a high molecular-mass linear positively charged polyvinylamine polymer, which also serves as stationary phase for ot-cec separation of peptides and proteins. a simple laboratory-made pressurized liquid junction nanoflow interface for ce-it-ms was developed and applied to separation and analysis of peptides and tryptic protein digests [ ] . in this device, the fs capillary and the spray tip were positioned in the electrode vessel containing the appropriate spray liquid. the electrospray potential was applied on the electrode inside the liquid junction. a stable electrospray was produced at nanoliter per minute flow rates generated in the emitter tip by hydrostatic pressure of the spray liquid, see fig. . another sheathless interface was developed with a graphite-coated esi tip attached to the separation capillary and has proved to be suitable for ce-esi-ms separation and characterization of therapeutic peptide hormones [ ] . maldi-ms with tof mass analyzer (maldi-tof-ms) is combined with ce and cec separations of peptides and proteins more in an off-line mode than with an on-line liquid sample delivery connection [ ] . the target plate specifically designed for the ce fraction collection and a semiautomatic fabrication of a prestructured maldi target coated with silicone has been developed for increased sensitivity in maldi-tof-ms analysis of hydrophobic peptides [ ] . the small spot size diameter (ca. . mm) and the spot pattern design were compatible with the fraction collection from ce. the matrix, a-cyano- -hydroxycinnamic acid, was applied on the target plate prior to the ce fractionation, and the fraction deposition was performed directly onto the maldi target. using this procedure, nine highly hydrophobic peptides from cyanogen bromide digest of integral membrane protein bacteriorhodpsin were ce-separated, fractionated and offline detected by maldi-tof-ms. the main advantage of maldi, soft ionization, and generation of predominantly single charged molecular ions of even polypeptide and protein macromolecules with m r up to is used for the exact determination of m r with an accuracy of . % and for identification of peptides and proteins isolated by micropreparative cze (see section . ). maldi matrices with high acid concentrations afford enhanced tolerance of cze buffers to be used for introducing peptides to the mass analyzer. the ms detection enables to detect and characterize also the nonpeptidic parts of peptides and proteins, such as nitration [ ] and glycosylation [ ] ; e.g., sialylated glycopeptides contained in hplc fractions of tryptic digest of bovine a -glycoprotein were separated from asialopeptides by ce. ce effluents were deposited directly onto a metallic target and their off-line maldi-tof-ms analysis allowed characterization of four glycosylation sites in the glycoprotein. in addititon, ms/ms was used to confirm peptide sequences and glycan content in the glycoforms. ms/ms detection systems are in general applied to determination of amino acid sequence of ce or cec separated peptides and proteins [ ] . a new technique, (ce-ms/ms)(n), in which multiple ce-ms/ms subanalyses (injections followed by analyses) are performed and experimental variables, such as bge composition and temperature, are manipulated during each ce-ms/ms subanalysis, has been developed in order to maximize amino acid sequence coverage of complex peptide and protein mixture [ ] . other detection schemes, such as electrochemical, contact or contacless electric conductivity, and nmr, are much less suitable and/or were much less applied to peptide detection in ce and cec in the reviewed period as compared to optical and ms detection. for that reason the reader is referred to the general recent reviews dealing with developments and applications of the former detectors, electrochemical [ , , ] , conductivity [ , ] , and nmr [ , ] , where few particular examples of application of these detectors for peptide ce analyses are mentioned. one of them is, e.g., the electrochemical detection of met-enkephalin, leu-enkephalin, and [d-ala- ]-leu-enkephalin separated by nace in mm ammonium acetate in acn/ methanol bge [ ] . using a pt microdisk electrode with disk diameter mm set to an actual potential . v (reference electrode ag/agcl), lods in the submicromolar range were observed, i.e., about one order of magnitude lower as compared to uv-detection. ce and cec separation modes performed on microfabricated microfluidic devices -microchips -represent the platform for a new generation of miniaturized analyzers. in these devices all operations, sample preseparation, preconcentration, mixing, derivatization, separation, and detection, are fully integrated and automated in the above mentioned mtas or "lab on a chip". the mtas undergo a period of a rapid and intensive developments, as demonstrated in the recent reviews [ , , ] . they are considered to become the most powerful tools of analytical chemistry in the coming years with a broad application in life sciences, biotechnology, and drug development, particularly in genomic, proteomic, and metabolomic research requiring fast, high-efficient, high-sensitive, and high-throughput separation and characterization of nucleic acids, proteins, peptides, and other biomolecules [ ] . an example of a functional model of mtas is a microchip ce device with on-line microdialysis sampling and on chipsample derivatization by nda and -me for lif detection, which was developed to monitor peptides and amino acids in samples of biological origin, such as microdialysates from brain [ ] . ce separations of fitc-derivatized peptide neuromediators, oxytocin, bradykinin, and enkephalins, performed in elastomer (pdms-pdms) and hybrid (pdms-glass) microfluidic devices have shown the perspective of chip ce for analysis of peptides and neuropeptides in small volumes [ ] . using the on-chip electrokinetic sample stacking and lif detection the fast and effective peptide separations were achieved within tens of seconds at injection volumes of about pl with plate numbers of up . applicability of microchips to affinity electrophoretic separation was demonstrated by investigation of the noncovalent interactions between neurotransmitters and sulfated b-cds in a commercially available quartz microchip with uv-absorption detection and in a home-made chip station with electrochemical detector [ ] . in spite of the fact that microchip ce provided less precise data than classical cae, in the future, more applications of chip cae for highthroughput screening and combinatorial chemistry can be expected. several types of microchips have been developed for integration of electrophoretic separations of peptides and proteins with esi-ms detection via special, low dead volume and liquid junction connection and for integrated preconcentration and derivatization procedures, micromanipulation and multidimensional separations, see sections and . , respectively. the microfabricated device has been constructed even for continuous free-flow arrangement of ze, itp and ief [ ] . however, due to the limited preparative capacity of theses devices, they are more suitable for continuous monitoring of selected analytes than for really preparative purposes. peptides are broadly utilized in many fields of biological, biochemical, and biomedical research as well as in biotechnology, pharmaceutical, food, and feed industry, e.g., in the investigation and modeling of the interactions of hormones with receptors, enzymes with substrates and inhibitors, antigens with antibodies, in the mapping of antigenic determinants (epitopes) of proteins, and in the production of peptide drugs and food and feed additives. this results in enlarged need for ce and cec application to quality control and purity determination of peptide preparations. in the majority of the above applications of synthetic, from natural material isolated or biotechnologically prepared peptides, ce and cec can be employed as sensitive control methods for the determination of their purity, or as a control method of the efficiency of the other, mainly chromatographic separation methods used for their purification. ce and cec provide rapid and accurate qualitative and quantitative data about the peptide preparations. further some recent ce and cec applications to peptide analyses will be given, in addition to those mentioned already in the previous sections on methodology and instrumentation. cze has proved to be powerful tool for the quality control/quality assurance of many types of drugs, including peptide and peptidomimetic drugs in the pharmaceutical industry. cze in acidic bge, mm phosphoric acid, mm tris, ph . , with uv-absorption detection at nm was applied to determination of purity degree of lecirelin, synthetic decapeptide analog of lhrh, used as verinary drug for ovulation in livestock, for treatment of ovarian cysts and for improvement of conception rates [ ] . example of cze analysis of a crude and hplc-purified preparation of lecirelin is shown on fig. . a macrocyclic glycopeptide antibiotic vancomycin in innovative microparticles and in commercial formulations has been analyzed by fast cze method using short-end capillary injection (effective length . cm, total length . cm) in . mm phosphate bge, ph . [ ] . purity of synthesized glutathione, and its related tetrapetide analogs and their stability and antioxidant activity in different solutions, water, a physiological solution, phosphate buffer, copper(ii) sulfate solution, and hydrogen peroxide solution, was checked by mekc using mm sodium phosphate as bge and mm sds as micellar pseudophase [ ] . mekc was found to be superior method for this application over rp-hplc, since the latter techniques failed in the separation of monomers and dimers of these peptides. microheterogeneity of peptaibol alamethicin f , -residue peptide isolated from the culture broth of the mold trichoderma viride, was analyzed by nace-ms using . mm ammonium formate in methanol, apparent ph . , as bge, and it and tof as mass analyzers [ ] . cze has been applied to purity determination and characterization of synthetic analogs and fragments of [leu- ]and [met- ]-enkephalins and dalargin, bioactive peptides with opiate activity [ ] . these oligopeptides (dipeptides to hexapeptides) were analyzed as cations in two acidic bges ( mm h po , mm tris, ph . , and mm iminodiacetic acid, ph . ), and some of them as cations and others as anions in alkaline bge ( mm tris, mm tricine, ph . ). purity degree of these peptides, expressed in three different ways (relative peak height, relative peak area, and relative corrected peak area), was evaluated, and their effective mobilities at standard temperature c were determined. series of opioid peptides, enkephalins, endomorphin i, and dynorphin a were analyzed by cze with on-line esi-ms detection in positive ion mode [ ] using volatile bge composed of mm acetic acid, mm formic acid, ph adjusted by ammonium hydroxide to . . the great advantage of ms detection is that from the obtained ms spectra their exact m r can be determined (see fig. ), their quality (identity) can be confirmed or in the case of ms/ms their structure can be elucidated. for the full characterization of peptide preparations, especially pharmaceuticals and peptides used in biological tests, it is necessary to know also the content of their lowmolecular mass ionic admixtures, e.g., fluorides, acetates, and trifluoracetates present as counterions in basic peptides, originating from the synthesis or purification of peptide products. one example of such application is the determi- nation of the contaminating anionic counter-ions (acetates, trifluoroacetates, and trifluoromethanesulfonates) of the strongly basic peptide drug, lecirelin, by anionic citp, using mm hcl/his, ph . as leading electrolyte, and mm mes, ph , as terminating electrolyte [ ] . thanks to a high sensitivity of some detection schemes, particularly lif, electrochemical, and ms, frequently enhanced by on-line sample enrichment techniques, ce and cec methods are applicable also to the analysis of peptides present at low concentration levels in complex biomatrices, such as biological fluids, cell lysates, and tissue extracts. several ce methods have been developed for determination of glutathione (gsh) [ ] , an extremely important biopeptide acting as antioxidant to prevent and limit oxidative damage of proteins in most living cells and participating in the reduction of disulfides and other molecules. hence, its determination in cells, tissue extracts, and body fluids is of great importance. a new ce method with lif detection was developed for the rapid separation and sensitive detection of gsh and glutathione-disulfide (gssg) after derivatization by -chloro- -nitrobenzo- -oxa- , -diazol (nbd-cl) [ ] . under the optimized experimental conditions, in mm sodium borate, mm b-cd, ph . bge, linear relationships between the peak height and concentrations of the analytes in normal and second-derivative electropherograms were obtained ( . - . mm). the lods for gsh and gssg in normal and second-derivative electropherograms were . and . mm and . and . mm, respectively. the sensitivity of the method was sufficient for determination of gsh and gssg in human plasma and tobacco leaves. the other ce-lif techniques were applied to simultaneous and rapid determination of gsh and reactive oxygen species in individual red blod cells [ , ] , in apoptotic leukemia cells [ ] , and in probiotic bacteria [ ] . validated cze methods, using mm or mm sodium borate, bges, ph , with uv-detection at nm, have been applied to determination of gsh and gssg in order to monitor oxidative stress and response to antioxidative treatments in an animal model, such as the rat made diabetic by streptozotocin injection [ ] . a new cze method has been developed for determination of gsh and gssg in microdialysis samples [ ] . the analysis was performed in reversed eof mode using cationic surfactant, . mm ttab in mm ammonium chloride bge, ph adjusted by naoh to . . a special, on-column preconcentration technique, ph-mediated base stacking, allowed that even medium-sensitive uv-absorption detection at nm was sufficient for detection of gsh and gssg in liver microdialysates of anesthetized rats with submicromolar detection limits. on the other hand, the content of gsh in must and wines was assayed by ce with lif detection [ ] . sample preparation involved conjugating of gsh and other potential thiols with monobromo-bimane (mbb) in ches buffer. the derivatized gsh and other main nonvolatile thiols were separated in mm phosphate bge and detected by lif using diode laser with excitation wavelength nm (close to absorption maximum of mbb-gsh adduct, nm) and measuring the emission of thiol mbbderivatives at nm. the method was used for monitoring the changes in the reduced gsh content in white wines during alcoholic fermentation and barrel aging. the detection limit for gsh was nmol/l, which is much lower than its average concentration in must and wine. mbb derivatization has been employed also for cze determination of oligo-and polypeptidic phytochelatins or class iii methallothioneins, and their precursors (cys, g-glu-cys, gsh), in the extracts from marine microalgae [ ] . the separation of thiol peptides and phytochelatins was improved when mm phosphate bge, ph . , was modified with . % v/v methanol. the lod of gsh was . mm with uv-detection at nm. two chip-based immunoaffinity ce systems have been applied to rapid concentration measurement of inflammatory neuropeptides in tissue fluids of patients with neuropeptide-associated muscle pain [ ] . one chip was designed to perform electrokinetic flow immunoassay while the other utilized an immunoaffinity port, containing an array of immobilized antibodies, to capture the analytes of interest. from the two systems the immunoaffinity capture system was found to be superior. using this system, twelve different inflammation-associated mediators could be determined in approximately min as compared to min when using the flow immunoassay chip. with the expanding array of commercially available antibodies, this chipbased system can be applied to a wide variety of different analytes. native and derivatized neuropeptides containing d-amino acids in individual neurons from the marine mollusk, aplysia californica, were characterized by ce with uv-absorption and lif detection and maldi-ms [ ] . the combination of peptide derivatization by fitc and fluorescamine followed by ce separation with lif detection proved to be well-suited for single cell analysis due to its ability to analyze peptides in such small sample volumes. competitive immunoassay using ce with lif detection was developed for monitoring of met-enkephalin [ ] . the method is based on competitive reaction between me and fluorescein-derivatized me (me-f) with anti-me antibody, ce separation of the me-antibody bound and free me-f, followed by lif detection of the fluorescent species. the assay specificity, sensitivity, and accuracy were excellent, allowing determination of me in the normal and cancer patients plasma on low ng/ml level. cze in sodium phosphate bge, ph . - . , with uvabsorption detection has been developed for baseline separation of ten enkephalin-related peptides [ ] . the method was used to analyze human csf samples spiked with these peptides after the samples were pretreated by spme, and the recovery was found to be in the range . - . %. the concentration lods of these peptides were in the range . - . mg/ml. cmekc method with mm borate/ mm phosphate, ph . , bge containing mm sds micellar pseudophase and % v/v methanol as organic solvent modifier, was elaborated for separation of zinc bacitracin and nystatin and applied for determination of these antibiotics as additives in animal feedstuff [ ] . the other applications of ce techniques to peptide analysis in complex matrices include determination of polyglutamyl -methyltetrahydrofolate forms in citrus products [ ] , analysis of microcystins, a group of hepatotoxic heptapeptides produced by various genera of cyanobacteria in contaminated drinking or recreational waters, by cec with two types of rp monolithic columns (c and c ) [ ] and analysis of para-k-casein and related peptides as indicators of milk proteolysis and ripening times of ewe's milk cheese [ ] . in addition to analysis and characterization of "static" peptide preparations, ce and cec are capable to monitor also the dynamic changes of peptide samples, such as their chemical and enzymatical reactions and modifications (e.g., oxidation, reduction, deamidation, hydrolysis, racemization), and physical changes -aggregation, denaturation, and folding/unfolding processes. hydrolysis, isomerization and enantiomerization of two aspartyl tripeptides, with isomeric amino acid sequences, gly-asp-phe-nh and phe-asp-gly-nh , incubated at c in acidic (ph ) and basic (ph ) solutions, was in detail studied by validated ce methods [ ] . most of the degradation products, including those arising from isomerization and enantiomerization of the asp residues were ce separated in mm sodium phosphate bge, ph . , see fig. . resolution of comigrating isomers was achieved by addition of cd-based chiral selectors into the bge. for tripeptide derivatives the assays were linear in the range of . - . mmol/l, for some dipeptides and amino acids the linear range was narrower due to their lower uv-absorption. the lods were in the range . - . mmol/l. at ph , the degradation of peptides proceeded via c-terminal deamidation and peptide backbone hydrolysis, whereas isomerization and enatiomerization were observed in combination with deamidation at ph . cleavage of peptides and proteins by a new method utilizing light-generated radicals from titanium dioxide was monitored by ce in mm acetate bge, ph . , with uv detection at nm [ ] . reproducible products patterns (electropherograms), consistent with cleavage of peptide bond at proline for angiotensin i, lys-bradykinin and myoglobin, were obtained, demonstrating that the cleavage procedure is rapid, specific and reproducible. monitoring and analytical characterization of degradation of a pseudopeptide composed of esterified tyrosine and lysine linked by urea bonds was carried out by ce hyphenated with it-tof-ms analyzer [ ] . several degradation species have been identified and a kinetic analysis of the variation of their concentration with time was obtained. several ce applications are dealing with enzymatic conversions of peptides, to study some details of these processes and/or activity of enzymes and kinetics of their acting on peptides and proteins and other compounds using the so called electrophoresis-mediated microanalysis (emma) [ , ] . two ce modes, cze and cmekc, were applied to the screening of combinatorial peptide libraries for their inhibitory activites to botulinum neurotoxin serotype a, a proteolytic enzyme that induces muscle paralysis [ ] . the peptide products in this enzyme assay were labeled with -( -carboxy-benzoyl)- -quinoline-carboxaldehyde (cbqca) and separated by cze or mekc with lif detection (ar ion laser, excitation/emission at / nm). the products were completely separated within min (cze) or min (mekc) as compared to their incomplete separation within h by hplc. a new cze method has been developed for the monitoring of glutathione s-transferase detoxification activity toward styrene oxide [ ] . the enzymatic reaction was carried out directly in a thermostatted autosampler vial and the formation of conjugates between gsh and syrene oxide was monitored by sequential mekc runs. the determinations were performed in mm phosphate/ mm tetraborate bge, ph . , with mm sds micellar pseudophase, with uv-detection at nm. monitoring of physical changes is represented by the application of cze to the studies of folding, unfolding, refolding, and conformational changes of proteins and polypeptides [ ] . ce and cec techniques are emloyed also in the area of peptide/protein characterization by their amino acid composition and sequence of amino acid analysis. survey of ce applications for amino acids analysis, including amino acid analysis of complete peptide hydrolysate, can be found in the recent review [ ] . different peptide sequencing platforms consisting of ce separation method coupled on-line or off-line to different ms/ms devices, such as maldi-tof-tof, esi-it, esi-tof, and ft-icr, were compared with a conclusion that each of these techniques has its own pros and cons [ ] . a computer-aided method has been developed to propose the amino acid composition and sequences of small peptides (m r , ) present in a complex vegetable protein hydrolysate [ ] . the method is based on predictive models for peptide hydrophobicity and charge/mass ratio. the former model is related to rp-hplc-ms analysis and the latter model is related to ce-ms analysis of the protein hydrolysate. the developed program determines the possible amino acid combinations as a function of the mass determination of the data provided by the model. in the hydrolysate of rapeseed proteins by alcalase the amino acid composition and sequence has been determined using the databank sequences of native proteins. amino acid sequences of alamethicins f , -residue peptaibol peptides isolated from the culture broth of the mold t. viride, forming voltage-dependent ion channels in bilayer lipid membranes and exhibiting antibiotic activities, have been determined by nace (in . mm ammonium formate bge in meoh) coupled to esi-it-ms and esi-tof-ms [ ] . ms/ms esi-it-ms was used for elucidation of the amino acid sequence based on the fragmentation pattern of the selected parent ions. an improved amino acid sequence coverage of complex peptide and protein mixtures, e.g., tryptic digests of a sixprotein mixture with an average m r around and a mixture of e. coli ribosomal proteins, was achieved by a new technique, (ce-ms/ms)(n) [ ] . in this approach multiple ce-ms/ms subanalyses (injections followed by analyses) are performed and experimental variables, such as bge composition and temperature, are manipulated during each ce-ms/ms subanalysis, utilizing the advantage of small sample volume (, nl) and short analysis time (ca. min) of ce. ms/ms was used to confirm amino acid sequences of tryptic peptides of bovine a -glycoprotein isolated by hplc and followed by ce-ms/ms analysis [ ] . high efficiency and high resolving power make ce and cec very useful methods for peptide mapping, i.e., separation of peptide fragments generated by specific chemical and/or enzymatic cleavage of proteins and polypeptides. peptide mapping serves as an important tool for protein identification, for sequence determination of internal parts of polypeptide chains, for monitoring of post-translational modification and structure elucidation of proteins. in addition, peptide maps can be obtained also as patterns obtained by separation of peptides present in complex biological fluids, such as serum, plasma, or csf. due to the high-complexity of peptide maps, namely of large proteins and biofluids, usually multidimensional separations, -de, de-ms, hplc-cze, hplc-ms, cze-ms, and hplc-cze-ms, are necessary for complete resolution of all peptides present in these mixtures (see section . ). monitoring of tryptic digestion of b-lactoglobulin a and b variants in a microscale by cze allowed developing optimized experimental conditions for specific hydrolysis of these proteins (enzyme/substrate ratio / , incubation in mm ammonium bicarbonate buffer, ph , at c for h) [ ] . highly reproducible and highly resolved peptide maps were obtained in bge consisting of mm formic acid, ph . ; they revealed the region where the aberrant peptides of b-lactoglobulin variants may be located. the highly selective separation of tryptic bsa peptides was achieved by a partial filling mekc with a mixed micelle system composed of a zwitterionic surfactant, mm - (n,n-dimethylhexadecylammonium) propanesulfonate, and a nonionic surfactant, . % m/v brij , in an optimized bge, mm ammonium-formate, ph . [ ] . some therapeutic (glyco)proteins and peptides have been characterized by ce-esi-it-ms, ce-uv, and ce-lif peptide mapping as a complement of rp-hplc-ms peptide mapping using endoproteinase lys-c for peptide/protein digestion [ ] . tryptic digests of cytochrome c were analyzed by ce-esi-it-ms system with simple on-line peptide preconcentration by dynamic ph-junction [ ] . in some advanced systems, ce and cec peptide mapping is on-line connected with previous protein digestion by enzymes (trypsin, pepsin) immobilized in microreactors directly coupled to separation capillary or to esi-ms [ , ] . in these arrangements, the advantages of application of immobilized enzymes for peptide mapping, high stability, and high activity of the enzyme and noncontamination of protein peptide maps by enzyme fragments due to the suppressed autolysis, have been confirmed. chiral compounds applied as drugs, food additives, and agrochemicals represent classes of compounds with high economical and scientific potential. hence, the separation of enantiomers is of paramount importance. due to the high efficiency, resolution power, speed, and miniaturization, ce and cec techniques have become very powerful and frequently used methods for chiral analysis and stereoisomer separation [ ] including chiral and stereoselective separations of peptides as demonstrated in the reviews [ , ] . with respect to the comprehensive survey of peptide stereoseparations in these reviews, only few representative examples of peptide stereoseparations are briefly mentioned in this section. the enantiomeric and diastereomeric ce separations of peptides were systematically studied in the group of scriba. enantiomers, diastereomers, and positional isomers of peptides originated from the degradation of aspartyl tripeptides, gly-asp-phe-nh , and phe-asp-gly-nh , were separated in acidic phosphate or formate-based bges with carboxymethyl-b-cd or sulfated b-cd as chiral selectors [ ] , see fig. . separation of the diastereomers of phosphinic pseudopeptides, i.e., peptides with one peptide bond substituted by phosphinic acid moiety, -p(o)oh-ch -, derived from the structure n-ac-l-val-l,d-alac(p(o)oh-ch )-l,d-leu-l-his-nh , has been investigated in achiral bges within a broad ph range, . - . [ ] . the best resolution was achieved in the acid ph region around the pk a values of the central phosphinic acid group of the pseudopeptides but successful separation of some diastereomers was achieved also in neutral and alkaline bges. peptides are not only the subject of ce chiral separations but they are used also as chiral selectors for separation of other classes of enantiomeric compounds. most important representatives of peptide chiral selectors are macrocyclic glycopeptides, vancomycin, ristocetin, and teicoplanin, used for a broad class of chiral separations both in electromigration and chromatographic techniques [ ] . these selectors are mostly used in the countercurrent, partial filling mode, where the solutes reach the detection cell window after the chiral selector has been displaced from the window region, minimizing the background absorbance from the chiral selector and improving sensitivity but they can be also used as immobilized chiral stationary phase in cec. new peptide chiral selectors are developed using the combinatorial peptide library approach [ ] . the application of ce and cec for preparative separations of peptides is much less compared to analytical separations owing to: (i) inherently low preparative capacity of the small id capillary columns, and (ii) more complicated adaptation of analytical capillary setup to preparative one than in chromatographic techniques. the latter problem, caused by the dipping of both capillary ends into the bge in the electrode vessels through which the electric field is applied over the capillary, has been solved by special modifications of ce systems. these systems and several procedures developed for fraction collections from the capillary have been described in the earlier review [ ] and book chapter [ ] . hence, ce and cec techniques are used for preparative purposes mostly in an indirect way, via the analysis of peptide fractions obtained by other methods (preparative chromatography, free-flow electrophoresis) and for evaluation of the efficiency of the separation method used for peptide purification. direct application is limited to microscale preparation. in commercial devices the autosamplers are used as fraction collectors and the electroelution is sometimes speeded up or completely replaced by hydrodynamic flow introduced at the inlet end of the capillary. such system has been used, e.g., for micropreparative fractionation of tryptic fragments of b-lactoglobulin a and b [ ] . peptides in collected fractions are later on concentrated by evaporation of the solvent and off-line characterized by (maldi) ms or by amino acid and sequence analysis, see section . . . for the continuous fraction collection in ce it is necessary to use special designs of the capillary outlet for completing the electrical circuit. this is achieved, e.g., by coaxial sheath liquid interface transporting the sample components leaving the exit of the capillary directly on a matrix-coated maldi target [ , ] . the problem of limited preparative capacity of ce (usually less than mg per run) can be partially solved by increasing the inner diameter of the capillary (compromising the separation efficiency), by repetitive fraction collection or by the use of multicapillary systems. multiple separations in a single narrow bore capillary and pooling of the fractions with the same mobility are suggested for isolation of peptides in the amounts sufficient for sequence analysis. principle solution of the preparative capacity problem is to convert analytical capillary separations to the continous free-flow arrangement on the basis of the earlier developed model of the correlation between capillary and free-flow electrophoresis [ , ] . free-flow electrophoresis, particularly the ff-ief mode has been used for prefractionation of complex peptide/protein mixtures in proteomic studies of cells [ ] [ ] [ ] and body fluids, such as human plasma [ ] and human saliva [ ] . ce and cec are not only high-efficient and high-sensitive analytical techniques but they are also more and more utilized as physicochemical methods, capable to provide important physicochemical characteristics of separated analytes, including peptides and proteins, such as effective and absolute (limiting) electrophoretic mobilities, effective charges, pi, m r , stokes radii, pk a of ionogenic groups, diffusion coefficients, and association (dissociation, binding) constants of peptide complexes [ , ] . in addition, ce and cec can be used also for monitoring of the physicochemical processes, e.g., conformation changes during unfolding/ folding processes and velocity of chemical reactions and physical changes. cze in sieving media can provide data on the size of separated analytes. m r of polypeptides and proteins can be estimated from cze of their complexes with sds (capillary version of sds-page) [ , ] . stokes radii and effective (net) charges of peptides can be assessed from effective mobilities measured by cze and from the different relations between peptide mobility and their charge-to-size ratio. due to the approximative character of all models correlating peptide mobility with their charge/size ratio the calculated values of charge and size (m r ) represent also only approximative characteristics. combination of cze measurement of electrophoretic mobilities of protein/polypeptide charge ladders and classical electrophoresis theory has been suggested to provide data for estimation of the effective (net) charge and size of larger (poly)peptides and proteins [ ] . net charge, hydrodynamic size, and shape of peptides were explored through cze measured effective mobilities and have been explored using the linderstrøm-lang electrophoresis model and its perturbed version [ ] . another important peptide/protein parameter related to charge, pi, can be determined by cief [ , ] , but also in this case one should have in mind that the determined pi value is dependent on the composition of the ampholytes and other experimental conditions used. diffusion coefficients of peptides can be estimated from their electrodriven motion in the capillaries and chip channels [ ] . effective and ionic mobilities of phosphinic pseudopeptides, peptide isosteres with one peptide bond substituted by a phosphinic acid moiety -p(o)oh-ch -, and pk a of this moiety and other ionogenic groups (c-terminal-, g-gluand b-asp-carboxyl, n-terminal-and e-lys amino, his-imidazolyl) have been determined from the precise measurements of the ph dependence of effective mobilities within a broad ph range . - . in bges with constant ionic strength ( mm) [ ] . effective mobilities, corrected to standard temperature c, were subjected to nonlinear regression analysis and the obtained apparent pk a values were recalculated to thermodynamic pk a values by extrapolation to zero ionic strength according to extended debye-hückel model. similar measurements of the dependence of effective mobilities on ph within a broad ph range - provided pk a values of ionogenic groups in peptide hormones [ ] and opioid peptides [ ] . in a comprehensive study, the effective mobilities and pk a values of a series of oligopeptides, oligoglycines, oligo(lalanines), and oligo(l-valines) with a number of residues up to ten, have been determined by cze in bges within a broad ph scale, . - . , at two ionic strengths ( and mm) and at temperatures from to c [ ] . for each peptide family, the pk a values were modeled as a function of the number of residues, temperature, and ionic strength. using this broad set of experimental data a semiempirical model was developed allowing to predict pk a values for any oligopeptide composed of amino acids with neutral lateral chains. the input parameters of this model are only the number of residues and the pk a of terminal amino acids in their free form. the model can predict the peptide pk a values at a given ionic strength and temperature and can also be used for selection of the optimum ph for the separation of mixtures of peptides the pk a of which are known or could be estimated. from the cze measurements of the dependence of effective mobilities of peptides on the charge/size ratio the probable secondary structure of similar peptides was determined, e.g., the random coil structure was predicted for peptide hormones [ ] , opioid peptides [ ] , and gnrhs [ ] . the electrophoretic mobilities of a series of dipeptides determined by cze allowed estimation of their transdermal iontophoretic mobility and to evaluate their suitability for iontophoretic delivery [ ] . cae is now widely used for determination of association or dissociation constants of peptide complexes with different types of both low-and high-molecular-mass ligands, for review see [ , ] . large series of studies has been performed to estimate receptor-ligand interactions using the system consisting of glycopeptide macrocyclic antibiotics (vancomycin, teicoplanin, ristocetin, and their derivatives) from s. orientalis and free or derivatized dipeptides d-ala-d-ala [ - , , ] . new methods, fluorescence anisotropy ce (face) and affinity probe ce (apce) with lif detection, have been developed for quantitative analysis of peptide-protein interactions, particularly for the interactions between the src homology domain of protein sh -bb and tyrosine-phosphorylated peptide corresponding to the binding sequence of jak [ ] . the separation time of s, achieved by high electric field strength of v/cm in a cm long capillary, was on the same time scale as complex dissociation and allowed determination of both dissociation constant, (k d = nm) and dissociation rate (k off = . . / s corresponding to a half-life . s). with lod - nm both apce and face were found to be suitable for investigation of binding interactions with rapid kinetics in a microscale. the assay was extended to a multipexed system involving the separation of three sh domain proteins, src, sh -bb and fyn [ ] . the interaction between c-like proteinase, key target for drug against severe acute respiratory syndrom (sars) coronavirus, and its octapeptide inhibitor have been studied by cae [ ] . from the mobility shift assay of the inhibitor, i.e., from the changes of its mobility in the bge with increasing concentrations of the proteinase, the binding constants k b of . /m at c and . /m c were determined. analysis of the thermodynamic parameters, changes of gibbs free energy, enthalpy, and entropy, obtained from temperature dependence of k b , indicated that hydrophobic interactions might play a major role in the binding process, along with electrostatic attractions. cae mobility shift assay has been found to be an efficient and sensitive method also for both qualitative and quantitative studies of the interactions between the peptides and oligo-and polynucleotides, it has been employed, e.g., for selection and identification of human gnrh promoter binding peptides using - /- region of hgnrh promoter and a phage-displayed peptide library [ ] and for selection of aptamers with high affinity to neuropeptide y from random-sequence nucleic acid libraries [ ] . as demonstrated in the above sections, ce and cec possess an extremely high potential for analysis, preparation, and characterization of peptides. nowadays, these techniques are regarded as accomplished complements and/or counterparts of other high-performance separation methods, particularly hplc. applications of ce and cec methods to analysis, isolation, and characterization of peptides are further broadening; ce and cec are being utilized not only as highly efficient and highly sensitive analytical techniques, capable to determine femtomole to zeptomole amounts of peptides in nano-to picoliter sample volumes of complex biological matrices, but also as valuable physicochemical methods, which can provide important physicochemical and biochemical characteristics of peptides, such as their effective mobilities and charges, m r 's, acidity constants of their ionogenic groups, diffusion coefficients, association constants of their complexes, conformation of their molecules, and rate constants of their reactions. peptides represent an extremely numerous class of vitally important biomolecules. many of them and a lot of their functions have been recognized but even more have to be revealed and elucidated. for a more detailed understanding of living processes, a comprehensive investigation of a whole peptide set of a cell, organ, or organism (peptidome), i.e., peptidomics, has to be performed. also the structure and functions of proteins in proteomic studies are mostly identified via their peptide fragments. hence, undoubtedly, the analysis, purification, and characterization of peptides will belong to the most challenging tasks of ce and cec methods also in the future. as concerns these techniques themselves, their development will proceed to implementation on microfluidic devices and integration into mtas, on-line coupled with high-sensitive lif or ms detection. multidimensional separation systems based on hyphenation of ce, cec, lc, and ms techniques will be necessary for analysis of complex peptide and protein mixtures in peptidomics and proteomics. the author has declared no conflict of interest. electrophoresis capillary electrophoresis, theory and practice electrophoresis electrophoresis electrophoresis electrophoresis electrophoresis electrophoresis electrophoresis analytical and preparative separation methods of biomacromolecules key: cord- -npijn co authors: esfandiyari, reza; halabian, raheleh; behzadi, elham; sedighian, hamid; jafari, ramezan; imani fooladi, abbas ali title: performance evaluation of antimicrobial peptide ll- and hepcidin and β-defensin- secreted by mesenchymal stem cells date: - - journal: heliyon doi: . /j.heliyon. .e sha: doc_id: cord_uid: npijn co peptides are secreted by different cell types and are trendy therapeutic agents that have attracted attention for the treatment of several diseases such as infections. antimicrobial peptides exert various mechanisms such as changing cell membrane permeability which leads to inhibition or death of bacterial cells. mesenchymal stem cells (mscs) are key to produce antimicrobial peptides and to inhibit the growth of pathogens. these cells have been shown to be capable of producing antimicrobial peptides upon exposure to different bacteria. as a result, antimicrobial peptides can be considered as novel agents for the treatment of infectious diseases. the purpose of this review was to investigate the targets and mechanisms of antimicrobial peptides secreted by mscs. antibiotics are used to treat bacterial infections through either inhibiting or killing the target bacteria. the first antibiotic was discovered by alexander fleming in , called penicillin and saved the lives of millions of people. however, the discovery of penicillin as a modern medication was preceded by the management of microbial infections in egypt, greece and ancient china [ ] . shortly after the discovery of penicillin, the bacterial resistance to this antibiotic became one of the challenges in the treatment of bacterial infections. therefore, new generation of beta-lactam antibiotics was developed to overcome antibiotic resistance and to treat bacterial infections. however, the first case of methicillin-resistant staphylococcus aureus (mrsa) was reported in england in [ , ] . the production and discovery of new antibiotics continue to this day and new antibiotics are marketed to counteract the drug resistance problem. yet, the point should be raised that one day antibiotics can no longer affect bacteria and will no longer be able to control bacterial infections. consequently, in recent years, researchers have devised other means to treat bacterial infections. one of such approaches is the use of antimicrobial peptides (amps) or "peptide antibiotics" to kill pathogenic bacteria and to treat bacterial infections [ ] . over the past decades, antimicrobial peptides (peptide antibiotics) have been shown to be effective in innate immunity of various species, such as plants, invertebrates and vertebrates. the intrinsic immune system is the first line of defense against the attack of microorganisms, among which the antimicrobial peptide molecules are the most important ones. the cathelicidin family is important antimicrobial agents in mammals [ , ] . these peptides are mainly stored in lysosomes of macrophages (mq) and polymorphonuclear neutrophils (pmns) [ ] . cathelicidins have been isolated from many cell types including neutrophils to coordinate the immune system, but have been found in other immune cells such as epithelial cells and macrophages and have been shown to combat against bacteria, viruses and fungi. cathelicidins have a variety of sizes ( - amino acids) and also have a wide range of structures [ ] . the molecular mechanism of antimicrobial peptides has been investigated [ ] . stem cells have been the focus of research because they have shown good potential in the field of therapy [ ] . one of the features of the stem cells referred to in this review is antimicrobial activity of mesenchymal stem cells that perform this action through antimicrobial peptides such as ll- , hepcidin and β-defensin- [ ] . the purpose of this study is to briefly review mesenchymal stem cells and antimicrobial peptides and how these peptides function. in recent years, stem cells have been widely used in the treatment of many diseases. one of the most important stem cells is mesenchymal stem cells (mscs) that have been shown to play a role in regulating the immune system and suppressing deleterious properties. mscs have the ability to differentiate into mesenchymal tissues such as cartilage, bone, muscle and fat. mscs have been obtained from bone marrow, umbilical cord, blood, placenta, skeletal muscle and adipose tissue. recent studies have found that mscs play an important role in the treatment of diseases, including infections, by producing antimicrobial peptides [ , , ] . cathelicidin is a carrier that has a wide range of functional molecules (i.e. cysteine or non-cysteine). the presence of this peptide has been proven in cattle, rabbits, pigs and humans [ ] . due to the unique characteristics of antimicrobial peptides, these peptides are one of the main candidates in the treatment of bacterial diseases and are effective on antibiotic resistant strains and even cancer cells. these properties include rapid killing and a wide range of activity that perform antimicrobial action by pore-forming the cell membrane [ ] . but these peptides can also be toxic to the cells of the body, so using peptides with a wide range of lethality and low side effects can help cure bacterial infections [ ] . in , agerberth et al [ ] based on the protected section of cathepsin, derived human bone marrow cdna clones from an unspecified antibacterial peptide named fa-ll- . the peptide constitutes of monocyte [ ] amino acids whose n-terminal is fall and the name of the peptide was coined for fall. the helical structure of this peptide was investigated in a saline environment containing vitamin e upon synthesis and antibacterial activity was investigated [ ] . the peptide is specifically secreted in the secondary granules of neutrophils. it is also produced by many types of cells, including macrophages, natural killer (nk) cells, epithelial cells of the skin, airways, eyes and intestinal tract. also, the expression of the peptide ll- is controlled by inflammatory pathways, similar to the pathway of vitamin d [ ] . in addition to antimicrobial activity, this peptide has immunomodulatory roles. for example, exposure to μg/ml of ll- peptide during a monocyte-macrophage differentiation leads to a positive inflammatory response, resulting in a decrease in the level of interleukin and an adjustment of p . in addition, the peptide ll- leads to the phenotype m , which suggests that this peptide has an important role in the development of macrophages and cytokine production [ ] . other chemical properties of ll- is as follows: migration of neutrophils and eosinophils through the formyl-peptide receptor; transactivation of the epidermis growth factors by the peptide causes the migration of keratinocytes, which results in wound healing; mcp- ccl- is a monocyte extraction factor that is secreted by ll- after stimulation. in addition, transforming growth factor beta (tgfβ) released from the epithelial cells of the intestine after exposure to ll- has an effect on the migration of epithelial cells and improved response. it is concluded that the peptide ll- is spread at the site of the infection, causing inflammatory response and wound healing [ , , ] . ll- peptide has different activities, all of which lead to antimicrobial activity. these include regeneration and angiogenesis, which is done by the formyl-peptide receptor-like expressed on the endothelial cells [ ] . chemotaxis is also one of the activities of this peptide, which causes the migration of mast cells to the environment [ ] . in addition, degranulation of mast cells and the release of inflammatory mediators takes place by the ll- peptide [ ] . ll- peptide, on the other hand, induces many cytokines and induces the production of antibodies [ ] . ll- peptide from primary human keratinocytes and human keratinocytes (hacat) cells protect from apoptosis [ ] . the results have shown that fluid membrane-associated peptides increases the plasma membrane of the subcutaneous glandular cells [ ] . this peptide also considered as an endotoxin, which inhibits the responses of inflammatory proteins to the bacterial lipopolysaccharides (lps) in human cells (fig. ) [ ]. this peptide performs its actions by connecting to cell receptors ( table ) . as previously mentioned, ll- is actively involved in physiological responses to eukaryotic cells, which is essentially an antimicrobial peptide. this peptide is effective in people whose immune system is low, so that through transactivation of egfr and angiogenesis by fprl receptor and chemotaxis causes cell migration [ ] . in addition, the expression of transgenic ll- leads to a significant increase in proliferation of corpuscular cells in hacat and hek [ ] . with the presence of lymph node metastases in estrogen-receptor positive or er positive cancer, clinical trials have shown an increase in erb-b receptor tyrosine kinase (erbb ) signaling, indicating an increase in peptide ll- activity, which plays an important role in the treatment of cancer [ ] . this peptide also interacts with p x purinoceptor, which is expressed mainly in monocytes, macrophages and dendritic cells (dcs) and stimulates and releases cytokine il- β in human gingival fibroblasts (fig. ) [ ]). another antimicrobial peptide produced by mscs is called hepcidin, which plays an important role in the clearance of pathogens. this antimicrobial peptide is rich in cysteine, the precursor of which is secreted by the liver (fig. ) . hepcidin is present in three forms of prehormone ( amino acids), prohormone ( amino acids) and hormones ( amino acids) [ ] . this peptide was discovered in and was called leap , but after a while due to presence in the liver, the peptide was named "hepcidin". this peptide is effective on a wide range of fungi, bacteria and viruses. one of the most important effects of hepcidin in the body is the regulation of iron hemostasis. this peptide prevents iron absorption from the small intestine and releases iron from reticuloendothelial cells. in infectious diseases, macrophages and bacteria compete to absorb iron [ ] . macrophages interfere with the absorption of iron by bacteria. eventually, the pathogen does not grow and replenish. factors that cause hepcidin production are increased in bone marrow and anemia. other factors that increase the production of hepcidin are iron accumulation and inflammation [ ] . hepcidin is effective on iron transfer from macrophages. in the presence of hepcidin, ferritin is transmitted into the macrophage and is destroyed by lysosomes, resulting in storage of iron inside the cell. in low concentrations of hepcidin, ferritin is present in the cell membrane, allowing the release of iron. after leaving the cell, iron oxide is rapidly oxidized by ceruloplasmin, a copper-rich ferroxidase and converted into ferric iron and then bound to transferrin [ ] . hepcidin is bound to plasma alpha- macroglobulin (alpha m). evidence suggests that other cells may express the hepcidin mrna at a much lower level than the hepatocytes; the biological significance of the extra hepatic production of hepcidin remains uncertain. plasma hepcidin is freely treated through glomeruli and in animals with normal kidney activity it quickly passes through the urine. in addition, a part of hepcidin is cleansed through degradation along with ferritin [ ] (fig. ). cysteine-rich cationic proteins are found in vertebrates, invertebrates and plants, which range from to amino acids [ ] . the role of these peptides is to defend the host against bacteria, fungi and viruses. these peptides play a role in destroying the bacterial cell membrane. defensins were discovered for the first time in . the basic genes produced by these peptides are highly polymorphic. generally, defensins are classified in three main groups of alpha, beta and theta, each of which has different genetic symbols and is produced from different cells [ ] , as shown in table . in immature individuals, defensins (present in breast milk) play a very important role in protecting the disease due to the weak immune system. the defensin is produced by epithelial cells and leukocytes, as indicated in table . alpha defensin exists only in mammals, while beta defensin exists in many vertebrates, invertebrates and plants, but theta table defensin types [ , , ] . defensin- alpha defensin- β -defensin β β-defensin secreted by leukocytes and epithelial cells of many kinds. have been found only in the leukocytes of the rhesus macaque and the olive baboon, papio anubis, being vestigial in humans and other primates. fig. . mscs produce microbial peptides including hepcidin, ll- and β-defensin to fight against bacteria. as shown in the figure above, the antimicrobial peptide ll- is effective against staphylococcus aureus and escherichia coli bacteria, while the effect of β-defensin peptide on the bacteria of escherichia coli has been proven [ ] . defensin has been found in several species of ancient monkeys, but not in humans and other primates. the important point is that the mrna manages to denote the theta defensin, but the mutations cause the end codon to stop peptide production. one of the key characteristics of defensins is their antimicrobial activity, which is active against grampositive and gram-negative bacteria, fungi and viruses [ ] . in addition to antimicrobial activity, defensin exerts other functions that includes chemical absorption for monocytes, cytokines production by monocytes and epithelial cells, angiogenesis activity and response to hormones. antimicrobial effect on gram-positives is different from gram-negative bacteria. the reason is that, first, the cell wall structure of gram-positive bacteria is different from gram-negatives (the cell wall of gram-negative bacteria is rich in lipopolysaccharide (lps), which has a negative charge, while in gram-positive bacteria the cell wall have teichoic acids (ta) with less negative charge. secondly, the peptidoglycan layer is thick in gram-positive bacteria and the formation of pores by defensin on the surface of the gram-positive cells requires more time. therefore, defensin has weaker anti-microbial activity in gram-positive bacteria [ ] . defensin functions on the bacterial cells via the l-arginine cationic chain that are attached by electrostatic absorption to the membrane's anionic structure, then defensin is introduced into the membrane by using the electromotive force of the membrane, creating channels inside the membrane. ultimately, materials that are surrounded by membranes are leaked outside or inside the cell and causes bacterial cell lysis [ ] . attempts to design novel treatment strategies as alternatives for the current treatment of diseases such as sepsis, bacteremia, bloodstream infections and other bacterial infections have become a major challenge. one of the most important treatments used to kill pathogenic bacteria is the use of antibiotics, which has some disadvantages in addition to its benefits. antibiotic resistance is one of the biggest treatment problems with antibiotics. antibiotic resistance is on the rise and the number of resistant are constantly circulating in the communities and it can be said that it will not last long as antibiotics will no longer be effective against bacteria [ , ] . an emerging approach to treat infections is the use of antimicrobial peptides produced from various cells, including mscs. research has shown that mscs have antimicrobial properties and this property is related to the production of antimicrobial peptides (fig. ) [ ] . there are several barriers to using stem cells in treatment, such as the immune response to stem cells that can reduce the function of these cells, as well as their use may cause infection, poisoning, cancer and even death [ ] . as a result, it is better to use mesenchymal stem cells in treatment if the treatments that have been used so far are no longer effective. on the other hand, the use of mesenchymal stem cells as a treatment has many benefits, one of which is the lack of damage that is usually caused by chemical treatments on the body. the use of mesenchymal stem cells in the treatment of bacterial infections can reduce the spread of antibiotic resistance [ ] . in conclusion, mscs are very capable of eliminating bacterial infections. the use of these cells and their antimicrobial products can be a potential alternative to current treatments, while bacterial resistance can be avoided. author contribution statement reza esfandiyari, raheleh halabian, elham behzadi, hamid sedighian, ramezan jafari, abbas ali imani fooladi: conceived and designed the experiments; analyzed and interpreted the data; wrote the paper. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. a brief history of the antibiotic era: lessons learned and challenges for the future the antibiotic resistance crisis: part : causes and threats molecular mechanisms of antibiotic resistance antimicrobial peptides: an introduction antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide ll- the role of cathelicidins in the innate host defenses of mammals cathelicidins, multifunctional peptides of the innate immunity human defensins and ll- in mucosal immunity the human gene fall and processing of the cathelin precursor to the antibacterial peptide ll- in granulocytes human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and stargardt's macular dystrophy: follow-up of two open-label phase / studies antibacterial effect of mesenchymal stem cells against escherichia coli is mediated by secretion of beta-defensin- via toll-like receptor signalling concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing mesenchymal stem cells in tissue repair antimicrobial activity of mesenchymal stem cells: current status and new perspectives of antimicrobial peptide-based therapies purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides a novel tool against multiresistant bacterial pathogens: lipopeptide modification of the natural antimicrobial peptide ranalexin for enhanced antimicrobial activity and improved pharmacokinetics membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides fall- , a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis solution structure and peptide binding of the sh domain from human fyn ll- directs macrophage differentiation toward macrophages with a proinflammatory signature induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide ll- little peptide, big effects: the role of ll- in inflammation and autoimmune disease an angiogenic role for the human peptide antibiotic ll- /hcap- a cathelicidin family of human antibacterial peptide ll- induces mast cell chemotaxis the cathelicidin ll- activates human mast cells and is degraded by mast cell tryptase: counter-regulation by cxcl serum levels of ll- and inflammatory cytokines in plaque and guttate psoriasis the human antimicrobial peptide ll- suppresses apoptosis in keratinocytes modulation by ll- of the responses of salivary glands to purinergic agonists aldose reductase mediates the lipopolysaccharide-induced release of inflammatory mediators in raw . murine macrophages new development in studies of formyl-peptide receptors: critical roles in host defense curcumin inhibits proliferation of interleukin- -treated hacat cells the human cathelicidin antimicrobial peptide ll- and mimics are potential anticancer drugs ll , a human antimicrobial peptide with immunomodulatory properties α- antitrypsin binds preprohepcidin intracellularly and prohepcidin in the serum iron homeostasis and the inflammatory response iron homeostasis in host defence and inflammation intracellular iron transport and storage: from molecular mechanisms to health implications hepcidin bound to α -macroglobulin reduces ferroportin- expression and enhances its activity at reducing serum iron levels antimicrobial proteins in intestine and inflammatory bowel diseases comparative genomics and evolution of the alpha-defensin multigene family in primates increased levels of α-defensin (- , - and - ) in type diabetic patients with nephropathy defensins. handbook of biologically active peptides rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membranelytic peptides the tlr -par axis regulates bone marrow mesenchymal stromal cell survival and therapeutic capacity in experimental bacterial pneumonia review of antibiotic resistance in china and its environment mesenchymal stem cells and immunomodulation: current status and future prospects antimicrobial design of titanium surface that kill sessile bacteria but support stem cells adhesion hepcidin regulation: ironing out the details the authors declare no conflict of interest. no additional information is available for this paper. key: cord- - rntyccn authors: wang, ke‐ming; kumar, senthil; cheng, yi‐sheng; venkatagiri, shripathi; yang, ai‐hwa; yeh, kai‐wun title: characterization of inhibitory mechanism and antifungal activity between group‐ and group‐ phytocystatins from taro (colocasia esculenta) date: - - journal: febs j doi: . /j. - . . .x sha: doc_id: cord_uid: rntyccn tarocystatin from colocasia esculenta, a group‐ phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. it is composed of a highly conserved n‐terminal region, which is homological to group‐ cystatin, and a repetitive peptide at the c‐terminus. the purified recombinant proteins of tarocystatin, such as full‐length (fl), n‐terminus (nt) and c‐terminus (ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. kinetic analysis revealed that fl peptide exhibited mixed type inhibition (k (ia) = . μm and k (ib) = . μm) and nt peptide showed competitive inhibition (k (i) = . μm), whereas ct peptide possessed weak papain activation properties. a shift in the inhibitory pattern from competitive inhibition of nt peptide alone to mixed type inhibition of fl peptide implied that the ct peptide has an regulatory effect on the function of fl peptide. based on the inhibitory kinetics of fl (group‐ ) and nt (group‐ ) peptides on papain activity, an inhibitory mechanism of group‐ phytocystatins and a regulatory mechanism of extended ct peptide have each been proposed. by contrast, the antifungal activity of nt peptide appeared to be greater than that of fl peptide, and the ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. the results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase. tarocystatin from colocasia esculenta, a group- phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. it is composed of a highly conserved n-terminal region, which is homological to group- cystatin, and a repetitive peptide at the c-terminus. the purified recombinant proteins of tarocystatin, such as full-length (fl), n-terminus (nt) and c-terminus (ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. kinetic analysis revealed that fl peptide exhibited mixed type inhibition (k ia = . lm and k ib = . lm) and nt peptide showed competitive inhibition (k i = . lm), whereas ct peptide possessed weak papain activation properties. a shift in the inhibitory pattern from competitive inhibition of nt peptide alone to mixed type inhibition of fl peptide implied that the ct peptide has an regulatory effect on the function of fl peptide. based on the inhibitory kinetics of fl (group- ) and nt (group- ) peptides on papain activity, an inhibitory mechanism of group- phytocystatins and a regulatory mechanism of extended ct peptide have each been proposed. by contrast, the antifungal activity of nt peptide appeared to be greater than that of fl peptide, and the ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. the results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase. they are usually - kda in size and show high homology with chicken egg white cystatin [ ] . the group- phytocystatins are approximately or greater than kda, such as those found in cabbage [ ] , soybean [ ] , taro [ ] , sesame [ ] and strawberry [ ] . they have a highly conserved n-terminal region, which is similar to that in group- , and are tailed by a repetitive peptide at the c-terminus, in which variation is possibly caused by gene duplication [ ] . the third group of phytocystatins, group- , is found in potato [ ] and tomato [ ] , and includes an kda multi-cystatin with eight cystatin domains. phytocystatins show variable expression patterns during plant development and defense responses to biotic and abiotic stresses [ ] [ ] [ ] . although at least two functions have been assigned to phytocystatins, such as regulation of protein turnover and protection of plants against insects and pathogens [ ] , their physiological functions remain obscure. the taro, colocasia esculenta, is an important staple food of taiwan aborigines, and is widely cultivated in local mountainous farms. this crop, especially kaohsiung no. , is popular for its high productivity and lower susceptibility to pathogens. it might be expected that such taro corms display the characteristic mechanisms regulating protein turnover, as well as defense barriers towards pathogens. in a preliminary survey of proteinase inhibitors from taro tuber, copious amount of a cysteine proteinase inhibitor were discovered [ ] . recently, we isolated a group- phytocystatin from taro corms, named cecpi, and demonstrated its anti-papain activity as well as anti-fungal activity [ ] . in the alignment data, we also found that the group- phytocystatin is like a group- phytocystatin with the addition of a c-terminal extension. moreover, the c-terminal region of the group- phytocystatin shares a high consensus sequence among the discovered species [ ] . the c-terminal part is probably responsible for regulating inhibitory activity and target specificity. to obtain a better understanding of the structure and biochemical function of tarocystatin cecpi, we amplified separately the intact full length (fl), n-terminal region (nt) and c-terminal region (ct) peptides by pcr and studied their relationship by in-gel anti-papain activity, inhibitory patterns and anti-fungal activity. based on a comparative study of group- (nt peptide) and group- (fl peptide), we discuss the inhibitory mechanism of group- phytocystatins and their evolutionary significance. in addition, both the inhibitory characteristics of the 'noncanonical' binding mode of group- phytocystatins towards papain and the 'canonical' binding mode of group- phytocystatins are addressed. purification of recombinant proteins from escherichia coli and in-gel inhibitory activity assay the fl peptide comprises of amino acids, including amino acids of nt peptide and amino acids of ct peptide. expressed recombinant fl, nt and ct peptides were further purified from the e. coli and analyzed by . % sds ⁄ page. purified proteins of both fl and nt peptides showed two bands, each with the lower band corresponding to a kda glutathione s-transferase (gst) protein, with the upper band corresponding to kda for gst-fl and kda for gst-nt peptide fusion proteins (fig. a) . the ct peptide showed only one band corresponding to kda (gst-ct). the free recombinant proteins of the three peptides ( fig. b) were obtained by digesting off gst peptide and performing chromatography [ ] for further biochemical analysis. the inhibitory activity of recombinant proteins was assessed by an in-gel activity assay and can be visualized by the clear zone of hydrolysis (fig. c ). by contrast, increasing the concentration of recombinant ct peptide acting on papain confirmed that the ct peptide enhanced its capacity (fig. d ). a previous study showed that tarocystatin (i.e. fl peptide) has effective activity on hyphal growth inhibition against several phytopathogenic fungi [ ] . in an attempt to compare the antifungal effect of different peptides of tarocystatin, a bioassay on mycelial growth of sclerotium rolfsii was carried out. fl (group- ) and nt (group- ) peptides exhibited apparent antifungal activity at a concentration > . nm, but no antifungal activity was observed in the ct peptide bioassay ( fig. a) . it appeared that nt peptide (group- ) was more effective than the fl peptide (group- ) (fig. b) . although antifungal activity of phytocystatins from taro, strawberry and chestnut has been reported previously [ , , ] , the mechanism of inhibitory activity of phytocystatins against phytopathogenic fungi remains unclear. the presence of the ct peptide in the fl peptide appears to be the cause of the reduction in antifungal activity. the hyphal morphology was also observed under low and high magnification microscopy. the growth-retarded mycelium exhibited swelling, less branches and blunt tips at an nt peptide concentration of . nm (fig. c) , and displayed swelling, no branches, very short tips and fragmentation at a concentration of . nm. before inhibition analysis, the recombinant protein was purified by being passed through affinity columns and subsequently cleaved by thrombin and identified by sds ⁄ page analysis. electrophoresis of free recombinant protein of fl, nt and ct peptides, showed maximum purity (fig. b) . to determine the inhibition constant, n a -benzoyl-d,l-arginine b-naphthylamide hydrochloride (bana) was used as a substrate at a concentration range of - lm for the assay with equimolar ( nmol) papain and inhibitor concentrations (fig. a) . the ct peptide curve appeared above the control (ck), indicating that the ct peptide enhances the enzyme activity, which is consistent with the anti-papain activity determined by the in-gel assay (fig. c,d) . both fl and nt peptides could inhibit papain activity by % and %, respectively, whereas ct peptide activated papain by % (table ) . therefore, fl peptide exhibited mixed inhibition, nt peptide exhibited competitive inhibition and ct peptide exhibited allosteric activation (fig. b ). further verification of the inhibition characteristics was performed by repeating the experiment after making a slight modification, with bana at a concen-tration in the range - lm, as well as varying the inhibitor level in the assay. a lineweaver-burk plot of the reactions with varied inhibitor levels again showed competitive inhibition for nt peptide and mixed inhibition for fl peptide (fig. a,b) . thus, the presence of two inhibition types was confirmed. the inhibition constants (k i values) could be calculated from the apparent k m and v max changes ( table ). the k i value of nt peptide (group- ) inhibition on papain was found to be . · ) m. this value is very close to the k i of rice oc-i ( . · ) m) [ ] . in addition, comparison of inhibitory activity with other group- species showed that k i for nt peptide of tarocystatin is lower than those for rice oc-ii ( . · ) m) [ ] , job's tears cystatin ( . · ) m) [ ] and soybean cystatin l ( . · ) m) [ ] , but higher than those for sesame ( . · ) m) [ ] and maize cci ( . · ) m) [ ] . nt peptide inhibitory activity appears to be intermediate among the group- phytocystatin family. in mixed inhibition, the k i value is separated into k ia and k ib . k ia is described as the dissociation of inhibitor from enzyme, whereas k ib is for that between the inhibitor and enzyme-substrate complex. a prominent characteristic of mixed inhibition compared to competitive inhibition is that the mixed inhibitors bind to enzymes as well as enzyme-substrate complexes, but competitive inhibitors bind only enzymes. thus, the ct peptide of tarocystatin may be able to dock onto the papain structure when the active site is occupied by a substrate. furthermore, the occurrence of the k ib value is always tailed with an unknown regulatory effect, indicating that the ct peptide functions to alter the target protein conformation and prevent product formation. the nt peptide functions like the entire oc-i and confers tarocystatin with an affinity for the competing active site. the d structural model of tarocystatin was predicted to infer the interaction between group- tarocystatin and papain. the ct peptide sequence shares % identity and % similarity with taro nt ( - amino acids), as solved by nmr spectroscopy [ ] . although there was no established template for ct peptide d structure prediction, it shares % identity and % similarity to group- oc-i (fig. ). therefore, the ct peptide structure was predicted using secondary structure estimation and a folding pattern simulation program with pseudo-energy minimization. subsequently, the entire tarocystatin d structure was obtained by combining the structures of two segments. its conformation resembled an earphone comprising two solid masses and a linear structure (fig. ) . a highly structural similarity between the nt and ct peptides was found and, presumably, the ct peptide compete with the nt peptide for binding to the active site (fig. ) . however, the assay using varied concentrations in the range - lm of ct peptide to compete with the nt peptide at a concentration of . lm did not demonstrate that the ct peptide reduced the inhibitory capacity of the nt peptide (fig. a) . instead, it revealed that the ct peptide does not act competitively. to determine whether the connection between the nt and ct peptides is important for inhibitory capacity of the fl peptide, equal amounts of nt and ct peptides were mixed and the inhibitory capacity of the mixture was then compared with that of only the nt or fl peptides. the curve of the mixture of nt and ct peptides did not tend to that of the fl peptide in the retrieve test (fig. b) . the pattern of competitive inhibition against papain by the nt peptide of group- is consistent with the previous findings obtained for many other group- phytocystatins [ , ] , whereas the mixed type inhibition against papain by fl peptides of group- has not been reported to date. information about mixed inhibition by other phytocystatins is scarce. a similar inhibition model, noncompetitive inhibition, was reported in strawberry facpi- [ ] and in soybean l and r [ ] . of these, only facpi- belongs to the group- phytocystatins and demonstrates a strong inhibitory activity ( . · ) m). the facpi- amino acid sequence is highly homologous with tarocystatin, but its mechanism cannot show mixed inhibition. to unravel the mechanism, a detailed investigation of the d structural interaction between group- phytocystatins and papain is necessary. in the present study we are the first to show the inhibition difference between group- and group- phytocystatins, and to examine the importance of the extended c-terminal region (ct peptide of tarocystatin) with respect to interaction with anti-papain activity. in the analysis of the primary structure of tarocystatin, we found that the group- tarocystatin (fl peptide) is a group- phytocystatin (nt peptide) possessing an additional ct peptide. moreover, the ct peptide of the group- phytocystatin shares a high consensus sequence among the discovered species [ ] . both the fl and nt peptides exhibit a good inhibitory property on papain activity, whereas the ct peptide exhibited papain activation that was also evident in an in-gel inhibitory assay (fig. c) . the inhibition constant demonstrated that the fl peptide exhibited mixed inhibition, and the nt peptide exhibited competitive inhibition, suggesting a canonical binding mode as with many other group- phytocystatin species previously reported ( table ). the enhancement of the proteinase activity by % (table ) implicates that the interaction between papain and the ct peptide possesses refolding in the conformation change of the papain protein. the mixed type inhibition against papain by the fl peptide might be due to the presence of the ct peptide, which plays an activation role on papain when used alone. therefore, the mechanism of the ct peptide with respect to enhancing papain activity presumably involves allosterically binding adjacent to the active ⁄ substrate binding site and altering the papain conformation to be more accessible for the substrate, which is defined as the 'noncanonical' binding mode, where these inhibitory characteristics are quite different from the 'canonical' binding mode of group- phytocystatins, as noted previously [ ] . this change may also shift the orientation of the nt peptide to bind with competitive inhibition and result in blocking the substrate from the approaching catalytic site. when the ct peptide was bound to papain and linked with the nt peptide, substrates still had the chance to bind to the active cleft. nevertheless, the nt peptide was so close to active cleft that allows nt peptide binding prior to any approaching substrates. thus, the enhancing effect of the ct peptide was followed by an immediate binding of the nt peptide. in this case, substrates still could reach the active cleft and be trapped by some inner pulling force, but could not be fixed in the catalytic site that the nt peptide blocked. this mechanism was like a noncompetitive inhibition, where substrates could bind to the enzyme-inhibitor complex but not to be turned to products. however, if the nt peptide bound to papain before the ct peptide, tarocystatin would simply exhibit competitive inhibition. the alternative binding pattern strongly supports the idea that tarocystatin is a mixed type inhibitor, and provides evidence for the difference between group- and group- phytocystatins. phytocystatin has been known for its defense function against attack by insects and pathogens. these proteins have received much attention from researchers due to their potential utilization as bioinsectides in agrobiotechnology [ , ] . to extend our previous study on antifungal activity [ ] , a bioassay on mycelial growth of s. rolfsii was performed, and revealed that the fl and nt peptides exhibited apparent antifungal activity at a concentration above . nm, but no significant antifungal activity was observed for the ct peptide ( fig. a,b) . microscopic observations indicated that the nt peptide appeared to be stronger than fl peptide (fig. b) . because the ct peptide alone does not show any antifungal effect, this implies that the antifungal activity might be connected to the nt peptide conformation. a reduction of antifungal activity in vitro by fl peptide may be due to a molecular mass difference. it has been speculated that the fl peptide is larger than the nt peptide, making it more inefficient to diffuse inside hyphal cells. the true mechanism responsible for the antifungal activity of tarocystatin still requires further investigation. to date, the physiological significance of the ct peptide remains unknown. accumulating evidence shows that this repeated domain may originate from gene duplication and be exploited for other functions [ ] . recent evidence also demonstrated that carboxy terminus-extended phycys have the capacity to inhibit human legumanin peptide due to the presence of the conserved motif snsl and act as a bifunctional inhibitor [ ] . our findings focused on the ct peptide on cysteine protease, which may function with three roles: (a) to endow the n-terminal domain with more specificity and inhibition to papain; (b) to prevent the n-terminal domain from rapid digestion by endogenous or exogenous peptidase; and (c) to enrich its molecular size as an ideal storage protein. due to these beneficial characteristics, the ct peptide could be reserved under evolutionary selection. further studies, including mutagenesis and structural studies, are required to better understand the molecular mechanisms involved in the tarocystatin binding to papain and to identify the regulatory cleft involved in the inhibition process [ ] . based on the characterization of inhibitory function of group- and group- phytocystatins, we suggest that fig. . competition and retrieve test of the ct peptide to nt peptide. (a) competition test: the y-axis is catalytic velocity of papain, which was measured as the change in optical density over time. each reaction had the indicated amount of ct and nt peptides that reacted with papain. an increasing ct level did not reduce the inhibitory capacity of the nt peptide, but instead maintained a steady intensity. (b) retrieve test: the mixture of nt and ct peptides of lm was compared with an equal amount of only fl or nt peptides in the reaction with varied substrate concentrations. the curve of nt plus ct peptides highly overlapped that of the nt peptide, indicating that the nt peptide cannot retrieve the inhibition efficacy of the fl peptide when it disconnects from the ct peptide. group- and group- both evolved from a common ancestor. the evolutionary direction from group- toward group- by gene duplication appears to be an adaptation resulting from an evolutionary 'arms race' of rapid change in both interacting proteins. construction of three dna regions of the cecpi gene the underlined bases in the primers indicate restriction sites for bamhi (ggatcc) or ecori (gaattc). the primer combination of f and r was used for amplification of the fl peptide; f and r was for the nt peptide, and f and r was for the ct peptide. the pcr products were digested with bamhi and ecori, and ligated to pgex- tk vector (amersham biosciences, piscataway, nj, usa) at the corresponding restriction sites. expression, purification and characterization of recombinant tarocystatin e. coli bl (de ) cells containing the appropriate construct were grown at °c in yta liquid medium until d of . was reached. the recombinant cecpi expres-sion was induced by addition of mm isopropyl-b-d-thiogalactopyranoside. two hours after induction, the recombinant proteins were extracted from ml of bacterial culture by using b-perÒ gst-fusion protein purification kit (pierce no. ; pierce biotechnology, rockford, il, usa). for the assay of inhibitory kinetics of cecpi fragments, the gst fusion protein was cleaved with units of thrombin for h at room temperature. finally, the recombinant proteins were collected by passing the extract through a glutathione sepharose b affinity column (amersham biosciences). the protein was quantitated with a bio-rad protein assay kit (bio-rad, hercules, ca, usa) using bsa as a standard. qualitative analysis of cecpi protein was performed according to michaud et al. [ ] on . % sds ⁄ page containing % gelatin. a mixture of cecpi proteins and papain was first incubated at °c for min in a mildly denaturing buffer ( . mm tris-hcl, ph . ; % sds, % sucrose; . % bromophenol blue), and then subjected to electrophoresis using a hoefer se system (hoefer, inc., holliston, ma, usa). after migration, the gels were transferred to a . % v ⁄ v aqueous solution of triton x- for min at room temperature to allow renaturation followed by incubating in reactive buffer ( mm sodium phosphate, ph . , containing mm edta, mm l-cysteine and . % triton x- ) for min at °c. subsequently, the gels were rinsed with water and stained with coomassie brilliant blue. proteinase inhibitor activity was visualized as clear zones on a blue background and the intensity of the clear band is inversely related to the inhibition level. k i values of papain inhibition were determined from lineweaver-burk plots, a double reciprocal plot of substrate concentration versus velocity. the velocity was determined by measuring the a of the chromophore, as described by pernas et al. [ ] . briefly, an appropriate amount of inhibitor was pre-incubated with lm of papain in ll of reaction mixture containing . m sodium phosphate buffer (ph . ), mm edta and mm -mercaptoethanol at °c for min. the reaction was started by the addition of ll of a varied concentration in the range - lm of bana (sigma, st louis, mo, usa) as substrate. the reaction mixture was incubated at room temperature for min and ll of % hcl in ethanol (w ⁄ v) was added to stop the reaction. the chromophore was then developed by addition of ll of . % p-dimethylaminocinnamaldehyde in ethanol followed by incubation at room temperature for min and measurement of a . corre-menguy et al. [ ] the inhibitory activity was recorded as the inhibition percentage (%) and the inhibition percentage (i%) of papain was calculated using the equation: where, t and t* are the velocities in the absence and presence of the inhibitor from reactions, respectively. the average inhibitory activity was calculated from i% values of varied substrate concentrations. the fungal activity assay was performed as described previously [ ] . five pieces of sclerotia of phytopathogenic fungus s. rolfsii were cultured in ml of half strength potato dextrose broth, which contained purified gst-tarocystatin segment fusion proteins at concentrations of . , . , . and . nm in four separate sets. the fungi were cultured at °c under continuous shaking ( r.p.m.) on an orbital shaker for h. hyphal growth inhibition by tarocystatin segment proteins was observed directly, as well as under a microscope. the tarocystatin primary sequence (aam ) was subjected to ncbi psi-blast with a threshold of . for searching for homologous sequences from various plants. the sequence similarities of amino acid sequences were distributed with the highest identities of % and a positive of % for soybean to the lowest identities of % and a positive of % for tomato, excluding nonplant and multidomain cystatin homologs. these sequences were aligned using clustalw [ ] and shaded with genedoc [ ] software. the secondary structure of these sequences was analyzed by two programs, psi-pred [ ] and yaspin [ ] . the results obtained by the two programs were consistent with each other and showed that both nt and ct peptide secondary structures were arranged in a similar pattern. this was also verified by aligning the oc-i with the tarocystatin nt and ct peptide regions. therefore, the stereofolding pattern of oc-i [ ] can be taken as a template for the cecpi folding prediction by modeler . [ ] . the two structural conformations were merged after analysis by the automatic docking system, zdock . [ ] , and then remodeled by modeler . [ ] . structural and phylogenetic relationships among plant and animal cystatins mechanism of inhibition of papain by chicken egg white cystatin papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central gln-val-val-ala-gly region conserved among cystatin superfamily members molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). homology with animal cystatins and transient expression in the ripening process of rice seeds characterization of a cdna encoding a cysteine proteinase inhibitor from chinese cabbage (brassica campestris l. ssp. pekinensis) flower buds soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (colocasia esculenta cv cloning, functional expression, and characterization of cystatin in sesame seed the strawberry gene cyf encodes a phytocystatin with antifungal properties evolutionary mechanism acting on proteinase inhibitor variability characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor purification and characterization of a cystatin form the leaves of methyl jasmonate-treated tomato plants trade-offs between pathogen and herbivore resistance differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and jasmonate the involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants the cystatins: protein inhibitors of cysteine proteinase a chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests two distinct cystatin species in rice seeds with different specificities against cysteine proteinases molecular cloning and functional expression of cdna encoding a cysteine proteinase inhibitor, cystatin, from job's tears (coix lacryma-jobi l. var ma-yuen stapf) two woundinducible soybean cysteine proteinases have greater insect digestive proteinase inhibitory activities than a constitutive homolog corn cystatin i expressed in escherichia coli: investigation of its inhibitory profile and occurrence in corn kernels three dimensional solution structure of oryza cystatin-i, a cysteine proteinase of the rice, oryza sativa l japonica structural basis of the endoproteinase-protein inhibitor interaction carboxy terminal extended phytocystation are bifunctional inhibitors of papain and legumanin cysteines proteinases assessing the stability of cystatin ⁄ cysteine proteinase complexes using mildly-denaturing gelatin polyacrylamide gel electrophoresis clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice genedoc: analysis and visualization of genetic variation the psi-pred protein structure prediction server a simple and fast secondary structure prediction algorithm using hidden neural networks comparative protein modelling by satisfaction of spatial restraints zdock: an initialstage protein-docking algorithm a cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, microdochium nivale characterization of the expression of a wheat cystatin gene during caryopsis development the present study was supported by the national science council, taiwan, under project nsc- - -b- - to kai-wun yeh. we thank dr michael conrad (university of north carolina at chapel hill) for critically reading the manuscript and for his helpful suggestions. key: cord- - hgrs authors: miazga-karska, malgorzata; michalak, katarzyna; ginalska, grazyna title: anti-acne action of peptides isolated from burdock root—preliminary studies and pilot testing date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: hgrs this work aimed to study the anti-bacterial, anti-biofilm and anti-oxidant potential effects of low molecular weight (lmw) peptides (br-p) isolated from burdock (arctium lappa l.) roots. we conducted a preliminary study to exclude or confirm the antibiotic activity of the lmw peptides fraction of this plant. br-p were isolated using gel filtration and a kda cut-off membrane. the obtained peptides were identified by maldi tof/tof. antibacterial activity was tested against acne strains using diffusion tests, mic and mbc. the fibroblast cytotoxicity of br-p was tested, and the selectivity index (si) value was determined. the fraction of br-p peptides isolated from burdock root with a molecular weight below da and theoretic pi (isoelectric point) of . – . showed a narrow spectrum of activity against gram-positive acne bacterial strains. one of the br-p peptides assessed on maldi rapiddenovo was lrcdygrffaskslydplkkrr cationic peptide. it was analogous to that contained in a. lappa protein, and theoretically it was matched as a peptide with antibiotic nature. br-p did not show toxicity to fibroblasts in the tested concentration up to mg/ml, obtaining cc( ) mg/ml. the si value for the tested propionibacterium strains ranged from to . finally, an active dressing based on chitosan/alginate/genipin was prepared using freeze-drying. the formed dressing was evaluated for its anti-acne activity. to sum up: preliminary biological studies confirmed the anti-acne properties of the isolated peptide fraction from burdock root and pointed to the possibility of using it to create an active dressing on the skin. the world health organization (who) warns against epidemiological threats. today it is coronavirus, tomorrow drug-resistant bacteria can cause a large number of complications and high mortality. irrational antibiotic therapy leads to microbial resistance, which is the ability of microorganisms (like bacteria, viruses, and some parasites) to stop antimicrobial drugs from resisting against them. as a result, standard treatments become ineffective, and infections persist and may spread to others. who reports that the misuse of antibiotics is putting us all at risk, and the world is heading towards a pre-penicillin era. however, if effective anti-infection medication runs out, most invasive medical procedures, from tooth extraction to complicated surgery, will again be at high risk of fatal infection [ ] . propionibacterium acnes is the most important skin inflammatory factor. an acne skin treatment lasting several years is a difficult and complex problem not only for dermatologists. it is also noticeable by surgeons; for patients with acne around the shoulders and the back, chest or thighs, it can cause postoperative infections and prolong healing due to the presence of propionibacterium acnes. in such cases, bactericidal treatment before and after surgery is recommended. in most european the burdock roots samples, isolated using ultrasound and salting out procedure, were fractionated using a sephadex g- gel filtration (materials and methods, section . .) the obtained fractions were evaluated for the amount of protein and antibacterial properties (the diffusion test in solid medium). the active fractions were combined and resulting in obtaining a peptide fraction named br-f (the amount of protein for the br-f fraction was µg/ml). br-f was initially tested for antibacterial activity (figure , table ), and the remaining br-f part was further ultrafiltrated in amicon ( kda cut-off), followed by freeze-drying to obtain final br-p peptide samples (the protein content was µg/ml). it turns out that the antimicrobial activity increased with purification steps, during which the protein levels decreased. the data in table and figure indicate that both peptide samples of burdock root (br-f and br-p) did not inhibit the growth of gram-negative bacteria. in contrast, there was a noticeable inhibitory effect on gram-positive bacteria by both peptide samples: br-f and the purified peptide fraction of br-p inhibited the actions of bacterial strains, both aerobic and anaerobic. however, the purified br-p peptide fraction worked almost twice as strong compared to br-f. these br-f created zones of gram-positive bacteria growth inhibition in the range of . - mm, while br-p peptides showed growth inhibition of the tested strains in the range of . - . mm (figure ). the above results were confirmed with a minimum inhibitory concentration (mic) evaluation. data in table indicate the most favorable, low mic concentration values were obtained by br-p peptide samples primarily against the acne strains p. acnes pcm (mic . µg/ml) and p. acnes pcm (mic . µg/ml). higher mic values were determined against other gram-positive staphylococcus aureus and s. epidermidis bacteria (mic and µg/ml, respectively). however, a twice as high br-f concentration was needed to achieve such mic values for s. aureus and s. epidermidis and up to eight times higher to reach mics for both p. acnes species. in the mic assay, both peptide burdock samples were not active against gram-negative strains. another mbc/mic test confirmed that the bactericidal or bacteriostatic nature of burdock samples depends on the step of their purification (table ). the br-p peptide fraction was bactericidal against all tested gram-positive strains, both aerobic and anaerobic. however, br-f fractionated using a sephadexg- gel filtration had only a bacteriostatic character to these gram-positive strains. due to the low br-p and br-f activity against gram-negative strains, the mbc/mic value could not be determined. in subsequent experiments, the br-p peptide sample was used because of its good antibacterial activity. in the next stage of the study, the ms spectrum of the peptide mixture was assessed by maldi-tof. the obtained spectrum was smoothed, and the list of peaks for the signal-to-noise ratio was digitally generated (figure , figure s ). obtained data confirmed that the br-p sample consisted of peptides with molecular masses lower than da. the major peaks (corresponding to peptides) with a signal-to-noise ratio greater than were loaded and subjected to biotools rapidenovo sequencing. the signal-to-noise parameter response to intensity of the peak corresponds to the number of certain peptides. these peaks were selected for analysis due to the supposition that they had the greatest effect on antibacterial action. the peptide sequences obtained from the br-p burdock root sample were further analyzed using the basic local alignment search tool (blast) homology base. this base allowed the peptides present in the br-p sample to be matched (in the homology field) with analogous peptides and proteins found in the asteraceae family. the selected peptides of a given amino acid sequence were included in the enzymatic (oxygenases, dehydrogenases, transferases) or plant immune proteins from the asteraceae family. they are specified in table . in the case of nwfkpg sequence fragment, there are suggestions of proteins from which they may originate. additionally, for a m/z two equally likely sequence fragments were selected (fsrerd, nkfsre). in the case of antimicrobial peptides, it was important to determine the pi value. in our mixture of peptides from burdock, ten occurring in the largest amount were distinguished. based on the data contained in table , it can be concluded that the br-p peptide mixture contained peptides with a pi in the range of . - . . among them there was a cationic peptide lrcdygrffaskslydplkkrr with theoretically inhibitory activity on bacterial permease and with a pi of . . thus, due to the type of activity and cationic nature, the peptide may be responsible for antibiotic activity. * signal-to-noise ratio on mass spectrum. ** protein sequences matched to sequence in databases in basic local alignment search tool (blast). *** activities determined in biopep-uwm database of proteins and biopep-uwm database of bioactive peptides. **** theoretical pi determined in peptidemass expasy. the next step was to determine the strength of scavenging the free radicals of br-p in the dpph assay. such an activity of the peptide sample would be desirable when preparing an anti-acne formulation. in the experiment, radical scavenging assays were used to evaluate the activity of br-p and glutathione (gsh) as a positive control ( figure ). the dpph free radical inhibition assay of the new peptide fraction br-p revealed the potent antioxidant activity as observed through the plot of effective concentration ( - µg/ml) ( figure ). the ic value of br-p for dpph inhibition was found to be . µg/ml. the results were comparable to that of the reference peptide gsh (tested at - µg/ml) and with ic of . µg/ml. in analyzing the sequence of peptides present in the sample, it can be seen that they contained fragments of peptides considered to be antioxidant, hence high antioxidant properties may result. additionally, during the isolation process there is a possibility of nonspecific breaking of amino acid chains, resulting in the occurrence of small di-or tripeptides that are not observed on the maldi spectrum. it may mean that there are many more peptide fragments with antioxidant activity in the br-p sample. the investigated br-p peptides sample was evaluated for its cytotoxic activity towards the human skin fibroblast cell line (bj) after hours of incubation. based on mtt assay results, the half maximal cytotoxic concentration (cc ) was determined and summarized in figure . data show that br-p peptides exhibited no cytotoxicity against bj cells in the entire tested concentration range ( . - mg/ml). the fibroblast viability was maintained with a tested br-p concentration range at a high level of over %. this result means that the cc values of br-p peptides were higher than mg/ml. thus, for biocompatibility and selectivity determination of extracts, the cc value of br-p was assigned as mg/ml (table ) . to estimate the in vitro therapeutic safety and efficacy of the br-p peptide sample, the selectivity index (si) was determined. si index is defined as the ratio of cytotoxic activity (cc ) to antibacterial activity (mic). high values of the selectivity index indicate that the tested sample may be more effective against bacterial strains and safer for eukaryotic cells. thus, si values below indicate lack of therapeutic safety, and si values higher than allow to perform in vivo evaluation [ ] . it is extremely therapeutically important that the br-p exhibited a favourable and highly safe therapeutic potential against acne strains p. acnes pcm and p. acnes pcm , with safety si values of and , respectively. however, towards the tested gram-negative bacteria (e. coli and p. aeruginosa) br-p did not meet the safety requirements. the last stage of research was the production of an exemplary functional anti-acne dressing modified with the br-p peptide fraction from burdock root. the dressing material was created with genipine cross-linked chitosan and alginate. obtained after freeze-drying, the hydrogel sheet composed of polysaccharides was a uniform, thin foam resistant to chipping or cracking. the control unmodified and modified with br-p peptide dressings were immersed in a medium with a plankton form of acne strains. it was compared whether such planktonic forms of bacteria could create colonies resulting in biofilm formation to the same extent on control and modified materials. the conducted test imaged in a confocal laser-scanning microscope showed the beneficial effects of modification with br-p ( figure ). namely, the confocal laser-scanning microscope (clsm) images showed p. acnes or s. aureus biofilms on the surface of the dressing with viable and dead colonies (green and yellow-red fluorescence, respectively). the control dressings group demonstrated viable p. acnes or s. aureus bacteria, which formed green fluorescent biofilm architecture indicating that bacteria were viable during the evaluation period. whereas, only red colonies of dead bacteria were seen on the tested dressings. images show weaker or no adherence of tested bacteria to the br-p-modified dressing in comparison to the unmodified control ( figure ). acne vulgaris is a chronic disease of the hair sebaceous gland accompanied by seborrhoea. it is not an infectious disease, but patients have a multiplication of propionibacterium acnes [ ] . achermann's team presented relative abundances of propionibacterium species in different skin areas, proving that these bacteria are not only present in the face area, resulting in not only cosmetic trouble, but make it difficult to carry out surgical procedures in a larger area [ ] . the roots of burdock (arctium lappa l.) are commonly used as herbal medicine based on its valuable phytochemical content. pereira et al. [ ] , gentil et al. [ ] and pirvu et al. [ ] showed antibacterial activity against gram-positive and gram-negative bacteria of crude extracts from burdock, and knott et al. and jingvi et al. [ , ] proved skin improvement when using burdock extracts. one of the molecules in burdock roots responsible for antibacterial properties can be peptides because every organism (from microorganisms to mammalia) has an innate defense system, which is determined by the presence of, among others, antimicrobial peptides (amps) [ ] . therefore, it was desirable in our work to purify the crude extract of burdock root to assess the biological activity of the peptide solution. the presence of fractions of peptides with molecular masses lower than in da br-p was determined by maldi tof. the major peaks were subjected to biotools rapiddenovo sequencing ( figure ). the determined theoretical pi values confirmed the presence of some cationic peptides with a preference for antibacterial activity with pi values of . , . , . , and . . based on table , we can theoretically assume that the peptide lrcdygrffaskslydplkkrr with pi of . and kk amino acid fragment, with activity indicated as a bacterial permease ligand, may be responsible for the antibiotic activity. in the mechanism of energy transduction in bacterial membranes, a bulk-phase, transmembrane electrochemical ion gradient is an important problem and concerns, e.g., secondary active transport, oxidative phosphorylation, and rotation of the bacterial flagellar motor. transport involves substrate-specific membrane proteins that catalyze equilibration or uphill translocation of solute across a membrane. these carrier proteins are named transporters, cotransporters, symporters, or permeases. thus, permeases are involved in active transport of energy sources, e.g., glucose, fructose, various sugars, and sugar alcohols. thus, permeases, by providing the bacterial cell with a source of energy, may be the target of molecular inhibition of bacterial growth [ ] . both burdock root samples (br-f, br-p) obtained in subsequent purification stages were evaluated for antimicrobial character. the data of bacterial growth inhibition show that burdock root polypeptide samples had the ability to inhibit the growth of acne bacteria (figure ) . the results indicate that the antibacterial nature of the samples increased with the number of purification steps, as the br-p-purified sample was at least twice as strong as br-f. the mic value confirmed that br-p had stronger antibacterial properties than the mixture of crude br-f polypeptides (table ) . br-p possessed bactericidal nature against all gram-positive strains, whereas br-f was only active against anaerobic gram-positive strains. the tested preparations from burdock roots did not inhibit the activity of gram-negative strains. the narrow spectrum demonstrated by burdock root samples, limited only to gram-positive bacteria, may be beneficial. it suggests that chemotherapy with a single drug may be used in the fight against acne, which is preferred for the identified pathogen, minimizes the impact on the body's physiological flora, and reduces side effects and toxicity. our tests also showed that a br-p sample in an acid environment ph (bhi broth and bhi agar ph . ) had antibacterial activity. this is a promising result and therapeutically useful for cosmetic formulations requiring acidic ph. besides, antibacterial peptides active under acidic conditions have a protective effect against bacterial infections (the skin, oral cavity, urinary tract, and vagina possess acidic ph). a korean team [ ] noted that the high antibacterial properties of nod and nod peptides isolated from nordotis discus in an acidic environment might be useful when these peptides are applied as skin therapy in ph of about . . the clinically important measurement results of the selectivity index si value are interesting. the high values of the selectivity index indicated that the tested sample may be more effective against bacterial strains and safer for eukaryotic cells. thus, si values below determine a lack of therapeutic safety, and si values higher than allow to perform in vivo evaluation [ ] . si values of br-p for gram-positive strains were at least and, even compared to acne strains, had an extremely favorable selectivity of up to . low toxicity and good antibacterial activity result in a safe value on therapeutic index, suggesting that the obtained purified burdock root peptides could be promising as an anti-acne agent. other authors also report favorable, high si values of tested antimicrobial peptides (amps) from plants and the possibilities of their application in the cosmetics, medical, food, or agriculture industries [ ] [ ] [ ] [ ] [ ] . on the other hand, kumar et al. [ ] showed that, although many amps have reached clinical trials, not many of them have been approved by the us food and drug administration (fda) due to issues with toxicity. authors have pointed out strategies to improve the activity and biocompatibility of amps, such as chemical modifications and the use of delivery systems [ ] . another georgian-american team of scientists working on de novo amp synthesis pointed out that the design of amps with high therapeutic indexes, low cost of synthesis, high resistance to proteases, and high bioavailability remains a challenge. authors have developed a digital tool to predict amp algorithms for the design of de novo amps, and short peptides with high therapeutic indexes against gram-negative bacteria have been designed [ ] . due to the nature of acne skin diseases, anti-acne drugs should also have an antioxidative nature. free radicals are not only dangerous for normal and acne skin, but they also can lead to a variety of various human diseases (cardiovascular diseases, rheumatoid arthritis, alzheimer's disease, and cancer). according to some of the authors, regardless of the solvent used, the crude burdock root extract possessed significant antioxidant activity, as determined with dpph; thus, crude extract seems to be a promising antioxidant [ , , [ ] [ ] [ ] . in our work the dpph free radical scavenging assay was performed to assess the antioxidant capacity of the br-p peptide samples against the free dpph radical. the experiment showed potent antioxidant activity (range - µg/ml) (figure ). the ic value of br-p for dpph inhibition was found to be . µg/ml. the possibility of scavenging free radicals through burdock peptides is promising when applied to dermatological cosmetic formulations. it is significant that also proteins, polypeptides and peptides are proper antioxidants because they can inhibit biomolecule oxidation though some pathways (including inactivation of reactive oxygen species, scavenging free radicals, chelation of pro-oxidative transition metals, and reduction of hydroperoxides). changes to the tertiary protein structure increase the accessibility of amino acid residues that can scavenge free radicals and chelate pro-oxidative metals. peptides, not proteins, show the most promise as proteinaceous antioxidants, and a number of studies have shown that they have a substantially higher activity than proteins with a whole, intact structure [ , ] . for diseases with keratinolytic dysfunction, it is beneficial to combat both free radicals and the bacteria that cause these diseases, with a lack of cytotoxicity. low-weight molecular fractions of burdock root peptides exhibit such combined characteristics. in the last stage of the experiments, we made an antibacterial and antioxidant active dressing for acne skin. the dressing base created de novo with polysaccharides (chitosan and sodium alginate) was crosslinked by genipin and modified with br-p peptides. the hydrogel thus obtained, after freeze-drying, had a dry foam structure that was tested for bacterial biofilm adhesion. the br-p-modified dressing was found to have beneficial antibacterial properties; it showed no adhesion or poor adhesion of the tested bacteria to the surface compared to the unmodified control material, as evidenced by clsm images (figure ) . to use the developed modification of active br-p peptide from burdock roots to create commercial dressings, in the future, research should be carried out with clinical bacterial strains isolated from dermatological patients. it would be advisable to carry out in vivo tests that would confirm or exclude the effectiveness of antibacterial dressings in the prevention and treatment of peri-operative acne skin infections. burdock roots (arctium lappa l, bardanae radix dried, kawon, gostyń, poland) ( g) were grated in a mortar, and proteins were extracted with buffer ( mm na hpo , mm nah po , mm kcl, pepsin . mg/g defatted powder, mm edta, ph . ) by sonication at • c in a series of × min. ammonium sulphate was added to get % saturation, and precipitate obtained after h at room temperature was removed after centrifugation ( min at × g). collected supernatant was adjusted to % ammonium sulphate saturation, and precipitated proteins (after centrifugation at × g for min at • c) were resuspended in distilled water and dialyzed against distilled water using da cut-off dialysis tubing (sigma-aldrich, saint louis, mo, usa). the peptide fraction after dialyse was next purified by size exclusion chromatography on a sephadex g- (sigma-aldrich) on a column ( × cm) using the same buffer as that used in extraction. each eluted fraction ( ml) was collected and monitored for peptides at λ = nm. additionally, fractions were collected and analyzed using a screening antibacterial test on solid medium (br-f fractions). the samples active against bacteria were combined together and ultrafiltrated through a kda cut-off membrane (amicon). the antibacterial activity was again controlled after freeze-drying and treated as an active peptide fraction from burdock roots (br-p fractions). the freeze-dried extract br-p sample was weighed, the overall process yield was calculated, and then mg/ml stock solutions were prepared. maldi technique was used to monitor the peptide purification. for measuring molecular masses of the peptides, the br-p sample was analyzed on a matrix-assisted laser desorption/ionization time of flight mass spectrometer ultraflextreme (maldi tof/tof; bruker, bremen, germany). maldi analysis was performed according to a previously proposed procedure [ ] . briefly, . µl of peptide sample was mixed with the same volume of matrix saturated solution containing α-cyano- -hydroxycinnamic acid (hcca, bruker, bremen, germany) and , -dihydroxy benzoic acid (dhb, bruker, bremen, germany). next, the obtained mixture was spotted on an anchorchip maldi plate (bruker, bremen, germany) with prespotted hcca in acetone. analyzed br-p peptides were subjected to mild ionization using maldi-tof in the linear mode and recorded in active positive reflector mode within the - m/z range with laser frequency hz and shot counts. the collected spectrum was smoothed using the savitzky-golay method, baseline corrected (top hat baseline algorithm), and the list of peaks for the signal-to-noise ratio of > was generated using flex analysis . software (bruker, bremen, germany). the major peaks with signal-to-noise ratios greater than were assigned to rapiddenovo sequencing using fragmentation spectra obtained in ms/ms tandem mode. in this aim, the ms/ms data were loaded and recognized automatically, b-and y-ions were detected, and the software predicted the sequence or its fragment based on the peaks which were presented. automatically predicted sequences were given with their probability of occurrence. the most probable sequence (or sequence fragment) was next analyzed by the blast algorithm for finding regions of amino acid similarity in order to assign them to proteins from which they originated. we selected "protein-protein blast" as a search algorithm which simply compares a peptide fragment to a protein database. we chose only proteins from asteraceae family to compare and designated peptide fragments were % identical to the matched proteins. knowing a sequence of a given protein enabled the determination of the peptide sequence with a given mass (m/z) using peptidemass expasy [ ] . the peptide sequence was then analyzed for active fragments in the biopep-uwm database, while theoretical biological activity and isoelectric point were indicated [ ] . antibacterial assays were done using the micro-aerobic strains propionibacterium acnes pcm and propionibacterium acnes pcm as well as the aerobic strains staphylococcus aureus atcc , staphylococcus epidermidis atcc , pseudomonas aeruginosa atcc , and escherichia coli atcc as a microbial cause of acne appearance. before setting up the experiments, micro-aerobic bacteria were grown separately on bhi agar (biomaxima s.a., lublin, poland), ph . the preliminary antibacterial activity of the burdock roots samples against pathogenic gram-positive micro-aerobic and aerobic bacterial acne was evaluated by measuring the zones of inhibition in the disk diffusion method [ ] . antibacterial disc diffusion assays were carried out on petri plates with solid medium (bhi agar (ph . [ ] , or m-h agar). appropriate strain cultures were separately spread over the agar surface with a sterile cotton swab. next, each sample ( µl) was placed on petri plates with an agar medium. after h of incubation at • c (for aerobic strains) or h at • c (for micro-aerobic strains), zones of microbial growth created around the tested burdock samples were measured and recorded as the diameters of inhibition (mm). a broth microdilution method was used to evaluate the minimum inhibitory concentration (mic) according to the clsi document (clsi performance standards for antimicrobial susceptibility testing, eighteenth international supplement, clsi document m -mic, clinical laboratory standards institute, wayne) with some modifications. the lowest concentration of the tested compound (µg/ml) which did not result in any visible growth of bacteria was considered as the mic value. a serial doubling dilution of the burdock samples was prepared in -well plates ( µl per well). a suitable medium (m-h broth, bhi broth) was used as a diluent. the final concentrations of burdock samples were - . µg/ml. finally, µl of inoculum of the tested bacterial strain ( . × cfu/ml) was added to each well. the tests were performed either at • c for h (aerobic strains) or at h (micro-aerobic strains). after incubation, the panel was digitally analyzed at nm using the microplate reader bio tech synergy (usa) with a dedicated software system. the growth intensity in each well was compared to the negative (clear broth) and positive (broth with inoculum) controls. additionally, minimal bactericidal concentration (mbc) was obtained by spreading on agar µl of medium from the clear well, which did not show any visible growth after incubation during the mic test. the plates were incubated at • c for h, and the mbc was defined as the lowest concentration of sample without bacterial growth. each experiment was repeated in triplicate. the cell culture experiment was performed using a human skin fibroblast cell line (bj cells from american type culture collection atcc, uk). bj cells were cultured in emem supplemented with % fbs, u/ml penicillin, and µg/ml streptomycin and maintained at • c in a humidified atmosphere of % co and % air. the cells were seeded in -well plates in µl of a complete growth medium (supplemented with % fbs) at a concentration of . × cells/well and incubated for h at • c in a humidified atmosphere of % co . after this time, the br-p sample was firstly diluted in dmso to obtain the stock solution at mg/ml, and the solution was further diluted in culture medium supplemented with % fbs. subsequently, the culture medium was replaced with µl of the serial dilutions of the br-p peptide sample. untreated cells were used as a control of cytotoxicity, and different concentrations of dmso were used as a solvent control. the cell cultures were incubated at • c for h. cytotoxic effects were estimated using the mtt test. the experiment was repeated in three separate measurements. the half-maximal cytotoxic concentration (cc ) was defined as the sample concentration required to reduce cell viability to %. the cc values were calculated via four-parameter nonlinear regression analysis (graphpad prism , version . ) and were presented as mean values ± standard deviation (sd). the present study evaluated the antioxidant activity of br-p samples by employing a diphenylpicrylhydrazyl (dpph • ) (sigma-aldrich, usa) radical scavenging assay based on the abdel moneim method [ ] . this assay was conducted spectrophotometrically in -wells plates using digital reader bio tech synergy (usa). color disappearance of dpph ( µl . mm in % ethanol) after min was monitored at nm. this dpph decolouration, or lack thereof, was caused by the presence of µl tested samples in the concentration range - µg/ml. due to the precipitate formed (in some samples) during incubation (as a result of the ethanol of dpph with the sample combination), after min of incubation the mixture had to be centrifuged, the sediment rejected, and the clear solution read in a clean -well plate. ascorbic acid and glutathione (gsh) were used as standards for the , -diphenyl- -picrylhydrazyl scavenging assay. the following equation was used to calculate the percentage of dpph decolorization of the sample: % dpph • radical inhibition = [(abs control − abs sample)/abs control] × ( ) where abs is absorbance at nm. radical scavenging activity was expressed as the inhibition concentration (ic ), i.e., concentration of sample required to inhibit % of dpph free radicals. ic was determined graphically from the curve plot between the percentages of dpph scavenging activity and the sample's concentration. dressing material for acne skin disease treatment was created using natural polysaccharides crosslinked with genipin (sigma-aldrich, usa). briefly, . % chitosan ( - % deacetylation degree, - kda, sigma-aldrich, usa) and . % sodium alginate (sigma-aldrich, usa) were separately dissolved in ml of . % acetic acid and water, respectively. then, alginate was poured in portions into the chitosan standing on a magnetic stirrer. after the two polysaccharides were completely mixed, calcium β-glycerophosphate ( mg/ml) used as inorganic filler was added and mixed for min at • c. the obtained hydrogel was divided into two parts: one as an unmodified control and another modified with br-p ( . mg/ml) as the active substance. finally, % genipin ( . µg/ml) was added as the bifunctional cross-linker, with further mixing until a homogeneous mixture was obtained. the resulting modified and unmodified gels were transferred quantitatively into appropriate forms and left for h at room temperature for crosslinking. the final stage of dressing formation was deep frozen (− • c) and freeze-dried (lyo gt basic srk systemtechnik, gmbh, germany), resulting in discs with a diameter of about . mm and thickness of about . mm. modified and unmodified dressings were sterilized using a plastic/paper peel pouch in ethylene oxide prior to further testing. dry dressing samples (unmodified dressing as a control; dressing modified by br-p) were washed in µl of pbs and then transferred to the bottoms of -well polystyrene plates (cytoone, usa). each well with a disc sample inside was filled up with µl of appropriate broth (bhi or m-h). finally, µl of inoculum ( . × cfu/ml) was added to each well. for mono-species biofilm assay it was p. acnes pcm and s. aureus atcc . sterility controls (only m-h or bhi broth) were included in all experiments. to allow biofilm formation, i.e., the adhesion of planktonic forms of bacteria in the colonies attached to the biomaterial, the plates were incubated twice as long. namely, plates with aerobic bacteria were incubated for h, and with micro-aerobic bacteria h, both at • c. the tests were performed using three replicates. the viability of bacteria and their adhesion to the material surface were determined using double fluorescent staining for both live and dead bacteria using viability/cytotoxicity assay kit for bacteria live/dead cells (biotium, hayward, ca, usa). dressing samples with bacterial suspensions were prepared for confocal microscopy assay after the incubation. first, the medium from wells was removed and gently washed twice with µl of . % nacl, to remove the loosely adherent planktonic bacteria and to leave only the biofilm attached to the material. next, samples were transferred into fresh wells and filled with µl of . % nacl and live/dead dye. the solution of this dye was prepared by mixing µl of dmao with µl of ethd-iii in µl of . % nacl. three microliters of such obtained live/dead dye solution was added to disc-containing wells with µl of pbs. the samples were incubated for min at room temperature in the darkness, and bacterial colonies attached to dressing samples were visualized using a confocal microscope (clsm) with dedicated software. the results are expressed as mean ± rsd. statistical analysis was performed by analysis of variance (anova) tests, and statistical significance was set accordingly at the p = . level. our research allowed us to obtain promising results regarding the antibacterial and antioxidant activities of the tested burdock peptides. the obtained solution containing br-p peptides with a value below da from burdock roots had anti-acne properties and were not toxic to fibroblast cultures. these properties of active peptides have allowed them to be used to produce therapeutic dressing material (br-p/chitosan/sodium alginate) for use in infections against gram-positive bacteria in acne skin disease. one of the peptides is lrcdygrffasksldplkkrr, whose theoretical pi and bacterial permease ligand activity may possess antibiotic, anti-oxidative and anti-seborrheic properties. however, in-depth research is necessary on the biochemical analysis of the isolated br-p peptide fraction. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : title: br-p sample analyse in maldi/tof. ms/ms fragmentation spectra for rapiddenovo sequencing. antibiotic-resistant acne: lessons from europe magainins, a class of antimicrobial peptides from xenopus skin: isolation, characterization of two active forms, and partial cdna sequence of a precursor cell membrane lipid composition and distribution: implications for cell function and lessons learned from photoreceptors and platelets peptide antimicrobial agents new type of antimicrobial protein produced by the plant pathogen clavibacter michiganensis subsp. michiganensis antimicrobial peptides. ups phytamp: a database dedicated to antimicrobial plant peptides a hevein-like protein and a class i chitinase with antifungal activity from leaves of the paper mulberry purification and antipathogenic activity of lipid transfer proteins (ltps) from the leaves of arabidopsis and spinach de la canal, l. misctletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi defensins-components of the innate immune system in plants, flowers and stems analysis of two novel classes of plant antifungal proteins from radish (raphanus sativus l.) seeds novel antifungal defensins from nigella sativa l. seeds expression of a novel small antimicrobial protein from the seeds of motherwort (leonurus japonicus) confers disease resistance in tobacco purification biochemical characterization and antifungal activity of a new lipid transfer protein (ltp) from coffea canephora seeds with α-amylase inhibitor properties an antimicrobial peptide ar-amp from amaranth (amaranthus retroflexus l.) seeds committee on herbal medicinal products (hmpc). assessment report on arctium lappa l. radix, european medicines agency antimicrobial assays of tyree native british plants used in anglo-saxon medicine for wound healing formulations in th century england antibiofilm effect of burdock (arctium lappa l.) leaf fraction and its efficiency in meat preservation antioxidant antifungal and anti-inflammatory effects of wild-growning romaniam native arctium lappa l. 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(burdock) and their structure-activity relationships (sars) of free radical scavenging activities antioxidant activity of proteins and peptides purification and identification of novel antioxidant peptides from enzymatic hydrolysate of ginkgo biloba seed proteins matrix-assisted laser desorption/ionization time of flight (maldi-tof) mass spectrometric analysis of intact proteins larger than kda biopep-uwm database of bioactive peptides current opportunities manual of clinical microbiology the neuroprotective effects of purslane (portulaca oleracea) on rotenone-induced biochemical changes and apoptosis in brain of rat this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -pq obtrc authors: capasso, domenica; del gatto, annarita; comegna, daniela; russo, luigi; fattorusso, roberto; saviano, michele; di gaetano, sonia; zaccaro, laura title: selective targeting of αvβ integrin in hepg cell line by rgdechi d peptide date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: pq obtrc recently, the research community has become increasingly concerned with the receptor αvβ , a member of the well-known integrin family. different ongoing studies have evidenced that αvβ integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. in this scenario, the availability of a selective αvβ antagonist without ‘off-target’ protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. we recently reported a cyclic peptide, rgdechi d, obtained by structure-activity studies. to our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvβ integrin and not cross react with αvβ . here we demonstrated the ability of the peptide to diminish both adhesion and invasion of hepg cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the pi k pathway. the peptide, also decreases the formation of new vessels in endothelial cells. taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma. integrins are transmembrane receptors able to dictate cellular responses to a variety of inputs thanks to their capacity to differentially recognize distinct environments. to allow for this flexibility, integrins are comprised of α and β subunits that pair to form at least different functional heterodimeric receptors. they can transduce extracellular stimuli resulting in an extensive range of downstream effects on cell adhesion, migration, proliferation, differentiation and apoptosis [ ] . in vivo studies have shown that in various types of cancers the expression of some integrins on the surface of neoplastic cells is frequently up-regulated. the αv subunit, which forms heterodimers with β , β , β , β or β subunits, is able to recognize more extracellular matrix (ecm) ligands and growth factors thanks to the rgd sequence. the availability of ligands able to discriminate among different integrin subtypes, sharing sequence and structure homology but different biological effects, is a desirable aim in the biomedical field. over several decades the scientific community has focused on different integrins such as αvβ , α β and αvβ and little attention has been paid to integrin αvβ whose relevance has been assessed from several ongoing studies. αvβ integrin binds mainly the ecm protein vitronectin [ , ] and has the ability to control different biological and pathological events, thus representing one of the more intriguing integrins. it has been known for years that integrins are commonly used as receptors by many human viruses [ ] , and αvβ actually seems to play a critical role in multiple aspects of infection pathogenesis [ , ] . viral proteins with the rgd motif promote infection by binding integrin heterodimers [ , ] , thus activating pi k/akt or mapk signaling pathways which promote virus entry and infection of the host cell [ ] . moreover, in addition to the well-established contribution of αvβ integrin to angiogenesis [ ] [ ] [ ] , much evidence supports the crucial role of this integrin in promoting cancer cell migration and invasion. it seems that αvβ mediates the early steps of liver metastasis formation of colon carcinomas through different mechanisms [ , ] . additionally, there is substantial evidence for interplay between αv integrins and tissue growth factor (tgf-beta) during the pathological epithelial-to-mesenchymal transition that occurs in many types of cancer [ ] [ ] [ ] [ ] [ ] [ ] , which is the reason why anti-integrin therapeutics are indeed under development as treatment for tgf-beta-related disorders [ ] . hepatocellular carcinoma (hcc) is a primary malignancy of the liver that usually develops from a background of liver fibrosis and inflammation. it is associated with a high propensity for vascular invasion and metastasis, which accounts for its poor prognosis [ , ] . the high mortality rate is due to the advanced stage and frequent recurrence after surgical resection, and most patients are limited to surgical treatment or chemotherapy [ ] . consequently, to improve the survival rate, novel and effective therapeutic strategies are needed. recently, hoshino et al. proved that exosomal β integrin regulates liver tropism associated with liver metastasis in several tumors [ , , ] . in a more recent study, lin et al. demonstrated that β integrin is highly expressed in hcc tissues, facilitating cancer cell growth and promoting cell migration [ ] . in human hcc patients, fibrinogen [ ] activates hepatic stellate cells (hsc) [ ] and liver specific mesenchymal cells by binding αvβ integrin [ ] widely expressed by neoplastic cells to promote liver disease progression and tumor metastasis [ ] . recently yan et al. stated that treatment with cilengitide peptide, an αvβ /αvβ integrin antagonist, significantly blocked hsc activation and function and reduced proliferation of oncogenic hepatocytes and progression of liver fibrosis [ ] . thus, it is definitively clear that β integrin can be considered a diagnostic biomarker and a potential therapeutic target in hcc. to date only αvβ /αvβ and αvβ /α β antagonists have been reported in the literature, whilst selective αvβ peptide antagonists are still missing. selective modulation of αvβ integrin is highly desirable to allow targeted specific therapy and potentially to limit side-effects. this aspect, despite the availability of structural model of the head group of αvβ integrin constructed by homology modeling, is further complicated by the lack of the high-resolution d structure [ ] . in the last few years, our research activities have mainly focused on the identification of integrin selective peptide ligands by rational design [ ] [ ] [ ] [ ] . in we reported nmr and computational studies on the αvβ selective peptide, rgdechi, aiming to identify the molecular basis that regulates receptor recognition mechanism. by this combined approach we also demonstrated that the substitution of the key homocitrulline residue with aspartic acid shifts the receptor binding selectivity from αvβ to αvβ integrin, thus identifying a novel and selective αvβ ligand, named rgdechi d [ ] . here we reported the in vitro characterization of this peptide on the hepg cell line, one of the well-known model systems for hcc [ ] , evaluating its ability to selectively bind αvβ integrin and to interfere with angiogenesis, cell migration, invasion and proliferation. the expression levels of αvβ and αvβ integrins on surface of hepg were determined by means of quantitative cytofluorimetric assay using phycoerythrin (pe) conjugated antibodies. to maintain surface marker integrity, cells were detached using . mm edta in pbs. data indicate that hepg cells show an average surface expression of . × ± αvβ (figure ), while they do not present a significant amount of αvβ receptors. the good level of αvβ expression together with the absence of αvβ make this cell line a suitable model to study the biological behavior of a selective αvβ molecule. molecules , , x of the expression levels of αvβ and αvβ integrins on surface of hepg were determined by means of quantitative cytofluorimetric assay using phycoerythrin (pe) conjugated antibodies. to maintain surface marker integrity, cells were detached using . mm edta in pbs. data indicate that hepg cells show an average surface expression of . × ± αvβ (figure ), while they do not present a significant amount of αvβ receptors. the good level of αvβ expression together with the absence of αvβ make this cell line a suitable model to study the biological behavior of a selective αvβ molecule. figure . quantification of αvβ and αvβ receptors on hepg cell surface. cells were stained with phycoerythrin (pe)-anti-αvβ antibody (magenta curve) or pe-conjugated anti-αvβ antibody (green curve) or pe-control isotype (black curve). the histogram is representative of three independent experiments. cell adhesion assays were performed in order to confirm the capability of the peptide to bind only αvβ and not to cross react with αvβ or α β , unlike the current peptide-based molecules reported the in literature which are not able to discriminate between these integrins. rgdechi d, rgdechi and scrambled peptides were synthesized as previously reported [ , ] . the effect of rgdechi d on hepg adhesion to vitronectin, a matrix able to recognize both αvβ and αvβ integrins [ ] [ ] [ ] , was analyzed by crystal violet assay. to prevent receptor internalization, the cells were pre-incubated at °c with peptides or an anti αvβ integrin antibody; the treatment occurred in an appropriate adhesion buffer containing divalent cations essentials for receptor binding [ ] . then, cells were seeded onto vitronectin. as shown in figure a , rgdechi d greatly reduces the adhesion of hepg plated onto vitronectin of %; this result is even better than that obtained from the monoclonal anti-αvβ antibody ( %), used as positive control. incubation of cells with the scrambled peptide (negative control) does not decrease cell adhesion. considering the already reported rgdechi d binding specificity towards αvβ with respect to αvβ [ ] , this result confirms the specificity of action of rgdechi d towards αvβ on the hepg cell surface and indicates the ability of the peptide to compete with vitronectin and to prevent hepg cell adhesion. furthermore, it is worth noting that the inhibition occurred in a concentration-dependent manner cell adhesion assays were performed in order to confirm the capability of the peptide to bind only αvβ and not to cross react with αvβ or α β , unlike the current peptide-based molecules reported the in literature which are not able to discriminate between these integrins. rgdechi d, rgdechi and scrambled peptides were synthesized as previously reported [ , ] . the effect of rgdechi d on hepg adhesion to vitronectin, a matrix able to recognize both αvβ and αvβ integrins [ ] [ ] [ ] , was analyzed by crystal violet assay. to prevent receptor internalization, the cells were pre-incubated at • c with peptides or an anti αvβ integrin antibody; the treatment occurred in an appropriate adhesion buffer containing divalent cations essentials for receptor binding [ ] . then, cells were seeded onto vitronectin. as shown in figure a , rgdechi d greatly reduces the adhesion of hepg plated onto vitronectin of %; this result is even better than that obtained from the monoclonal anti-αvβ antibody ( %), used as positive control. incubation of cells with the scrambled peptide (negative control) does not decrease cell adhesion. considering the already reported rgdechi d binding specificity towards αvβ with respect to αvβ [ ] , this result confirms the specificity of action of rgdechi d towards αvβ on the hepg cell surface and indicates the ability of the peptide to compete with vitronectin and to prevent hepg cell adhesion. furthermore, it is worth noting that the inhibition occurred in a concentration-dependent manner with an ic of . µm ( figure b ). with the aim to evaluate the capability of the peptide to discriminate between αvβ and α β , cell adhesion assays on k cells, displaying α β at high levels and αvβ and αvβ at very low levels [ ] , were carried out. the cells were seeded both onto fibronectin, the main ecm protein that binds α β receptor, and onto α β antibody coated plates. the results obtained indicate that rgdechi d is not able to inhibit k adhesion neither on fibronectin nor that of the α β specific antibody; in this experiment the rgdechi peptide [ ] was used as a control to recognize αvβ integrin [ ] thus proving the validity of experimental data ( figure a ,b). the competition experiments here reported are all in the direction of corroborating rgdechi d specificity towards αvβ with respect to αvβ or α β integrins. with an ic of . µm ( figure b ). with the aim to evaluate the capability of the peptide to discriminate between αvβ and α β , cell adhesion assays on k cells, displaying α β at high levels and αvβ and αvβ at very low levels [ ] , were carried out. the cells were seeded both onto fibronectin, the main ecm protein that binds α β receptor, and onto α β antibody coated plates. the results obtained indicate that rgdechi d is not able to inhibit k adhesion neither on fibronectin nor that of the α β specific antibody; in this experiment the rgdechi peptide [ ] was used as a control to recognize αvβ integrin [ ] thus proving the validity of experimental data ( figure a ,b). the competition experiments here reported are all in the direction of corroborating rgdechi d specificity towards αvβ with respect to αvβ or α β integrins. the major life-threatening event in cancer patients is the metastasis formation which involves different events, such as cell migration and invasion into blood or lymphatic vessels in distal organs. firstly, to investigate whether rgdechi d could interfere with these mechanisms, an in vitro scratch assay was performed on hepg cells. after h of seeding, the cell monolayers were scratched linearly and incubated with the peptide rgdechi d ( µm). then, images were taken at , , and h after wounding ( figure a ). the results show that in the presence of the peptide, the wound healing was delayed compared to scrambled peptides and untreated cells. remarkably, rgdechi d k were pre-incubated with peptides ( µm) for min at • c then seeded on anti-α β coated plates. the results are presented as the percentage of adherent cells respect to the control (untreated cells) and are expressed as means ± se of at least three independent experiments performed in triplicate. statistical significance was analyzed using student's t test, unpaired, two-sided (* p < . ). the major life-threatening event in cancer patients is the metastasis formation which involves different events, such as cell migration and invasion into blood or lymphatic vessels in distal organs. firstly, to investigate whether rgdechi d could interfere with these mechanisms, an in vitro scratch assay was performed on hepg cells. after h of seeding, the cell monolayers were scratched linearly and incubated with the peptide rgdechi d ( µm). then, images were taken at , , and h after wounding ( figure a ). the results show that in the presence of the peptide, the wound healing was delayed compared to scrambled peptides and untreated cells. remarkably, rgdechi d inhibits closure of the gap both at and h after the scratch ( figure b ). afterwards, to evaluate the cell invasion inhibition, hepg cells were incubated with µm peptide for h and seeded on trans-well chambers coated with ecl cell attachment, an efficient model system that better mimics in vivo cell invasion. as shown in figure a , significant decrease in tumor cell invasiveness is observed when cells are treated with the rgdechi d peptide with respect to untreated cells (control) or scrambled peptides. the decrease in invasiveness was quantified and resulted in %, as indicated in the graph ( figure b ). the effect on cell adhesion, migration, and invasion, events closely correlated with the metastatic cascade, highlight promising features of this peptide in inhibiting critical steps of cancer progression. molecules , , x of inhibits closure of the gap both at and h after the scratch ( figure b ). afterwards, to evaluate the cell invasion inhibition, hepg cells were incubated with µm peptide for h and seeded on trans-well chambers coated with ecl cell attachment, an efficient model system that better mimics in vivo cell invasion. as shown in figure a , significant decrease in tumor cell invasiveness is observed when cells are treated with the rgdechi d peptide with respect to untreated cells (control) or scrambled peptides. the decrease in invasiveness was quantified and resulted in %, as indicated in the graph ( figure b ). the effect on cell adhesion, migration, and invasion, events closely correlated with the metastatic cascade, highlight promising features of this peptide in inhibiting critical steps of cancer progression. angiogenesis is required for invasive tumor growth and metastasis and constitutes a key process in the control of cancer progression, thus elucidating its tight correlation with tumor cell invasion and migration. the involvement of αvβ integrin in all these events together with its high expression in hcc tissues are well documented. since this tumor is highly aggressive and often culminates in extensive metastasis [ ] [ ] [ ] , ] , the targeting of integrin αvβ is a promising approach to improve the survival rate. the ability to inhibit the generation of new capillary blood vessels by the rgdechi d peptide was examined by an angiogenesis in vitro assay. human umbilical vein endothelial cells (huvec) angiogenesis is required for invasive tumor growth and metastasis and constitutes a key process in the control of cancer progression, thus elucidating its tight correlation with tumor cell invasion and migration. the involvement of αvβ integrin in all these events together with its high expression in hcc tissues are well documented. since this tumor is highly aggressive and often culminates in extensive metastasis [ ] [ ] [ ] , ] , the targeting of integrin αvβ is a promising approach to improve the survival rate. the ability to inhibit the generation of new capillary blood vessels by the rgdechi d peptide was examined by an angiogenesis in vitro assay. human umbilical vein endothelial cells (huvec) were used as specific system to evaluate tube assembly. the cells were incubated on a gel containing various matrix proteins such as laminin, collagen type iv, heparan sulfate. cellular network structures were already developed by h. in figure a the formation of new capillary tubes is evident and, interestingly, in the sample treated with rgdechi d results to be significantly reduced. the branch points were counted to better evaluate the percentage of inhibition. the results indicate that the rgdechi d peptide is able to inhibit the branch point formation of about % ( figure b ). were used as specific system to evaluate tube assembly. the cells were incubated on a gel containing various matrix proteins such as laminin, collagen type iv, heparan sulfate. cellular network structures were already developed by h. in figure a the formation of new capillary tubes is evident and, interestingly, in the sample treated with rgdechi d results to be significantly reduced. the branch points were counted to better evaluate the percentage of inhibition. the results indicate that the rgdechi d peptide is able to inhibit the branch point formation of about % ( figure b ). to assess if rgdechi d has cytotoxic activity besides ability to interfere with the initial stages of tumor dissemination such as adhesion, invasion and angiogenesis, we evaluated the peptide effect on cell proliferation and on related pathways. to this aim, hepg were thus incubated with the peptide at different concentrations ( - µm) for h; furthermore, h after the first incubation, one cell aliquot was incubated with a second addition of peptide ( µm). as indicated in figure , rgdechi d inhibits cell proliferation in a dose-dependent manner; in particular, at the higher concentration used, rgdechi d induces inhibition of proliferation ( %) with respect to the untreated cells. the scrambled peptide has no effect on cell proliferation. an interesting result was to assess if rgdechi d has cytotoxic activity besides ability to interfere with the initial stages of tumor dissemination such as adhesion, invasion and angiogenesis, we evaluated the peptide effect on cell proliferation and on related pathways. to this aim, hepg were thus incubated with the peptide at different concentrations ( - µm) for h; furthermore, h after the first incubation, one cell aliquot was incubated with a second addition of peptide ( µm). as indicated in figure , rgdechi d inhibits cell proliferation in a dose-dependent manner; in particular, at the higher concentration used, rgdechi d induces inhibition of proliferation ( %) with respect to the untreated cells. the scrambled peptide has no effect on cell proliferation. an interesting result was obtained by the double addition of the peptide, which results in a greater decrease in cell proliferation ( %) suggesting a probably poor serum stability of rgdechi d. akt is an important effector of cell survival whose phosphorylation determines the activation of pi k pathway triggered by integrin signaling [ ] . the effect of rgdechi d binding on the activation of the integrin receptor was analyzed in light of the results obtained with the anti-proliferative assay. for this purpose, the phosphorylation of akt was evaluated by western blotting analysis performed on hepg cell lysates after treatment with µm rgdechi d for h. in figure , a decreased signal level of pakt with rgdechi d treatment compared to not treated cell lysates can be observed. no effect on the signal is induced by scrambled peptide treatment. this result clearly suggests that rgdechi d can reduce akt phosphorylation in hepg cell line in good agreement with its capability to reduce the cell proliferation as well. akt is an important effector of cell survival whose phosphorylation determines the activation of pi k pathway triggered by integrin signaling [ ] . the effect of rgdechi d binding on the activation of the integrin receptor was analyzed in light of the results obtained with the anti-proliferative assay. for this purpose, the phosphorylation of akt was evaluated by western blotting analysis performed on hepg cell lysates after treatment with µm rgdechi d for h. in figure , a decreased signal level of pakt with rgdechi d treatment compared to not treated cell lysates can be observed. no effect on the signal is induced by scrambled peptide treatment. this result clearly suggests that rgdechi d can reduce akt phosphorylation in hepg cell line in good agreement with its capability to reduce the cell proliferation as well. molecules , , x of obtained by the double addition of the peptide, which results in a greater decrease in cell proliferation ( %) suggesting a probably poor serum stability of rgdechi d. akt is an important effector of cell survival whose phosphorylation determines the activation of pi k pathway triggered by integrin signaling [ ] . the effect of rgdechi d binding on the activation of the integrin receptor was analyzed in light of the results obtained with the anti-proliferative assay. for this purpose, the phosphorylation of akt was evaluated by western blotting analysis performed on hepg cell lysates after treatment with µm rgdechi d for h. in figure , a decreased signal level of pakt with rgdechi d treatment compared to not treated cell lysates can be observed. no effect on the signal is induced by scrambled peptide treatment. this result clearly suggests that rgdechi d can reduce akt phosphorylation in hepg cell line in good agreement with its capability to reduce the cell proliferation as well. finally, to investigate if cell death induced by the rgdechi d effect is due to the apoptosis process, a caspase- activity assay was carried out. hepg cells were treated with µm peptide for h and, as shown in the figure , the examined peptide induces a remarkable increase in caspase activity, the differently scrambled peptide does not induce apoptosis, thus suggesting that rgdechi d has a direct effect on cell viability mediated by an apoptotic pathway. these results are in good agreement with several reports demonstrating that cell detachment caused by the treatment with rgd-based peptides determines cell death by apoptosis, a process known as anoikis [ ] . treatment was carried out on hepg . cells were treated with the rgdechi d peptide for h. (a) total cellular extracts ( µg) were resolved by sds page and analyzed by western blotting with anti-pakt (ser ) and anti-akt antibodies. the western blotting is representative of three independent experiments. (b) bar graph illustrates the percentage of band quantification respect to the control. = control, = rgdechi d, = scrambled peptide. the results are expressed as mean ± se of percentage of pakt normalized to the density of akt. finally, to investigate if cell death induced by the rgdechi d effect is due to the apoptosis process, a caspase- activity assay was carried out. hepg cells were treated with µm peptide for h and, as shown in the figure , the examined peptide induces a remarkable increase in caspase activity, the differently scrambled peptide does not induce apoptosis, thus suggesting that rgdechi d has a direct effect on cell viability mediated by an apoptotic pathway. these results are in good agreement with several reports demonstrating that cell detachment caused by the treatment with rgd-based peptides determines cell death by apoptosis, a process known as anoikis [ ] . figure . apoptosis assay. caspase- activity on hepg was determined after incubation with peptides ( µm) for h. caspase- activity (calculated as nmol -amino- -trifluoromethylcoumarin (afc)/min/µg protein) was expressed as the percentage of caspase- activity with respect to control (untreated cells). the results are expressed as means ± se of at least three independent experiments performed in triplicate. statistical significance was analyzed using student's t test, unpaired, twosided (* p < . ). here, we demonstrate that in hepg cell line the rgdechi d peptide shows an interesting effect in some steps of the metastatic pathway. in addition, the peptide displays cytotoxic activity, ascribable to the interference with the pi k pathway, mediated by an apoptotic process, in accordance with other rgd based peptides that, through cell detachment, send cells into apoptosis. collectively, the obtained results give indications about a potential role of rgdechi d as a selective ligand for hcc targeting. moreover, considering that αvβ is an interesting player in different pathological events, ranging from tumor to viral diseases, selective antagonists could be considered appealing candidates for the treatment of a wide spectrum of disorders. scrambled peptide rgdechi d caspase- activity (% vs control) * figure . apoptosis assay. caspase- activity on hepg was determined after incubation with peptides ( µm) for h. caspase- activity (calculated as nmol -amino- -trifluoromethylcoumarin (afc)/min/µg protein) was expressed as the percentage of caspase- activity with respect to control (untreated cells). the results are expressed as means ± se of at least three independent experiments performed in triplicate. statistical significance was analyzed using student's t test, unpaired, two-sided (* p < . ). here, we demonstrate that in hepg cell line the rgdechi d peptide shows an interesting effect in some steps of the metastatic pathway. in addition, the peptide displays cytotoxic activity, ascribable to the interference with the pi k pathway, mediated by an apoptotic process, in accordance with other rgd based peptides that, through cell detachment, send cells into apoptosis. collectively, the obtained results give indications about a potential role of rgdechi d as a selective ligand for hcc targeting. moreover, considering that αvβ is an interesting player in different pathological events, ranging from tumor to viral diseases, selective antagonists could be considered appealing candidates for the treatment of a wide spectrum of disorders. human hepatocarcinoma cell line (hepg ) (atcc u.s.) were grown in dmem supplemented with % fetal bovin serum (fbs), % glutamine, u/ml penicillin and µg/ml streptomycin (euroclone, milan, italy). human umbilical vein endothelial cells (huvec) were purchased from lonza. cells were grown in endothelial cell growth medium (egm- ) from lonza, (basel, switzerland). all experiments were performed using low passage cell cultures [ ] . chronic myelogenous leukemia cells line (k ) (atcc, manassas, va, usa.) were grown in rpmi with added heat-inactivated % fbs (fetal bovine serum), mm glutamine, u/ml penicillin and µg/ml streptomycin (euroclone, milan, italy). the cells were maintained in humidified air containing % co at • c. for facs analysis adherent cells at about % confluence were detached using . mm edta in pbs (sigma aldrich, darmstads, germany), centrifuged and re-suspended in pbs containing . % bsa. cell aliquots ( . × cells) were treated with primary monoclonal antibody pe conjugate, (millipore, burlington, ma, usa) or isotype control (santa cruz biotechnology, dallas, tx, usa), at the same concentration ( µg/ml), whole a reaction volume of µl for min at • c. after washing, the cells were analyzed by using a flow cytometer equipped with a nm argon laser (facscan, becton dickinson, franklin lakes, nj, usa). a total of , events per sample were collected. integrin quantification with quantibrite pe beads (bectondickinson biosciences) was evaluated as previously described [ ] . values of fluorescence intensity were obtained from the histogram statistic of cellquest software. the effect of peptides on hepg or k adhesion was tested on nunc maxisorp well plates (dasit sciences, milan, italy) coated with µg/ml vitronectin (for hepg ), fibronectin or anti-α β antibody (for k ) (millipore, burlington, ma, usa). , cells/well were incubated in the presence of peptides ( µm) or antibodies ( µg/ml) at • c for min as previously reported [ ] and then cells were seeded on coated plates for h. non adherent cells were gently removed by repeated washing and adherent cell number was evaluated by crystal violet (sigma aldrich) assay, which correlates optical density with cell number. for the ic calculation, the peptides were added at different concentration starting from µm to µm. the mean value ± standard error (se) of adherent cells for each treatment was expressed as relative percentage of cell number vs. untreated cells (control). statistical differences were determined by student's t test, paired, two-sided. all experiments were performed in triplicate and repeated at least times; a p value < . was considered to be significant. the ic value was calculated by graphpad prism software. hepg cells were cultured in well plates until they reached the confluence, and were linearly scratched with a plastic pipette tip to create a wound [ ] . after washing with pbs, necessary to remove loose cells, the medium containing µm of peptides was then added, and cells were incubated at • c in a humidified incubator in % co . each scratch area was photographed at , , and h. the wound size for the different peptides was calculated as width at each end-points with respect to their value at h as follows: wound closure (%) = − (wound width t = x/wound width t = ) × . cell invasion was assayed using transwell chambers coated with ecl cell attachment matrix (millipore corporation) used according to manufacturer's instructions. two duplicates were set for the assay. cells at the logarithmic phase were detached using mm edta in pbs and washed twice with serum-free dmem. a total of × cells/ µl were seeded in the upper chamber. in the lower chamber, µl of dmem medium containing % fetal bovine serum was added for incubation at • c in a % co incubator for h. after removal of the upper chamber, they were washed twice with pbs and migrated cells were fixed with % formalin for min. next, the cells on the bottom surface of the membrane were stained with crystal violet for min. the cells which did not migrate were removed with cotton swabs [ ] . cell images were obtained under a phase contrast microscope (zeiss, oberkochen, germany) and the cells were counted by axiovert software (zeiss). transwell migration assays were repeated three times and performed in duplicate. all measurement data are presented as the mean ± se. statistical analysis was performed using a t-test where p < . was considered to indicate a statistically significant difference. ecmatrix tm solution ( µl) was plated in a pre-cooled well plate and incubated for h at • c. huvec cells were harvested, incubated ( cells) with µm peptide and seeded onto solidified matrix. after h of incubation the tube formation was inspected under an inverted light microscope at × magnification [ ] . imagines were acquired by axiovert zeiss microscopy fluorescence. the experiments were performed two times in duplicate. the effect of peptides on hepg proliferation was analyzed with an rtca icelligence™ instrument (acea, san diego, ca, usa) with a method that uses label-free, real time, non-invasive analysis [ ] . the instrument records a signal of electrical impedance due to the cell covering of gold electrodes located on the surface of a special microplate. the variation of the electrical impedance is named cell index (ci). ci values are proportional to the area of cells adherent and, as a consequence, to their numbers [ ] . in detail, , cells/well were seeded and incubated in the presence of peptides for h. the ci was calculated by measuring the slope of the proliferation curve between two selected time points for each curve, considering that the maximum ci values observed is the end-point of the experiments. statistical differences were determined by student's t test, paired, two-sided. all experiments were performed in triplicate and repeated at least three times; a p value less than . was considered to be significant. hepg cells were incubated with peptides ( µm) for h at • c. whole cell lysates were obtained by using lysis buffer ( mm hepes, ph . , mm nacl, % triton) supplemented with phosphatase (sigma aldrich, milan, italy) and protease inhibitor cocktails (roche, milan, italy). cell lysates were incubated on ice for min and then centrifuged at . rpm for min to remove cell debris. protein concentrations were determined by the bradford method using bio-rad reagent (bio-rad laboratories, hercules, ca, usa). proteins ( µg) were resolved by sds-polyacrylamide gel electrophoresis (sds-page) and transferred to a pvdf membrane (millipore). the membrane was probed with the primary antibodies (anti pakt (ser ) or anti akt (cell signaling technologies, boston, ma, usa)) over night at • c. proteins were visualized with an enhanced chemiluminescence detection system (euroclone, milan, italy) and images were acquired with chemidoc xrs system (bio-rad laboratories, italy) and analyzed with the quantityone software. reblot was performed using a harsh stripping buffer containing % sds, mm tris hcl, ph . , . % β-mercaptoethanol, incubating the membrane at • c for min with some agitation. determination of caspase- activity was performed by a fluorometric assay based on the proteolytic cleavage of the carbobenzoxy-asp-glu-val-asp- -amino- -methylcoumarin (acetyl-devd-afc alexis biochemicals, san diego, ca, usa) as described elsewhere [ ] . in detail, hepg were treated with the peptides ( µm) for h at • c. after h, cells were processed with reaction buffer ( mm hepes, ph . , . mm edta, . % nonidet p- , . % chaps and mm dtt) and µg of lysates were incubated with µm ac-devd-afc at • c for h. samples were analyzed using a microplate reader (biotek, winooski, vt, usa) (excitation wavelength nm; emission wavelength nm). an afc standard curve was determined, and caspase-specific activity was calculated as nmol of afc produced per min per µg proteins at • c at saturating substrate concentration ( µm). the diverse roles of integrins and their ligands in angiogenesis purification and functional characterization of integrin alpha v beta . an adhesion receptor for vitronectin ligand binding to integrins beyond rgd: virus interactions with integrins beta integrin is the major contributor to the alphavintegrin-mediated blockade of hiv- replication integrin alphavbeta internalizes zika virus during neural stem cells infection and provides a promising target for antiviral therapy a multi-targeting approach to fight sars-cov- attachment definition of two angiogenic pathways by distinct alpha v integrins integrin alphavbeta regulates lung vascular permeability and pulmonary endothelial barrier function src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta in vascular endothelial growth factor signaling elevated expression of integrin alphav and beta subunit in laryngeal squamous-cell carcinoma associated with lymphatic metastasis and angiogenesis alphavbeta -integrins mediate early steps of metastasis formation significance of integrin alphavbeta and erbb in enhanced cell migration and liver metastasis of colon carcinomas stimulated by hepatocyte-derived heregulin mesenchymal to epithelial transition in development and disease inflammation and emt: an alliance towards organ fibrosis and cancer progression role of beta -integrin in epithelial-mesenchymal transition in response to tgf-beta p -activated kinase interacts with integrin alpha v beta and regulates alpha v beta -mediated cell migration increased expression of the coxsackie and adenovirus receptor downregulates alphavbeta and alphavbeta integrin expression and reduces cell adhesion and migration integrin beta contributes to the tumorigenic potential of breast cancer cells through the src-fak and mek-erk signaling pathways integrin targeted drug and gene delivery practice guidelines committee, management of hepatocellular carcinoma early and late recurrence after liver resection for hepatocellular carcinoma: prognostic and therapeutic implications tumour exosome integrins determine organotropic metastasis integrin-beta , a mir- -targeted gene, promotes hepatocellular carcinoma tumorigenesis by regulating beta-catenin stabilit elevated serum plasma fibrinogen is associated with advanced tumor stage and poor survival in hepatocellular carcinoma patients. medicine (baltimore) , , e hepatic stellate cells and the reversal of fibrosis fibrinogen induces cytokine and collagen production in pancreatic stellate cells expression, regulation, and function of alpha v integrins in hepatocellular carcinoma: an in vivo and in vitro study activation of hepatic stellate cells during liver carcinogenesis requires fibrinogen/integrin alphavbeta in zebrafish human integrin alphavbeta : homology modeling and ligand binding deciphering rgdechi peptide-α β integrin interaction mode in isolated cell membranes novel and selective alpha(v)beta receptor peptide antagonist: design, synthesis, and biological behavior conformational studies of rgdechi peptide by natural-abundance nmr spectroscopy imaging of alpha(v)beta( ) expression by a bifunctional chimeric rgd peptide not cross-reacting with alpha(v)beta( ) a combined nmr and computational approach to determine the rgdechi-hcit-alphav beta integrin recognition mode in isolated cell membranes anticancer effects of echinacoside in hepatocellular carcinoma mouse model and hepg cells affinity of integrins for damaged extracellular matrix: alpha v beta binds to denatured collagen type i through rgd sites matrix metalloproteinase and alphavbeta integrin-dependent vascular smooth muscle cell invasion through a type i collagen lattice dioscin inhibits the invasion and migration of hepatocellular carcinoma hepg cells by reversing tgf-beta -induced epithelial-mesenchymal transition cross-talk mechanism between endothelial cells and hepatocellular carcinoma cells via growth factors and integrin pathway promotes tumor angiogenesis and cell migration specific beta integrin site selectively regulates akt/protein kinase b signaling via local activation of protein phosphatase a rgdechi-hcit: alphavbeta selective pro-apoptotic peptide as potential carrier for drug delivery into melanoma metastatic cells unveiling a vegf-mimetic peptide sequence in the iqgap protein functional binding surface of a beta-hairpin vegf receptor targeting peptide determined by nmr spectroscopy in living cell chemical modification for proteolytic stabilization of the selective alphavbeta integrin rgdechi peptide: in vitro and in vivo activities on malignant melanoma cells therapeutic potential of a novel alphavbeta( ) antagonist to hamper the aggressiveness of mesenchymal triple negative breast cancer sub-type a selective αvβ integrin antagonist hidden into the anophelin family protein ce from the malaria vector anopheles gambiae application of real-time cell electronic sensing (rt-ces) technology to cell-based assays this article is an open access article distributed under the terms and conditions of the creative commons attribution we are grateful to maurizio amendola (institute of biostructures and bioimaging-cnr, naples) for technical assistance and luca de luca (institute of biostructures and bioimaging-cnr, naples) for computer assistance. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -ejvv lxv authors: bowdish, d. m. e.; davidson, d. j.; hancock, r. e. w. title: immunomodulatory properties of defensins and cathelicidins date: journal: antimicrobial peptides and human disease doi: . / - - - _ sha: doc_id: cord_uid: ejvv lxv host defence peptides are a conserved component of the innate immune response in all complex life forms. in humans, the major classes of host defence peptides include the α- and β-defensins and the cathelicidin, hcap- /ll- . these peptides are expressed in the granules of neutrophils and by a wide variety of tissue types. they have many roles in the immune response including both indirect and direct antimicrobial activity, the ability to act as chemokines as well as induce chemokine production leading to recruitment of leukocytes to the site of infection, the promotion of wound healing and an ability to modulate adaptive immunity. it appears that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, t cells and epithelial cells. the importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides showgreater susceptibility to bacterial infections. in the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents. that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, t cells and epithelial cells. the importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides show greater susceptibility to bacterial infections. in the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents. human neutrophil peptide hbd human beta defensin tlr toll-like receptor lps lipopolysaccharide lta lipoteichoic acid tnf-α tumour necrosis factor alpha mhc major histocompatibility complex class bal bronchoalveolar lavage overview host defence peptides are small (generally less than amino acids), positively charged peptides that are an evolutionarily conserved component of the innate immune response. originally characterised as natural antimicrobial agents, it is becoming increasingly apparent that these peptides have a wide range of immunomodulatory properties that are either complementary to, or independent of, antimicrobial activity. interest in the immunomodulatory functions of these peptides is increasing, and indeed many peptides and proteins with similar characteristics to host defence peptides have been found to have either antimicrobial or immunomodulatory properties in addition to their primary functions. in humans the best-characterised host defence peptides are the defensins and the sole cathelicidin, hcap- /ll- . the amino acid sequences of these peptides are summarised in table . in general, the defensins are between and amino acids long (approximately . kda) and contain three conserved disulphide bridges (white et al. ) . the genes for all human defensins are clustered on chromosome (sparkes et al. ; maxwell et al. ) . they are further subdivided to include the αand β-defensins, a distinction based on the organisation of the three characteristic cystine disulphide bonds. the canonical sequence of the α-defensins is x - cxcrx - cx ex gxcx gx ccx - , where x represents any amino acid residue. these peptides are rich in cysteine, arginine, and aromatic residues . the cysteines are linked - , - , and - . initially these peptides were isolated from the neutrophils and are thus called human neutrophil peptides (hnp)- to - . the hnps are expressed at the transcriptional level in the bone marrow, spleen and thymus, where they co-localise with peripheral blood leukocytes (zhao et al. ) . the three hnps are highly homologous, differing by only one amino acid at the nh terminus. because of the high sequence similarity and difficulties in purifying the individual peptides as well as the high degree in functional similarity, the hnp - are often studied as a group, although certain studies have demonstrated differences in their antimicrobial ) and immunomodulatory activities (chertov et al. ) . hnp was identified as a hnp due to its structural homology to the hnp - (wilde et al. ). this gene differs from the other genes of this family by an extra -base pair segment that is apparently the result of a recent duplication within the coding region (palfree et al. ) . as with the other hnps, hnp is found in the neutrophils, but is also called corticostatin because it exhibits corticostatic activity and inhibits corticotrophin-stimulated corticosterone production (singh et al. ) . despite its variation from the conserved sequences of hnp - , it appears to have much more potent antimicrobial activity (wilde et al. ) . two other α-defensins, hd and hd , are found solely in the intestinal tract. hd and hd were found to be expressed at the transcriptional level solely in the small intestine and in situ hybridisation demonstrated that this expression occurs in the paneth cells bevins , ) . southern blot analysis using a nucleotide probe for the conserved signal sequence of the defensins indicated that a number of genes with high homology to hnps exist within the human genome. the β-defensins are expressed in a variety of tissue types, including epithelial cells from the trachea and lung, in the salivary and mammary glands, in a variety of organs such as in the plasma and skin (bensch et al. ; zhao et al. ; harder et al. ; reviewed in lehrer and ganz ) . the expression of certain β-defensins is inducible upon stimulation with bacterial components or pro-inflammatory cytokines and thus these peptides are presumed to be an important component of host defence to infection or inflammation. the canonical sequence for the beta defensins is x - cx - (g/a) x cx - cx - c x - ccx n . the best characterised members of the β-defensin family are hbd - ; however, the antimicrobial properties of hbd have been recently published (garcia et al. ) and over potential β-defensin homologues have been identified in the human genome based on sequence similarity to hbd - (schutte et al. ) . the cathelicidins are an evolutionarily conserved family of host defence peptides which are found in cows, sheep, guinea pigs, rabbits, mice, and primates (reviewed in zanetti ) and are characterised by having an evolutionarily conserved n-terminal domain called the cathelin domain. in addition, these peptides have a signal sequence, which is believed to target their delivery to the secondary granules of neutrophils. the c terminal domain, which is released by cleavage of proteases, has both antimicrobial and immunomodulatory properties. despite the conserved nature of the cathelin domain, its function remains unclear, although it has been proposed to block the antimicrobial activity of the cleaved product, presumably as a mechanism which allows storage of the peptide in its inactive form , and there is some evidence that it has anti-protease activity . the sole human cathelicidin ll- /hcap- is found at high concentrations in its unprocessed form in the granules of neutrophils and is processed upon degranulation and release (sorensen et al. ) . consequently, it is found at sites of neutrophil degranulation. it is also produced by epithelial cells and is found in a number of tissues and bodily fluids, including gastric juices, saliva, semen, sweat, plasma, airway surface liquid and breast milk (bals et al. c; murakami et al. b; hase et al. ; murakami et al. ) . generally epithelial cells have been shown to produce the hcap- form. although the hcap- has been shown to be cleaved by the neutrophil protease, protease , when released from neutrophils, it is not entirely clear how or when hcap- is cleaved when it is produced by epithelial cells. there are a variety of processed forms of hcap- that result from as-yet uncharacterised cleavage processes. for example, a -kda form is found in gastric juice (hase et al. ) , while numerous cleavage products are found in the sweat (murakami et al. b) . as well, hcap- from semen is cleaved to a -amino acid antimicrobial peptide all- in the vagina, thus potentially providing some antimicrobial protection after sexual intercourse ). there appears to be some overlapping, complementary and possibly even enhanced antimicrobial activity of these isoforms (murakami et al. ) ; however, to date there is no information about their immunomodulatory properties. due to the high homology of hnp - , they are often classed as a group. the hnps are found exclusively in leukocytes and at their highest concentrations in neutrophils where they are localised to azurophilic granules. the recently described hnp is also found in azurophilic granules but at much lower concentrations (wilde et al. ) . to date there has been no indication that their expression levels can vary substantially. neutrophils stimulated with il- , fmlp or phorbol -myristate -acetate causes degranulation and release of hnp - (chertov et al. ) , thus it is believed that relatively high concentrations of these peptides are present at sites of infection or inflammation. interestingly, in the course of infection hnp levels increase systemically. although the mechanism is not entirely clear, the plasma levels of defensins increase significantly in patients experiencing septicaemia or meningitis (from approximately ng/ml to as much as , ng/ml). the concentration of neutrophil defensins generally correlates with that of il- , a potent chemoattractant for neutrophils (ashitani et al. ) , as well as the presence of other neutrophil components such as elastase (zhang et al. ) . presumably this occurs due to neutrophil degranulation in response to bacterial or pro-inflammatory stimuli as concentrations decrease after antibiotic treatment (panyutich et al. ) . hd and hd are expressed throughout the gastrointestinal tract, with the highest levels of expression occurring in the jejunum and ileum (dhaliwal et al. ) . specifically, immunohistochemical studies have shown that hd is expressed by paneth cells, some villous epithelial cells and in the terminal ileal mucosa (cunliffe et al. ) . although there is great variability between individuals, levels of these defensins are increased in certain disease states such as acute coeliac sprue (frye et al. b) and are decreased in others such as hiv-related cryptosporidiosis (kelly et al. ). in patients with crohn's disease or ulcerative colitis, but not in healthy individuals, hd immunoreactive cells are present in the crypt region of a large proportion of colonic samples, indicating that expression in these disease states might be dysregulated (cunliffe et al. ) . limited studies have demonstrated that hd and hd may also be present at low levels in airway epithelial cells (frye et al. a) or the female reproductive tract (quayle et al. ). the patterns of expression of the β-defensins are markedly different. in general hbd is not up-regulated during the course of infection or inflammation or by stimulation with pro-inflammatory cytokines or bacterial components and in many cases the presence of hbd is detectable at the transcriptional level but is undetectable at the protein level (o'neil et al. ; frye et al. b) . however its constitutive presence in airway surface liquid, in intestinal and colon cell lines and in other tissues and bodily fluids implies that it may be involved in maintenance and homeostasis of these areas (salzman et al. ) . the expression levels of hbd appear to be quite low, ranging from the pg/ml to ng/ml levels in most bodily fluids. hbd is an inducible host defence peptide whose expression is altered under both infectious and inflammatory conditions. it has been found to be up-regulated by pro-inflammatory stimuli in oral epithelial cells and keratinocytes (krisanaprakornkit et al. ) , in intestinal and colonic epithelial cell lines (o'neil et al. ; ogushi et al. ; vora et al. ) and in various lung epithelial cell lines (singh et al. ; becker et al. ) . hbd is believed to be an important component of host defences, since hbd expression is depressed in patients with atopic dermatitis who often present with cases of acute and chronic colonisation by staphylococcus aureus (ong et al. ) but is increased in psoriatic skin, a disease in which patients are fairly resistant to bacterial infection. it has been demonstrated to be upregulated by both commensal and pathogenic bacteria in the oral mucosa and keratinocytes, although by different mechanisms (krisanaprakornkit et al. ; chung and dale ) . hbd is inducible during the course of inflammation and infection in the gastrointestinal system, as observed at both the mrna and protein levels. hbd expression in intestinal and colonic epithelial cell lines is increased upon stimulation with il- α, flagellin or bacteria, in an nf-κb-dependent manner (o'neil et al. ; ogushi et al. ) , and by either lipopolysaccharide (lps) or lipoteichoic acid (lta) in a tlr and tlr -dependent manner (vora et al. ) . interestingly, other inflammatory mediators such as tnf-α and lps do not induce hbd up-regulation (o'neil et al. ) . this may reflect a predominantly intracellular expression pattern of tlr in these cells, which has been suggested to be an evolutionary adaptation to the high bacterial load in the intestine (naik et al. ; hornef et al. ) . increases in hbd expression have been detected in inflamed intestinal and colonic tissues by rt-pcr and immunohistochemistry, in crohn's disease and ulcerative colitis (fahlgren et al. ) , and in the stomach of patients suffering from helicobacter pylori-induced gastritis (wehkamp et al. ) . in the lung, hbd has been found to be down-regulated in a variety of infectious and inflammatory diseases including cystic fibrosis and infections (chen et al. ) . in lung transplant patients, it has been found to be present at greater than ten times the concentration in patients suffering from bronchiolitis obliterans syndrome, a consequence of rejection of the transplant, compared to transplant patients without signs of rejection (ross et al. ) . less is known about the newly characterised hbd . hbd expression and activity is not well characterised; however, it has been demonstrated to be inducibly expressed at the transcriptional level in bronchial epithelial cell lines stimulated with tnf-α, bacteria, or live rhinovirus (harder et al. ; duits et al. ). in addition, this peptide can be induced in amnion cells in response to bacterial components and is found at high concentrations in human fetal membranes, by immunohistology, indicating that it may be involved in maintaining the sterility of the intra-amniotic environment (buhimschi et al. ) . the pro-cathelicidin hcap is found at high concentrations in the granules of neutrophils and is produced by epithelial cells. the camp-gene promoter that directs the expression of this peptide contains many transcription factor binding sites (larrick et al. ) , including a vitamin d response element (wang et al. ) . consistent with this, hcap , and/or its processed product ll- , has been shown to be up-regulated in sinus epithelial cells, at the transcriptional level, by the bacterial products lps and lta (nell et al. ) . it is also increased in bronchial airway cells by il-α (erdag and morgan ) . in other cell types, pro-inflammatory cytokines do not increase the expression of this peptide, implying that other signalling pathways may be involved (hase et al. ). its expression is increased in gastric epithelial cells upon stimulation with a wild-type strain of h. pylori, but not a type iv secretion mutant (hase et al. ) , and is found at increased levels in other forms of bacterial infection. it is not entirely clear under what circumstances neutrophils release ll- . the antimicrobial activity of all host defence peptides is highest in media of low ionic strength, and the activity of most peptides is sensitive to the presence of physiological concentrations of ions such as na + , mg + and ca + . the antimicrobial properties of the β-defensins, for example demonstrate profound salt sensitivity, and in some cases their antimicrobial activity is completely lost at concentrations of mm nacl (bals et al. a (bals et al. , b garcia et al. ) . for example, hbd is antimicrobial towards gram-negative bacteria at concentrations of - µg/ml (singh et al. ), but its antimicrobial activity is almost completely abrogated by the presence of mm sodium ions. although hbd is slightly less sensitive to the presence of sodium ions, its antimicrobial activity is reduced from . - . µg/ml in conditions of low ionic strength to . µg/ml or more in the presence of mm sodium (singh et al. ) . of the human β-defensins, hbd is the most potent antimicrobial. this peptide is more basic, has a broader spectrum and stronger bactericidal activity against gram-positive and gram-negative bacteria, as well as yeast, and is salt-insensitive at concentrations less than mm na + ions (harder et al. ) . most biological fluids, including sputum (halmerbauer et al. ) , airway surface liquid (baconnais et al. ) and serum/plasma (hoshino et al. ) contain mg + and ca + at free concentrations between and mm, and the presence of these ions is generally more detrimental to antimicrobial activity than na + alone. the α-defensins are susceptible to concentrations of ca + and mg + as low as . mm ). in the presence of mm na + ions, the antimicrobial activity of ll- is decreased two-to eightfold (turner et al. ) , and in the presence of standard tissue culture media, which contains mm nacl and - mm of mg + and ca + , ll- has no killing activity against s. aureus or salmonella typhimurium even at concentrations as high as µg/ml . in some cases, for example in the granules of neutrophils, the concentrations of host defence peptides are estimated to be as great as mg/ml, and there is no doubt that upon ingestion of bacteria these concentrations are sufficient to cause direct antimicrobial activity, despite the presence of divalent cations or other inhibitory substances. however, it is questionable whether these concentrations are reached at mucosal surfaces. for example, in patients suffering from inflammatory lung disease or infection, the concentration of hnps in the bronchoalveolar lavage (bal) has been estimated to be . - . and µg/ml in two different studies (cole et al. ; spencer et al. ). this contrasts with antimicrobial activity in low salt medium in vitro at greater than µg/ml for s. aureus and escherichia coli (nagaoka et al. ) , and reduction of the infectivity of adenoviruses in an airway epithelial model at concentrations of between - µg/ml (spencer et al. ) . interestingly, the antimicrobial activity of the host defence peptides may also be inhibited by components of serum. for example, hnp has also been demonstrated to possess antiviral activity towards enveloped viruses, but this activity is abrogated by the presence of serum or albumin (daher et al. ). it is believed but has not been conclusively shown that the high concentrations of α-defensins in neutrophils would overcome any localised serum effects (daher et al. ). moreover, the antibacterial activity of ll- has been demonstrated to be abrogated by the presence of apolipoprotein . in contrast to the antimicrobial activity of these peptides, their immunomodulatory properties are generally studied in the presence of standard tissue culture media, which contains physiologically relevant concentrations of ions and serum proteins. under these conditions, host defence peptides have been demonstrated to induce chemokine production, reduce pro-inflammatory cytokine production, alter transcription, induce proliferation and angiogenesis, induce chemotaxis and alter dendritic cell differentiation. thus the immunomodulatory properties of host defence peptides are unaffected by physiological ion concentrations and it seems possible that they may be the predominant function of these peptides in vivo. this perspective is quite controversial, and will remain so since it is extremely difficult to discriminate between direct and indirect (i.e. through stimulation of innate immunity) mechanisms of killing. one often used argument for the likely antimicrobial function of these peptides is their substantial variation over evolution (e.g. the mouse cramp and human ll- peptides share only % homology), which could have arisen from the evolutionary pressure of dealing with different pathogens. however, not all antimicrobial proteins are as divergent, and we note that a number of proteins involved in immunity and reproduction show similar "rapid evolution" to the antimicrobial peptides (emes et al. ). there has been some debate about whether host defence peptides might be directly antimicrobial in vivo. certainly, neutrophil granules and the crypts of the lumen contain sufficiently high concentrations of peptides to ensure substantial antimicrobial activity; however, it is less clear that antimicrobial activity occurs at the lower concentrations in such sites as mucosal surfaces, and it is worth noting that such sites are often heavily colonised by a rich and diverse collection of commensal bacteria. the evidence for antimicrobial activity in certain body sites is in our opinion inconclusive. on the one hand, certain bodily fluids such as sinus fluid (cole et al. ) and gastric fluids (hase et al. ) can directly kill certain micro-organisms, and this antimicrobial activity is ablated or reduced by removal of proteins or immunodepletion with a peptide-specific antibody. however, in certain animal models in which peptides and bacteria are instilled simultaneously, bacterial counts are often not significantly different from mice treated with bacteria alone, despite improved outcome or reduced pro-inflammatory responses (sawa et al. ; giacometti et al. ) . the difficulties in assessing the role of host defence peptides in vivo are profound, as it is almost impossible to account for synergistic interactions between peptides and other factors, to assess the actual concentrations at the sites of infection and to discriminate the direct antimicrobial activity of peptides from other less direct effects such as enhancement of inflammatory mechanisms (chemotaxis and recruitment of effector cells, enhancement of nonopsonic phagocytosis, etc.). nonetheless, creative experiments and animal models have begun to elucidate the roles of these peptides in vivo. in transgenic mouse model studies in which the expression of certain host defence peptides is ablated, these mice are somewhat more susceptible to infection and carry increased bacterial loads when challenged nizet et al. ) . although this was interpreted as being due to direct antimicrobial activity, other components of host defences must be considered. for example, in a mouse model of peritoneal klebsiella pneumoniae infection, small doses of hnp ( ng- µg) caused an increase in leukocyte accumulation. in this model, it was the leukocyte accumulation which was linked to hnp induced antimicrobial activity, as the reduction in bacterial counts was significantly diminished in leukocytopenic mice. similar results were observed in s. aureus thigh infections (welling et al. ) . gain of function studies have found that introducing or increasing the expression of a host defence peptide can reduce bacterial loads in certain animal models of infection. for example, adenovirus transfer of ll- /hcap- into the lungs of mice that were subsequently challenged with pseudomonas aeruginosa led to a reduction in both the bacterial load and in production of the pro-inflammatory cytokine, tnf-α (bals et al. ) , and intriguingly, similar gene therapy decreased susceptibility to sepsis induced by lps in the complete absence of bacterial infection. in other models, the simultaneous instillation into the mouse lung of p. aeruginosa and either of hbd or a ll- derivative led to reduced lung damage and pro-inflammatory cytokine production, but did not affect bacterial counts (sawa et al. ). there are very few human diseases that are characterised by defects in host defence peptide production, perhaps emphasizing their importance. however, the neutrophils of individuals with specific granule deficiency, a disease characterised by frequent and severe infections, have a reduction in the size of the peroxidase positive, defensins-containing granules (parmley et al. ) and are deficient in defensins ). however, it is difficult to assess the extent to which these infections result from the lack of defensins, as these patients are also deficient in other neutrophil components. it is believed that the constitutive production and deposition of neutrophils is of crucial importance to maintaining the immunological balance of the mouth. patients who suffer from morbus kostman, a severe congenital neutropenia, and are treated with g-csf to restore neutrophil level, do not express ll- in these cells. one of the manifestations of this disease is frequent and severe infections and periodontal disease (putsep et al. ) . it has been proposed that the absence of ll- may give a selective advantage to bacteria that at low levels are commensals but at higher levels are responsible for periodontal disease. it is unclear, however, whether ll- is directly microbicidal towards common pathogens of the mouth or marshals other defences. although a number of oral bacteria are susceptible to ll- (< µg/ml) at mm nacl in vitro, far fewer bacteria are susceptible in physiologically more relevant isotonic environments (tanaka et al. ) . although ll- has been detected in saliva, the actual concentration was not determined (murakami et al. a) . other indirect evidence for the in vivo antimicrobial activity of host defence peptides is that a decreased level of expression often correlates with frequency or severity of disease. for example, hbd and ll- expression is depressed in patients with atopic dermatitis who often present with cases of acute or chronic colonisation by s. aureus (ong et al. ) . in contrast to atopic dermatitis, hbd expression is increased in psoriatic skin, a disease in which patients are fairly resistant to bacterial infections (harder et al. ; nomura et al. ) . whether host defence peptides are directly or indirectly antimicrobial, it is apparent that it is of advantage for bacterial pathogens to subvert their expression or activity. for example, streptococcus pyogenes binds to α -microglobin and secretes a small proteinase which inhibits ll- from interacting with the bacteria and thus prevents ll- mediated killing (nyberg et al. ). ll- expression has been shown to be decreased in shigella infection, consistent with a proposed mechanism of evasion by this bacterium (islam et al. ) . however, it is not clear whether this is a direct down-regulation of expression, or a consequence of denuding the epithelium, with reduced expression in the replacement cells. early experiments with host defence peptides demonstrated that many of these peptides have mitogenic effects on a variety of cells and cell lines. since modest to high concentrations of host defence peptides are found at sites of infection and inflammation, it has been hypothesised that this proliferative effect might be involved in wound healing and re-epithelisation. consistent with this hypothesis, both the human and mouse cathelicidins are up-regulated at sites of incision or wounding, even if the wound is sterile. the appearance of cathelicidins in the skin has been ascribed to both synthesis within epidermal keratinocytes, and deposition from granulocytes that migrate to the site of injury . upon incision, hcap- (the precursor to ll- ) has been shown to be up-regulated in the epidermis bordering the wound. this increase in expression at both the rna and protein levels was clearly evident at the migrating front of the wound during re-epithelialisation. levels of hcap- decreased following wound closure and eventually returned to baseline levels when the wound was intact and reepithelisation was complete. hcap- was found to be an active component in the process of re-epithelisation since antibodies specific for the peptide decreased the rate of re-epithelisation in a concentration-dependent manner (heilborn et al. ) . consistent with this observation, low levels of ll- (as low as ng/ml) have been demonstrated to increase proliferation in an endothelial cell line (koczulla et al. ) . the importance of this peptide in re-epithelisation has been further inferred from its presence in wounds which are healing normally, but its absence in chronic ulcers (heilborn et al. ) . hnps are potent mitogens for epithelial cells, squamous cell carcinoma cell lines and fibroblasts in vitro at low concentrations (murphy et al. ; . interestingly, in one of the earliest studies of these effects it was demonstrated that the hnps acted synergistically with insulin to induce proliferation (murphy et al. ) . in general it has been hypothesised that the mitogenic properties of the neutrophil defensins on non-myeloid cells is an important component of the healing process. however, certain tumours and tumour cell lines have been demonstrated to inappropriately express neutrophil defensins, and in such cases it is believed that this expression might lead to inappropriate proliferation. for example, in a renal carcinoma cell line, the α-defensins hnp - are expressed at both the transcriptional and protein levels. at moderate levels (i.e. ≤ µg/ml), the defensins had mitogenic activity on a subset of these cell lines. by influencing tumour cell proliferation, α-defensins could potentially modulate tumour progression of renal carcinoma cells (muller et al. ) . moderate concentrations (e.g. ≤ µg/ml ) of neutrophil defensins (hnp - ) induce proliferation of a lung epithelial cell line in vitro (aarbiou et al. ) . consistent with these observations, a combination of hnp - caused a dose-and time-dependent increase in cell migration and wound closure of an airway epithelial cell line, possibly due to an ability to induce the expression of genes involved in proliferation (aarbiou et al. ). the mitogenic activity of the hnps and cathelicidins does not appear to be shared by the β-defensins. although hbd has been demonstrated to be up-regulated in chronic ulcers (butmarc et al. ) , it has not been demonstrated to be involved in re-epithelisation. in addition, the β-defensins investigated did not increase the proliferation of epithelial cells, squamous cell carcinoma cell lines or fibroblasts (m. ). an interesting phenomenon which has been observed to occur in response to two cathelicidins, human ll- and its mouse homologue cramp, is the induction of angiogenesis, which is the process of blood vessel formation and/or growth. the formation of new blood vessels results in restoration of tissues increasing the oxygen supply and the provision of blood substances and cells to these tissues. as such it is a requirement for tissue repair and wound healing as well as for the marshalling of innate immunity. thus this function is consistent with a role for host defence peptides in the maintenance and repair of tissues. in a chorioallantoic membrane assay, µg of ll- induced an increase in blood vessel growth, while in a rabbit hind limb model of angiogenesis, collateral vessel growth and blood flow were increased (koczulla et al. ) . interestingly however, despite the known chemotactic properties of this peptide, no inflammatory infiltrate was detected. the angiogenic properties of ll- appear to stem from its direct interaction with endothelial cells rather than induction of growth factors. these data are consistent with the observation that cramp knockout mice have reduced vascular structures at the wound edge at the site of injury (koczulla et al. ) . a mouse β-defensin, defbd , has been shown to be involved in vasculogenesis, which is the differentiation of endothelial cells from progenitor cells during blood vessel development, leading to the de novo formation of blood vessels and tubes. tumours expressing defbd recruit dendritic cell (dc) precursors via ccr and result in enhanced vascularisation and growth in the presence of the cytokine vegf-a (conejo- ). interestingly, these dcs differentiate to express both dc and endothelial cell markers in response to vegf, indicating that these cells undergo endothelial cell-like specialisation after or during migration to newly formed vessels. this implies that host defence peptides may play important roles in vascular development. it has been observed that there are similarities between chemokines and host defence peptides. indeed, many chemokines have modest antimicrobial activity (hieshima et al. ; yang et al. ) , while a derivative of the highly active antimicrobial peptide, horseshoe crab polyphemusin is a potent antagonist of cxcr (tamamura et al. ) . indeed it has been proposed that certain host defence peptides have evolved from duplication of chemokine genes, although this connection is controversial (durr and peschel ; yang et al. ) ; consistent with this, certain peptides have chemotactic activity. interestingly, unlike the chemokines characterised to date, many host defence peptides appear to have chemotactic activity over a wide range of species, and generally speaking these activities are often observed at concentrations fold or more higher than observed with the classical chemokines. hnp and - have been demonstrated to induce chemotaxis of t cells in vitro at concentrations of between . - ng/ml, with maximal activity occurring at less than ng/ml (chertov et al. ) . hnp is a more potent chemoattractant of monocytes that hnp , with optimal activity at concentrations of - - - m, while hnp failed to induce significant chemotaxis (territo et al. ) . conversely, these peptides were not chemotactic for neutrophils (territo et al. ) , and indeed a subsequent study demonstrated that hnp actually suppressed polymorphonuclear (pmn) migration to formyl-methionyl-leucyl-phenylalanine but not to interleukin (grutkoski et al. ) . in balb/c mice, h after subcutaneous injection, a mixture of hnp - was demonstrated to induce infiltration of pmns and mononuclear cells, while in hupbl-scid mice the defensins-induced infiltrate consisted of modest numbers of cd + cells (chertov et al. ) . interestingly, in contrast to in vitro results, in this animal model study, the infiltration of pmns was observed. however, it is unclear whether pmn infiltration was caused by direct chemotaxis or indirect effects of the peptide treatment. further studies demonstrated that these peptides specifically lead to chemotaxis of immature dendritic cells and naïve, but not memory, t cells (yang et al. a ). collectively, these data indicate that neutrophil granules contain important chemotactic factors which promote the infiltration of cells of both the innate and adaptive immune responses. the β-defensins hbd and hbd are chemoattractants for immature dendritic cells and memory t, cells with peak activities occurring at µg/ml (yang et al. ) . these activities are mediated through the chemokine receptor ccr , which also binds the chemokine larc. hbd , but not hbd , has also been demonstrated to be a chemotactic agent for tnf-α treated human neutrophils (niyonsaba et al. ), a response that is also mediated through ccr . ll- has been demonstrated to be chemotactic for rat mast cells (niyonsaba et al. b) , mouse mononuclear cells and pmns (chertov et al. ) , as well as human neutrophils, monocytes and t cells (de et al. ) . as ll- has been demonstrated to induce a number of chemokines, there has been some debate as to whether it induces chemotaxis directly or indirectly by induction of classical chemokines. in the rat mast cell model, it appears as though this chemotaxis is a direct effect: as when mast cells are cultured with ll- and the supernatants are used for the chemotaxis assay, chemotaxis can be blocked by anti-ll- antiserum (niyonsaba et al. b) . host defence peptides may also indirectly enhance chemotaxis by inducing the production of chemokines from a variety of different cell types, including epithelial cells and monocytes. the hnps, for example, have been demonstrated to induce il- from lung epithelial cells and cell lines (van wetering et al. ; sakamoto et al. ) and to induce the production of il- β and il- mrna production (sakamoto et al. ) from a lung epithelial cell line. it is unclear whether the β-defensins have similar chemokine-inducing activities. hbd , for example, does not induce il- expression in bronchial epithelial cells (sakamoto et al. ). however, in bal from patients with diffuse panbronchiolitis, the hbd concentration correlated significantly with the numbers of cells recovered from the bal fluid (total cells, neutrophils, and lymphocytes) (hiratsuka et al. ) , implying that there might be a link between this peptide and cellular infiltration to the site of infection. ll- has been demonstrated to induce mcp- and il- release in a mouse macrophage and a human bronchial epithelial cell line, respectively, and both chemokines were increased upon stimulation with ll- in whole human blood (scott et al. ) . ll- has also been demonstrated to induce chemokine transcription (il- , mcp- , mcp- ) and release (il- ) in a mitogen-activated protein kinase (mapk)-dependent manner in human peripheral blood derived monocytes . both ll- and the hnps are neutrophil-derived peptides which are released upon neutrophil degranulation. these peptides induce the transcription and release of chemokines, specifically il- , which preferentially attract neutrophils. consequently the presence of these peptides correlates well with that of il- , a potent chemoattractant for neutrophils (ashitani et al. ) , as well as the presence of other neutrophil components such as elastase (zhang et al. ). it appears that host defence peptides induce chemotaxis in two ways: first through direct chemotactic activity of pmns and mononuclear cells mediated through ccr and other as yet to be identified receptors and second through inducing chemokine production which would hypothetically increase the numbers of neutrophils and monocytes at sites of infection. this then would have the net effect of promoting or marshalling cells important in innate immunity to the sites of excessive production (through induction) or deposition (through neutrophil degranulation) of these host defence peptides. this then begs the question as to whether host defence peptides are overtly pro-inflammatory. early experiments determined that a number of host defence peptides from various sources bound to lps from diverse gram-negative bacteria and reduced lps-induced release of pro-inflammatory cytokines (e.g. tnf-α, il- , il- ) and nitric oxide from monocyte or macrophages and protected mice from lps lethality (larrick et al. (larrick et al. , vandermeer et al. ; kirikae et al. ) . initial studies focussed on the unprocessed form of cathelicidin, hcap- (kirikae et al. ); however, it was later found that the lps-binding properties of the peptide were contained within the processed -amino acid c-terminal domain, ll- (turner et al. ). it has been proposed that the anti-endotoxic properties of these peptides are the result of the inhibition of binding of lps to cd ) and lipopolysaccharidebinding protein (lbp) (scott et al. ) , and/or indirect effects on cells (scott et al. ) . ll- has been shown to block a number of lps-induced inflammatory responses, including contractility and (nitric oxide) no release in aortic rings (ciornei et al. ) , pro-inflammatory cytokine production in a macrophage cell line and in animal models (scott et al. ) (ohgami et al. ) , suppression of leukocyte infiltration in a model of endotoxin-induced uveitis (ohgami et al. ) and lethality in animal models of sepsis (scott et al. ) . these effects occur at concentrations in the physiological range for ll- ( - µg/ml) and may reflect a natural role for ll- in the body (e.g. balancing of the potential stimulus by endotoxin from commensals). this anti-endotoxin activity appears to correlate with an ability to dampen the pro-inflammatory effects of the gram-positive surface molecule lipoteichoic acid (scott et al. ) . it appears that there may be marked differences in the ability of ll- and the defensins to inhibit the pro-inflammatory effects of endotoxin. for example, hnp and hbd are not potent inhibitors of lps-lbp binding (scott et al. ) . in ex vivo whole blood experiments, hnp was approximately , -fold less potent than bpi at reducing tnf-α in response to gramnegative bacteria and is much less potent in blocking endotoxin activity, as assessed by a surrogate assay, the limulus amoebocyte lysate assay, or in priming pmn for arachidonate release or stimulating leukocyte oxidase activity (levy et al. ) . thus, the ability to bind to and neutralise endotoxininduced activity in humans may be more evident for ll- and other proteins such as bacterial permeability-inducing protein (bpi) and lbp (weiss ) . natural killer cells are cd -cd lymphocytes that are an important component of the innate immune response. they kill transformed and infected cells, but unlike t cells they are active against cells that have decreased or ablated expression of major histocompatibility complex class (mhc ) molecules. the cytolytic properties of nk cells are increased in the presence of cytokines produced by cells of the innate immune response. nk cells themselves produce cytokines, such as ifn-γ, which are involved in the enhancement of both the innate and adaptive immune responses. nk cells contain a wide variety of cytotoxic peptides of which granulysin (nk-lysin) is considered to be the most important (kumar et al. ) . recently nk cells have also been demonstrated to express the transcripts for ll- and hnp - , and these peptides were found in the supernatants of il- -treated cells consistent with an involvement in the cytotoxic properties of these cells (agerberth et al. ) , or alternatively an immunomodulatory role. consistent with these observations, it has been shown that both tlr and tlr agonists induce the release of hnp - from nk cells into the supernatant and that this release is increased synergistically in the presence of other cytokines found at the site of inflammation (chalifour et al. ). it is not entirely clear what the role of defensins may be in modulating nk-induced cytotoxicity. in one study it was found that nk mediated cytolysis of the transformed cell line kn is decreased in the presence of hnp - in a dose-dependent manner. as well, nk cells treated with hnps had a decreased expression of both cd and cd ). this study also demonstrated that there are high concentrations of hnps due to the infiltration of neutrophils in colorectal tumours, but not in surrounding healthy tissue. thus the authors hypothesised that the presence of hnps might actually protect cancerous cells from nk cytolysis. a conflicting study demonstrated that pbmcs, treated with opsonin-coated zymosan particles, induced the release of substances that enhanced nk-mediated cytotoxicity. these substances were identified as neutral serine proteases and hnps. of the peptides tested, hnp was the most potent, increasing nk-mediated cytolysis optimally at a concentration of . µg/ml (lala et al. ) . clearly, further studies are required to fully elucidate the role that host defence peptides have on nk mediated cytoxicity. monocytes and macrophages do not express high levels of defensins or cathelicidins unless stimulated by lps or pro-inflammatory mediators (agerberth et al. ; duits et al. ) . however, when thus stimulated, they secrete as yet unidentified factors that stimulate epithelial cells and keratinocytes to produce host defence peptides . monocytes and macrophages are, however, quite responsive to stimulation with these peptides and both ll- and the defensins have been demonstrated to induce chemotaxis (territo et al. ; de et al. ) . it has been noted that host defence peptides are strong inducers of chemokine activity in monocytes (chaly et al. ; . interestingly it has been demonstrated that the hnps are able to prevent hiv replication in monocytes and monocyte-derived macrophages and that this property may be due to their ability to induce chemokine production and/or receptor antagonism (guo et al. ). hnp and hnp were both demonstrated to induce production of mip-α and mip- β, the ligands for ccr in monocyte-derived macrophages and to prevent replication of a ccr tropic strain of the virus, presumably by blocking virus binding to ccr (guo et al. ). there has been some evidence that host defence peptides might work as opsonins (fleischmann et al. ; sawyer et al. ) . although this property would be predicted to generally enhance that antimicrobial activity associated with these peptides, one study demonstrated that an ll- derivative actually promoted infectivity of coxiella burnetii, an intracellular pathogen of macrophages (aragon et al. ) . generally, host defence peptides are thought to possess anti-inflammatory properties, as described above. however, in some cases, they may actually enhance some aspects of a pro-inflammatory response. ll- , for example, has been demonstrated to enhance il- β processing and release in lps-primed primary human monocytes (elssner et al. ) . this property appears to be conserved across a range of host defence peptides from a number of different species (perregaux et al. ) . mast cells are distributed throughout the body and are also found in low amounts in the blood. these cells rapidly accumulate at sites of infection, and upon encountering certain bacterial components or pro-inflammatory stimuli they promote the inflammatory response by releasing histamine, which causes vasodilation and thus assists in the recruitment of cells and substances from the blood. two host defence peptides, ll- and hbd have been demonstrated to be chemotactic for rat mast cells, although they may work by different mechanisms (niyonsaba et al. b (niyonsaba et al. , . thus mast cells may accumulate at sites of high concentrations of host defence peptides such as at sites of neutrophil degranulation or at epithelial surfaces in vivo. hbd and ll- as well as the hnp - and hnp homologues from rabbits and guinea pigs have also been demonstrated to induce histamine release (befus et al. ; niyonsaba et al. ) . this property may be especially important in the development of host defence peptides as drugs, as mast cell degranulation is a potentially detrimental side effect. host defence peptides have been demonstrated to interact with epithelial cells. neutrophil peptides have been demonstrated to induce proliferation (m. , induce chemokine production (van wetering et al. ) and stimulate cell signalling pathways . ll- has been demonstrated to bind to a lung epithelial cell line in a manner which suggests that it may have more than one receptor . it has also been demonstrated that binding and subsequent internalisation is required in order to induce il- production . host defence peptides in the adaptive immune response in addition to apparently having multiple roles in innate immunity, it is becoming clear that host defence peptides can modulate the adaptive immune response, and several studies have now demonstrated adjuvant activities of host defence peptides in vivo. the mechanisms involved remain unclear, although these activities could reflect the innate immunity modulating activity of host defence peptides and the fact that there appears to be a strong interconnection between innate and adaptive immunity. the relatively non-immunogenic model antigen ovalbumin (ova) is widely used to study adaptive immune responses. intranasal co-administration of human α-defensins hnp - with ova was shown to enhance the production of ova-specific igg antibodies and ova-specific cd + t cells, which produced significantly more ifnγ, il- , il- , and il- (lillard et al. ). this indicated the capacity of α-defensins to alter the host response to ova, acting as adjuvants to promote a mixed t helper (th) cell response. in two other recent studies, hnp , the human β-defensins hbd and hbd (brogden et al. ) , and a simple synthetic peptide klkl klk (fritz et al. ) were also demonstrated to be effective adjuvants. the observation that such effects are observed with the model peptide klkl klk suggests the possibility of a relatively non-specific mechanism, and that such activities may therefore be seen with a broad range of host defence peptides. however, the nature of the enhanced responses may depend both on the antigen and the peptide used. in contrast to the mixed th- /th- response enhanced in the hnp - -treated animals (lillard et al. ) , ova-stimulated splenic lymphoid cell cultures were found to produce significantly decreased levels of ifn-γ, when taken from hbd -treated mice (brogden et al. ). on the other hand, although klkl klk induced a strong th- type response when co-administered with ova, it enhanced a mixed response when the trivalent influenza split-vaccine fluvirin was used as antigen, with the production of both igg and igg antibodies (fritz et al. ). interestingly, this report also demonstrated that a peptide could markedly enhance antigen association with a monocytic cell line in vitro, and that co-administration in vivo could result in the formation of a transient depot of antigen at the site of injection. these observations indicate that antigen uptake by antigen-presenting cells (apcs) might be enhanced in the presence of the peptide, and thus influence responses in the presence of klkl klk in vivo. although these studies clearly showed altered humoral and th responses to antigens, the functional consequences of these alterations were not clearly demonstrated. in another study, mice were given an intraperitoneal vaccination combining a b-cell lymphoma idiotype antigen and daily µg injections of human α-defensins. this study also demonstrated adjuvant activity, whereby the defensins led to increased levels of antigen-specific igg antibodies and enhanced ifn-γ production by splenic cells (tani et al. ) . moreover, defensins showed mitogenic properties (with a significant increase in the number of splenic b cells) and led to an increase in resistance to tumour challenge. the latter observation raised the possibility that an antigen-specific cytotoxic t cell response was being generated in addition to a humoral response. these studies collectively demonstrate that co-administration of host defence peptides with antigens can enhance and perhaps alter the nature of the host's specific adaptive immune responses in vivo. this raises the question of whether host defence peptides might naturally act as endogenous adjuvants to enhance normal immunological responses, since many peptides can be up-regulated or secreted at sites of infection and inflammation. it is unclear whether the doses used in such studies to assess in vivo immunological processes are within relevant physiological ranges. the physiological significance should be addressed by examining transgenic mice with defective production of host defence peptides, although the issue of possible functional redundancy amongst the many murine defensins must be considered when examining single gene knockouts. the published characterisations of such mice have concentrated on innate responses and have generally not described defects in adaptive immune responses morrison et al. ; moser et al. ) . however, one mbd- knockout model was found to display a defect in generating antibodies to the carbohydrate capsule of pneumococci (c. moser, personal communication) . while this is consistent with an in vivo role for this constitutively expressed defensin in generating an effective humoral response, it is clearly an area requiring further study. regardless of possible physiological significance, the adjuvant effects of host defence peptides are clearly of interest from an immunotherapeutic and vaccinology perspective. in contrast to the studies that have co-administered host defence peptides and antigens, other groups have taken an alternative dna-vaccine approach. this methodology involved immunizing mice with dna plasmids encoding non-immunogenic lymphoma antigens fused to murine β-defensins (biragyn et al. ) . successfully transfected cells of an undefined nature should then express the peptide/lymphoma antigen fusion proteins. this strategy represents an attempt to target antigen to immature dendritic cells (idcs), by exploiting the affinity of the β-defensin portion of the fusion proteins for the chemokine receptor ccr , expressed on idcs. this approach also demonstrated an adjuvant capacity for host defence peptides; however, igg responses were only observed when the plasmid encoded a fusion of the antigen and peptide, and not observed after simple co-administration of peptide and antigen. interestingly, anti-tumour activity was also generated in these mice (most effectively with murine β-defensin ), but did not correlate with the amplitude of the humoral response (superior with murine β-defensin ). furthermore, this anti-tumour activity could be transferred to other mice with the delivery of splenocytes, but not serum, from vaccinated animals, indicating the generation of cytotoxic t cells in response to non-immunogenic antigens when fused to peptides. in another recent study, using a similar approach, immunisation of mice with a plasmid fusing the human cathelicidin ll- to m-csfr (acting as a tumour antigen in this model) also generated enhanced antigen-specific humoral and cytotoxic responses, and prolonged survival in a tumour model ). ll- fusion plasmids were found to be significantly more effective than the m-csfr plasmid alone, or co-administration of separately encoded m-csfr and ll- plasmids. these animal studies all demonstrate the adjuvant capacity of host defence peptides in vivo, but the mechanisms underlying these observations have not been fully elucidated. a variety of hypotheses can be proposed, including direct modulation of lymphocyte responses, mitogenic effects, chemotactic capacity, increased apc antigen uptake and consequently enhanced presentation, activity as endogenous danger signals, alterations to the apc cytokine environment, or direct modulation of apc function (fig. ) . the most obvious mechanisms might include altered antigen uptake (fritz et al. ) and direct modulation of lymphocyte activity and proliferation (tani et al. ) , boosting apc presentation, cellular and humoral responses. this could be further enhanced by direct chemotactic effects of host defence peptides, resulting in the chemotaxis of monocytes, neutrophils, macrophages, idcs, mast cells and t lymphocytes (territo et al. ; chertov et al. ; yang et al. yang et al. , a yang et al. , b niyonsaba et al. a niyonsaba et al. , b , and the enhancement of chemokine receptor expression on these cells (scott et al. ) . in addition, host defence peptides could act indirectly to stimulate the release of potent traditional chemokines (such as il- ) from epithelial cells (van wetering et al. ) , and/or cause mast cell degranulation , enhancing vascular permeability. these direct and indirect chemotactic effects could amplify the inflammatory response and bring key cells of the adaptive immune response to the location of the antigen. while recruiting memory t cells to an infection site may induce a more rapid cellular response to previously encountered antigens, the recruitment of monocytes and idc is likely to be critical to generating the initial response. dendritic cells are sentinel leukocytes that capture antigen in the peripheral tissues and then initiate and orchestrate t cell helper (th- ) responses, the nature of which determines the character of the adaptive immune response (moser and murphy ) . this process is critical to generating a successful defence against harmful microbial non-self antigens while maintaining tolerance to self. it is dependent upon the antigen-capturing capabilities of idcs, and antigen-presenting capabilities of mature dendritic cells (mdcs). idcs are derived from circulating haematopoietic precursor cells and predc populations (monocytes and plasmacytoid cells) under the influence of specific cytokines and growth factors (liu ; pulendran et al. ). in the tissues, these cells encounter and take up antigen. stimulation of idcs by conserved structures on certain microbial antigens, acting via the toll-like receptors (tlrs) of the innate immune system (medzhitov and janeway ) or by signals from host cytokines, results in dc activation. these activated cells mature to become effective antigen-processing and presenting mdcs, migrate to the secondary lymphoid organs and interact with naïve t-lymphocytes (banchereau et al. ) . the characteristics of the mdcs determine the nature and consequences of this interaction, resulting in proliferation and differentiation, or deletion of t cells, and determine the polarisation of the th response (lanzavecchia and sallusto ) . whereas steady-state trafficking of non-activated idcs carrying self-antigen is thought to help maintain tolerance, it has been proposed that sustained trafficking of large numbers of highly stimulatory mdcs to the t cell areas is necessary for the generation of an effective t cell proliferative response (lanzavecchia and sallusto ) . this would require extensive, repeated recruitment of circulating predcs to the site of infection, with rapid differentiation to replace the "first-line" resident idcs. thus, at the simplest level, it is conceivable that the in vivo effects of host defence peptides on the adaptive immune response are the result of direct and indirect chemotaxis of idcs and monocytes to the site of inflammation. thus, chemotaxis, altered antigen uptake, and mitogenic effects on lymphocytes offer potential mechanisms by which host defence peptides may enhance responses to immunogenic antigens. however, these explanations do not account for the generation of humoral and cytotoxic t lymphocyte responses to non-immunogenic antigens observed in vivo. in these examples, an increase in the number of dcs encountered and the amount of antigen taken up should make no difference to the response in the absence of an activating signal. indeed, theoretically, this might serve to increase host tolerance to these antigens. despite this, host defence peptides clearly enhance an adaptive immune response to non-immunogenic antigens in vivo. on the basis of current literature, we propose three hypotheses that might explain these observations. the first two theories propose that these peptides directly or indirectly provide an activating signal to differentiated idcs concurrent with these cells encountering antigen, while the third proposes peptide modulation of dc differentiation from precursor cells. in one intriguing report, it was demonstrated that murine β-defensin fusion proteins were capable of activating idcs directly in a tlr- dependent manner, to produce t helper (th- ) polarised responses . in the context of these dna plasmid vaccines, stimulation of the innate pattern recognition pathways through tlr would occur in close spatial and temporal conjunction to an otherwise non-immunogenic antigen. this suggests that host defence peptides might be capable of functioning as endogenous ligands of innate pattern recognition receptors. however, activation of idcs was not observed with murine β-defensin in the absence of fusion to lymphoma antigen. although this appears to make it improbable that this mechanism is responsible for most of the above-described in vivo observations, it is possible that peptide and antigen concentrations are much higher in co-administration studies, than when relying on dna plasmid expression. thus, if the temporal coordination of tlr stimulation and antigen presentation are critical, this may be achieved by high concentration co-administration and depot formation at the site of delivery but, when utilizing a dna vaccine approach, require peptide fusion. however, despite proving effective as an adjuvant for humoral responses (biragyn et al. ) , murine β-defensin fusion proteins did not have tlr- dependent idc-activating capabilities . furthermore we have seen no evidence of an ability to directly mature human monocyte-derived dc in vitro when studying a range of peptides at or above the putative physiological concentrations, (dj davidson, aj currie,, rew hancock, dp speert, unpublished data). these data indicate that direct activation of idcs may not be an inherent property of host defence peptides. thus, although direct activation of idcs is unlikely to be the basic mechanism underlying host defence peptide adjuvant activities, we cannot rule out a peptide-specific effect in which temporal coordination of tlr ligation and chemokine receptor-directed antigen uptake by the same cell are critical. an alternative mechanism to explain altered idc activation is to suggest that the effects of host defence peptides might be indirect, acting to alter the milieu in which these cells encounter antigen. defensins have been shown to increase expression of various cytokines, including il- , il- , mcp- and gm-csf , in different airway epithelial cells, while ll- can induce il- and mcp- expression in epithelial and monocytic cells (scott et al. ) . changes to the cytokine environment may induce a myriad of effects, from the chemotactic activities of mcp- and il- , and cellular differentiation effects of gm-csf, to the enhancement of b cell proliferation and blockade of the suppressive effects of regulatory t cells by il- (pasare and medzhitov ) . possibly other factors are induced that might activate idcs even in the presence of non-immunogenic antigens. following activation of monocytes with s. aureus or phorbol myristate, human α-defensins at con-centrations as low as nm, can increase the expression of tnf-α and il- β (chaly et al. ) . these cytokines have the potential to directly induce dc maturation, sharing components of activating pathways with tlr, and thus potentially enhancing the generation of highly stimulatory mdcs. however, while such mechanisms might therefore be proposed at sites of inflammation, similar activities in the presence of non-immunogenic antigens are only speculative. the third hypothesis relates to our recent discovery that the human cathelicidin ll- can modulate the differentiation of idcs from precursor cells, with consequent impact on th cell polarisation ). the stimulatory nature of dcs is subject to dynamic temporal regulation (langenkamp et al. ) and can be modified by precursor cell lineage, the specific antigen captured, the receptors engaged, and the microenvironment for both differentiation and maturation (liu ; pulendran et al. ; de jong et al. ; boonstra et al. ) . we demonstrated that ll- has the potential to act as an endogenous environmental modifier of dc differentiation ). ll- -primed dcs displayed significantly upregulated endocytic capacity, modified phagocytic receptor expression and function, up-regulated co-stimulatory molecule expression, enhanced secretion of th- -inducing cytokines, and promoted th- responses in vitro. these results suggest the potential for host defence peptides to exert effects on the adaptive immune system by priming newly differentiating dcs to enhance their antigen uptake and presentation capabilities and influence the nature of the response they will subsequently generate. according to this hypothesis, host defence peptides would not simply affect "first-line" resident idcs, but act upon the differentiating "second-line" dcs that can sustain highly stimulatory presentation of antigen to generate an effective t cell proliferative response. in the context of a physiological role, ll- -primed idcs might be generated at sites where ll- is up-regulated in response to infection or inflammation, be matured by immunogenic antigens, and promote a more robust adaptive immune response. however, ll- -primed idcs also have increased expression of the co-stimulatory molecule cd in vitro in the absence of activating stimuli and any other signs of maturation. if such cells were generated in vivo in the presence of a high concentration depot of host defence peptides at the site of vaccination, or in an area of peptide overexpression by host cells transfected with a dna vaccine, they might be capable of presenting non-immunogenic antigen in a stimulatory context. although enhanced humoral and cytotoxic t-lymphocyte (ctl) responses in dna vaccinated mice are dependent on the fusion of ll- and the antigen , this might again relate to issues of local concentration and co-presentation to the same cells. interestingly however, enhanced splenocyte ifn-γ responses were observed not only in mice vaccinated with the ll- fusion plasmid, but also in those given separately encoded ll- and antigen plasmids. this might reflect the enhanced ifn-γ responses of t cells stimulated with ll- -primed dcs in vitro. however, further research is required to establish the in vivo significance of the ll- -priming of dcs observed in vitro. furthermore, the effects of other host defence peptides on dc differentiation have not been described, raising uncertainty about this hypothesis in the context of the in vivo studies using defensins, or synthetic peptides as adjuvants. in conclusion, the potential for host defence peptides to modulate the adaptive immune response is evident, but remains largely undescribed. in addition to further exploration of the effects in vitro, innovative in vivo modelling is a priority to dissect the mechanisms underlying these observations. a clear understanding of the extent and mechanisms of the immunomodulatory effects of host defence peptides will be fundamental to their future development as novel therapeutic agents. however, these early in vivo studies demonstrate great potential for targeting tumours, recalcitrant, antibioticresistant pathogens, infections for which effective vaccines do not exist, and vaccines, which generate suboptimal responses of an inappropriate nature. mammalian host defence peptides were originally discovered as components of the non-oxidative killing mechanisms of neutrophils. in the granules of neutrophils, these peptides are found at sufficiently high concentrations to be antimicrobial. however, it is less clear that this is the case at mucosal surfaces or in other body fluids, especially at sites that already support a rich and diverse normal flora. certain body fluids, including sinus fluid and gastric juices, have innate antimicrobial activity against certain bacteria, and the components that appear to contribute to this include a variety of antimicrobial proteins (e.g. lysozyme, secretory phospholipase a ), as well as peptides (e.g. defensins) (cole et al. ) . however, the specific contributions of each of these components to overall antimicrobial activity has not been determined, and given the moderate levels of peptides often found in these fluids, synergy between individual agents working in combination may be important, as been demonstrated for some peptides, and combinations of lysozyme and peptides in vitro (singh et al. ; yan and hancock ) .this is still further complicated at sites such as the mucosa when considering the abilities of host defence peptides to modulate innate immunity, as discussed extensively in this review. one approach to trying to resolve these mechanisms is to use genetic strategies using either knockout models in animals or specific genetic deficiencies in humans nizet et al. ; putsep et al. ; salzman et al. ) . such studies have clearly demonstrated that defensins and cathelicidins are integral components of host defence in mammals, and that these peptides are required to reduce bacterial load and inhibit infection. however, they do not always permit the discrimination between the various potential mechanisms of host defence peptides, namely direct antimicrobial activity, synergistic activity with other antimicrobial components and/or the broad range of abilities to modulate immunity. indeed, these distinctions may be unimportant, as they all have the same net result, namely the control of potentially dangerous microbes. we hypothesise that all of these mechanisms operate in the body of mammals, and that any given peptide may have different roles in anti-infective immunity according to the body site it is found, its local concentration, the prevailing physiological conditions, and the other antimicrobial and cellular components of immunity at that site. a clear illustration of this complexity is provided by a study of the use of human defensin (hnp- ) to treat experimental peritoneal k. pneumoniae infections (welling et al. ). in this model, hnp injection was shown to markedly reduce bacterial numbers, but the antibacterial effect was associated with an increased influx of leukocytes into the peritoneal cavity, and this was strongly related to the antibacterial effect, as no such activity was observed in leukocytopenic mice. a further challenge to our thinking, and possibly the most profound question in innate immunity, is how mammals manage to support a complex normal flora while retaining the ability to respond to potentially dangerous pathogens. the toll-like receptors (tlrs), which represent one of the major "triggers" of innate immunity, do not really distinguish between the conserved surface molecules from pathogens and commensal organisms. thus it is of interest as to whether host defence peptides may play a role in this delicate dance between symbiosis and pathogenesis. as shown by e. nishimura et al., many commensal bacteria from the oral cavity are quite susceptible to hbd , while chung and dale indicated that both commensals and pathogenic bacteria can induce this defensin (chung and dale ; e. nishimura et al. ). conversely, putsep et al. compared germ-free and normal mice to conclude that an intestinal microflora does not have a major influence on the production or processing of defensins (putsep et al. ) . however, we consider at least one activity of these peptides might play a role for host defence peptides in homeostasis, namely anti-endotoxic activity, which in our experience is expressed at lower concentrations of ll- than other immunomodulatory activities. it seems possible that this would provide a mechanism for balancing the po-tential stimulation of tlrs by surface molecules from commensal organisms. innate immunity would then be triggered by local perturbations of peptide concentrations though mechanisms such as degranulation of phagocytes, or specific up-regulation by certain cytokines. in addition, the efficiency of these peptides might the increased by other local factors, for example phagocytes that enter the local site (welling et al. ) or local cytokines such as gm-csf that has been shown to enhance mapk signalling by ll- . the full spectrum of the immunomodulatory properties of these peptides is not yet known and each new report demonstrates that the range and importance of these immunomodulatory effects is greater than initially suspected. most likely both antimicrobial and immunomodulatory activities are to some degree involved, as this is consistent with the redundant and efficient nature of evolution, and with the concept of innate immunity as a network of overlapping mechanisms. understanding the interplay between host defence peptides and innate and adaptive immunity will expand our knowledge of immunity in general and allow us to develop anti-infective therapies adapted from nature's design that will enhance the efficiency of immune defences. human neutrophil defensins induce lung epithelial cell proliferation in vitro neutrophil defensins enhance lung epithelial wound closure and mucin gene expression in vitro the human antimicrobial and chemotactic peptides ll- and alpha-defensins are expressed by specific lymphocyte and monocyte populations ll- enhances adaptive immune response in murine model challenged with tumour cells a cationic antimicrobial peptide enhances the infectivity of coxiella burnetii elevated levels of alpha-defensins in plasma and bal fluid of patients with active pulmonary tuberculosis ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts mouse beta-defensin is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract human beta-defensin is a salt-sensitive peptide antibiotic expressed in human lung the peptide antibiotic ll- /hcap- is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide immunobiology of dendritic cells cd -dependent lipopolysaccharide-induced beta-defensin- expression in human tracheobronchial epithelium neutrophil defensins induce histamine secretion from mast cells: mechanisms of action hbd- : a novel beta-defensin from human plasma mediators of innate immunity that target immature, but not mature, dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens toll-like receptor -dependent activation of dendritic cells by beta -defensin flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing t helper type and cell development: dependency on antigen dose and differential toll-like receptor ligation the human cationic peptide ll- induces activation of the extracellular signal-regulated kinase and p kinase pathways in primary human monocytes impact of ll- on anti-infective immunity defensin-induced adaptive immunity in mice and its potential in preventing periodontal disease the novel antimicrobial peptide beta -defensin is produced by the amnion: a possible role of the fetal membranes in innate immunity of the amniotic cavity human beta-defensin- expression is increased in chronic wounds direct bacterial protein pamp recognition by human nk cells involves tlrs and triggers alpha-defensin production neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells beta-defensins and ll- in bronchoalveolar lavage fluid of patients with cystic fibrosis identification of defensin- , defensin- , and cap /azurocidin as t-cell chemoattractant proteins released from interleukin- -stimulated neutrophils innate immune response of oral and foreskin keratinocytes: utilization of different signaling pathways by various bacterial species effects of human cathelicidin antimicrobial peptide ll- on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro innate antimicrobial activity of nasal secretions determinants of staphylococcus aureus nasal carriage cationic polypeptides are required for antibacterial activity of human airway fluid tumor-infiltrating dendritic cell precursors recruited by a betadefensin contribute to vasculogenesis under the influence of vegf-a human defensin is stored in precursor form in normal paneth cells and is expressed by some villous epithelial cells and by metaplastic paneth cells in the colon in inflammatory bowel disease direct inactivation of viruses by human granulocyte defensins the cationic antimicrobial peptide ll- modulates dendritic cell differentiation and dendritic cell-induced t cell polarization microbial compounds selectively induce th cell-promoting or th cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals ll- , the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like (fprl ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells intestinal defensin gene expression in human populations cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group a streptococcus rhinovirus increases human beta-defensin- and - mrna expression in cultured bronchial epithelial cells expression of beta-defensin and mrna by human monocytes, macrophages and dendritic cells chemokines meet defensins: the merging concepts of chemoattractants and antimicrobial peptides in host defense a novel p x receptor activator, the human cathelicidin-derived peptide ll , induces il- beta processing and release comparison of the genomes of human and mouse lays the foundation of genome zoology interleukin- alpha and interleukin- enhance the antibacterial properties of cultured composite keratinocyte grafts increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis opsonic activity of mcp- and mcp- , cationic peptides from rabbit alveolar macrophages the artificial antimicrobial peptide klklllllklk induces predominantly a th -type immune response to co-injected antigens expression of human alpha-defensin (hd ) mrna in nasal and bronchial epithelial cells differential expression of human alpha-and beta-defensins mrna in gastrointestinal epithelia defensins. natural peptide antibiotics of human neutrophils microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: chediak-higashi syndrome and "specific" granule deficiency human betadefensin : a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity cathelicidin peptide sheep myeloid antimicrobial peptide- prevents endotoxin-induced mortality in rat models of septic shock alpha-defensin (human neutrophil protein ) as an antichemotactic agent for human polymorphonuclear leukocytes alpha-defensins inhibit hiv infection of macrophages through upregulation of cc-chemokines the relationship of eosinophil granule proteins to ions in the sputum of patients with cystic fibrosis a peptide antibiotic from human skin isolation and characterization of human beta-defensin- , a novel human inducible peptide antibiotic expression of ll- by human gastric epithelial cells as a potential host defense mechanism against helicobacter pylori the cathelicidin anti-microbial peptide ll- is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium ccl has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity increased concentrations of human betadefensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis tolllike receptor resides in the golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells influence of heart surgery on magnesium concentrations in pediatric patients downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial dna as a potential regulator paneth cells of the human small intestine express an antimicrobial peptide gene defensin- mrna in human paneth cells: implications for antimicrobial peptides in host defense of the human bowel paneth cell granule depletion in the human small intestine under infective and nutritional stress protective effects of a human -kilodalton cationic antimicrobial protein (cap )-derived peptide against murine endotoxemia an angiogenic role for the human peptide antibiotic ll- /hcap- inducible expression of human beta-defensin by fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier regulation of human beta-defensin- in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the nf-kappab transcription factor family granulysin: a novel antimicrobial the differential effects of polymorphonuclear leukocyte secretion on human natural killer cell activity kinetics of dendritic cell activation: impact on priming of th , th and nonpolarized t cells regulation of t cell immunity by dendritic cells human cap : a novel antimicrobial lipopolysaccharide-binding protein a novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity structural, functional analysis and localization of the human cap gene interaction and cellular localization of the human host defense peptide ll- with lung epithelial cells defensins of vertebrate animals modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations antibacterial proteins of granulocytes differ in interaction with endotoxin. comparison of bactericidal/permeability-increasing protein, p s, and defensins mechanisms for induction of acquired host immunity by neutrophil peptide defensins by il- signaling, monocyte-derived cells dramatically enhance the epidermal antimicrobial response to lipopolysaccharide dendritic cell subsets and lineages, and their functions in innate and adaptive immunity rapid sequence divergence in mammalian beta-defensins by adaptive evolution innate immunity characterization of the mouse beta defensin , defb , mutant mouse model beta-defensin contributes to pulmonary innate immunity in mice dendritic cell regulation of th -th development human alpha-defensins hnps- , - , and - in renal cell carcinoma: influences on tumor cell proliferation cathelicidin antimicrobial peptides are expressed in salivary glands and saliva cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense expression and secretion of cathelicidin antimicrobial peptides in murine mammary glands and human milk defensins are mitogenic for epithelial cells and fibroblasts cathelicidin family of antibacterial peptides cap and cap inhibit the expression of tnf-alpha by blocking the binding of lps to cd (+) cells synergistic actions of antibacterial neutrophil defensins and cathelicidins absence of toll-like receptor explains endotoxin hyporesponsiveness in human intestinal epithelium bacterial products increase expression of the human cathelicidin hcap- /ll- in cultured human sinus epithelial cells oral streptococci exhibit diverse susceptibility to human beta-defensin- : antimicrobial effects of hbd- on oral streptococci effect of defensin peptides on eukaryotic cells: primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines evaluation of the effects of peptide antibiotics human beta-defensins- /- and ll- on histamine release and prostaglandin d( ) production from mast cells epithelial cellderived human beta-defensin- acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase c-dependent pathway a cathelicidin family of human antibacterial peptide ll- induces mast cell chemotaxis epithelial cell-derived antibacterial peptides human beta-defensins and cathelicidin: multifunctional activities on mast cells human beta-defensin- functions as a chemotactic agent for tumour necrosis factor-alpha-treated human neutrophils innate antimicrobial peptide protects the skin from invasive bacterial infection cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes alpha -macroglobulin-proteinase complexes protect streptococcus pyogenes from killing by the antimicrobial peptide ll- salmonella enteritidis flic (flagella filament protein) induces human beta-defensin- mrna production by caco- cells effect of human cationic antimicrobial protein peptide on endotoxin-induced uveitis in rats expression and regulation of the human beta-defensins hbd- and hbd- in intestinal epithelium endogenous antimicrobial peptides and skin infections in atopic dermatitis the gene encoding the human corticostatin hp- precursor contains a recent -base duplication and is located on chromosome plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis abnormal peroxidase-positive granules in "specific granule" deficiency toll pathway-dependent blockade of cd +cd + t cell-mediated suppression by dendritic cells antimicrobial peptides initiate il- beta posttranslational processing: a novel role beyond innate immunity modulating the immune response with dendritic cells and their growth factors germ-free and colonized mice generate the same products from enteric prodefensins deficiency of antibacterial peptides in patients with morbus kostmann: an observation study gene expression, immunolocalization, and secretion of human defensin- in human female reproductive tract increased bronchoalveolar lavage human beta-defensin type in bronchiolitis obliterans syndrome after lung transplantation differential effects of alpha-and betadefensin on cytokine production by cultured human bronchial epithelial cells protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin evaluation of antimicrobial and lipopolysaccharide-neutralizing effects of a synthetic cap fragment against pseudomonas aeruginosa in a mouse model interaction of macrophage cationic proteins with the outer membrane of pseudomonas aeruginosa discovery of five conserved beta -defensin gene clusters using a computational search strategy the human antimicrobial peptide ll- is a multifunctional modulator of innate immune responses cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (lps) to lps binding protein primary structures of three human neutrophil defensins structure of a novel human granulocyte peptide with anti-acth activity production of beta-defensins by human airway epithelia synergistic and additive killing by antimicrobial factors found in human airway surface liquid human cathelicidin, hcap- , is processed to the antimicrobial peptide ll- by extracellular cleavage with proteinase processing of seminal plasma hcap- to all- by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina assignment of defensin gene(s) to human chromosome p the role of human neutrophil peptides in lung inflammation associated with α -antitrypsin deficiency pharmacophore identification of a chemokine receptor (cxcr ) antagonist, t ([tyr( , ),lys ]-polyphemusin ii), which specifically blocks t cell-line-tropic hiv- infection sensitivity of actinobacillus actinomycetemcomitans and capnocytophaga spp. to the bactericidal action of ll- : a cathelicidin found in human leukocytes and epithelium defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens monocyte-chemotactic activity of defensins from human neutrophils activities of ll- , a cathelin-associated antimicrobial peptide of human neutrophils effect of defensins on interleukin- synthesis in airway epithelial cells neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone protective effects of a novel -amino acid c-terminal fragment of cap in endotoxemic pigs ) β-defensin- expression is regulated by tlr signaling in intestinal epithelial cells cutting edge: , -dihydroxyvitamin d is a direct inducer of antimicrobial peptide gene expression apolipoprotein a-i binds and inhibits the human antibacterial/cytotoxic peptide ll- defensin pattern in chronic gastritis: hbd- is differentially expressed with respect to helicobacter pylori status bactericidal/permeability-increasing protein (bpi) and lipopolysaccharide-binding protein (lbp): structure, function and regulation in host defence against gram-negative bacteria antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation structure, function, and membrane integration of defensins purification and characterization of human neutrophil peptide , a novel member of the defensin family regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense synergistic interactions between mammalian antimicrobial defense peptides beta-defensins: linking innate and adaptive immunity through dendritic and t cell ccr human neutrophil defensins selectively chemoattract naive t and immature dendritic cells ll- , the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like (fprl ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells mammalian defensins in immunity: more than just microbicidal many chemokines including ccl /mip- alpha display antimicrobial activity antimicrobial and protease inhibitory functions of the human cathelicidin (hcap /ll- ) prosequence cathelicidins, multifunctional peptides of the innate immunity conventional mechanical ventilation is associated with bronchoalveolar lavage-induced activation of polymorphonuclear leukocytes: a possible mechanism to explain the systemic consequences of ventilator-induced lung injury in patients with ards regulation of activities of nk cells and cd expression in t cells by human hnp- , - , and - widespread expression of beta-defensin hbd- in human secretory glands and epithelial cells we would like to acknowledge support for the authors' research from the applied food and materials network, the canadian bacterial diseases network and the functional pathogenomics of mucosal immunity program funded by genome prairie and genome bc, with additional funding from inimex pharmaceuticals. rewh holds a canada research chair, dmeb is supported by a cihr studentship, and djd was funded by a wellcome trust uk, international prize travelling research fellowship ( ). key: cord- - s ok uw authors: nan title: abstracts of the th annual symposium of the protein society date: - - journal: protein science doi: . /pro. sha: doc_id: cord_uid: s ok uw nan c-terminus glutamine-rich sequence deleted) to elucidate the role of metalloprotein hpn-like by fluorescence resonance energy transfer (fret) (figure ) [ ] . we found the selective coordination of ni(ii) and zn(ii) to the purified sensors and in e. coli cells. surprisingly, specific interaction between the fret sensors and bi(iii) was observed. our fret analysis confirmed the role of hpnl for ni(ii) storage and revealed the potential association of hpnl with bi-based antiulcer drugs in cells. pb- rna fate is controlled by highly-regulated rna binding proteins molecular mechanism that links the mrna-degradation pathway with extracellular signaling networks through the reversible unfolding of a rna binding domain (rbd). rna binding is also controlled by ph conditions. this finding becomes relevant for rbps such as t-cell intracellular antigen (tia- ), which shuttles between two cellular compartments (nucleus and cytoplasm) with slightly different ph values. in fact, rna binding by tia- is modulated by slight environmental ph changes due to the protonation/deprotonation of tia- histidine residues [ , ] . the ph dependence of the tia- /rna interaction provides a new insight into the function of tia- in recognizing new rna targets [ ] , like the ' terminal oligopyrimidine tracts ( tops) of translationally-repressed mrnas. along with tia- , the rbp hu antigen r (hur) is involved in the assembly/disassembly of cytoplasmic stress granules (sg), which arise as a protective mechanism by preventing mrna decay under stress situations. despite wide acceptance that rbps harboring aggregation-promoting prion related domains (prds), such as tia- , stimulate rapid self-association and formation of sgs, we propose that scaffolding sgs may be driven by rbds, since prd-lacking rbps, like hur, often form oligomers [ , , ] and are included in sgs. under continuous stress, the transition from the physiological to pathological aggregation of rbps in sgs may depend on post-translational modifications of rbds. rna-binding proteinopathies, characterized by the nucleation of irreversible sgs, are often found in neurodegenerative diseases. altogether, resulting insights into rna biology suggest that highly-regulated rbps determine mrna fate from synthesis to decay. a threat to million people in underdeveloped nations around the world, african trypanosomiasis (sleeping sickness) is a neglected tropical disease (ntd) caused by the protozoan parasite trypanosoma brucei (t. brucei). t. brucei is transmitted to humans via the tsetse fly, and replicates in the blood before crossing into the brain, causing death for the infected individual. current treatments that are available for african sleeping sickness are highly toxic and usually difficult to administer past the blood-brain barrier. it is our belief that coupling less toxic compounds with efficient drug delivery systems will contribute to the development of the most effective drug against african sleeping sickness. our goal was to determine a novel and effective chemical inhibitor with the potential to prevent the replication of t. brucei in the human body. the enzyme target for inhibition studied in this research was -phosphogluconate dehydrogenase ( pgdh), a cytosolic enzyme in the pentose phosphate pathway (ppp) of t. brucei. pgdh is essential in the ppp due to its ability to oxidize -phosphogluconate into ribulose- -phosphate, which is essential for the formation of nucleotides. primer overlap extension polymerase chain reaction (pcr) was used to synthesize the coding dna sequence of the pgdh gene, which was then cloned into a pnic-bsa inducible expression plasmid with an n-terminal histidine tag, by way of ligation independent cloning. the protein was then expressed in bl (de ) escherichia coli (e. coli) cells and purified via nickel column affinity and size exclusion fast protein liquid chromatography (fplc) to perform inhibition assays. through virtual screening, various ligands obtained from the chembridge library and nih clinical collection) were docked into the active site of the crystal structure of tb pgdh (pubchem identification pgj) using gold molecular docking software. the top scoring compounds were selected by utilizing parameters such as hydrophobic interactions, hydrogen bonds, and van der waals forces. the compounds with the best scores that also satisfied lipinski's rule of criteria for druggability were then tested in spectrophotometric enzyme inhibition assays monitoring the absorbance of nadph at nm. compounds that show inhibitory activity in the assays will be taken to higher levels of testing to determine their effect on t. brucei in other organisms. nmr studies of the structural influence of phosphopantetheinylation in nonribosomal peptide synthetase carrier proteins and impact on binding affinities andrew goodrich , dominique frueh nonribosomal peptide synthetases (nrpss) are modular enzymatic systems responsible for the production of complex secondary metabolites in bacteria and fungi. each module is comprised of (at least) three core domains whose combined action leads to the selection, activation, and incorporation of a single small molecule into a growing peptide. central to each module is the carrier protein (cp), which is first primed via attachment of a '-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active holo form. an adenylation (a) domain then covalently attaches an amino or aryl abstract acid onto the ppant arm via formation of a thioester. the cp then shuttles activated monomers and growing peptides between the active sites of catalytic domains in both the same and adjacent modules. during cp priming and peptide elongation, a cp thus exists in multiple different post-translational states and interacts with numerous catalytic domains. understanding how nrpss are able to efficiently orchestrate this series of sequential protein-protein interactions between a cp and its partner catalytic domains is key to unraveling the molecular mechanism of nrp synthesis. using a combination of isothermal titration calorimetry and nuclear magnetic resonance (nmr) titrations, we found that converting a cp from the apo to holo form alters its affinity for its partner a domain. this change in binding suggests a means by which directionality in protein-protein interactions is achieved in nrpss. however, we also found that a domain binding affects the same subset of residues in both the apo and holo forms. in order to identify the molecular features underpinning this difference in affinity, we solved the nmr solution structures of the apo and holo forms of the cp. here, we present the solution structures of an apo and holo cp and discuss them in light of their differential binding to an a domain. functional analysis of of conditional analog-sensitive alleles of essential protein kinases in the fission yeast schizosaccharomyces pombe. juraj gregan , mfpl/imp, the genome of the fission yeast schizosaccharomyces pombe encodes for protein kinases that are essential for viability. studies of the essential kinases often require the use of mutant strains carrying conditional alleles. to inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. the mutation of a single residue in the atp-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. using this approach, we constructed conditional analog-sensitive alleles of essential protein kinases in the fission yeast s. pombe. i will present the functional analysis of these mutants during meiosis. peptide conjugates: from self-assembly towards applications in biomedicine ian hamley university of reading, dept of chemistry self-assembling peptides and their conjugates offer exceptional potential in nanomedicine. i will present some of our recent work on nanoscale assembled peptides and their conjugates, focussing on lipopeptides [ , ] and peg-peptide conjugates [ ] . pegylation is an important technique in the development of conjugates for applications in therapeutics. it is found to greatly influence self-assembly of peptides and proteins -one example from our own work is a peptide which itself forms twisted fibrils but when peg is attached, self-assembly of the conjugate leads to spherical micelles [ ] . the conjugate can be enzymatically degraded using alpha-chymotrypsin, releasing the peptide. this nanocontainer delivery and release system could be useful in therapeutic applications. thermoresponsive telechelic peg/peptides with hydrophobic dipeptide end groups (di-tyrosine or di-phenylalanine) were developed, one of which shows a de-gelation transition near body temperature and which may be useful in bioresponsive delivery systems [ ] . examples from our recent work on self-assembling lipopeptides will also be outlined. our focus is to investigate potential relationships between self-assembly and bioactivity, in particular in the fields of regenerative medicine [ ] [ ] [ ] [ ] [ ] , antimicrobial systems [ , ] and immune therapies [ ] . been shown to become derivatized with argpyrimidine, a prominent nem that occurs on arginine residues [ ] , in certain human cancer tissues and cell lines [ , ] . this nem was linked to the elevated antiapoptotic activity of the protein [ , ] , whereby modification of arg- appeared to be of particular significance [ ] . in this work, hsp homogeneously modified with argpyrimidine at position is generated for the first time. using expressed protein ligation [ ] , the first semisynthesis of the unmodified protein is achieved as well. our approach, which combines organic chemistry, peptide synthesis and protein synthesis, enables complete control over protein composition and thus can provide previously unattainable insight into the properties of this vital chaperone following nonenzymatic modification. the synthesis of argpyrimidine-modified hsp and the progress towards structural and functional characterization of the protein will be presented herein. kunitz-type protease inhibitors belong to a widespread protein family present in many plant species and play an important role in plant defense against insect pests and pathogens. members of this family are typically inhibitors of proteases of serine class. interestingly, a few members were identified as inhibitors of proteases of cysteine class, however, they have not been functionally and structurally characterized. our study is focused on kunitz-type inhibitors of cysteine proteases (pcpis) from potato (solanum tuberosum). a series of kda pcpis was purified using a multi-step chromatographical protocol, and two most abundant and effective isoinhibitors named pci - and pci were characterized in detail. they were screened against a broad panel of model cysteine proteases and digestive cysteine proteases from herbivorous insects. pci - and pci exhibit different inhibitory specificity pattern and potency up to the nanomolar range. both isoinhibitors were crystallized and their spatial structures were solved and refined at . Å (pci - ) and . Å (pci ) resolutions. a position of reactive sites against cysteine proteases on the conserved b-trefoil fold scaffold was proposed. the work provides the first analysis of pcpis with respect to the structure-function relationships and evolution within the kunitz-type inhibitor family. role of the abcc transporter in the mode of action of the bacillus thuringiensis cry ac toxin in the diamond back moth plutella xylostella protonation pattern influence actively properties of molecules and play an essential role in biochemical mechanisms. for an accurate determination of the protonation equilibria, the absolute proton solvation free energy needs to be known. the determination of this energy represents one of the most challenging problems in physical chemistry. this is particularly difficult for protons solvated in water, where the solvation is dynamically performed by different water clusters and the proton is not attached to a single solvent molecule. the proton solvation is notably important in order to quantify mechanisms of proton transfer and such processes have been investigated for a long time based on different approaches, often leading to contradictory conclusions. a rigorous and accurate protocol for computing proton solvation in solvents of different nature is of prime importance for applied (pharmaceutical and material science) and fundamental sciences. in this study, proton affinities, electrostatic energies of solvation and pka values of a reference set of organic molecules are computed in protic and aprotic solvents. proportional to the free energy of proton dissociation, the pka value calculation is therefore strongly dependent on the free energy of proton solvation. such energy is then determined in acetonitrile (acn), methanol (met), water and dimethyl sulfoxide (dmso) in order to obtain the best possible match between measured and computed pka values. the computation of these values is based on a combination of quantum chemical (qc) and electrostatic approaches by using a thermodynamic cycle connecting gas-phase and solvent-phase of proton dissociation. the computed proton solvation energies in acn, met, water and dmso of the present study are very precise (rmsd much lower than ph value). they will be a basis for better understanding of proton solvation and help to predict pka values of organic compounds in different solvents more precise. biochemical characterization of two evolutionary distant ten-eleven translocation enzymes and their utility in -methylcytosine sequencing in the genomes at single-base resolution subtypes leading to an inability to perceive pain and painful neuropathies, respectively. however, as nav ion channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors would be suitable as drug leads. disulfide-rich venom derived mini-proteins from cone snails and spiders are being actively pursued as novel therapeutics for pain, because of their high selectivity and potency at human ion channels, including sodium channels (nav). two main strategies of inhibition have been identified; blocking the pore and interacting with the voltage-sensor domains (vsd) surrounding the pore. the ion-conducting pore is highly conserved between all sodium channel subtypes whereas the voltage-sensor domain binding sites are less conserved. therefore, inhibition of a specific nav isoform is more achievable using inhibitors that modulate vsds than with pore blockers. gating modifier toxins from spider and cone snail venom inhibit nav . and nav . by interacting with the vsd. they appear to reach their target by partitioning into the lipid membrane surrounding the ion channel, thus enabling access to the vsd. toxin pharmacology may therefore not only be driven by the peptide-ion channel interactions, but also including the lipids surrounding the channel protein, a feature that is very much under explored. it is therefore apparent that peptide-lipid interactions in combination with peptide-channel interactions need to be considered when designing potent inhibitors. using a range of biophysical techniques, including surface plasmon resonance and nuclear magnetic resonance, we are studying the interactions underpinning the mechanism of action between toxins and membranes and toxins and ion channels. initial results show that the lipid composition surrounding ion channels play a major role in terms of toxin:lipid interaction and that these interactions can be used in combination with traditional structure-activity relationship studies to design selective and potent nav inhibitors, which will be discussed. we believe that our studies will ultimately delineate what drives toxin pharmacology and nav subtype selectivity and will lead to improve rationally engineering of novel therapeutics for the treatment of pain. micelles promote aß assembly into pore-forming oligomers montserrat serra-batiste , mariam bayoumi , margarida gair ı , mart ı ninot-pedrosa , giovanni maglia , nat alia carulla institute for research in biomedicine (irb barcelona), biochemistry, molecular and structural biology section, university of leuven, the formation of amyloid-b peptide (ab) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in alzheime rs disease (ad). - therefore, it is important to understand how oligomers form within a membrane environment. using solution nuclear magnetic resonance (nmr) spectroscopy, combined with size exclusion chromatography (sec), we have studied the two major ab variants-ab and ab , the latter having a more prominent role in ad than the former-under carefully selected micelle conditions intended to mimic a membrane environment. our results indicate that after an incubation period, ab , but not ab , assembles into oligomers with specific structural properties, which we have named stabilized micelle oligomers (smos). smo complexes incorporate into lipid bilayers as well-defined pores, a feature linked to neurotoxicity. these results have important implications in the ad field as they provide a new perspective on how ab oligomers cause neurotoxicity. indeed, our findings constitute a first step towards the establishment of a new therapeutic target for ad. dimer formation. it should be noted that this nb peptide contains the autophosphorylatable ser- associated with phk activation, and phosphorylated nb; peptide was considerably less effective in promoting b-dimer formation than non-phosphorylated peptide. these results suggest a role for ser- autophosphorylation in mediating homodimeric b subunit interactions within the phk complex, and augment previous studies on the activation of phk by phosphorylation in which changes at the nterminus of b are critical in the activation of the catalytic g subunit. summing these results leads to a new model of activation. in this model, in the inactive state, the nonphosphorylated n-terminus of b interacts directly or indirectly with the regulatory c-terminal domain of the g subunit, inhibiting catalytic activity. upon phosphorylation of the n-terminus of b, three important events occur: ) the interaction between b and g is disrupted, ) the b subunits of the holoenzyme self-associate, and ) the catalytic domain is activated. thus, we envision that the n-terminus of b acts as an allosteric switch, with activation triggered by phosphorylation of this region, causing disruption of its previously inhibiting interactions with g and promotion of b b dimerization to stabilize the activated conformation of g . the research was supported financially by the university of kansas medical center biomedical research training program and nih grant dk . pb- hssb is involved in the cellular response to oxidative dna damage christine touma , nicolas paquet , derek j. richard , roland gamsjaeger , , liza cubeddu , school of science and health, university of western sydney, queensland university of te chnology, school of molecular bioscience, university of sydney cellular dna is subject to oxidative damage in the presence of reactive oxygen species. the , -dihydro- -oxoguanine ( -oxog) adduct is the most common form of oxidative damage and results in g:c to t:a transversions; these lesions are normally processed by the base excision repair (ber) pathway. singlestranded binding (ssb) proteins of the oligonucleotide binding domain family are heavily involved in dna repair processes, which involve the detection of dna damage and recruitment of repair proteins to the site of damage. using immunofluorescence we demonstrate that hssb (a novel human ssb) levels increase in response to oxidative damage (h ). cells depleted of hssb are hypersensitive to oxidative damage and are also unable to efficiently remove -oxog adducts. we show that hssb forms dimers and tetramers under oxidative conditions and that this oligomerisation is likely mediated by inter-domain disulfide bond formation. furthermore, using surface plasmon resonance, we also show that oxidised hssb binds to -oxo-g damaged ssdna with higher affinity than non-damaged ssdna, indicating a direct role for oxidised hssb in the recognition of -oxo-g lesions. as oxidative stress is associated with aging, cancer and alzheimer's disease, understanding the molecular mechanisms of how cells repair oxidative dna damage will be crucial in the development of potential therapeutic treatments. epidemic typhus, which is caused by the bacterial pathogen rickettsia prowazekii, is a menacing disease world wide that the nih lists as one of america's greatest biological weapons threats. this research seeks to find novel inhibitors of b-ketoacyl-acp-reductase (fabg), an enzyme that catalyzes one of the reactions in the fatty acid synthesis type ii system in bacteria. this pathway is essential for survival in bacteria. the fabg enzyme uses nadph as a substrate, which facilitates the binding of the second substrate, acetoacetyl-acp into the active site. the acetoacetyl-acp is subsequently reduced into b-hydroxyacyl-acp. the coding dna sequence for the rpfabg protein was cloned into a pnic vector and transformed into e.coli bl (de ), then the protein was expressed and purified using metal affinity and size exclusion chromatography methods. high throughput molecular docking software (gold) was used to screen a commercial library of ligands against the acetoacetyl-acp region of the active site. the ligands with the best gold scores were selected to be tested in vitro. spectrophotometric enzyme inhibition assays were performed to determine whether the drugs could inhibit rpfabg activity. chlorogenic acid, a previously known inhibitor of homologous fabgs, was tested along with the other potential drugs, and was determined to have moderate inhibitory effects on rpfabg. loop modeling using icm software was performed in order to create a prediction of the complete rpfabg structure, including the disordered loops that are not a part of the f i pdb structure. co-crystallization of rpfabg with both substrates was carried out in order to obtain a structure, but only nondiffracting crystals resulted. further inhibition assays and crystallography trials are being performed in order to continue the search for a novel inhibitor of rpfabg and ultimately a treatment for epidemic typhus. the university of hong kong bioconjugation of proteins has emerged as a useful tool in the study of biological systems. there is an increasing need to develop new synthetic technologies for the bioconjugation reaction of proteins, and metal-catalyzed site-selective modification of proteins has attracted considerable interest in recent years. we have developed a ruthenium glycosylated porphyrin-catalyzed carbenoid transfer reaction for the site-selective modification of proteins. we firstly applied the catalysis to the selective modification of the n-terminus of peptides. by using ruthenium glycosylated porphyrin as catalyst, the n-terminus of a number of peptides can be modified through carbenoid n-h bond insertion in aqueous media with moderate to excellent conversion. the reaction is highly selective, for example, the reaction with ytsssknvvr, which contains various types of oxygenhydrogen and nitrogen-hydrogen bonds possibly available for carbenoid insertion, catalyzed by the ruthenium glycosylated porphyrin gave the n-terminal-modified product with > % conversion and without the formation of other modified peptides including doubly modified and oxygenhydrogen bond insertion products. we next extended the n-terminal modification method to proteins. eventually success was attained in the modification of rnase a and insulin. the reaction of rnase a with a diazoacetate mediated by ruthenium glycosylated porphyrin gave corresponding n-terminal-modified protein with % conversion. we also achieved a bioconjugation to ubiquitin via ruthenium glycosylated porphyrin-catalyzed alkene cyclopropanation in aqueous solution in two steps: ( ) incorporation of an alkenic group by the reaction of n-hydroxysuccinimide ester with ubiquitin and ( ) cyclopropanation of the alkene-tethered lys ubiquitin with the fluorescent labeled diazoacetate in the presence of a catalytic amount of ruthenium glycosylated porphyrin. the corresponding cyclopropanation product was obtained with % conversion based on maldi-tof mass spectrometry. in conclusion, we developed a ruthenium porphyrin-catalyzed siteselective modification of peptides and proteins in aqueous media. the method provides an entry to new bioconjugation reactions for protein modifications using metalloporphyrins as catalysts. uridine monophosphate synthase: architecture versatility in the service of late blight control francisco tenjo castaño , , manuel garavito , , leonor garc ıa , , silvia restrepo , barbara zimmermann biochemistry and molecular biology research group, universidad de los andes., mycology and plan pathology laboratory, universidad de los andes uridine monophosphate synthase (umpase), a bifunctional enzyme in the de novo pyrimidine biosynthetic pathway, is a protein comprised of orotate phosphoribosyl transferase (oprtase) and orotidine monophosphate decarboxylase (odcase). different fusion orders of the two domains have been documented to exist in nature. in some organisms oprtase and odcase are monofunctional proteins, and act as a complex. here, umpase from solanum tuberosum (potato) and from phytophthora infestans (an oomycete) were examined. p. infestans causes late blight disease in s. tuberosum, destroying crops and increasing production costs. since pyrimidines are fundamental cellular components, we have proposed that umpase could serve as a target to control p. infestans infection. the enzymes from p. infestans and s. tuberosum differ in their fusion order of oprt and odc. the study of these two umpase could facilitate the design of species-specific inhibitors, and might shed light on the effect of fusing umpase domains in one order or the other. to this end we carried out bioinformatic and biochemical characterization of the enzymes. sequence analyses showed residue differences among the p. infestans umpase sequences from three strains: , and t - . strain t - was found to have a duplicated umpase, but neither sequence corresponded to the ones predicted previously from the genome. a recombinant umpase from strain was expressed in bacteria and purified but it showed low solubility and was inactive in vitro. the recombinant umpase from the strain complemented both oprtase and odcase deficient e. coli strains. a soluble, active, recombinant protein was expressed and purified in the presence of high salt and the product ump (specific activity . lmol min- mg- ). the sequence skq was found at the c-terminus of the p. infestans umpase sequences and resembles a peroxisome signal peptide (skl). the predicted hydrophobicity of this umpase and its architecture (oprt at the c-terminus and odc at the n-terminus) resembles that of the umpase from leishmania donovani, which has been localized to the peroxisome. we suggest that p. infestans umps could also be located in this organelle. in contrast to the oomycete enzyme, s. tuberosum umpase is highly soluble, and has a higher specific activity (vmax . lmol min- mg- ). we measured the kinetic parameters km(orotate) . lm, km(prpp) . lm, and found that it exhibited product inhibition by pyrophosphate. in conclusion, the different architectures of the two umps might be related to distinct biochemical characteristics, further supporting this protein as a good candidate for p. infestans control. we present computer simulation studies of three different antimicrobial peptides we have been studying by md computer simulation in collaboration with experimentalists. the first is daptomycin, a potent lipopeptide currently licensed to treat infections caused by multi-drug-resistent bacteria. the mechanism of action of daptomycin is currently not completely understood. we have solved the nmr structure of this molecule, and attempted to determine the size of its oligomer by small angle neutron scattering (sans) supported by computer simulation. feglymycin is a -amino-acid peptide with a high percentage of unusual amino acids such as -hydroxyphenylglycine and , -dihydroxyphenylglycine. feglymicin inhibits mura and murc enzymes which are involved in bacterial peptidoglycan synthesis, while also displaying anti-hiv activity by interaction with the viral envelope protein gp . a previous x-ray structure shows the molecule forming a dimer. here, the molecule was studied by nmr in water and dmso. in water, the molecule is clearly at least a dimer, while in dmso it is a monomer. we have performed noe refinement simulations in order to elucidate a structure, however, due to a lack of long-range noe contacts, a unique structure cannot be determined. labyrinthopeptin a is a lantibiotic that contains labionin, a unique carbacyclic posttranslationally modified amino acid that links the protein backbone in three different locations. labyrinthopeptin a has shown promising activity as a pain killer. starting from the x-ray structure, we present results from the first md simulation studies of this unique peptide. because of the extensive cross-linking, this peptide is observed to be highly rigid in its native form. simulation results of mutants are also presented. antibiotics with new mechanism of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. here we report the discovery of a new peptidomimetic antibiotic (l - ), which is active with a minimum inhibitory concentration (mic) in the low nanomolar range, only against pseudomonas sp., and with a non-membrane-lytic mechanism of action. a drug target identified both in a forward genetic screen for resistance determinants and by photoaffinity labeling is the ß-barrel protein lptd, which plays an important role in lps transport and the outer membrane biogenesis. the x-ray structure of lptd in complex with lpte from shigella flexneri shows a stranded b-barrel linked to a periplasmatic n-terminal jelly-roll domain. interestingly the homology model structure for lptd from pseudomonas shows a significant difference: an insertion of around amino acids in the n-terminal domain. the results of our attempts to purify and characterize this large outer membrane protein and to determine the binding site of the peptidomimetic antibiotic will be shown. the theory of how life on earth begun still remains unclear. nevertheless, according to some theories, at the beginning level proteins did not emerge as a complex globular forms as know today. at the times, when solely rna molecules stored both genetic information and catalyzed the chemical reactions in primitive cells, peptides acted as a proteins nowadays [ , ] . literature postulate that the possible role of primordial short peptides was to catalyze reactions in rna-world, as they possess an excellent ability to self-assemble into well-ordered nanostructures [ , ] . elementary functional loops (efls) can be considered as a small structures (blocks) having specific signatures and providing functional residues important for binding/activation as well as principal chemical transformation steps of the enzymatic reaction [ ] . p-loop efl is a widespread structure across vast majority of protein families such as motor domains, aaa , reca, pepck and many others. sequential alignment of these protein families reveals existence of a conserved p-loop motif, that is able to bind atp molecule. we investigated the structure and atpase activity of peptides, which sequences possessed strongly conserved gxgk[t/s] motif from ploop. the goal of our work was to check if peptides corresponding to the most conserved p-loop motif fragment are able to bind and hydrolyze atp molecule. all peptides under study were chemically synthesized and their structures was investigated by nmr spectroscopy. the ability to bind atp molecules was analyzed by using hplc chromatography. results of our study show, that peptides with conserved p-loop motif have a suitable structures to promote binding of the molecules with phosphate group, but cannot accelerate pyrophosphate hydrolysis process. conference participation for w. _ z. supported by the fp project mobi health (grant agreement no ). computational resources were provided by the informatics center of the metropolitan academic network (ic man task) in gdansk, poland. ck is a ubiquitous serine/threonine protein kinase, being one of the most pleiotropic of all protein kinases . ck plays a key role in cell growth, differentiation, cell death and survival, and become the therapeutic target in cancer treatment, since its level is significantly increased in cancer cells . halogenated ligands have been widely developed as potent inhibitors of protein kinases. among them , , , -tetrabromobenzoteriazole (tbbt) is one of the first potent and selective inhibitor of ck a, directed towards the conserved atp binding site . to assess contribution of electrostatic interactions to the specificity and strength of binding of multi halogenated inhibitors by a protein kinase, we have studied interaction between ck a and nine benzotriazole derivatives, representing all possible patterns of halogenation on the benzene ring. herein, we present results that support existence of two alternative regions that are involved in ligand binding. aspartic acid is known for its function in coordination of a mg ion, which is required for atp binding . asp has been identified in crystal structure of ck :tbbt complex (pdb j , fig. ) as the charged residue closest to tbbt. there is also lys proximal to tbbt, interaction with which may favor anionic form of ligands (pk for tbbt < ), however it is involved in the intramolecular salt bridge, and thus its mutation may significantly change stability of the protein. crystal structure of tbbt complexed with ck (pdb: j ). residues with a distance to tbbt (magenta) shorter than a are shown. red residue is negatively charged, blue ones are protonated. abstract comparison of kdiss values determined for ligands at ph and at ph shows that strength of the complex significantly varies upon deprotonation of the triazole ring. this confirms former hypothesis that a negatively charged ligands cluster at the atp binding site region proximal to lys , which is beneficial both to the specificity and to strength of the binding. we have also observed for the tested ligands variations in their binding to either wild type protein and its d n mutant (with less negative charge distributed over atp binding site). all ligands displaying higher pka for dissociation of the triazole proton bind to the mutant visibly weaker than to the wild-type protein. altogether reveals the predominance electrostatic intermolecular interactions. although, negatively charged ligands most probably cluster at the atpbinding site proximal to lys , beneficial for the strength of binding, the less dissociated forms are favored due to unfavorable interactions of the anionic form of ligands with asp . there are many virulence factors produced by these strains, many of which are encoded on mobile genetic elements. psms are of specific interest because these virulence factors are encoded on the core genome of the bacteria and therefore all strains of staphylococci bacteria produce some variation of psms with a variety of biological functions. the specific mechanism by which psms act as virulence factors has been poorly understood until recently. biological functions of psms include cell lysis, biofilm formation and the ability to kill neutrophils after phagocystosis. these toxins are of special interest to our research group due to their genetic similarities to certain bacteriocins, namely leaderless bacteriocins. both groups of peptides are ribosomally synthesized with a n-terminal formyl methionine and secreted from the bacteria by atp-binding cassette (abc) transporters without any leader sequence or signal peptide. abc transporters may also play a role in immunity towards psms and leaderless bacteriocins. these similarities led our group to investigate the solution structure of these peptides through nuclear magnetic resonance (nmr). isolating psms from the producer organisim, s. aureus, typically involves lengthy extractions and low yields. for these reasons, we opted to chemically synthesize the desired peptides using solid phase peptide synthesis (spps). utilizing a variety of spps techniques, psm a and psm a were successfully synthesized, however, due to the hydrophobic nature of psm b , an alternate genetic approach was devised to isolate psm b . formation of a fusion protein between psm b and the small ubiquitin like modifier (sumo) protein allowed for heterologous expression. upon cleavage of the fusion protein with sumo protease, and subsequent purification and isolation of the cut peptide, psm b was obtained. as previously reported, the psms were found to be alpha-helical in structure inducing solvents. a series of dimensional ( d) nmr experiments were ran to determine chemical shift assignments and to obtain noe data. importing the chemical shift assignments and noe data into the structure calculating software, cyana, we were able to elucidate the solution structure of psm a and psm a and we are currently working towards the elucidation of psm b . the synthesis, isolation, characterization and solution structures of the aforementioned psms will be discussed here. transition metals are critical for enzyme function and protein folding, but their excess can mediate neurotoxic oxidative processes [ ] . as, energy production involves oxidative phosphorylation, a process requiring a continuous flow of electrons, mitochondria are particularly vulnerable to oxidative damage [ ] . as such, mitochondria are the major sites of reactive oxygen species (ros) generation, which are produced as byproducts of the electron transport chain. since free iron and certain ros can engage into potentially deleterious processes such as fenton reaction, mitochondrial iron homeostasis must be tightly controlled, and dysregulation of iron metabolism in this organelle has been associated with various diseases, including friedichs ataxia (fa), alzheimer's, and other neurodegenerative disorders [ ] . engineering an efficient mitochondriatargeting, cell-permeable vector is a challenge due to the fact that mitochondrion is impermeable to a wide range of molecules. the development of delivery vectors has been made possible by a greater understanding of mitochondrial structure and chemical features of molecules that selectively localize within this organelle. from these findings, two generalized requirements for mitochondrial localization are delocalized positive charge and lipophilicity [ , ] . targeting iron in this organelle is proposed as a means to ameliorate fa symptoms. desferrioxamine (dfo) is a bacterial siderophore with high affinity for iron, but low cell penetration. we prepared conjugates of dfo with mitochondria penetrating peptides and studied their iron-binding characteristics in vitro. the lipophilic and charged peptides tat - (h-arg-lys-lys-arg-arg-gln-arg-arg-arg-oh) [ ] , a (h-cha-arg-cha-lys-cha-arg-cha-lys-nh ) [ ] , ss- (h-dmt-arg-phe-lys-nh ) [ ] and ss- (h-phe-arg-phe-lys-nh ) [ ] , are known to permeate cytosolic and mitochondrial membranes. they were prepared and conjugated to dfo in solid-phase [ ] , an alternative synthetic route. once detached from the resin, fully deprotected, purified and characterized by means of lc/ms and aminoacid analysis, it was observed that the dfo-conjugated peptides displayed iron-binding abilities identical to the free chelator dfo. dfo-conjugated peptides were also able to quench the iron-catalysed oxidation of ascorbate (a model of oxidative stress in plasma of iron-overloaded patients), as probed by a high throughput fluorimetric method [ , ] . these results indicate that our synthesis and conjugation strategy were successful in preserving the iron-binding moiety and the antioxidant ability of the free chelator dfo. the proteolytic activity and oligomerization status of the human htra protease functioning as a tumor suppressor of an n-terminal domain not required for proteolytic activity, a central serine protease domain and a cterminal pdz domain. the latter serves as a substrate or regulator binding domain and may participate in oligomerization. htra s, its short natural isoform, lacks the pdz domain which is substituted by a stretch of c-terminal amino acid residues, unique for this isoform. down-regulation of htra in tumors, shown by other groups and us, suggests htra s involvement in oncogenesis [ ] . htra acts as a proapoptotic protein and is suggested to function as a tumor suppressor. it promotes cytotoxicity of etoposide and cisplatin in lung cancer cell lines [ , ] . to date, htra has been poorly characterized from the biochemical point of view, mainly due to the fact that it is difficult to purify recombinant htra . we were able to express in bacterial system and purify htra in quantities sufficient to perform structural studies. the aim of this study was to characterize and compare the proteolytic properties and quaternary structure of the htra isoforms. both studied isoforms lacked the n-terminal domain. htra with the pdz domain removed (htra -dpdz) and htra s (htra s) were fully active at a wide range of temperatures and their substrate affinity was not impaired. this indicates that the pdz domain is dispensable for htra activity. as determined by size exclusion chromatography, htra formed stable trimers while both htra -dpdz and htra s were monomeric. this suggests that the presence of the pdz domain, unlike in other human htras (htra and htra ), influences htra trimer formation. the unique c-terminal sequence of dn-htra s appeared to have little effect on activity and oligomerization [ ] . cyclodextrins (cds) are cyclic oligosaccharides that have been recognized as useful pharmaceutical excipients. in aqueous solution cds are capable to form complexes with various ligands, hosting inside their cavity either a whole molecule, or part of a ligand. inclusion complexes with cds offers a variety of physicochemical advantages over the biologically active ligands, including the improved aqueous solubility, solution stability or an increase of bioavailability. ck is an ubiquitous, highly pleiotropic and constitutively active ser/thr protein kinase. halogenated benzotriazoles have been developed as potent and selective inhibitors of this enzyme. the interaction of the catalytic domain of human protein kinase ck with a series of brominated ligands, which represent all possible patterns of halogen substitutions to the benzene ring of benzotriazole, was previously studied by microscale thermophoresis (mst) [ ] . this method alloweddetermination of binding affinities for seven ligands, all of which were found consistent with the values determined independently by isothermal titration calorimetry (itc). however, a very limited aqueous solubility of some brominated benzotriazoles may decrease their bioavability, thus affectingtheir apparent activity [ ] . to overcome this limitation, the aqueous solubility of halogenated benzotriazoles in the presence of cyclodextrins has been tested. the formation of inclusion complexes with b-cyclodextrin (b-cd), hydroxypropylb-cyclodextrin (hp-b-cd) and g-cyclodextrin (g-cd) in aqueous solutions, followed by uv-vis spectroscopy, substantially improved the solubility of tbbt and its derivatives. the interaction between protein kinase ck and cyclodextrins, and also with their inclusion complexes with halogenated benzotriazoles, was followed with the aid of the microscale thermophoresis. the results obtained clearly demonstrate that the binding of halogenated benzotriazoles by ck is only moderately affected by cyclodextrins. oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. here we introduce bivalent peptide-dna conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toeholdmediated strand displacement reactions. employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nm concentrations of peptide-dna locks and oligonucleotide displacer strands. introduction of two different toehold strands on the peptide-dna lock allowed signal integration of two different inputs, yielding logic orand and-gates. the range of molecular inputs could be further extended to protein-based triggers by using proteinbinding aptamers. insights of a novel kind of cell wall binding domain that cleaves the peptidoglycan muropeptide: the cw_ motif noem ı bustamante , , manuel iglesias, noella silva-mart ın, isabel uson, pedro garc ıa, juan hermoso, marta bruix, margarita men endez institute of physical-chemistry 'rocasolano', csic, institute of physical-chemistry 'rocasolano', csic, ciber of respiratory diseases (ciberes), center of biological research (cib), csic, enzybiotics constitute a hopeful alternative to current treatments to fight against bacterial infections. phage endolysins are consider as enzybiotics due to their capacity to cleave the peptidoglycan (pg) of gram-positive bacteria in a generally species-specific manner and kill bacteria when exogenously added ( , ) . the cpl- endolysin, a lysozyme encoded by the pneumococcal cp- bacteriophage, is a remarkable exception among all the pg hydrolases produced by streptococcus pneumoniae and its bacteriophages due to its capacity of degrading pneumococcal cell walls containing either choline or ethanolamine ( , ) . this fact confers to cpl- the advantage of displaying a broader microbicide spectrum comparing to choline binding proteins ( ) . this behavior results from the acquisition of a cell wall binding module (cwbm) made of three identical repeats of amino acids each (cw_ motifs), with unknown specificity and totally unrelated with the choline-binding motives present in pneumococcal hydrolases. interestingly, cw_ repeats have been identified in many putative proteins potentially involved in cell wall metabolism (pfam entry: pf ) from different species of gram positive and gram negative bacteria, and some bacteriophages ( ) . preliminary studies of thermal stability in presence of a small cell wall structural-analogue (glcnac-murnac-l-ala-d-isogln) point to the muropeptide as the cell wall target recognized by cw_ motifs ( ) . in this communication we have gone in depth in the characterization of cw_ repeats. we present the first crystal structure of the cw_ motif, which reveals a three-helical bundle folding. using std_nmr spectroscopy the epitope of binding of the disacharide dipeptide to this repeats has been identified. interestingly, the b anomer of the murnac moiety, the form present in the peptidoglycan, seems to be preferentially recognized with respect to the a anomer. finally, a docking model of the complex cw_ /gmdp compatible with std results was built allowing to identify the major contacts between the protein and the muropeptide and to propose the relevant role of a conserved arginine residue in this interaction. energy-dependent aaa proteases carry out regulated proteolysis to ensure protein quality control and post-translational regulation of many cellular processes. control of proteolysis occurs primarily at the level of substrate recognition, which can be modulated by adaptor proteins. the clps adaptor protein enhances and inhibits degradation of different classes of substrates, and thus triggers a specificity switch in clpa. whereas the mechanism for substrate delivery by clps has been described in detail, the inhibition mechanism is poorly understood. we show that clps inhibits ssra substrate recognition and processing, instead of simply preventing substrate binding. we demonstrate that clpa engagement of the clps n-terminal extension (nte) is necessary, and may even be sufficient, for inhibition. in addition, we find that inhibition of substrate processing requires a longer nte, as compared to inhibition of substrate recognition. interestingly, the nte length required for inhibiting substrate processing is also necessary for suppression of the clpa atpase rate. furthermore, preliminary data suggests that clps slows down substrate translocation. these results support a model where there is an ssra•clpa•clps inhibitory complex in which the clpa pore engages the clps nte. this engagement of the nte causes suppression of atpase activity, and therefore slower substrate translocation and processing. this model illustrates how an adaptor protein can inhibit recognition of one type of substrate while efficiently promoting degradation of a different substrate. single-molecule assay development for studying human rna polymerase ii promoter-proximal pausing rna polymerase ii (polii) pausing has been shown to play a significant role in transcription regulation of elongating polii complexes in a large number of metazoan and mammalian genes ( ) . the traditional understanding of transcription regulation in mammals involved controlling polii recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. most works investigating promoter-proximal polii pausing have employed chromatin immunoprecipitation followed by sequencing to determine polii localization or in vitro transcriptional assays using nuclear extracts analyzed with radio-active gel electrophoresis. in order to gain greater mechanistic insight into the regulation of promoter-proximal polii pausing, we have been developing a diffusion-based single-molecule method using alternating laser excitation on the micro-second timescale (msalex). the method detects rna transcripts generated by a reconstituted human polii system in vitro using complementary doubly dye-labeled single-stranded dna (ssdna) probes. the human gene hspa b for heat shock protein (hsp ) is used as a model system due to its extensive characterization in drosophila. the method would provide a rapid, sensitive and robust avenue to screen for protein factors regulating promoter-proximal polii pausing. controlling of the pic composition using the reconstituted system allows for dissection of the functional roles of different pic components in facilitating regulation of polii pausing. we have demonstrated the hybridization of double dye-labeled ssdna probe to complementary ssdna mimicking rna transcripts and to transcripts generated with bacterial rna polymerase. also, a functional reconstituted human polii system has been verified using radioactive polyacrylamide gel electrophoresis of transcripts from in vitro transcription assays. malaria is a major global health problem. in , there were an estimated million case of malaria and deaths, most of them children under years old [ ] . among the malaria species that affect humans, plasmodium falciparum is the most deadly form. since no efficient vaccine is available yet, the fight against malaria includes vector control, protection from mosquito bites and artemisinin combined therapy. however, resistances to all known treatments have been observed. therefore, new antimalarial strategies involving novel targets and new mechanisms of action are needed. during its life cycle, in erythrocytic stage, which causes all the malaria symptoms, plasmodium falciparum relies on phospholipids to build the membranes necessary for daughter cell development. approximately % of parasite phospholipids consist of phosphatidylcholine (pc) and phosphatidylethanolamine (pe) synthesized by the parasite through the de novo kennedy pathways. in the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by ctp:phosphocholine cytidylyltransferase [ec . . . ] is rate limiting and appears essential for the parasite survival at its blood stage [ ] [ ] . we are focused on the structural characterization of this enzyme, the identification of effectors by fragment-based drug design approach (fbdd) and then their optimization to eventually design a lead. the first reported crystal structure of the catalytic domain of the enzyme target (pfcct) has been solved at resolution . Å, enzyme-substrates complexes (cmp-, phosphocholine-and choline-bound forms) at resolutions . - Å and an enzyme-product (cdp-choline) complex structure at resolution . Å that give detailed images of binding pocket, demonstrate conformational changes between apo-and holo-protein forms and provide the information about the mechanism of the catalytic reaction at atomic level. the fbdd method uses a library of small molecules (fragments) with molecular weight that does not exceed da to explore target binding sites. although fragments often have too low affinities to evoke a biological response, their probability of binding is high because they are small enough to prevent unfavorable interactions with target protein-binding sites. moreover, they represent more attractive and synthetically tractable starting points for medicinal chemistry compared to more complex compounds. as the affinity is low, fragment screening usually depends on detecting binding rather than inhibition. screenings of a fragment library ( molecules) has been performed by fluorescence-based thermal shift assay and nuclear magnetic resonance saturation transfer difference (nmr std) [ ] . this combination of techniques identified so far fragment hits that are currently evaluated for their binding modes and affinities. co-crystallization of the protein-fragments complexes is carrying out to provide accurate information on the molecular interactions. topology of interactions will be used to rationally monitor every iterative round of the optimization process allowing subsequent rational design. [ ] world health organization, world malaria report (who press, geneva, switzerland), http://www. who.int/malaria/publications/world_malaria_report_ /wmr- -no-profiles.pdf?ua protein scaffolds play a crucial role in signaling pathways by generating signal specificity and increasing signal efficiency and amplitude. engineered protein scaffolds can be used as key regulators for signal transduction in artificial signal transduction cascades where they can regulate in-and output of the network. in this research a - - protein scaffold is developed which induces dimerization of proteins mediated by the small molecule stabilizer fusicoccin. as proof of principle caspase is used to constitute proximity induced dimerization. dimerization of caspase leads to its activation and consecutively initiates the caspase cascade involved in the programmed cell death pathway. caspase does not naturally bind to - - proteins, therefore the caspase monomer is conjugated to a - - binding motif which is known to bind into the binding grooves of a - - dimer. this interaction can be stabilized by the small molecule fusicoccin. we showed that upon addition of small molecule fusiccocin caspase dimerization is induced, resulting in caspase activity which is measured using a synthetic caspase substrate. moreover the biphasic effect of the - - scaffold could be proven. additionally, the activated caspase is also able to cleave its natural substrate caspase , downstream in the caspase cascade. these results indicate that the - - platform is a versatile small molecule induced dimerization platform which can be used as tool for engineering of a synthetic signaling network. the g e variant of the apoptosis inducing factor, responsible of a rare encephalopathy, is hampered in nad /h binding luca sorrentino , laura rigamonti , mirvan krasniqi , alessandra calogero , vittorio pandini , maria antonietta vanoni , alessandro aliverti the apoptosis inducing factor (aif) is a highly conserved mitochondrial flavoprotein known to play two opposite roles in eukaryotic cells: in mitochondria it is required for efficient oxidative phosphorylation (oxphos), while, when released into the cytoplasm, it triggers caspase-independent apoptosis ( ) . the mechanism of aif-induced apoptosis was extensively investigated, whereas its mitochondrial role is poorly understood. there are many evidences of aif importance for mitochondrial correct morphology and functions and recently the discovery of its direct interaction with chchd , a key regulator of respiratory complexes subunits import and folding in mitochondria, was reported ( ) . a unique feature of aif, probably pivotal for its vital function, is the ability to form a tight, air-stable charge-transfer (ct) complex with nad and undergo dimerization. although some aspects of aif interaction with nad / h have been analyzed, its precise mechanism is not fully understood. we investigated the effect of the pathogenic g e replacement, associated with oxphos defect and neurodegeneration ( ) , to understand how it could alter aif properties at the molecular level. to do so, we analysed how the wild type and the g e forms of murine aif interact with nad /h and nicotinamide mononucleotide (nmn / h), finding that the pathogenic replacement resulted in a dramatic and specific decrease of the rate for ct complex formation and consequent protein dimerization only in the case of the physiological ligand. our results demonstrate that the adenylate moiety of nad /h is crucial for the ligand binding process and that the g e replacement causes an alteration of the adenylate-binding site of aif that drastically decreases the affinity for and the association rate of the ligand. in addition, we shed new light on the mechanism of the dimerization process, demonstrating that fad reduction rather than nad /h binding initiates the conformational rearrangement of aif that leads to quaternary structure transitions. taken together, our results contribute to define how aif works at the molecular level in binding nad /h and undergoing dimerization and also point out that the g e replacement, responsible of a rare neurodegenerative disease, has the selective effect of slowing down the formation of aif dimeric ct complex. dipartimento di bioscienze, universit a degli studi di milano, dipartimento di scienze veterinarie e sanit a pubblica, universit a degli studi di mical, from the molecule interacting with casl, indicates a family of conserved cytoplasmic multidomain proteins that catalyze a nadph-dependent f-actin depolymerization activity through their essential n-terminal fad-containing monooxygenase-like domain (mo) in response to semaphorin signaling [ ] . this domain is followed by calponin homology (ch) and lim domains, proline-and glutamate-rich regions and a c-terminal coiled-coil motif that mediate the interaction with various proteins (e.g: crmp, casl, plexin, g proteins, ndr) [ ] . to contribute to establish the catalytic properties of mical mo and their modulation by the additional domains and by the interacting proteins, we have produced and are characterizing the human mical (mical-fl) and forms containing the mo [ ] , mo-ch and mo-ch-lim domains. all mical forms contain stoichiometric amounts of fad in the mo domain and zn ions in the lim domain. mical-mo catalyzes a nadph oxidase (h o -producing) activity. the ch, lim and c-terminal domains lower its catalytic efficiency (kcat/km, nadph) mainly due to an increase of km for nadph. the kcat is similar for all forms excepted for mical-fl where a -fold drop is observed, in agreement with the proposed autoinhibitory function of the c-terminal domain [ ] . the ph dependence of the kinetic parameters of mo, moch and mochlim is complex suggesting that it does not reflect the ionization state of individual groups, but rather the overall protein charge. mical-mo, -moch and -mochlim catalyze a nadph-dependent f-actin depolymerization with a similar apparent km for actin. f-actin (but not g-actin) stimulates the rate of nadph oxidation by increasing kcat and lowering knadph. the extent of nadph oxidation exceeds total f-actin which is in contrast with the proposal of specific modification of actin met or met reported for drosophila and mouse moch [ ] [ ] , but it suggests that f-actin stimulates the nadph oxidase activity or a case of substrate recycling. accordingly, with hmical mo and moch several actin residues are oxidized beside met and met . thus, the ch and lim domains do not seem to be important for the mical-actin interaction and actin modification may be mediated by in situ h o production. in hek t and cos- cells mouse collapsin response mediator protein- (mcrmp ) interacts with mical inhibiting h o production [ ] , suggesting that crmp could be a hydroxylatable substrate of mical-mo. we have produced the same mcrmp form ( - aa) and we have shown that under conditions that limit non specific interactions a mild stimulation (up to %) of nadph oxidation is observed. f-actin reversed the effect of mcrmp suggesting their competition for mical. these results suggest that crmp , a major microtubules regulator, is not the substrate of the mo domain, but actin and microtubules cytoskeleton components may be linked through the formation of crmp-mical complex in response to semaphorin-plexin signaling. experiments are in progress to complete the characterization of mochlim and full length mical forms. green fluorescent protein (gfp), owing to its genetically encoded strong fluorescence, has become one of the most important tools in modern biology [ ] . enhanced gfp (egfp, f l/s t-gfp), frequently used variants of this protein, is thermodynamically more stable and -times brighter than gfp [ ] . due to the improved fluorescent properties, egfp is commonly used as a fluorescent intracellular marker in bio-imaging in vitro and in vivo. despite sustained interest of the scientific community and numerous practical applications, the actual biological role of gfp remains elusive. recent reports put forward a hypothesis of antioxidant and photo-protective functions of gfp [ ] . in this study, we focused on the photo-protective role of egfp against reactive oxygen species (ros) photo-generated by visible light in water suspensions of nano-particular nitrogen-doped titanium oxide (n-doped nano-tio ), that is in the system: 'n-doped nano-tio )/visible light'. n-doped nano-tio (sumitomo tp-s ) was chosen as a photo-catalyst, since it is widely accepted that nitrogen doping enhances visible light photoactivity of tio . -hydroxy- , , , -tetramethylpiperidine-n-oxyl (tempol), a paramagnetic water-soluble compound, belonging to the nitroxide class o superoxide dismutase (sod) mimetics, was used as a target for photo-generated ros. a solar simulator, with the flux output intensity of kw/m , was used as a visible light source. electron spin resonance (esr) was employed to monitor the changes in the paramagnetic signal of tempol exposed to the action of ros in the absence and presence of egfp. in the absence of egfp and after min of illumination, due to a combined action of superoxide (o •-) and hydroxyl (oh•) radicals generated by the system 'n-doped nano-tio )/visible light, the esr signal of um tempol decayed by %. moreover, the growth of a new signal, interpreted as -oxo- , , , -tetramethyl- -piperidinyloxy (tempone), resulting from the attack of oh•radicals on tempol, was also observed. in contrast, in the presence of egfp ( . um) , the ros-induced decay of the esr signal of tempol was markedly smaller, not exceeding %. concomitantly, the growth of the esr signal of tempone was also partially inhibited ( % smaller amplitude), as compared to the process performed in the absence of egfp. in summary, our results point to a significant inhibition of the photodecomposition of tempol in the presence of egfp and support the hypothesis of the protective role of this fluorescent protein against ros generated by the system 'n-doped nano-tio )/visible light'. school of chemistry, national university of ireland galway, school of biochemistry and immunology, trinity college dublin by studying a variety of anionic ligands and their interactions with cationic cytochrome c, we are building knowledge of protein recognition geared towards regulating activity. in previous work it was shown that psulfonatocalix [ ] arene selectively binds to, and encapsulates, three lysine side chains on cytochrome c . here, the binding of two small molecule ligands to cytochrome c was investigated. nmr spectroscopy was used and in one case, a crystal structure of the complex was obtained (fig ) . the calixarene bound to cytochrome c, reveals a crystal packing assembly that suggests it is a key mediator of crystal formation. nmr data analysis indicates the calixarene's binding site on cytochrome c. the pillararene, a relatively new class of compound, is a symmetrical arrangement with a p-rich cavity , related structurally to calixarenes. this suggests good host-guest complexation properties. previously, the carboxylatopillararene showed selective binding to arginine, lysine and histidine . with this ligand, an interaction with cytochrome c was observed and a complex formed. additionally, biphasic binding behaviour was observed through analysis of the chemical shift perturbations. this may indicate more than one binding event taking place. the data from these studies indicate that recognition is occurring and again that lysine side chains play an essential role. the enzyme dihydrofolate reductase (dhfr) is necessary for the growth and development of all organisms. the structure and function of escherichia coli dhfr have been characterized in buffer. however, dhfr exists in living cells, where the protein concentration can exceed g/l. we know that weak, non-specific chemical interactions with cytosolic proteins alter protein conformation and dynamics, , both of which are expected to influence dhfr catalysis. investigators have examined steady-state enzyme kinetics under crowded conditions, but conclusions can be conflicting. , here, the effects of crowding on e. coli dhfr catalysis are assessed through specific activity measurements in solutions of synthetic polymers. these kinetics studies are complemented by in-cell and in vitro f nmr data from fluorinated tryptophan residues. preliminary results suggest that the effects of polymeric crowders on dhfr activity are non-monotonic, which may arise from the polymer's transition from the dilute to semi-dilute regime. the data suggest that synthetic polymers are not a valid representation of the cellular interior. biotechnology department, university of verona calcium (ca ) is one of the most important second messengers in eukaryotes. ca binding proteins can be subdivided into two categories: "ca buffers" that modulate ca ion concentrations in cells, and "ca sensors" that decode ca signals in a wide array of physiological processes in response to external stimuli. calmodulin (cam) is the prototypical example of ca sensor proteins in both animals and plants. in addition to conserved cam, plants possess a unique family of cam-like proteins (cmls). many of these cmls still remain uncharacterized and the investigation of their biochemical and biophysical properties will provide insight into ca signalling in plants. herein, a detailed characterization of arabidopsis thaliana cml is reported. cml is a protein of amino acids with a theoretical molecular weight of , da and % amino acid sequence identity with atcam . cml is predicted to have one functional ca binding site despite the presence of three ef-hand motifs (prosite). we overexpressed cml in e. coli and analyzed its biochemical and biophysical characteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation, thermal stability and proteolytic susceptibility upon ca binding. isothermal titration calorimetry (itc) and nuclear magnetic resonance (nmr) spectroscopy identified one ca binding site in cml and showed that ca and mg compete for the same binding site. the kd values determined by itc established that cml has higher affinity for ca than for mg . our data were consistent with the sequence based prediction of one functional calcium binding site. differential scanning calorimetry (dsc) showed that ca and mg have the same stabilizing effects on protein folding. apo-cml undergoes two thermal unfolding transitions, but in the presence of ca or mg only one unfolding event at an intermediate temperature occurs. limited proteolysis experiments showed that ca binding affords protection against cml digestion by trypsin. surprisingly, cml exhibits very few conformational changes upon calcium binding, which were evaluated by ans fluorescence and stokes radius measurements in the apo-and ca bound-forms. these results suggest that cml does not show the characteristics of a classical ca sensor protein. to better understand the physiological role of cml in plants, in vivo analysis will be performed. pb- fbp controls the hepatocyte morphology through rho signaling jun zhang , mingming ling , qianying zhang , yunhong wang , deqiang wang the department of cell biology and genetics, the formin-binding protein (fbp ) widely expressed in eukaryotic cells was previously identified to play a role in morphological maintenance in hepatocyte, but the molecular mechanism keeps still unclear so far. in the present investigation, it was found that rho family proteins cdc /rac signaling was involved in the morphological regulation controlled by fbp . knockdown of endogenous fbp expression with rnai technique or dominant negative mutant of fbp could trigger the cell morphological remodeling from the epithelioid to fibroid following the significant down-regulation of cdc / rac activities and dephosphorylation of paxillin. while the rho protein specific activator could restore the cdc /rac activities, and in turn abrogated the silence effect. overexpression of wild type fbp could not result in any of the morphological transition. furthermore, withdrawal of the silence could induce morphological recovery when the fbp expression, cdc /rac activities and paxillin phosphorylation were restored to the normal level. the experimental evidences strongly indicated that fbp was implicated in morphological control probably via rho signaling pathway in hepatocyte. key words: fbp ; rho signaling; paxillin; morphological control; hepatocyte this work was supported by a grant from national natural science foundation of china (nsfc, no. cytochrome c oxidase (cco) is the final enzyme in the respiratory chain of mitochondria but also an integral part of the metabolism of many types of bacteria. in a complex, stepwise redox-reaction, cco catalyzes the reduction of molecular oxygen to water and utilizes the resulting free energy to pump protons across the membrane thereby creating an electrochemical gradient [ , ] . to investigate proton pumping spectroscopically it is possible to label the entrance of the proton entrance channel with fluorescein, a ph sensitive dye, which allows determining time resolved local changes in proton concentration at the cytoplasmic cco surface and related properties. it has already been shown that the redox state of copper and heme centers affects such properties at the cytoplasmic surface. [ ] this study is a theoretical approach to investigate changes of pka values of the fluorescein label at the entrance of the k-channel for different protonation pattern in both oxidized and reduced cco by performing molecular dynamics (md) simulations. further work is based on calculations of pka values of the fluorescein using software karlsberg [ , ] . methods for genetically and synthetically manipulating protein structure enable a greater flexibility in the study of protein function. we have shown that using inteins as traceless, cleavable purification tags enables the separation of full length unnatural amino acid (uaa) containing proteins from their corresponding truncation products. this method has been used to incorporate uaas in previously unattainable positions in a variety of proteins using a myriad of uaas, inteins, and purification tags. in other applications, we have used e. coli aminoacyl transferase (aat) to selectively modify the n-termini of proteins with uaas in denaturing conditions and conditions that maintain folding. applications of particular interest include overcoming the need for an n-terminal cys residue in expressed protein ligation, transfer of reactive handles for "click" chemistry labeling of proteins, and transfer of fluorogenic molecules for photophysical experiments. we have found that aat can transfer protected cysteine, homocysteine, and selenocysteine to expressed proteins. after ligation, these residues can be converted to met or ala, making the ligation traceless. we continue to develop variants of aat to broaden the substrate scope of both its transferred substrate and n-terminal recognition element. in addition, expressed protein ligation is being used to incorporate backbone modifications, such as the thioamide, into various positions in the protein calmodulin to determine how these modifications can impact the structure and function of an ordered protein. in general, by working at the interface of several protein modification technologies, we have made beneficial discoveries that might be missed by more focused approaches. function and modularity of cw_ motives in the c-terminal region of the endolysin cpl- encoded by the cp pneumococcal bacteriophage manuel iglesias-bexiga , , noelia bernardo-garc ıa , rub en mart ınez-buey , noem ı bustamante , , guadalupe garc ıa , , marta bruix , juan hermoso , margarita men endez , dept. of biological physical-chemistry, iqfr-csic, ciber of respiratory diseases (ciberes), department of crystallography and structural biology, iqfr-csic, bacteriophage lytic murein-hydrolases have been proposed as enzybiotics, an efficient way to fight bacterial infections. however, the use of these enzymes is normally restricted to gram-positive bacteria since the outer membrane of the gram-negative bacteria hampers the access of the hydrolases to the peptidoglycan substrates. all the murein hydrolases reported in the pneumococcal system, both from host or phage origin, depend on the aminoalcohol choline to be fully active. there is only a unique exception to this rule, the cpl- lysozyme. this hydrolase is encoded by the lytic pneumococcal phage cp- and, instead of the common cell wall binding module (cwbm) that recognizes choline, cpl- harbors a completely different cell wall binding structure. recent studies have revealed that reducing the net charge of the cwbm, from . to . , leads to an improvement in the antibacterial activity of cpl- ( ) . the cwbm of cpl- is composed by three identical repeats of amino acids, the cw_ motives, and it folds both in the presence and in the absence of the n-terminal catalytic module ( ) . this module shows the capacity of recognize the glcnac-murnac-l-ala-d-isogln muropeptide (gmdp), structurally related with the peptidoglycan basic unit ( ) . here, we report the high resolution structure of the cell wall binding module of the cpl- endolysin. each cw_ repeat is composed of a bundle of three a-helices with a highly negative electrostatic charge at the surface. the strong inter-repeat interactions and the high ionic strength used in the crystallization conditions allow them overcoming the electrostatic repulsions inducing a closed-packed structure with a three-fold symmetry. the module dimensions ( x x Å) and the repeat arrangement in the crystal structure are inconsistent with the gmdp binding characterization, the activity displayed by cpl- truncated variants with one or two cw_ repeats, or the experimental determined hydrodynamic properties. using the small angle x-ray scattering (saxs) technique and the atsas computational platform ( ), a different arrangement of the cw_ repeats is envisaged in solution (fig. ) , whose rather opened structure ( x x Å) is consistent with the experimental data. additionally, employing the saxs-based structure and the honeycomb structure proposed for the peptidoglycan, a model, where each cw_ repeat of the cell wall binding module fit in adjacent glycan chains, has been derived. in , the protein structure initiative (psi) was started as to determine three-dimensional structures of proteins within every family. once solved, structures are deposited into the protein data bank (pdb) and termed structural genomics (sg) proteins. as of june , there are over , sg proteins deposited in the pdb and most of them are of unknown or uncertain biochemical function. in addition, many of these sg proteins have a putative functional assignment based on their sequence and structural similarities with proteins of known function; such comparisons can be made against large databases using programs such as blast or dali. however, these putative functional assignments are often incorrect. this project analyzes members of the crotonase superfamily (cs). the cs consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity, including hydrolase, isomerase, hydratase, and dehalogenase activities. this superfamily also contains at least sg proteins, so it is ideal to test predictions of protein function. our approach is based on local structure matching at the computationally predicted active site. first, partial order optimum likelihood (pool) is used to predict the functionally important residues of each sg protein and of the proteins of known function in the superfamily. next, structurally aligned local sites of activity (salsa) is used to align the predicted catalytic residues of the well-characterized members in the superfamily. from this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the sg proteins. we demonstrate based on these computational methods that the majority of the putative annotations in the cs superfamily are likely incorrect. currently, biochemical assays are being used to test these predictions. preliminary biochemical results show that one sg protein, thermus thermophilus q sls _thet , classified as a probable enoyl-coa hydratase, possesses hydrolase activity as predicted by our methods. the outcomes of this project will be to successfully classify the biochemical functions of sg proteins based on their local structure at the predicted active sites and to provide a conceptual framework for the functional classification of the remaining sg proteins within the pdb. this work is supported by nsf-che- . directly observing the synergistic dynamics in f-actin and microtubule assembly jun zhang , deqiang wang the department of cell biology and genetics, key laboratory of molecular biology on infectious disease although important in cellular activities, little attention was paid to the synergistic effects of actin and microtubule cytoskeleton assembly. with the time-lapse atomic force microscope (tl-afm), we directly observed the large-scale dynamic structure of actin filaments formed in the presence or absence of microtubulin in solution. in absence of microtubulin, the g-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single f-actin to giant filament in tree-like branched aggregates. the polymerized actin filaments, to which our most intense attention was attracted, was discretely arranged and showed obvious polymorphism in structures completely distinct from those in the presence of microtubulin. the supra-molecular complex structures of the latter were mainly composed of single f-actin and/or multifilaments clearly consisting of several single f-actin and regularly cross-linked with the assembled microtubular bundles. the experimental results demonstrated that the f-actin dynamics could be coordinated by microtubule assembly. further analyses implied that the interactions between f-actin and microtubule could prevent the emergence of structural polymorphism of f-actin alone, and give rise to organization of specific complex structures instead. it was suggested that dynamic synergy between the f-actin and microtubule would be implicated in living cells. the adaptor protein - - is found in a diverse range of pathologically relevant protein-protein interactions (ppis). as - - is a hub protein with very diverse interactions, it is able to influence the intracellular localization of their binding partners and they are key regulators of signal transduction processes as well as regulators of cell cycle functions.nevertheless, there are only few examples of - - acting extracellularly. one of the extracellular targets for - - is aminopeptidase n (apn). apn is an extracellular trans-membrane enzyme that acts as a receptor for - - . binding to apn, - - excreted by keratinocytes can upregulate the excretion of matrix metalloproteinase- (mmp ) in fibroblasts. mmp , by breaking down collagens, is key in the remodeling of the extracellular matrix. modulation of the - - /apn interaction thereby may play a crucial role in the fundamental understanding and ultimately treatment of wound healing, respiratory diseases and tumor growth. in the eukaryotic cell, the - - dimer operates as an adapter platform for binding partners. a wide range of classes of (small) molecules, natural products and peptides has been used to modulate the ppis, providing either stabilization or inhibition of the interactions of - - with its binding partner. binding partner fragments or peptides are known to bind to the - - binding groove via arecognition motif containing a phosphorylated serine or threonine. making use of the dimeric structure of - - , novel small-molecule inhibitors may be tethered to exploit the bivalent effect. from a large virtual screening and experimental validation, a scaffold containing a phenyl phosphonic moiety was identified, showing inhibitory properties for - - ppis. potent derivatives of this scaffold were bridged by polyethylene glycol (peg) linkers of varying lengths, thereby facilitating the compound to reach both binding sites of the - - dimer and concurrently increasing the compound's solubility in aqueous solution. similar bivalent inhibitors have been proven to synergistically increase their efficacy. biophysical evaluation by means of fluorescence polarization (fp) inhibition competition assays, revealed an increase of the half maximal inhibitory concentration (ic ) from approximately lm for the monomeric phenyl phosphonate to approximately . lm for the bivalent inhibitor with a Å linker. this demonstrates a -fold increase of inhibitory effect towards - - and its binding partner peptide mimic. extensive thermodynamic, kinetic and structural analysis of the interaction is in progress.phosphonic moieties have been shown to pass the cell membrane poorly, due to their highly charged character. by being able to specifically inhibit the extracellular interaction between - - and apn, these inhibitors are prevented from interfering with the extensive intracellular - - interactome. hence, these bivalent phenyl phosphonate inhibitors provide a promising strategy towards extracellular application. the mre complex is an oligomeric assembly comprising of dimmers of mre and rad proteins in archea and additionally nbs subunit present in eukaryote. it is the central player in the dna damage response -a functional network comprising dna damage sensing, signal transduction, cell cycle regulation and dna double strand breaks (dsbs) repair [ ] . recent structural studies revealed that rad hinge domain is rather a short kink in the coiled-coil region and adopts unusual dimerization mode by intermolecular coordination of zn(ii) and formation of so-called zinc hook domain [ ] . to date, very limited structural data on the zinc hook domain have been reported, the only known structure was resolved for rad homologue from hyperthermophilic archaeon -p. furiosus. unusual zn(ii) coordination mode in zinc hook domain raises question of how zinc hook domain assembles to form interprotein zinc binding site with sufficient stability to function at low intracellular free zn(ii) concentrations [ ] . our study on minimal zinc hook domain fragment ( aa) indicated low femtomolar affinity towards zn(ii) [ ] . extended zinc hook domain fragment ( aa) reveals even zeptomolar affinity. therefore, our main goal was to probe the thermodynamic and structural effects that are hidden in the small interprotein interface and are responsible for the dimerization of the large and critical protein machinery. probing of those effects was achieved by detailed biophysical characterizations (including potentiometry, nmr, hdx ms and cd spectroscopy) of protein fragments of zinc hook domains with a number of point mutations. we showed that extremely high stability of zinc hook domain from p. furiosus is achieved by the formation of hydrogen bond network in b-hairpins and interprotein hydrophobic core. eindhoven university of technology dna-based molecular circuits have become a very attractive tool in molecular imaging, synthetic biology, molecular diagnostics and biomolecular computing. the highly modular and predictable nature of watson-crick base pairing allows the construction of complex circuits using a limited set of logic gates and building blocks. however, the lack of generic approaches to interface dna-based molecular circuits with protein activity limits their application in biomedicine and molecular diagnostics. here we present a new, highly modular approach to control the activity of a reporter enzyme based on the dna-directed assembly and disassembly of a complex between tem -b-lactamase and its inhibitor protein blip. both proteins are conjugated to a unique oligonucleotide, allowing the assembly of the enzyme-inhibitor pair and inhibition of enzyme activity by the addition of a complementary template strand. addition of an oligonucleotide that is complementary to a loop sequence in the template results in the formation of a rigid dsdna spacer that disrupts the enzyme-inhibitor complex, restoring enzyme activity. using this noncovalent approach allowed easy tuning of the template and target sequences with only a single set of oligonucleotide-functionalized enzyme and inhibitor. to show the modularity of the system, a panel of different template sequences were selected. only in the presence of their complementary viral dna sequences restoration of enzyme activity was observed. in addition to this excellent specificity the system showed to by higly sensitive towards its target, since the presence of as little as fmol of target resulted in an observable increase in enzyme activity. the use of a stable and well-characterized enzyme-inhibitor pair, complemented by the modular design of our reversible dna-directed protein switch make it an attractive system to implement in dna-based molecular circuits. several studies demonstrated important roles of human carbonic anhydrases (hcas) in a variety of physiological and pathological processes. consequently, in recent years the catalytically active hca isoforms have become an interesting target for the design of inhibitors with biomedical applications [ ] . derivatized sulfonamides of type r-so nh represent the class of ca inhibitors (cais) mostly used and best characterized. the large number of crystallographic studies so far available on these molecules clarified the main factors responsible for the binding of the sulfonamide moiety to the ca active site. in particular, it has been highlighted that even though these molecules generally behave as very potent cais, they do not show selectivity for the different isoforms. indeed, the sulfonamide moiety plays a predominant role in the interaction with the enzyme, while any change in the nature of the r substituent has generally a rather marginal effect on the enzyme-inhibitor affinity. these characteristics make difficult the design of sulfonamide derivatives selective for the different ca isoforms. consequently, much efforts were dedicated in last years to the development of new inhibitors that, although presenting lower affinity for the ca active site, would be able to be more selective toward the different isoforms. carboxylic acids have been recently investigated as cais, showing that these molecules can adopt different binding modes to the enzyme active site. in particular, they can coordinate directly to the zinc ion or be anchored to the zinc-bound water molecule. however, the structural reasons responsible of this peculiar behavior have not been clarified yet. in a general research project aimed at providing insights into the binding mode of these molecules to cas, we have undertaken the characterization of two carboxylic acids, namely an ortho-substituted benzoic acid [ ] and a saccharine derivative, by means of kinetic, crystallographic and theoretical studies. exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants ana bernardo gancedo 'exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants' human lysozyme is a widely characterised protein whose mutational variants misfold into fibrils that are associated with systemic amyloidosis ( ) . although the process of aggregation for human lysozyme has been well studied, the details of early events within this process are not fully characterised. single molecule fluorescence microscopy has been used to determine the oligomeric distributions present in the aggregation process of a number of disease-related intrinsic disordered proteins (idps) ( ) . recent advances in site-specific labelling of human lysozyme ( ) have made this protein amenable to these single molecule fluorescence studies. we have introduced alexa-fluorophores into the i t variant of human lysozyme and have demonstrated that the process of in vitro fibril formation is not significantly altered. using these fluorophore-labelled proteins we can apply single molecule fluorescence to study the early aggregation events within this system, allowing us to compare protein aggregation in a globular protein and with the aggregation process of idp's. abstract protein structure, folding and function, while specific interactions with lipid molecules can also contribute towards the biological activity of some membrane proteins. improving understanding of the interactions has resulted in the development of artificial lipid systems that allow the bilayer properties to be rationally manipulated in vitro to control protein behaviour. the bacterial transporter lacy is a well known integral membrane protein from the major facilitor superfamily, responsible for the protondriven uptake of d-lactose in e. coli. with a high resolution structure available and considerable understanding of mechanistic detail, and with observed changes to both structure and function in different bilayer environments, lacy is a good model system for examining the behaviour of a major class of membrane proteins in these lipid systems. purified lacy has been reconstituted into liposomes and droplet interface bilayer systems of varying lipid composition and the effect on protein function and bilayer properties examined. targeting abeta oligomers by trehalose-conjugated peptides: a molecular dynamics study alzheimer's disease (ad) is currently one of the most common and devastating forms of dementia correlated with beta-amyloid peptide (abeta) accumulation in human brain tissue [ , ] . inhibiting abeta selfoligomerization in brain tissue remains one of the main strategies to prevent or treat this disorder. as a consequence, in recent years much efforts have been spent in the understanding of the amyloid fibril growth process and its modulation by putative drug molecules. an interesting class of compounds able to prevent abeta fibrillogenesis, is represented by beta-sheet-breaker (bsb) peptides [ ] . although these molecules are thought to recognize in a self-complementary manner the abeta hydrophobic core region, however their precise mechanism of interaction is still unclear. in this context, we have studied the structural basis underlying the inhibitory effect of abeta( - ) fibrillogenesis explicated by two promising trehaloseconjugated bsb peptides (ac-lpffd-th (thct) and th-succinyl-lpffd-nh (thnt)) [ ] using an all-atom molecular dynamics (md) approach [ , ] . the pentameric nmr structure [ ] of abeta has been used to model amyloid protofibril, and the two protofibril ends have been investigated as putative binding sites. our simulations suggest that the interaction with the two protofibril ends occurs through different binding modes. in particular, binding on the odd edge (chain a) is guided by a well defined hydrophobic cleft, which is common to both ligands (thct and thnt). moreover, targeting chain a entails a significant structure destabilization leading to a partial loss of b structure and is an energetically favoured process, as assessed by mm/pbsa calculations. a significant contribution of the trehalose moiety to complexes stabilities emerged from our results. the basic structural unit of chromatin is the nucleosome, which is composed of histone proteins forming a scaffold with about base pairs of dna wrapped around. chromatin compacts eukaryotic genomes and regulates gene activity, which is mediated in part by posttranslational modifications (ptms) on the n-terminal tails of the histones. uncovering the detailed relationship between histone tail modifications and gene activity is a major topic of biomedical sciences and general techniques for generating nucleosomes with defined modification patterns in large numbers would greatly facilitate such investigations. to this end we are establishing a chemical toolbox for designer chromatin with defined histone ptm patterns. a protein semysinthesis approach is used that bases on "ligation-ready nucleosomes" with truncated histone h that can be ligated with the corresponding synthetic histone tail. we resorted to sortase-mediated ligation as chemoselective ligation method. here we report our recent developments in establishing the envisioned chemical toolbox for designer chromatin. evaluating cation-pi and pi-pi interaction in proteins using various biophysical methods in proteins the aromatic residues phenylalanine (phe), tyrosine (tyr), and tryptophan (trp) can be involved in aromatic interactions known as cation-pi and pi-pi interactions (dougherty ). compared to other non covalent interactions in proteins, like h-bonds, dipole-dipole, or van der waals interactions, relatively little is known about the pi-pi and the cation-pi interactions. the strength of both aromatic interactions is dependent on the pi-electron density in the aromatic residues. a lowering of electron density can be created by introducing strong electron-withdrawing substituents like fluorine atoms in the aromatic ring (dougherty ). in this way a nearly isosteric change in the aromatic system results in a marked change in electron density. substitution with methyl groups is known to slightly increase the electron density. the response to low cellular oxygen levels in humans and other animals is induced by the hypoxia inducible transcription factors (hifs). these transcription factors are regulated by hypoxia inducible factor prolyl hydroxylases (phds), which act as 'oxygen sensors' by hydroxylating hifs, thus leading to the proteomic degradation of the transcription factors. over the last years, there have been multiple reports that describe additional phd substrates other than hifs. among them are the large subunit of rna pol ii, several transcription factors, and components of signalling pathways. validating these reports is of major medicinal relevance given that phd inhibitors are now in the late stage phase clinical trials. in order to investigate the selectivity of phds, the reported proteins have been tested as substrates for hydroxylation by mass spectrometry, and as binders or competitors of the phds. initial work on peptides that contain the putative hydroxylation sites has indicated that the phds are much more selective for their well-established substrate hif. however, in ongoing work these initial results are going to be validated on protein level by co-expressing phds with the reported substrates. additionally, peptides of reported substrates were screened for their ability to alter the kinetics of hif-hydroxylation by phd . an inhibitory effect of at least two different peptides on phd was observed, suggesting that there is an interaction between the prolyl hydroxylase and these peptides. in order to investigate the mode of binding and inhibition, nmr studies have been carried out and binding of the two inhibitory peptides on phd has been shown. altogether, these results indicate that, although phds might be more selective for hif as a substrate as it was initially thought, the enzymatic activity of the prolyl hydroxylases is possibly influenced by a number of other proteins that can directly bind to phds. non-natural aminoacids via the mio-enzyme toolkit alina filip , judith h bartha-v ari , gergely b an oczy , l aszl o poppe , csaba paizs , florin-dan irimie biocatalysis and biotransformation research group, department of chemistry, ubb, department of organic chemistry and technology an attractive enzymatic route to enantiomerically pure to the highly valuable a-or b-aromatic amino acids involves the use of aromatic ammonia lyases (als) and aminomutases (ams). all these enzymes have in common an auto-catalically formed -methylene- , -dihydroimidazole- -one (mio) electrophilic prosthetic group, and show high structural and sequence similarities. the recent advances in improving the functional properties of these enzymes increased both their biocatalytic and therapeutic applications. we aimed to create a library of recombinant mio-enzymes consisting of the pals and pams with large substrate promiscuity in order to provide access to various non-natural aminoacids through enzymatic ammonia addition and/or ammonia elimination reactions of the substrate library already available in our researchgroup. the developed complementary substrate and enzyme library would provide the mio-enzyme toolkit useful for the synthesis of nonnatural aminoacids. the synthetic gene of the enzymes (pcpal, rtpal, avpal, papam) were cloned into pet b_j expression vector using xhoi and bpu i cloning sites. the plasmid dna was transformed to several e.coli host strains (rosetta, bl , origami ) in order to optimize the expression yields. the enzymes containing an n-terminal his -tag were purified with affinity chromatography, followed by ion-exchange or/and size-exclusion chromatography, obtaining pure and homogenous proteins, in their tetrameric, presumably native fold. the enzyme activity and the kinetic parameters of the purified enzymes was determined towards the natural substrate l-phenylalanine, as well as towards novel bulkier aromatic substrates (heteroaryl alanines, styryl alanines, biphenylalanines). furthermore to enhance their biocatalytic applicability we covalently immobilized the enzymes to carboxylated single-walled carbon nanotubes (swcnt cooh) using linkers with different lengths, and tested the activity and recycling of the immobilized enzyme. antibodies that bind protein antigens are indispensable tools in biochemical research and modern medicine. utilizing a phage display selection strategy, we have obtained synthetic antigen binders (sabs), based on a fab fragment of igg, to a wide array of proteins as distinct as membrane proteins, structural proteins, scaffold proteins and nuclear targets. here we demonstrate the applicability of the sabs towards the native, full-length proteins in cells. we show that the generated sabs are able to pull-down endogenous proteins from mammalian cell extracts along with their natural binding partners. we developed a method of utilizing our high affinity and specificity binders as fluorescently labeled tools to visualize target proteins in their native environment in the cells without the need of secondary antibodies or blocking reagents. our system also includes a method of efficient delivery of generated antibodies to living cells, where they can perform their function. the sabs have been successfully used for altering biological processes in a controllable manner. in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors tai kubo , mohammed naimuddin , seigo ono national institute of advanced industrial science and technology (aist) in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors small molecule natural products are precious resources for drug discovery. during millions of years of evolution, natural products must have been exposed to various selection pressures and have been refined in structure and function to obtain the present features. in some peptide neurotoxins, however, the basic molecular scaffold mainly configured by disulfide (s-s) bridges and/or alpha/beta structures, is strictly conserved within each family even under the evolution pressure. on the other hand the loop regions, which are not heavily involved in scaffold formation, are highly diverged. this mode of molecular evolution named 'accelerated evolution', is reasonable to quickly adapt to the vigorous change of the environment. the evolutionally selected scaffold is compact harboring both rigidity and flexibility in nature, and it may support a topology appropriate for target recognition and selective interaction. inspired by the system, we designed random peptide libraries from the peptide neurotoxins of the accelerated evolution. a three-finger ( f) shaped snake neurotoxin consists of huge family evolved by accelerated gene evolution. we prepared a f-peptide library by introducing random sequences in each fingertip. another random peptide library with an ick (inhibitor cystine knot) motif was prepared based on a neurotoxin gtx - from spider; originally identified as a t-type ca channel modulator. each library was subjected to in-vitro evolution directed to specific target molecules. for the f-peptide library cdna display method was applied to select binders. when interleukin- (il- ) receptors were targeted, the selected f peptides showed binding affinities (kd nm) comparable to the native ligand il- . when trypsin was targeted, peptides with serine protease inhibitor activities similar to sti and bpti (ki nm) were isolated. specific binders to a trophic factor vegf were also generated from the f library. to target membrane proteins, we developed a unique in-vitro evolution system, and named it as the periss (intra periplasm secretion and selection) method. in the system, target membrane proteins are expressed in inner membrane of e. coli and peptides are secreted to the periplasmic space, in between the inner and outer membranes; and the space is served for interaction and selection. the periss method enabled us to identify a peptide specific to muscarinic receptor m subtype from the ick peptide library. in conclusion, it was proved that the library designed from the scaffold of peptide toxin, which evolved in the mode of accelerated gene evolution, has pluripotency in target recognition, interaction and even bioactivity. phenylalanine ammonia lyase from petroselinum cripsum (pcpal) belongs to the class of enzymes containing -methylideneimidazole- -one (mio) as a prostetic group and it is responsible for the conversion of l-phenylalanine into trans-cinnamic acid. this reaction is reversibile under high ammonia concentration. we analyzed several factors that can influence the enantioselective synthesis of nitrophenylalanine mediated by whole cells as well as purified mio-containing and mio-less pcpals. first we investigated the behaviour of the enzymes depending on the ammonia concentration. we also inspected the influence of the ph on the pcpal catalyzed biotransformations. based on our results, we concluded that variation of ammonia concentration and the ph leads to decrease of enantioselectivity, suggesting that pcpal is able to catalyze the formation of both l-and d-enantiomers of electron-deficient structures. all microbial cellulase appears to have a conserved 'sg' amino acid sequence at an identical position in the n-terminal domain. the properties of the n-terminal amino acid sequence were also predicted computationally. this analysis showed that n-terminal sequence of the enzyme is unstable. the nterminal sequence also showed potential cleavage sites by different proteases which may contribute to its instability. the secondary structure analysis showed that the n-terminal sequence has % of the a.a. sequence in extended strand and % in random coil conformation. the n-terminal sequence was also analyzed for potential phosphorylation sites. while no potential serine and threonine sites were predicted, two tyrosine phosphorylation sites were predicted in the n-terminal sequence. the n-terminal sequence was also examined for the presence of kinase specific phosphorylation sites. the results showed the presence of one potential site which may be phosphorylated by pkc at position of the n-terminal sequence. the analysis for the prediction of the presence of oglcnac sites revealed that two such sites may potentially be present in the sequence. we have also predicted the ligand binding site in the n-terminal sequence of the protein. protein arginine methylation catalyzed by protein arginine methyltransferases (prmts), is a pivotal protein post-translational modification involved in a growing number of physiological and pathological processes including signal transduction, proliferation, differentiation and malignancy. prmt accounts for the majority of protein arginine methyltransferase activity in mammalian cells and, in consistence, a large amount of cellular substrates have been identified. several studies have reported that the activity of prmt changes upon stimulation in various cellular processes. in mammalian cells, prmt exists in a high molecular weight complex. the interacting partners of prmt , such as antiproliferative proteins btg and btg , protein phosphatase a, the orphan receptor tr , and ccr -associated factor (hcaf ) are shown to play a role in modulating the methyltransferase activity and the substrate selectivity of prmt . due to the pivotal roles of prmt in physiological and pathological conditions, intensive efforts have been put on the search of small synthetic chemical molecules which can efficiently modulate the activity of prmt for the potential development of therapeutics. in light of this, the intracellular small molecules that either transmit extracellular stimulation or act as cofactor to dictate the activity of prmts in cells are still poorly understood. our study focused on examining how cellular ions might affect the activity of prmt and found that divalent and monovalent ions differentially modulated the catalytic activity of prmt toward different substrates. oligomerisation properties of light-dependent protochlorophyllide oxidoreductase prothoracicotropic hormone (ptth) is one of the most important neuropeptide regulators for insect molting and metamorphosis. however, preparation of its recombinant protein has hardly been successful, because it is a homodimer protein with very complicated disulfide-bond structure. for example, silkworm ptth has three intramolecular disulfide bonds in its -residue polypeptide chain, and the two chains are further linked by an additional intermolecular disulfide bond to form the homomeric dimer. although the recombinant silkworm ptth was previously expressed in escherichia coli, the product was obtained only in precipitation fractions, and refolding of the precipitated protein provided the active dimer ptth in very poor yield. under such reductive conditions as in cytosol of the e. coli cells, formation of the correct disulfide-bond arrangement must be difficult. alternatively, for the heterologous expression of the silkworm ptth, we employed brevibacillus choshinensis (formally referred to as bacillus brevis), which has achieved good results in expression of various disulfide-bond-containing proteins. in this study, the silkworm ptth was expressed in the brevibacillus cells with an additional his -tag sequence at the c-terminus, for easier detection and purification. first of all, since the brevibacillus bacteria are equipped with a secretory system of the expressed proteins, a secretory signal sequence to be attached before the silkworm ptth was carefully selected. among four candidates in a commerciallyavailable kit, a signal sequence derived from an intrinsic cell-wall protein mwp gave better results in expression levels of the protein. second, incubation time of the cells was optimized, because an oligomerization state of the secreted ptth in the cell culture medium changed with the time. in the medium, various ptth oligomers including a monomer and a dimer were initially observed, but higher oligomers became a major portion of the secreted product after longer incubation than h. incubation for - h may be suitable for obtaining the native dimer form of the silkworm ptth. to remove the undesired monomer and higher oligomers, which mostly retained free sulfhydryl groups, the secreted proteins were treated with maleimide-peg -biotin. in the purification using a ni -nta column, the dimer of the his -tagged silkworm ptth was eluted with an imidazole gradient, separately ahead of other biotinylated proteins, probably due to interaction of the peg spacer with the ni -nta groups of the resin. after the reversed-phase hplc purification, the final product showed a single band on the nonreductive sds-page, and it had adequate ecdysone-releasing activity from isolated silkworm prothoracic glands. the brevibacillus bacteria are most promising host cells for the heterologous production of the insect ptth. role of the disulfide bridges in the transmembrane region of the insect prothoracicotropichormone receptor, torso torso is an insect cellular-membrane protein, which was recently identified as a receptor for prothoracicotropic hormone (ptth). although ptth is one of the important regulatory molecules in insect molting and metamorphosis, activation mechanism of torso by the ligand has not been elucidated yet. in this study, an oligomerization manner of the silkworm torso was examined, using heterologous expression in drosophila s cultured cells, because torso is a single-polypeptide receptor tyrosine kinase (rtk), and activation of such rtks is often triggered by the ligand-induced receptor dimerization on the cellular membrane. when activated with silkworm ptth, dimerization of the silkworm torso in the s cells was observed, using a cross-linking reagent bs , and the subsequent receptor autophosphorylation and downstream erk phosphorylation were also detected. surprisingly, however, the torso dimerization was revealed to occur even without the ligand stimulation, while the autophosphorylation and the erk phosphorylation were held in response to the stimulation. when fractionated by non-reductive sds-page, the silkworm torso showed an obvious dimer band, in addition to a faint monomer band, both with and without the ptth simulation, even though the receptor was not treated with the cross-linking reagent. this indicates that the torso protein is expressed originally as a disulfide-bond-linked dimer. in addition, by examining oligomerization states of several truncation and substitution mutants, cysteine residues in the transmembrane region were found to participate in the intermolecular disulfide bridges, linking the two receptor molecules in the dimer. when all of the three cysteines in the transmembrane region were replaced by phenylalanines, the disulfide-bond-linked torso dimerization was not observed, but spontaneous, ligand-independent association of the torso molecules was detected using the crosslinker bs . this spontaneous dimerization caused the apparent torso autophosphorylation, but it could not induce the downstream erk phosphorylation. consequently, without the intermolecular disulfide bridges, torso loses its responsiveness to the ptth stimulation. in conclusion, the disulfide bridges in the transmembrane region may play a role to preserve suitable relative position between the two torso molecules, which could induce ligand-dependent autophosphorylation leading to activation of the downstream signaling pathways in the cells. the yeast enzyme neutral trehalase (nth , ec . . . ) from saccharomyces cerevisiae hydrolyses the non-reducing disaccharide trehalose which serves as an energy source and a universal stress protectant in many different organisms. enzymatic activity of nth is enhanced by the yeast - - protein (bmh and bmh ) binding in a phosphorylation-dependent manner. nth activity is also regulated by ca binding to the ef-hand-like motif containing domain of nth [ ] .the native tbe page and analytical ultracentrifugation show that nth forms very stable complexes with bmh and bmh [ ] . to study the structure of nth alone and its complex with the - - protein we used circular dichroism, h/d exchange coupled to mass spectrometry, chemical cross-linking [ ] and small angle x-ray scattering (saxs) [ ] . at the same time protein crystallography of nth alone and its complex with bmh is performed.the low resolution structure of pnth :bmh protein complex revealed that binding of bmh induces a rearrangement of the whole nth molecule and that the region containing the ef-hand motif forms a separate domain which interacts with both bmh and catalytic domain of nth . we proved that integrity of the ef-hand motif is crucial for the bmh mediated activation of nth and ca binding. our data suggest that the ef hand-like motif functions as the intermediary through which bmh modulates the function of the catalytic domain of nth . these structural changes probably enable the substrate entry into the enzyme active site [ ] . our study of - - protein complex with the fully active enzyme nth offers a unique structural view of nth activation enabling us to better understand the role of the - - proteins in regulation of other enzymes. the assembly of self-regulating synthetic biochemical pathways in vitro has great potential as alternative catalysts for the high-yield production of low value/high volume commodity chemicals from biomass. high yields of low-value/high volume compounds that are required for economic viability is particularly difficult via traditional in vivo metabolic engineering of microbes due to competing biochemical pathways and toxicity. we have developed an alternative approach, called synthetic biochemistry, where the glycolysis pathway of central metabolism is reconstituted in vitro with an anabolic pathway that can produce useful compounds at high yield. in the specific synthetic biochemistry system described, reducing equivalents, atp, and carbon from glycolysis are funneled through the anabolic mevalonate pathway to produce the monoterpene limonene from glucose. the successful implementation of the in vitro pathway required development of a molecular purge-valve consisting of an nad and nadp specific reductase (ie wild-type and mutant pyruvate dehydrogenase), and nadh oxidase, noxe, to maintain proper nadp /nadph cofactor balance while allowing continuous carbon flux. we find that the purge-valve concept is readily transportable to other nad(p)h generating steps in central metabolism and can be used to convert glucose to limonene at high yield. chitinases (ec . . . ) are enzymes that randomly hydrolyze b- , glycosidic bonds of chitin and produce n-acetylchitooligosaccharide ((glcnac)n) that has various physiological functions such as immunostimulatory activity. most of fish takes crustacean such as shrimp and crab as food. therefore, the fish has chitinase in the stomach to chemically disrupt the chitinous envelope of crustacean. four chitinase isozymes ( - kda), pachia [ ] and pachib [ ] , and ptchia and ptchib, [ ] were purified from the stomach of silver croaker pennahia argentatus and threeline grunt parapristipoma trilineatum, by ammonium sulfate fractionation and column chromatographies, respectively. all the chitinases were stable and showed activity in the acidic ph range (ph - ). pachia and ptchia preferentially degraded the second glycosidic bond from the non-reducing end of (glcnac)n and pachib and ptchib had a preference for the third glycosidic bond of those. all the chitinases showed different substrate specificity toward insoluble long substrates. moreover, chitinase cdnas (pachi- and pachi- ) encoding pachia and pachib, and cdnas (ptchi- and ptchi- ) encoding ptchia and ptchib were obtained by cdna cloning using the rt-pcr and race method. the deduced amino acid sequences of all the chitinase cdnas contained n-terminal signal peptide, gh family catalytic domain, linker region, and chitin-binding domain. phylogenetic tree analysis of vertebrate chitinase revealed that fish stomach chitinases form unique chitinase isozyme groups, acidic fish chitinase- (afcase- ) including pachia and ptchia, and acidic fish chitinase- (afcase- ) including pachib and ptchib, which was different from an acidic mammalian chitinase (amcase) group. [ , ] the previously reported purified fish stomach chitinases [ ] can also be classified into two chitinase isozyme groups, afcase- and afcase- , by the n-terminal amino acid sequence. this study suggested that fish have excellent chitin degrading enzymatic system in which two different chitinases isozyme groups, afcase- and afcase- , with different degradation patterns are expressed in the stomach. recently, the enzymes produced by psychrophilic organisms have gained huge interest especially in the studies of temperature adaptation of the protein. previously, a cold-adapted yeast, glaciozyma antarctica pi was isolated from a marine environment in antarctica and the yeast was known to produce lipolytic and proteolytic enzymes. a gene encoding a unique recombinant bifunctional enzyme (lippi ) with cold active lipase with protease activity was successfully expressed, purified and characterized. temperature profile of the bifunctional lippi enzyme showed that the lipase functions optimally at c whereas the protease was more active at c. ph profile showed that both lippi lipase and protease were active at near neutral condition. activity of lippi lipase and protease were also activated in the presence of cacl but its protease counterpart seemed to be more active in the presence of zncl . effect of surfactants showed lippi lipase was activated by tween and sls and in contrast, lippi protease was almost deactivated in all surfactants tested. the presence of organic solvents did not affect both the lipase and protease activities. the lipase was more stable at solvents with higher log p value whereas the protease was slightly activated at low log p value particularly with dimethylsulfonyl. inhibitor studies revealed that lippi lipase was partially inhibited with edta and pmsf whereby the lippi protease was inhibited by pepstatin, edta and pmsf. lippi enzyme was successfully crystallized via vapour diffusion method. crystal of lippi enzyme was diffracted via synchrotron radiation. the three-dimensional structure of cold-adapted pi provided insight into cold adaptation and better understanding of the structural properties of lippi enzyme. the bifunctional properties of the enzyme could be potential candidate for low temperature industrial application. conformation-specific antibodies as enhancers and inhibitors of phosphatase activity of dep malgorzata nocula-lugowska , mateusz lugowski , anthony a. kossiakoff the university of chicago dep- (cd /ptp-h) is a transmembrane receptor-like protein tyrosine phosphatase (ptp) that has been implicated in the density-dependent regulation of cell growth, differentiation and transformation. it counteracts protein kinases by dephosphorylating a number of their substrates as well as the kinases themselves, thus potentially controlling the specificity of signals. for example egfr, vegfr , met, pdgf b receptor have been shown to be dephosphorylated by this phosphatase. dep- has been shown to act as a tumor suppressor and it has been proposed as a molecular target in antiangiogenesis therapy. as a result, both enhancers and inhibitors of dep- activity have the potential of elucidating pathways responsible for abnormal cell behavior. we generated synthetic antibodies against intracellular catalytic domain of dep- that act as modulators of the enzyme's phosphatase activity. by applying a combination of selection pressures an array of antibodies has been raised from phage display libraries of fab fragments which are capable of either enhancing or inhibiting dep- activity. in phosphatase assays with catalytic domain of dep- the antibodies demonstrate non-competitive or mixed kinetics. the crystal structure of dep- -inhibitor complex shows that this antibody binds to the part of the protein that is distant from the active site and acts by locking the enzyme in the nonnatural catalytically inactive state by hindering the closure of the wpd loop which is crucial for the reaction to occur. by contrast, as judged from the crystal structure of a complex of dep- with the antibody that enhances its phosphatase activity, this antibody seems to act by stabilizing the naturally found active state of dep- with wpd loop in the closed conformation. the antibodies are also able to recognize dep- in cells, as they stain dep- in immunofluorescence experiments. to test the applicability of raised antibodies in cells the activator was additionally used to pull down full-length endogenous dep- after being delivered to live cells. inhibition and enhancement of dep- activity by locking the enzyme in conformations which are either natural or imposed by allosteric binding of antibodies seems to be a mechanism that can be utilized to modulate activity of other tyrosine phosphatases. investigating acinetobacter baumannii pathogenesis: crystal structure of wbjb epimerase from a polysaccharide biosynthesis cluster oxygen homeostasis is regulated by hypoxia inducible factor, a transcription factor. when the oxygen level becomes too low (hypoxia), hypoxia-inducible-factor (hif- a) activates the expression of over a hundred genes, associated with angiogenesis, erythropoiesis, vegf (vascular endothelial growth factor), cell migration, and energy metabolism etc. hif- a cellular level is highly dependent on oxygen concentration and regulated by oxygen sensor enzyme, hif prolyl hydroxylase (phd plant sulphite reductase (sir) forms an electron transfer complex with ferredoxin (fd) for the reductive conversion of sulphite to sulphide. although previous studies have highlighted electrostatic interactions between oppositely-charged residues of the two proteins, detailed thermoenergetics of the intermolecular interaction for the complexation remains unknown. we herein carried out isothermal calorimetry of fd:sir complex formation at various nacl concentrations. driving force plot constructed from calorimetry showed that the complex was thermodynamically stabilized by both enthalpy and entropy through favourable electrostatic and non-electrostatic interactions. increasing nacl concentrations weakened interprotein affinity and contribution of the negative enthalpy changes became decreased, while no such significant decrease was found in the contribution of positive entropy changes. furthermore, a negative heat capacity change obtained from the enthalpy changes at distinct temperature indicated a contribution of hydrophobic interactions. these findings suggested that both electrostatic and nonelectrostatic interprotein interactions were energetically important for the complex formation. fddependent sir activity assay revealed a bell shaped activity curve with a maximum under a certain nacl concentration, while the methyl viologen-dependent assay of sir exhibited a profile of saturating curve, suggesting that an optimized interprotein interaction is a crucial factor in control of fd-dependent-sir activity. a residue-based nmr measurement of n-labeled fd upon complex formation with sir revealed that charged and non-charged residues were differentially contributed in the complex formation depending on nacl concentrations. we proposed that non-electrostatic forces were also critical for forming the fd:sir complex, and an optimized complex conformation for maximum enzymatic activity was achievable by a delicate balance among non-covalent intermolecular forces. these results may be extended for understanding of complexation between redox proteins containing biased charge clusters. ornithine transcarbamylase has a spatially extended active site as computationally predicted lisa ngu , kevin ramos , nicholas delateur , penny beuning , mary jo ondrechen understanding how an enzyme catalyzes a reaction is a fundamental problem in protein science. biochemical experimentation has revealed catalytic mechanisms of many enzymes; however these studies have focused almost exclusively on amino acid residues in direct contact with the reacting substrate molecule(s). here we report on the computational prediction and experimental verification of the importance of distal residues in enzyme catalysis, using e. coli ornithine transcarbamylase as an example. partial order optimum likelihood (pool), developed at northeastern university, is a machine learning technique that only requires the tertiary structure of a protein to predict important catalytic residues, based on computed, residue-specific electrostatic and chemical properties. pool has been shown to predict accurately the catalytic residues and to discern between compact and spatially extended active sites. dynamic conformational changes during catalysis and strong electrostatic interactions give rise to significant coupling between remote residues and the canonical active site residues of an enzyme. this suggests that at least some enzyme active sites are spatially extended, with second-and third-shell residues playing significant roles in catalysis. in this project, we focus on ornithine transcarbamylase (otc), for which dynamic processes are believed to play a role in its catalytic mechanism. otc is reported to undergo induced-fit conformational changes upon binding carbamoyl phosphate, which affects the subsequent binding of ornithine. residues predicted by pool to be catalytically important include five in direct contact with the substrate, r , h , d , c and r . pool also predicted remote residues to form a spatially extended, triple-layer active site. guided by computational predictions and using site-directed mutagenesis and kinetics assays of asp , his , glu and arg variants, we show that these pool-predicted remote residues, located in the second and third layers, are important for catalysis. alternative energy is a major focus of current research efforts. biodiesel, a mixture of fatty acid alkyl esters, is one of the most versatile alternative fuels currently in use. this is due to the fact that it is similar to gasoline and compatible with diesel engines found throughout the existing global infrastructure. biodiesel precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. however, the standard process for converting oil to biodiesel is heat-intensive and requires complete removal of water, reducing the overall net energy gained in its production. our work constitutes an attempt to explore enzymatic synthesis of biodiesel from lipids such as those derived from emerging fuel crops. previous literature describes fatty acid alkyl ester formation in human patients with mrsa staphylococcus aureus wound lesions. these esters are formed by partially characterized esterase activity from an unidentified source. we have identified two mrsa enzymes responsible for this activity by using a combination of size exclusion chromatography, gas chromatography-mass spectrometry, and mass spectrometric protein sequencing. these two highly similar enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous conditions at or near room temperature. we have demonstrated that other non-staphylococcal lipases do not exhibit this behavior. we have expressed these staphylococcal esterases in e. coli, and shown via gas chromatography that the expressed proteins catalyze the formation of fatty acid alkyl esters. based on sequence similarity to homologous proteins that have already been crystallized, we have predicted a structure for these enzymes and have engineered mutant fusions with higher rates of catalysis. our design hypothesis is that increased avidity for substrate molecules will yield a higher substrate concentration in the vicinity to the enzyme. to increase substrate concentration we have designed and expressed one of the enzymes as a chimeric fusion with the drosophila melanogaster alcohol-binding protein lush. gc-ms determination of biodiesel production rate indicates that the chimeric fusion has a lower-order rate constant with respect to ethanol. in other words, the fusion enzyme is less dependent on substrate concentration and is a superior catalyst at low ethanol concentrations. this result indicates that the rationally designed modification of binding avidity constitutes a potential avenue for improving the ability of enzymes to catalyze reactions with low-concentration or low-solubility substrates. functional elements of a human antizyme essential for binding and inhibiting human ornithine decarboxylase proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with -dimensional shapes and complex conformational dynamics that are not well represented by -dimensional sequence alone. these structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. in this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the kunitz-bpti family of canonical serine protease inhibitors by mesotrypsin. bovine pancreatic trypsin inhibitor (bpti), an archetypal serine protease inhibitor of the kunitz family, has a high affinity interaction with trypsin, yet its peptide bond hydrolysis is many orders of magnitude slower than other peptide substrates. mesotrypsin, a trypsin variant, has been shown to hydrolyze kunitz family inhibitors at accelerated rates; this is especially true of human kunitz domain inhibitors. amyloid precursor protein inhibitor (appi) and amyloid precursor like protein- (aplp ), two human kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than bpti. here, we present a new, unpublished crystal structure of a cleavage intermediate aplp bound to mesotrypsin, refined to . Å resolution, revealing a dramatic substrate conformational change we hypothesize to be required during cleavage of a kunitz domain. using this structure along with published structures of appi and bpti complexes, we have modeled acyl-enzyme intermediates of mesotrypsin, and we have carried out molecular dynamic simulations that explore the transition of the initially formed native-like acyl-enzyme through the conformational transformation that allows the progression of the hydrolysis reaction. we further identify a specific hydrogen bond, present in bpti but not appi, which forms a stabilizing feature of the bpti scaffold. using site directed mutagenesis, we probe the contribution of this bond to the proteolytic stability of bpti. collectively our data for these highly structured substrates show that proteolysis rates are limited by a necessary conformational change in the substrate as the reaction progresses. rigid substrates possessing stabilizing features that render them highly resistant to this conformational change are proteolyzed more slowly than more flexible substrates of similar structure. lpmos are copper metalloenzymes that carry out the oxidative cleavage of the b- , -glycosidic bond, generating new chain ends that can subsequently be processed by cellulases, boosting the cellulose degradation. lpmos have a b-sandwich conformation with a flat binding surface, allowing for the enzyme to bind to crystalline cellulose. the cu ion, required for activity, is located in a so-called "histidine brace", in which the n-terminal histidine is highly conserved. regioselectivity according to the carbon atom being oxidized, lpmo types are identified: type and type oxidizing at the c and the c respectively, type lpmos oxidizing both the c and the c adjacent to the glycosidic linkage. we were able to express a type- lpmo (phanerochaete chrysosporium gh d) and a type- lpmo (trichoderma reesei cel a) in p. pastoris. this has proven to be very challenging, as lpmo activity requires a perfect cleavage of the signal sequence. after activity assays on pasc, characteristic hpaec-pad traces were obtained which will serve as a reference for engineering experiments. enzyme engineering using the dm database, a structure based multiple sequence alignment tool, it is possible to identify residues specifically conserved in subsets of protein sequences. by defining a subset for each lpmo type, we were able to identify residues contributing to regioselectivity. these positions are now being rationally engineered in subsequent rounds of mutagenesis, using trcel a as a template. the effect of the mutations will be determined by analyzing the hpaec-pad trace released from pasc. the main goal is to investigate the possibility of deleting the c specificity in a type lpmo. folding topology determines substrate binding order in the ribokinase superfamily alejandra herrera-morand e , victor castro-fern andez , madrid, españa ribokinase superfamily comprises three enzyme families: the adp-dependent sugar kinases family, the atpdependent coenzyme kinases family and the atp-dependent sugar kinases family. in all these families there is a large domain composed by a rossmann motif but only the atp-dependent enzymes have a b-meander motif in the c-terminal end. interestingly, these enzymes display an ordered kinetic mechanism where the substrate that will be phosphorylated binds first to the enzyme. the adp-dependent enzymes present a topological re-ordering of the secondary structural elements which produces an equivalent tertiary structure, which can be thought as a non-circular permutation (ncp) of the bmeander region. these enzymes also display an ordered kinetic mechanism but with an inversed order being the nucleotide the first substrate to bind to the enzyme. as this b-meander region of the proteins constitutes almost entirely the nucleotide binding site, and given that the permutation is the major structural difference between adp and atp-dependent kinases, it could the responsible for the nucleotide specificity. to test this hypothesis we introduce, by permutation, an atp-dependent topology in the homologous adp-dependent glucokinase from t. litoralis (pergk). size exclusion chromatography and circular dichroism spectra show that both the wild type and the permutated enzyme eluted as monomers with similar hydrodynamic behavior, and have the same secondary structure content. kinetic assays employing atp or adp as substrate demonstrate that even in the presence of mm atp, the pergk enzyme is not able to carry out the phosphoryl transfer. to test if the ncp has an impact in the kinetic constants and substrate binding order we determine the kinetic mechanism through classical protocols, involving initial velocity studies, product inhibition and dead end inhibitors. the results demonstrate that the pergk enzyme presents an altered substrate binding order compared to the wild type enzyme, where glucose was the first substrate to bind to the enzyme and glucose- -p the last product to be released. also, ligand-induced conformational changes were determined in the crystal structures. the apo, the enzyme-glucose and enzyme-glucose-adpbs structures were determined at . Å, . Å and . Å resolutions, respectively. structure analysis reveals that glucose binding provokes major conformational changes in the pergk enzyme, whereas adp binding does not cause further changes in the conformation of the protein. the results show that although the permutation has no effect on the nucleotide preference it provokes a change in the substrate binding order that correlates well with that those observed in the crystal structures. also, they demonstrate that during the evolutionary history of the ribokinase superfamily folding topology dictates the substrate binding order (fondecyt ). background: human ceruloplasmin (cp) is a circulating copper-containing glycoprotein produced in the liver and first described as a component of alpha -globulin fraction of human plasma. cp belongs to the multicopper oxidase family and it is nowadays regarded as a "moonlighting" protein, because it changes its function according to substrate, localization and expression. cp plays a key role in copper transport and iron metabolism and it is also a potent inhibitor of leukocyte myeloperoxidase (mpo) (kd nm), a major source of oxidants in vivo. the protein is extremely susceptible to proteolysis. in fact, cp is a structural homolog of coagulation factors v and viii, that are physiological substrates of thrombin (fiia). interestingly, thrombin participates in both haemostatic and inflammatory responses: in some focus of inflammation, such as rheumatoid arthritis (ra), the high activity of fiia has been documented. it was demonstrated that fiia can promote the chemotaxis of neutrophils and monocytes and their adhesion to endothelial cells, to increase vascular permeability. all these effect are mediated by par- interaction, that are abundantly expressed in inflamed rheumatoid synovial tissues. aims: in this study the interaction of cp with thrombin was investigated to confirm the participation of fiia in "spontaneous" proteolytic degradation of cp. in fact, in vivo the integrity of cp is essential for its role in the transport or metabolism of copper. results: our results indicated that thrombin cleaves cp in vitro at arg-ser and lys-val bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the mpo inhibitory function of cp is abrogated. analysis of the synovial fluid of ra patients reveals that cp is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with fiia in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. we demonstrate that fiia has intrinsic affinity for cp (kd - nm), independently of proteolysis, and inhibits cp ferroxidase activity (ki nm). mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, fibrinogen gamma-peptide) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of thrombin binds to the negative upper region of cp, while the protease active site and exosite-i remain accessible. these results suggest that thrombin can exacerbate inflammation in ra by impairing via proteolysis the mpo inhibitory function of cp and by competitively inhibiting cp ferroxidase activity. an artificial pathway for isobutene production by direct fermentation: combining metabolic engineering and protein engineering benoit villiers , franc¸ois stricher the purpose of global bioenergies is to develop innovative metabolic pathways for the production of light olefins from renewable resources, by direct fermentation. light olefins (ethylene, propylene, linear butylene, isobutene and butadiene) are the core of the petrochemical industry. however, microorganisms do not naturally produce light olefins and no bioprocess to convert renewable resources to these molecules has been industrialized so far. global bioenergies has developed an artificial metabolic pathway including all the necessary enzymatic reactions from feedstock to isobutene. the metabolic route leading to isobutene can be divided in three parts, the first one being the use of natural reactions occurring in the host microorganism. second, heterologous natural reactions were introduced into the same host microorganism. finally, in contrast with most former approaches, non-naturally occurring reactions as enzymatic key steps were used, for example the decarboxylation of hydroxyisovaleric acid into isobutene. such non-natural critical steps were made possible by taking advantages of the natural catalytic and substrate promiscuity of exogenous enzymes. candidate enzymes are then evolved using systematic, random and semi-rational approaches in successive rounds in order to reach the desired catalytic efficiency. since all these reactions are enzymatic, isobutene can be obtained by direct fermentation, e.g. a process wherein all the chemical transformations are carried on by the host microorganism. the scale-up of this process began in november in a pilot plant installed in pomacle-bazancourt, france, with an annual capacity of tons of oxidation-grade isobutene. importantly, production of a volatile compound such as isobutene (and other light olefins) by direct fermentation presents two major advantages: first, the product is spontaneously removed from the culture broth, which alleviates the limitations linked with titer issues. second, the purification process is considerably easier and cheaper since no energy consuming methods such as distillation or phase separation are necessary to purify the end product. for the first time, batches of industrially produced isobutene from renewable resources have been obtained in the first half of . this isobutene has been in turn converted into isooctane, an additive currently used to improve gasoline quality, which could also be used as a standalone fuel. a demonstration plant is planned in leuna, germany, with an annual capacity of tons of polymer-grade isobutene and ibn-one, a joint venture with cristal union ( th european beet processor), has been formed to build and operate the first plant in france converting renewable resources into isobutene. finally, while the isobutene process is progressing towards industrial scale, global bioenergies is also developing new artificial metabolic pathways enabling direct bio-production of butadiene and propylene. the development of a coupled enzyme assay to detect isochorismate pyruvate lyase activity protein folding is typically defined in terms of the spatial arrangement of structural elements, i.e. helices, sheets and loops. we have, however, been developing an alternative and complementary paradigm based on conserved hydropathic interaction networks within proteins. these networks can be viewed as environments comprised of a mixture of polar and hydrophobic interaction fields, and may be the most important factor driving protein folding. this concept applies even to the lowest structural level within a protein: the sidechain conformations (or rotamers). exhaustive statistical analysis of existing crystallographic structures of proteins showed rotameric preferences and led to the creation of rotamer libraries frequently used in multiple aspects of structural biology, e.g., crystallography of relatively low-resolution structures, homology modeling and biomolecular nmr. however, little is actually known about the forces and factors driving the preference or suitability of one rotamer over another. in our study, tyrosine was analyzed since its sidechain has a comprehensive set of hydropathic properties that made it ideal as a proof of concept residue. construction of d hydropathic interaction maps of tyrosine residues in our dataset, reveals the environment around each, in terms of hydrophobic (p-p stacking, etc.) and polar (hydrogen bonding, etc.) interactions. after partitioning the tyrosines into backbonedependent bins, a map similarity metric based on the correlation coefficient was applied to each mapmap pair to build matrices suitable for clustering. notably, the first bin representing tyrosines, reduced to unique hydropathic environments with most diversity arising from favorable hydrophobic interactions with many different residue partner types. polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic oh and somewhat surprisingly a handful of unique environments for the tyrosine backbone. all but one of the environments are dominated by a single rotamer, the exception being an environment defined by a paucity of interactions with the tyrosine ring and as a consequence its rotamer is indeterminate. this is consistent with it being composed of mostly surface residues. each tyrosine residue attempts to fulfill its hydropathic valences and thus, structural water molecules are seen in a variety of roles throughout these environments. alanine was analyzed using the same protocol as well. having the smallest sidechain (and small hydropathic interaction maps), alanine allowed us to investigate a significantly larger database, permitting us to examine the correlation between hydropathic maps and various structural features. in conclusion, the analysis of hydropathic environments strongly suggests that the orientation of a residue in a three-dimensional structure is a direct consequence of its hydropathic environment, which leads us to propose a new paradigm, interaction homology, as a key factor in protein structure. it is not the surrounding residues that direct sidechain conformations, but rather the hydropathic "field" of the surrounding atoms. folding studies of independent domains of lysine, arginine, ornithine binding protein (lao) protein folding problem has been addressed from the past years until nowadays, however, we still can not explain how proteins acquire their native structure from their amino acid sequence. different approaches has been taken in order to study protein folding, for example, the comparative study of folding mechanism between homologues proteins with high identity of sequence and structure, and the study of independent regions within a single protein. previously in our laboratory, thermodynamic and kinetic folding properties of lysine, ornithine, arginine binding protein (lao), a amino acid periplasmic binding protein (pbp), composed by two rossmann fold domains (one continuous and the other discontinuous) attached by a hinge region, has been studied. even there is a functional research about binding characteristics of histidine binding protei ns (his j) domains of when expressed independently (chu, b. ); there are no folding studies in these conditions for this or another pbps. it should be noted that his j shares % of sequence identity and tertiary structure (rmsd Å) with lao. in order to know the folding effect of encoding different domains in the same poly peptidic chain, as well as its influence in function, we are studying the thermodynamic and kinetic characteristics of folding of independently expressed lobes of lao, and comparing with those of native protein. by now, we expressed and purified the discontinuous domain. circular dichroism (cd) and fluorescence intensity spectra show that this independent domain has primary and tertiary structure. thermal denaturation has a single cooperative transition, which indicates this domain is folded. thermodynamic analysis of temperature and urea-induced experiments suggest that lao's folding characteristics are not just the addition of those from independent domains. furthermore, folding and refolding kinetics suggest the presence of a burst phase intermediate. a hypothesis to reconcile the physical and chemical unfolding of proteins a comprehensive view of protein folding is crucial for understanding how misfolding can cause neurodegenerative diseases and cancer. when using physical or chemical perturbations, nmr spectroscopy is a powerful tool to reveal a shift in the native conformation toward local intermediates that act as seeds for misfolding. high pressure (hp) or urea is commonly used to disturb folding species. pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the proteinsolvent system. however, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. here, we provide nmr spectroscopy and d reconstructions from x-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by hp. in studying mpnep from moniliophthora perniciosa, we tracked two cooperative units using hp-nmr as mpnep moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. at subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by nmr intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle x-ray scattering (saxs)]. this state has a higher susceptibility to pressure denaturation (lower p / and larger dvu); thus, urea and hp share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. these observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. zinc: a promoter or inhibitor for iapp aggregation? feng ding , praveen nedumpully-govindan zinc ions have been found to play an important and yet complex role in human islet amyloid polypeptide (hiapp) aggregation, which is associated with b-cell death in type-ii diabetes (t d). both concentration-dependent promotion and inhibition of iapp aggregation by zinc ions have been observed in vitro. similarly, at the population level, both positive and negative correlations were reported between the activity of a b-cell specific zinc transporter and t d risk. zinc ions are able to bind a single histidine in hiapp and coordinate the formation of zinc-bound hiapp oligomers. we hypothesize that the relative zinc/hiapp concentration determines the population of zinc-bound hiapp oligomers with different molecular weights. we have applied molecular dynamics (md) simulations to systematically study the structure and dynamics of a range of zinc-coordinated hiapp oligomers, including monomers, dimers, trimers, tetramers, and hexamers. our computational results suggest that different zinc-bound oligomers have distinct aggregation propensities. high-molecular weight oligomers ( peptides) have higher aggregation propensity than zinc-free and zinc-bound hiapp monomers at mm concentration in silico. therefore, our results provide a molecular insight into the complex role of direct zinc binding on hiapp aggregation. at low zinc/hiapp stoichiometry, zinc binding promotes aggregation. as the stoichiometry increases and zinc ions bind to single hiapp peptides, the aggregation of hiapp is inhibited due to electrostatic repulsion between the charged zinc ions. our computational study sheds light on the complex role of zinc on hiapp aggregation and t d development. biomolecules function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of % with macromolecules [ ] . often, artificial macromolecular crowding agents are used to mimic these conditions in vitro and the excluded volume theory is applied to explain the observed effects [ ] . however, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [ , ] . we study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, polyethylene glycol, potassium chloride) [ ] . in contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. the enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. further, it remains unclear how such cosolutes reflect the physicochemical properties of the complex cell environment as a characterization of the in-cell crowding effect is lacking. thus, we developed a fret-based macromolecular crowding sensor to study the crowding effect in living cells [ ] . the averaged conformation of the sensor is similar to dilute aqueous buffer and cell lysate. we find that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. the presented method allows to systematically study in-cell crowding effects and understand them as a modulator of biomolecular function. the stability of biomolecules under co-solvent conditions is dependent on the nature of the co-solvent [ ] . this can alter a protein's properties and structural features through biomolecular interactions between its functional groups and the co-solvent molecules. ionic liquids (ils) represent a rather diverse class of co-solvents. the design flexibility of these molten salts is an attractive feature, allowing the properties of the il to be tuned to meet the requirements of different applications [ ] . particularly, the modulation of reaction pathways between folding states, offering possibilities to control irreversibility in non-native protein aggregation [ ] . this has led us to investigate the impact of ils as co-solvents with the well-known protein denaturant urea. urea is considered to be a non-ionic chaotrope disturbing considerable the grid of hydrogen bonds with the protein backbone. urea interacts preferentially with the protein surface, mainly apolar residues and that dispersion, rather than electrostatic interactions, is the main energetic contribution to explain the stabilization of the unfolded state of the protein and the irreversibility of the unfolding process in the presence of urea [ ] . a large body of multidomain protein folding work has been devoted to study monomeric proteins. how do multidomain multimeric protein fold, avoiding accumulation of stable intermediate is yet to be studied in detail. our present study is focussed on understanding the folding and assembly of the domains of a homodimeric l-aspraginase from a hyperthermophile pyrococcus furiosus (pfa). each monomer of pfa consists of distinct n-and c-terminal domains (npfa and cpfa, respectively), connected by a linker. the folding mechanism of each domain with respect to full length protein was studied by mutating one out of two tryptophans, one in each domain. domains were purified and studied individually to obtain parallel account of the folding of each domain in isolation. subunit assembly was studied by analytical size exclusion chromatography (sec), multiangle light scattering and functional activity. through far uv cd, intrinsic trp fluorescence and sec, we demonstrated that domain folding and subunit association were intimately linked in full length pfa. interestingly, en route to its folding there was complete absence of hydrophobic intermediates as probed by ans fluorescence. folding of npfa was highly cooperative and, it provides interacting surfaces for cpfa to fold and also facilitates subunit assembly. the folding cooperativity of isolated domains was very less compared to the folding cooperativity of their full length counterparts, as indicated by equilibrium m values. to our surprise, during ph induced denaturation, at ph and , the dimer dissociates into highly hydrophobic folded monomers which readily underwent amyloidogenesis. we showed that at such extreme conditions, cooperativity in folding process in multidomain multimeric protein is not solely governed by the folding of individual domains, rather by concomitant folding and association of domains directly into a quaternary structure. in other case, where subunit folding occurred prior to association, protein readily underwent extensive aggregation. groel assisted folding of multiple recombinant proteins simultaneously over-expressed in e.coli megha goyal , tapan kumar chaudhuri aggregation prone recombinant proteins very often form inclusion bodies and also exhibits poor yield of functional protein during in vitro refolding process from chemically denatured form. bacterial chaperonin groel provides folding assistance to several proteins, when over-expressed with one of the recombinant proteins. there are instances that groel in presence of few other co-expressed chaperones like dnaj, dnak etc provides better yield of folded protein during homologous and heterologous expression. considering the ongoing events in the cells, it is known that molecular chaperone groel assists in the folding of various proteins in the cytoplasm. hence attempt to fold multiple recombinant proteins over-expressing simultaneously with the co-expression of chaperones can be worth trying. this approach may cut down various complexities in the functional recombinant protein preparation, including time and effective cost. keeping this view in mind, folding of two simultaneously expressed aggregation prone proteins, kda e.coli maltodextrin glucosidase (malz) and kda yeast mitochondrial aconitase have been investigated with the co-expression of groel and groes in e.coli cytosol. it has been previously reported that both the chosen proteins undergo co-expressed groel-groes assisted folding in e.coli cytosol, when they over-express alone. in this study we have optimized the overexpression of malz and aconitase simultaneously in e.coli. further optimisation was carried out to coexpress groel along with malz and aconitase. based on the basic philosophy that soluble protein mainly contains folded fraction, the event of groel/es assisted folding of simultaneously overexpressed proteins, malz and aconitase was monitored through the attainment of soluble proteins under various sets of conditions such as temperature. the major outcome of the present study is that, with the groel-groes assistance, the yield of soluble proteins (malz and aconitase) together constitutes higher percentage of folded protein in contrast to the percent yield when a single protein was overexpressed. significance of this type of study relies on the fact that the cells can over-produce higher amount of recombinant proteins, when multiple over-expression takes place. not only pushing up cell's capability of over-expression, co-expression of groel and groes efficiently assists in the folding of multiple proteins simultaneously over-expressed in e.coli. amyloid fibrils associated with serious diseases including alzheimer's, parkinson's, and prion diseases promoted the challenge of studying protein misfolding, leading to the development of amyloid structural biology. amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. here, with b -microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (itc). the spontaneous fibrillation after a lag phase was accompanied by exothermic heat. the thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. we also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. in order to obtain general thermodynamic properties of protein aggregations, we further investigated aggregation of glucagon and insulin, two of the most famous amyloidogenic peptide hormones, using itc. we also observed characteristic heat of spontaneous amyloid fibrillation of both proteins after a lag time. taken all together, we showed that thermodynamic studies on amyloid fibrillation and amorphous aggregation were indeed possible by means of itc-based qualitative and quantitative calorimetric analyses. itc will become a promising approach for clarifying the thermodynamic properties of protein aggregates. the more case studies are required toward the establishment of thermodynamics of protein misfolding and aggregation when hydrophobic proteins are, for any reason, exposed to the cytosol they are rapidly captured by protective complexes which shield them from the aqueous surroundings and decide their fate (by either targeting them to their correct membrane homes or marking them for degradation by the ubiquitin/proteasome system). the bag holdase is a heterotrimeric protein complex, comprising bag , ubl a and trc , which works closely with the cochaperone sgta to triage hydrophobic proteins and pass them along the appropriate pathway. sgta also interacts with viral proteins and hormone receptors and is upregulated in numerous cancer types. these functions require further investigation to determine the scope of sgta as a therapeutic target. our lab has solved the solution structure of the n-terminal dimerization domain of sgta and characterised its interaction with two different ubiquitin-like (ubl) domains in the bag holdase (one from ubl a and the other from bag itself) using nmr chemical shift perturbation data and other biophysical techniques including isothermal titration calorimetry and microscale thermophoresis. at this meeting i will report on the progress we have made in structurally characterising further key players that participate in this quality control, with the aim of clarifying the intricate network of molecular interactions that governs these processes in health and disease. ensemble, ribbon and electrostatics spacefill views of the sgta dimerization domain structure. the final panel shows the structure overlaid with its yeast homologue. alpha synuclein is a small protein ( kda) expressed at high levels in dopaminergic neurons. fibrillar aggregates of a-synuclein inside the dopaminergic neuron are the major components of lewy bodies and lewy neuritis inclusion, which are considered as potential hallmark of parkinson's disease (pd). both in vitro as well as in vivo studies suggest that the soluble, oligomeric forms of a-syn are the more potent neurotoxic species, responsible for neuronal injury and death in pd. therefore, molecules that inhibit the toxicity of oligomers either by reducing their formation or by converting their more toxic oligomeric state to less-toxic fibrillar state would be effective agents for the drug development against pd. curcumin is one of the asian food ingredients which has shown a potential role as therapeutic agent against many neurological disorders including pd. however, the instability and low solubility makes it less attractive for use as potential therapeutic agent. the present work focuses on screening of the compounds similar to curcumin but having better effects on the morphology and toxicity of oligomeric and fibrillar assemblies of a-syn, which could be used as therapeutic agent preferentially over the naturally occurring curcumin. we synthesized and analyzed the effects of nine compounds, which are structurally similar to curcumin, on different stages of a-syn amyloid aggregation. here, we showed that curcumin and its analogs accelerate a-syn aggregation to produce morphologically different amyloid fibrils in vitro. however, there is no significant effect of curcumin and its analogs on the secondary structure of preformed a-syn fibrils. furthermore, these curcumin analogs showed differential binding affinities with the preformed a-syn aggregates, possibly due to difference in their chemical structures. the present data suggest the promising role of curcumin analogs in the treatment of a-synucleinopathy disorders. in vitro folding mechanisms determine the forces applied during co-translational folding there is currently much debate as to whether experiments conducted in vitro describe the folding of proteins in vivo. in particular, it is often suggested that the co-translational folding of nascent protein chains is dominated by the presence of the ribosome and associated chaperones, and that folding mechanisms will be affected by the vectorial nature of translation. here we use an arrest peptide assay to investigate the co-translational folding of a number of all-a spectrin domains that exhibit a range of thermodynamic stabilities and in vitro folding rates. our unexpected finding is that that the force exerted on the ribosome by these domains is not related to either the thermodynamic stability of the domain, or to the folding (loading) rate, but rather to the in vitro folding mechanism. we infer that the in vitro folding mechanisms of these domains are unaffected by the presence of the ribosome -even when part of the nascent chain is retained within the ribosome exit tunnel. there has been much work to date investigating the intermediates present in stalled translation complexes -but now, for the first time, we can begin to directly explore the rate limiting transition state in the co-translational folding of homologous proteins. can the structure of a protein (h . ) depend on the treatment of a solvent medium (explicit vs effective) in a coarse-grained computer simulation? ras pandey , barry farmer university of southern mississippi, air force research laboratory solvent medium plays a critical role in orchestrating the structure and dynamics of a protein. in computer simulation modeling of protein structure in a solvent medium, explicit, implicit, effectivemedium, approaches are often adopted to incorporate the effects of solvation. because of the complexity in incorporating all atomic and molecular details, the multiple components, reaching the large-scale, etc. implicit solvent or effective medium approach is generally more viable than the explicit solvent methods. some of the pertinent characteristics such as excluded volume of the solvent constituents, its concentration, and the underlying fluctuations which may be important in probing some issues are generally ignored in effective medium or implicit solvent approaches. using a coarse-grained approach, we investigate the structure and dynamics of a protein (a histone, h . ) in the presence of both effective as well as explicit solvent media over a range of temperatures with the monte carlo simulations. the protein is represented by a coarse-grained chain of residues whose interactions are described by knowledge-based residue-residue and hydropathy-index-based residuesolvent interactions. in effective medium approach, each empty lattice site around the protein structure acts as a solvent. only a fraction of lattice sites are occupied by mobile solvent constituents along with the protein chain in explicit solvent medium. large scale simulations are performed to analyze the structure of the protein for a range of residue-solvent interactions and temperature in both explicit and effective solvent media. we study a number of local (e.g. solvation and mobility profiles) and global (radius of gyration and structure factor) physical quantities as a function of temperature. we find that the response of the radius of gyration of the protein in explicit solvent is different from that in effective medium solvent. thus, the presence of fluctuations in explicit solvent approach have considerable effects on the structure and dynamics of protein h . . differences due to type of solvent on the response of some of these quantities as a function of temperature as well as general similarities will be presented. single-molecule vectorial folding and unfolding through membrane pores david protein folding and unfolding in vivo is frequently vectorial. for example, proteins are synthesized at the ribosome and emerge n-terminal first. as the polypeptide chain emerges from a nm wide pore is free to fold, interact with partners or misfold . in another example, proteins are unfolded at the proteasome by pulling from either the n or c terminus against a - nm wide pore, applying a tension on the residues surrounding the terminus of the protein . under this conditions, proteins may behave differently than when unfolded/refolded with temperature or urea. this may have important implications, as protein folding and unfolding in vivo is related to both function and disease. we noticed that vectorial folding is inherently linked to nanometer size pores. making use of nanopore technology we developed a method to monitor protein unfolding during membrane translocation at the single-molecule level . briefly, an oligonucleotide attached at either end of a protein threads a single protein nanopore inserted in a lipid membrane. in response to an applied membrane potential, the oligonucleotide pulls the protein through the pore and as it is forced to translocate it unfolds. analysing the ionic current we obtain the unfolding pathway and information on the polypeptide sequence. this methodology has shown that proteins unfold with different kinetics when pulled from one terminus or the other . remarkably, it is also possible to say whether the protein has been phosphorylated or not, and where . we have recently advanced our model system to study protein folding after translocation at the singlemolecule level . a single-protein molecule was translocated through a pore and forced to translocate back at predetermined times. we measured the stability of the refolded state at different times and we obtained the vectorial folding pathway of the protein. further, we observed that the protein was capable of co-translocational folding and that this premature folding contributed to the complete translocation of the protein. our results show that nanopore technology applied to proteins can be used to describe the vectorial folding and unfolding of proteins, providing insight to how these processes may work in vivo. further, single-molecule protein sequencing is a possibility that could revolutionise our knowledge on biological processes. thermodynamics studies of oligomeric proteins, which are the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. there is not a single report on the reversible equilibrium thermal unfolding of proteins composed by (b/a) barrel subunits, albeit this "tim barrel" topology is one of the most abundant and versatile in nature. the eponymous tim barrel, triosephosphate isomerase (tim) is a ubiquitous glycolytic enzyme that catalyzes the isomerization of glyceraldehyde- -phosphate and dihydroxyacetone phosphate. the unfolding of several tims, mainly of eukaryotic organisms, has been extensively studied. regarding thermal unfolding, eighteen tims, mainly from eukaryotes, as diverse as amoebozoa, euglenozoa, ascomycota and chordata, have been studied. even though a full thermodynamic characterization has been hampered by irreversible aggregation and/or the presence of hysteresis in all of them, the activation parameters that describe the kinetic control of five eukaryotic tims have been reported. we characterized the structure, catalytic properties, association state and temperature-induced unfolding of the eponymous tim barrel, triosephosphate isomerase (tim), belonging to five species representative of different bacterial taxa: deinococcus radiodurans (drtim), nostoc punctiforme (nptim), gemmata obscuriglobus (gotim), clostridium perfringens (cptim) and streptomyces coelicolor (sctim). irreversibility and kinetic control were observed in the thermal unfolding of nptim and gotim, while for drtim, sctim and cptim, the thermal unfolding was found to follow a two-state equilibrium reversible process, a behavior not observed previously for others tims. shifts in the global stability curves of these three proteins are related to organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. reversibility appears to correlate with low isoelectric point, the absence of residual structure in the unfolded state, small cavity volume in the native state structure, low conformational stability and a low melting temperature. furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce the possibility of aggregation and favor reversibility. it appears that there is a delicate balance between several contributions whose concerted interplay is necessary to achieve thermal reversibility in oligomeric enzymes. furthermore, the finding that the three reversible proteins come from organisms from different phyla suggests that unfolding reversibility may be more common than what is currently known supported by a critical step in the late phase of human immunodeficiency virus type (hiv- ) infection is targeting of the virally encoded gag proteins to the plasma membrane (pm) for assembly. prior to assembly, the hiv- gag polyprotein adopts a compact "folded over" conformation and exists in the monomeric or low-order oligomeric states. whereas it is established that the nucleocapsid domain of gag specifically recognizes motifs in the viral rna genome for packaging, there is compelling evidence that the myristoylated matrix (ma) domain also binds to cellular rna to prevent premature gag targeting to intracellular membranes. upon transport of gag to the pm, the interaction of ma with rna is exchanged for an interaction of ma with pm components. this molecular switch induces an extended conformation of gag, leading to formation of high-order gag oligomers on the pm. because gag is anchored and therefore captured by its interaction with the available phospholipids, the intracellular targeting of gag is likely to be determined by the relative strength of its interaction with the dominant lipids composing each membrane subcompartment. the key to understanding this essential molecular switch is elucidating at the molecular level the interaction of ma with specific pm components. for over two decades, biochemical, in vivo, in vitro and genetic studies have focused on factors that modulate binding of retroviral gag proteins to membranes but only recently the structural and molecular determinants of gag assembly have begun to emerge. in addition to the electrostatic interactions between a highly conserved basic region of ma and acidic phospholipids, it is now believed that the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. we employ nmr methods to elucidate the molecular determinants of gag binding to the membrane. our structural studies revealed that phosphatidylinositol- , -bisphosphate (pi ( the production of functionally antibodies depends on the transition of immature b cells to mature plasma cells and is tightly linked to several "quality control" check points. during b cell development, the pre-b cell receptor (pre-bcr) is the first checkpoint which determines the viability and proliferation of the pre-b cell. the pre-bcr is composed of an immunoglobulin (ig) heavy chain molecule associated with an ig light chain-like molecule called the surrogate light chain (slc). the slc is composed by two proteins k and vpreb which possess a unique region at the n-or c-terminus, respectively. vpreb lacks a b-strand which is provided by the k protein allowing the non-covalent interaction essential for formation of the slc heterodimer. our understandings of the molecular mechanism of slc function and assembly are still at an early stage. in particular, we do not know how the slc associates and forms the pre-bcr for the selection of all heavy chains (hcs). our study focuses on dissecting the "fab fragment" of the pre-bcr to study the effect of the unexpected structural features of the slc to gain insight in hc selection. the analysis of the assembly of the slc revealed a significant difference between the single domains and the complexes in terms of stability and assembly. the folding behavior of the ch domain in the presence of the slc is key for the first quality control mechanism in the endoplasmic reticulum (er) prior to surface expression. our results show that the slc interacts with ch domain in a similar manner to the cl domain. thus, the folding of the naturally disordered ch domain upon interaction with the slc releases the hc retention in the er by bip. taken together, our study provides new insights into the folding and assembly of the "fab fragment" of the pre-bcr and paves the way for a detailed mechanistic understanding of hcs selection by the unique slc. though the - (sfgailss) region of human islet amyloid polypeptide (hiapp) has long been known to be crucial for amyloid fiber formation, lack of b-ordering of this region in structures of the final fiber as determined by both nma and x-ray has been puzzling. new evidence now suggests that the fgail region forms ordered b structures only in early intermediates. we present new dir studies on the fgail region of hiapp, with uniformly c o labeled amides, along with spectral and kinetic modelling. evolution of the peak frequency and d lineshape of the labeled region clearly present a transition from random coil to a stable b sheet, a conclusion which is substantiated by simulation of the d ir spectra. as determined from kinetic modeling, the fgail b-sheet creates a free energy barrier that is the cause of the lag phase during aggregation. these findings help to rationalize a broad range of previous fragment and mutation studies as well as provide a mechanism for fiber formation that has self-consistent kinetics and structures. the temperature dependence of protein stability in living cells studies addressing the consequence of crowding that exist in the interior of cells have reached an interesting stage. experimental data so far, predominantly from, small to medium sized proteins are indicating that, in general, natively folded proteins including, intrinsically disordered, gain structure and stability under conditions mimicking cell interior. however, on the other hand, a few studies on small proteins indicate destabilization of the native state. in very few instances, crowding resulted in compaction and aggregation of the unfolded and partially folded states. experimental data on the consequences of cell-like crowding situation on relatively large proteins with complex folding free energy landscape are absent. alpha subunit of tryptophan synthase, a kda tim barrel protein, provides a unique opportunity to address the consequence of crowding on the structure and stability of the native state and also on a partially folded state stable equilibrium intermediate populated in its (un)folding reactions. in the presence of increasing amounts the most commonly used crowding agent, ficoll- , a non-monotonous increase in the far uv-cd is observed for the native state. a steady increase up to mg/ml ficoll followed by a decrease in far-uv cd region is observed, indicating loss of structure at increased concentrations of the crowding agent. h- n hsqc nmr and fluorescence (fl) spectra confirm the of loss of structure at higher concentrations of ficoll- . loss of native base line in the urea induced unfolding reaction monitored by cd and fl clearly confirms the destabilization of the native state. similar to the structural changes observed for the native state, for the equilibrium intermediate state maximally populated at m urea also, non-monotonous changes in the far uv cd and fluorescence spectra are observed. the highly populated equilibrium intermediate shows an initial steady increase in the far uv cd signal followed by a sudden decrease. our results suggest that the structure of both native and partially folded states may be affected under crowding conditions. alpha- antitrypsin (aat) is a -kda serine protein inhibitor (serpin), which acts as an inhibitor of neutrophil elastase within the lungs. during inhibition, the protein undergoes a dramatic conformational change in which its exposed reactive centre loop (rcl) is cleaved and inserts into the central a-sheet as an extra beta-strand. this highly dynamic protein is also susceptible to mutations, resulting in misfolding and the accumulation of ordered polymers as intracellular inclusions within the endoplasmic reticulum of hepatocytes, where aat is synthesized. despite much knowledge of the folding and misfolding properties of aat as an isolated protein, very little is understood of how aat acquires its structure during biosynthesis. like all proteins, the biosynthesis of aat takes place on the ribosome, and protein folding occurs in a co-translational manner as the nascent polypeptide chain emerges from the ribosome's exit tunnel. this study aims to develop the biochemical and nmr structural strategies to characterize the co-translational folding characteristics of aat as it is being synthesized on the ribosome. for these studies, we have designed a series of secm-stalled ribosome nascent chain complexes (rnc) of aat of different lengths, which mimics the "snapshots" of the protein synthesis, capturing the folding process of the nascent chain during its emergence from the ribosome. using this library, we have recently developed a strategy to produce large quantities of the rncs both in vitro and in vivo within e. coli, a prerequisite for detailed biochemical and structural studies. using the aat-rncs, we are developing a suite of biochemical strategies to probe the capacity for aat nascent chains to adopt native structure on the ribosome. we have combined protease inhibition assays, western blot and native-page analysis to demonstrate that aat can fold while bound to the ribosome. in addition, we have employed a cysteine-based modification "pegylation" assay to probe lowresolution structural information of aat-rnc and this will guide our structural studies by nmr spectroscopy to provide a detailed understanding of aat folding on the ribosome at high resolution. thermodynamic properties of proteins vary with the environmental solvent condition (temperature, ions, ph, denaturants, etc.). although the effect of each environmental factor on proteins has been well studied, the complex effect of more than two environmental factors was not studied thoroughly. in this study, we investigate the simultaneous effect of urea denaturation (disruption of non-covalent bonds in proteins) and acid denaturation (titration of protein residues) on the nature of the folding transition for cyu protein. we performed the molecular dynamics simulations of bbl (pdb code: cyu) protein in various urea concentration at k. we calculated ph-dependent free energy landscape using the extended munoz-eaton model and described the phase diagram for the folding transition of bbl at various ph value and urea concentration. we mapped out the phase diagram of the folding transition of cyu, which clarifies the condition with which it undergoes the cooperative folding transition or the barrierless folding transition. biophysical analysis of partially folded states of myoglobin in presence of , , -trifluoroethanol paurnima talele , nand kishore the protein folding process involves one or more distinct populated intermediates. one such partially folded structure of particular importance observed during protein folding pathway is molten globule state. the properties of a molten globule state are intermediate between those of native and unfolded protein molecules. the importance of studying equilibrium molten globule is in its greater stability and flexible structure which has been shown to bind a variety of substrates and play a definite role in certain human diseases via aggregation, misfolding or some other mechanism. a protein must assume a stable and precisely ordered conformation to perform its biological function properly. the stability of a protein under specific conditions depends on its interactions with the solvent environment. therefore it is essential to understand protein folding intermediates, protein solvent interactions and protein stabilization. we have made attempts to thoroughly investigate the formation of stable molten globule state of the protein induced by alcohol using combination of calorimetric and spectroscopic techniques. the presentation will cover the topic on biophysical studies on partially folded states of myoglobin in presence of , , -trifluoroethanol. the thermal denaturation of myoglobin was studied in the presence of , , -trifluoroethanol (tfe) at various ph values using differential scanning calorimetry and uv-visible spectroscopy. the most obvious effect of tfe was lowering of the transition temperature with increasing concentration of tfe up to . mol•dm- , beyond which no thermal transitions were observed. the conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. at ph . and . , partially folded states of myoglobin were confirmed by cd spectroscopy. quantitative binding of ans to the tfe induced molten globule state of myoglobin was studied by using isothermal titration calorimetry (itc). the results enable quantitative estimation of the binding strength of ans with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. the results also suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ans molecules which has been widely used as a fluorescence probe to characterize partially folded states of proteins. modules. each cbr comprises a b-hairpin core followed by a short linker sequence. choline molecules are bound between two consecutive repeats through hydrophobic and cation-p interactions with aromatic side chains. apart from its biotechnological applications as an affinity tag for protein immobilization and purification, clyta is useful as a model for understanding the folding and stability of repeat proteins. in this sense, we proposed to get minimal peptides encompassing the sequence of a single cbr or even only its b-hairpin core able to maintain the native fold and the ability to bind choline. to that end, we first proceeded to analyze the peptide comprising the third b-hairpin core, denoted as clyt . based on cd and nmr data we demonstrate that the peptide clyt conserves its native bhairpin structure in aqueous solution, but forms a stable, amphipathic a-helix in detergent micelles and as well as in small lipid vesicles [ ] . considering the great differences in the distribution of hydrophobic and polar side chains shown by clyt b-hairpin and a-helix, we propose that amphipathic structures are stabilized in micelles or lipid vesicles. this "dual" behavior is the only up-to-now reported case of a micelle-induced conformational transition between two ordered peptide structures. to check whether other cbr repeats also undertake b-hairpin to a-helix transition in the presence of micelles, so that it represents a general tendency ascribed to all pneumococcal choline-binding modules, we will show new experimental evidences based on cd and nmr structural studies on peptides derived from the bhairpin cores of other clyta repeats, as well as in modified clyt peptides. continuing our studies of the effect of like-charged residues on protein-folding mechanisms, in this work, we investigated, by means of nmr spectroscopy and molecular-dynamics simulations, two short fragments of the human pin ww domain [hpin ( - ); hpin ( - )] and one single point mutation system derived from hpin ( - ) in which the original charged residues were replaced with non-polar alanine residues. results, for both original peptide fragments of hpin demonstrate the presence of ensembles of structures with a tendency to form a b-chain reversal. understanding the biology of huntington's disease via the pathogenic huntingtin monomer huntington's disease (hd) is caused by an abnormal extension of the polyglutamine (polyq) region within exon of the protein huntingtin from typically glutamines to over . disease onset correlates with the huntingtin misfolding and causing the formation of aggregates, however recent studies have postulated that pathogenic huntingtin monomer may form compact structures that are responsible for neuronal toxicity in hd. we sought to examine the conformation of huntingtin monomers, how polyq sequence length affects monomer structure and which protein-binding partners in the cell may exert a gain-of-toxic mechanism in pathology. hydrogen-deuterium exchange mass spectrometry was used to measure the degree of structure in both non-pathogenic ( q) and pathogenic ( q) huntingtin, with results showing that both forms exchanged % of potential nh hydrogen bond donors within seconds (n ), with little to no further exchange over the following ten minutes. this result suggested that the pathogenic conformations are not stabilized by slow exchanging hydrogen bonds. binding partners to the monomer were assessed in neuro a cell culture by immunoprecipitation and quantitative ms/ ms proteomics approaches after depletion of aggregates by pelleting. proteins that more prevalently co-precipitated with pathogenic huntingtin included fused in sarcoma (fus), glycine-trna ligase (gars), peroxiredoxin (prdx ), phosphatidylethanolamine-binding protein (pebp /rkip), and histone subunit hist h a, all of which were significantly enriched by two-fold or greater. rna-seq analysis indicated that none of these proteins had altered expression levels, suggesting that the binding interactions are not due to changes in background abundance. overall we found that the conformational differences are subtle, yet are sufficient to generate several specific proteome interactions that offer clues to a toxic gain-of-function mechanism in pathology. work is ongoing to probe the more subtle changes in conformation and the importance of these interactors to mediating mechanisms of dysfunction. hereditary tyrosinemia type i is an autosomal recesive disorder caused by deficiency of fumarylacetoacetate hydrolase (fah) enzyme. deficiency of fah leads to cellular accumulation of toxic metabolites which include mainly, succinylacetone (sa), maleylacetoacetate (maa) and fumarylacetoacetate (faa) in many body tissues. fah is mainly expressed in hepatocytes and renal proximal tubular epithelium. therefore, liver and kidney are the two primary organs affected by this disorder, and development of hepatocellular carcinoma is the major symptom. missense mutations leads to a loss of enzymatic efficiency which, in a high number of mutations, correlates with loss of kinetic and thermodynamic stability of the enzyme. in our ongoing project, we are trying to elucidate the molecular basis of tyrosinemia by means of biophisical and structural characterization of fah wild type along with its mutations. this knowledge should help us design new therapies based on the identification of pharmacological chaperones that could restore the altered enzymatic stability of the enzyme. human fah wild type and selected mutants were synthesized and inserted in an expression vector for e. coli. the proteins were purified in a fplc and, their thermodynamic and kinetic stability investigated using circular dichroism. our preliminary results confirm the loss of termodinamic stability of different mutants and its variability compared to wild type protein. repulsion between net charges of subunits during ferritin assembly daisuke sato , hideaki ohtomo , atsushi kurobe , satsuki takebe , yoshiteru yamada , kazuo fujiwara , masamichi ikeguchi department of bioinformatics, graduate school of engineering, soka university, jasri/spring- the organisms have a lot of spherical shell-shaped supermolecules consisting of identical or distinct subunits (e.g., ferritin, virus capsid, lumazine synthase and encapsulin). such multimeric proteins spontaneously assemble into their native structures from the subunits to acquire the specific functions. however, the assembly mechanism of such supermolecules has not been understood in detail. hence, to investigate the assembly mechanism is biologically important. escherichia coli non-heme ferritin (ftn) consists of identical subunits, which are assembled into a spherical shell-shape with / / symmetry. ftn is able to store iron inside cavity. the subunit includes a-d helices forming -helix bundle, a long bc-loop between b and c-helices and a short e-helix at the c-terminal. ftn dissociates into dimers at acidic ph. the dimer was shown to maintain the native-like secondary and tertiary structures by circular dichroism spectra and small angle x-ray scattering (saxs). the acid-dissociated ftn is able to reassemble into the native structure when ph increases. to clarify ftn assembly mechanism, we performed the stopped-flow time-resolved saxs (tr-saxs) experiments. the saxs profiles could be acquired every ms after the initiation of reassembly. the initial velocity calculated from the forward scattering intensity increment was proportional to the square of the protein concentration, implying that the reaction is second-order. we propose the sequential bimolecular reaction, in which two dimers bind to form tetramer, then another dimer attaches to the tetramer to form a hexamer, and so on. the assembly rate depended on ph and ion strength, indicating that the electrostatic interaction plays an important role in the assembly reaction. the assembly rate decreased with increasing ph in the range from . to . and increased with increasing nacl concentration. this indicates that there are repulsive electrostatic interactions between assembly units and that they increases with increasing ph from . to . . a possible interaction is the repulsion between net charges of dimers since pi of ftn is expected to be . . to test this possibility, we made several mutants with different net charges. as mutational sites, we selected charged residues that are far from the subunit interface. selected sites were glu , glu , glu , glu and glu . we constructed the mutants with one, two, three or four glu -> gln substitutions of selected sites. the structures of those mutants were similar to that of wild-type ftn. if aforementioned hypothesis is correct, the assembly rate is expected to increase with increasing the number of substitution. the result agreed well with this expectation and strongly suggested that the electrostatic repulsion between dimers is an important factor determining the assembly rate of ftn. improved modeling of protein unfolding rates and pathways through solvation and modeling of beta-barrels benjamin walcott , , lu ıs garreta , christopher bystroff , , department of biology, rensselaer polytechnic institute, center for biotechnology and interdisciplinary studies, department of computer science, universdad del valle, department of computer science, rensselaer polytechnic intitute an understanding of the folding and unfolding pathways of proteins is integral to improving our ability to associate the structural impact of point mutations and disease etiology. information gained here can also be used for protein structure prediction and design. to model unfolding pathways in proteins we utilize a computational method called geofold. this approach uses recursive hierarchical partitioning of protein structure and finite elements simulation. geofold considers three types of partitioning operations: translational motion (break), single point revolute joints (pivot), and rotation around two points (hinge). from these operations, a directed acyclic graph (dag) is constructed where nodes correspond to the substructures created by these operations and the edges represent the operations. for each operation in the dag, its dissociation and reassociation rates are determined as a function of solventaccessible surface area, hydrogen bonds, voids, and conformational entropy. finite element simulations are carried out to simulate the kinetics of unfolding. this model accurately predicts changes in unfolding pathways due to disulfides in a four-protein case-study, but it fails to produce a realistic pathway for b-barrel proteins such as green fluorescent protein (gfp). to better model these barrel proteins, a new partitioning operation is introduced involving the breaking of all contacts between an adjacent set of b-strands, called a seam. in addition, to improve the accuracy of kinetic modeling, several updates have been made to the energy function, including an improved solvation model and a contact-orderbased estimation of the reassociation rates. the predicted unfolding rates and pathways using this improved geofold are compared with experimentally measured values in kineticdb for proteins with multi-state unfolding kinetics, point mutations, circular permutations, and engineered disulfides. the presence of multiple domains in a protein can result in the formation of partially folded intermediates, leading to increased aggregation propensity. this can be reduced by cooperative, all-or-nothing folding of the multi-domain protein. in good agreement with ensemble folding experiments, a coarsegrained structure-based model of e. coli adenylate kinase (ake) folds cooperatively. ake has three domains, nmp, lid and core. we examine the role of the interfaces between these domains in facilitating folding cooperativity in ake. mutants in which these interfaces are deleted exhibit similar folding cooperativities as wild-type ake. on closer inspection, we observe that unlike a typical multi-domain protein in which one domain is singly-linked to its adjacent domain, nmp and lid are inserted into core, i.e. they are both connected to core by two linkers each. we create circular permutants of ake in which the inserted domains are converted to singly-linked domains, and find that they fold less cooperatively than wild-type ake. domain insertion in wild-type ake facilitates folding cooperativity even when the inserted domains have lower stabilities. the n-and c-termini of nmp and lid are constrained upon the folding of core and this facilitates their folding. thus, nmp and lid which undergo large conformational changes during catalysis can be smaller with fewer stabilizing interactions. in addition, inter-domain interactions need not be optimized for folding, and can be tuned for substrate binding, conformational transition and catalysis. analysis of protein domains using structural bioinformatics suggests several examples of multi-domain proteins in which domain insertion is likely to facilitate folding cooperativity. tuning cooperativity on the free energy landscape of protein folding pooja malhotra , jayant udgaonkar national centre for biological sciences, tata institute of fundamental research the mechanism by which a protein explores the free energy landscape during a folding or unfolding reaction is poorly understood. determining whether these reactions are slowed down by a continuum of small ( kbt) free energy barriers or by a few large (> kbt) free energy barriers is a major challenge. in this study the free energy landscape accessible to a small protein monellin is characterized under native-like conditions using hydrogen exchange in conjunction with mass spectrometry. cooperative and noncooperative opening processes could be directly distinguished from the mass distributions obtained in the ex limit. under native conditions, where the native state is maximally stable, the unfolded state is transiently sampled in an entirely non-cooperative and gradual manner. under conditions which stabilize the unfolded state or destabilize the native state of the protein, the slowest structure opening event becomes cooperative. the present study provides an understanding of the relationship between stability and folding cooperativity. it suggests that the cooperative transitions observed in unfolding reactions maybe a consequence of the changes in the stabilities of the unfolded state and the transition state. it also provides rare experimental evidence for a gradual unfolding transition on a very slow timescale. role of electrostatic repulsion between unique arginine residues on the assembly of a trimeric autotransporter translocator domain eriko aoki , kazuo fujiwara , masamichi ikeguchi haemophilus influenzae adhesin (hia) belongs to the trimeric autotransporter family. the autotransporter consists of an n-terminal signal peptide, an internal passenger domain and a c-terminal translocator domain. the signal peptide directs to export across the inner membrane via the sec system and is cleaved, the passenger domain is a virulence factor, and the translocator domain (hiat) is embedded in the outer membrane. the crystal structure of hia translocator domain (hiat) has shown that hiat forms a transmembrane b-barrel of b-strands, four of which are provided from each subunit. the b-barrel has a pore that is traversed by three a-helices, one of which is provided from each subunit. the protein has a unique arginine residue at . arg side chains from three subunits protrude from the b-strand toward the center of the barrel and are close to each other. these residues seem to have an unfavorable electrostatic effect on the assembly and decrease the trimer stability. to investigate the role of this residue on the trimer assembly and stability of hiat, we replaced this arginine with the neutral amino acid, methionine (r m) or the positively charged residue, lysine (r k), and properties of these mutants were investigated. hiat and two mutants were dissociated by formic-acid treatment, and they were able to reassemble in the presence of the detergent. to measure the time course of trimer reassembly, amounts of reassembled trimer and monomer were quantified by sds-page at different assembly times. although the neutralized mutation increased the rate of reassembly, the final amount of reassembled trimer decreased, especially at higher protein concentration. these suggest that the neutralized mutation cause the incorrect oligomer formation. the far-uv cd spectrum of reassembled wt hiat was nearly identical with that of the native wt hiat. however, the spectrum of the reassembled r m mutant was more intense that of the native r m mutant, although the proportion of trimer was much lower than that of the wt hiat. this suggests that the incorrect oligomer has a secondary structure different from the wt hiat. r k mutant showed assembly properties similar to those of the wt hiat. therefore, the repulsion between positively charged residues seems to be important for preventing hiat from misassembly. similar proximity of arginine residues is observed for hiv capsid protein, carboxysome shell protein, lumazine synthase and so on. the electrostatic repulsion between arginine residues may be a general mechanism for protein assembly. department of veterinary pathobiology, kagoshima university, institute for food sciences, hirosaki university, faculty of fisheries, kagoshima university, department of veterinary histopathology, kagoshima university, veterinary clinical training center, kagoshima university, department of veterinary anatomy, kagoshima university, sakamoto kurozu inc., the united graduate school of agricultural sciences, kagoshima university kurozu is a traditional japanese rice vinegar. during fermentation and aging of the kurozu liquid in an earthenware jar over year, solid residue called kurozu moromi is produced. in the present study, we evaluated whether concentrated kurozu or kurozu moromi could ameliorate cognitive dysfunction in the senescence accelerated p mouse. senescence accelerated p mice were fed . % (w/w) concentrated kurozu or . % (w/w) kurozu moromi for or weeks. kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while kurozu moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. we hypothesize the effect is caused by the antioxidant effect of concentrated kurozu, however, the level of lipid peroxidation in the brain did not differ in senescence accelerated p mice. dna microarray analysis indicated that concentrated kurozu increased hspa a mrna expression, a protein that prevents protein misfolding and aggregation. the increase in hspa a expression by kurozu was confirmed using quantitative real-time pcr and immunoblotting methods. therefore, the suppression of amyloid accumulation by concentrated kurozu may be associated with hspa a induction. however, concentrated kurozu could not increase hspa a expression in mouse primary neurons, suggesting it may not directly affect neurons. young-ho lee although amyloid fibrils are associated with a number of pathologies, their conformational stability remains largely unclear. we herein investigated the thermal stability of various amyloid fibrils. a-synuclein fibrils, freshly prepared at c at neutral ph, cold-denatured to monomers at - c and heat-denatured at - c. meanwhile, the fibrils of b -microglobulin, alzheimer's ab - /ab - peptides, and insulin exhibited only heat denaturation, although they showed a decrease in conformational stability at low temperature in the presence of chemical denaturants. a comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in the fibril cores contributed to the cold denaturation of a-synuclein fibrils. reinforced electrostatic repulsion at low temperatures may promote cold denaturation, leading to a unique thermodynamic property of amyloid fibrils. we propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation due to the unfavorable burial of charged side-chains in fibril cores. key structural differences between tbtim and tctim revealed by thermal unfolding molecular dynamics simulations angel piñeiro , miguel costas , andrea guti errez-quezada dept of applied physics, university of santiago de compostela, lab. of biophys. chem., dept of physical chemistry, fac. of chemistry, unam the thermal unfolding pattern obtained by differential scanning calorimetry for trypanosoma cruzi and trypanosoma brucei triosephosphate isomerase (tcim and tctim) are significantly different although the crystal structure of both proteins is almost indistinguishable and the sequences are highly homogolous. in order to explain these differences at molecular level a set of molecular dynamics simulations were performed at different temperatures between and k. the obtained trajectories were analyzed in detail and the residues that showed to be key in the unfolding pathway of each species were identified. a set of residues that behave significantly different between both proteins were selected and proposed for mutations. the general aim is to identify the minimum amount of residue mutations that allow providing tbtim with the behaviour of tctim and vice versa. experimental complementary work is also being performed on the same protein. repositioning som as a potent inhibitor of transthyretin amyloidogenesis and its associated cellular toxicity salvador ventura , ricardo sant'anna , maria ros ario almeida , nat alia reixach , raul insa , adrian velazquez-campoy , david reverter , n uria reig universitat aut onoma de barcelona, instituto de biologia molecular e celular, icbas, the scripps research institute, som-biotech, universidad de zaragoza transthyretin (ttr) is a plasma homotetrameric protein implicated in fatal amyloidosis. ttr tetramer dissociation precedes pathological ttr aggregation. despite ttr stabilizers are promising drugs to treat ttr amyloidoses, none of them is approved by the food and drug administration (fda). repositioning existing drugs for new indications is becoming increasingly important in drug development. here, we repurposed som , an fda-approved molecule for neurodegenerative diseases, as a very potent ttr aggregation inhibitor. som binds specifically to ttr in human plasma, stabilizes the tetramer in vivo and inhibits ttr cytotoxicity. in contrast to most ttr stabilizers, it exhibits high affinity for both ttr thyroxine -binding sites. the crystal structure of som -bound ttr explains why this molecule is a better amyloid inhibitor than tafamidis, so far the only drug in the market to treat the ttr amyloidoses. overall, som , already in clinical trials, is a strong candidate for therapeutic intervention in these diseases. neurometals as modulators of protein aggregation in neurodegenerative diseases s onia s. leal , joana s. crist ovão , cl audio m. gomes protein misfolding and aggregation is a hallmark across neurodegenerative diseases such as alzheimer's disease and amyotrophic lateral sclerosis (als). since these diseases are mostly sporadic, the formation of protein amyloids in the nervous system depends of chemical and biological triggers within the neuronal environment, such as metal ions [ ] . in this communication i will overview the metallobiology of neuronal calcium, zinc and copper, which are key players in brain function and have altered homeostasis in most neurodegenerative conditions. our recent work will illustrate how this allows establishing molecular mechanisms in neurodegenerative diseases [ ] [ ] [ ] [ ] [ ] . in the pursuit of this goal, in the last years we have been investigating superoxide dismutase (sod ), a cu/zn metalloenzyme that aggregates in the fatal neurodegenerative disorder als, as a model. in sod -als cases, this ubiquitous protein selectively aggregates in motor neurons, implicating a local biochemical factor in the process: interestingly, zn and ca levels are upregulated in the spinal and brain stem motor neurons of als patients, and increased ca triggers multiple pathophysiological processes which include direct effects on the sod aggregation cascade [ , ] . recently we established that calcium ions promote sod aggregation into non-fibrillar amyloid, suggesting a link to toxic effects of calcium overload in als [ ] . we showed that under physiological conditions, ca induces conformational changes on sod that increase sod b-sheet content and decrease sod critical concentration and nucleation time during aggregation kinetics. we also observed that calcium diverts sod aggregation from fibrils towards amorphous aggregates. interestingly, the same heterogeneity of conformations is found in als-derived protein inclusions. we thus hypothesized that transient variations and dysregulation of cellular ca and zn levels contribute to the formation of sod aggregates in als patients [ , ] . in a follow up study we combined experimental and computational approaches to show that the most frequent ligands for ca are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. calcium interactions thus diminish gatekeeping roles by shielding repulsive interactions via stacking between aggregating b-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in als inclusions. interestingly, many fals mutations occur at these positions, disclosing how ca interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic als pathomechanisms [ ] . the amino acid proline is well-known by its disorder promoting and helix breaking properties. prolines can be accommodated within transmembrane (tm) alpha-helices and participate in important biological tasks like signal transduction, ligand binding and helix-helix packing. x-ray crystallography and nmr indicate that proline residues in membrane proteins induce distortions of the helix geometry to different extents ranging from small bends to severe kinks. however, such studies provide essentially a static snapshot of membrane-embedded helices. therefore, the link between proline dynamics and function is not completely understood. in this work we have used singlemolecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs) to probe the structure and dynamics of the tm domain of human glycophorin a (gpa), a widely used model membrane protein for oligomerization studies. a fluorescent dye pair has been attached to both ends of the membrane-spanning region of gpa, which allowed monitoring the average distance and distance fluctuations between the attachment points. site-specifically double-labeled gpa has been reconstituted into two membrane-mimetic systems: sds micelles and phospholipid bilayers assembled into nanodiscs. using proline-scanning mutagenesis we have systematically evaluated the impact of proline residues in different positions along the membrane normal on transmembrane helix length and lateral packing. furthermore, we have investigated the distance distribution in tm helices containing native prolines, namely the insulin receptor and the nesprin protein. our results shed light into the relation between proline dynamics and the folding and function of tm helices. thermodynamic contributions of specific mutations of l e protein in the rna: protein interface region measured by analytical ultracentrifugation and gel shift assay bashkim kokona , , sara kim , margaret patchin , britt benner , susan white in saccharomyces cerevisiae, ribosomal protein l e acts as an autoregulator by inhibiting the splicing of its pre-mrna and translation of its mrna. the l e protein-rna binding site has been previously studied, revealing a rna kink-turn motif, which is characterized by a sharp bend in the phosphodiester backbone due to unpaired nucleotides and internal tertiary interactions. l e structural flexibility at the rna-binding interface makes such interaction an excellent model to explore the energetics of rna protein binding. we made l e k a, f a, and f w mutants to quantify the thermodynamic contributions of such interactions to the protein-rna complex. we used analytical ultracentrifugation sedimentation equilibrium (se) and sedimentation velocity (sv) to investigate conformational changes and protein-rna binding free energy changes due to mutations. our computed changes of binding free energy based on the sedimentation equilibrium experiments were consistent with the gel shift assay results. in addition, sedimentation velocity experiments on the l e wild type indicate that protein-rna interaction is highly dynamic and involves conformational changes of the kink-turn rna induced by l e protein. our results provide new insights on understanding the binding between ribosomal proteins and their rna molecules counterpart, which can be used to complement the x-ray structure. role of a non-native a-helix in the folding of equine b-lactoglobulin takahiro okabe , toshiaki miyajima , kanako nakagawa , seiichi tsukamoto , kazuo fujiwara , masamichi ikeguchi equine b-lactoglobulin is a small globular protein ( residues). although elg adopts a predominantly b-sheet structure consisting of nine anti-parallel b-strands (a-i) and one major a-helix in the native state, it has been shown that a non-native a-helical intermediate accumulates during the burstphase of folding reaction from the unfolded state in the concentrated denaturant. to ask whether the non-native helix formation is important for acquiring the native b-sheet structure, we determined first where the non-native a-helix is formed. a stable analogue of the burst-phase folding intermediate was observed at acid ph (a state). the amide hydrogen exchange experiment and proline-scanning mutagenesis experiment have shown that the non-native a-helix is formed at the region corresponding to the h strand in the a state. to investigate the role of this non-native a-helix on refolding reaction of elg, we constructed several mutant proteins, which were designed to destabilize the nonnative a-helix in the folding intermediate without perturbation on the native structure. a mutant, a t, fulfilled this requirement, that is, a t showed a native structure similar to that of the wildtype protein, and largely reduced cd intensity in the a state. then, the refolding kinetics were investigated by the cd and fluorescence stopped-flow method. a t mutation resulted in reduction of the burst-phase cd intensity, which confirmed that the non-native a-helix is formed around the h strand region. subsequent to the burst-phase, four kinetic phases were observed for a t and the wildtype protein. importantly, the folding rate constants of the four kinetic phases were similar between both proteins. furthermore, interrupted refolding experiments demonstrated that the native state was formed in the two parallel pathways in the two slower phases of the four kinetic phases. the relative amplitudes of the two pathways were similar between a t and the wild-type protein. these results clearly showed that the formation of the non-native helix has little effect on the folding rates and pathways, and suggested that the non-native helix formation may not be a severe kinetic trap for protein folding reaction. impact of the chaperonin cct in a-synuclein(a t) amyloid fibrils assembly ahudrey leal_quintero , javier martinez-sabando , jose mar ıa valpuesta , begoña sot centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic) and fundaci on imdea-nanociencia cct is a eukaryotic chaperonin that uses atp hydrolysis to encapsulate and fold nascent protein chains. moreover, it has recently been shown that cct is able to inhibit amyloid fibers assembly and toxicity of the polyq extended mutant of huntingtin, the protein responsible of huntington disease. although this opens the possibility of cct being also able to modulate other amyloidopathies, this has not addressed yet. the work presented here intends to determine the effect of cct in the amyloid fibers assembly of a-synuclein(a t), one of the mutants responsible of parkinson disease. it is demonstrated that cct is able to inhibit a-synuclein(a t) fibrillation in a nucleotide independent way, suggesting that this effect is based on binding rather than on active folding. furthermore, using deletion mutants and assaying the interaction of cct with monomers, soluble oligomers and fibres, it has been possible to unravel the mechanism of this inhibition: cct interferes with fibers assembly by interacting with a-synuclein(a t) nac domain once soluble oligomers are formed, thus blocking the reaction before the fibers start to grow. amyloid-like aggregation of nucleophosmin regions associated with acute myeloid leukemia mutations daniela marasco , concetta di natale , valentina punzo , domenico riccardi , pasqualina scognamiglio , roberta cascella , cristina cecchi , fabrizio chiti , marilisa leone , luigi vitagliano department of pharmacy, cirpeb: centro interuniversitario di ricerca sui pepti, section of biochemistry, department of biomedical experimental and clinical scie, institute of biostructures and bioimaging nucleophosmin (npm ) is a multifunctional protein involved in a variety of biological processes and implicated in the pathogenesis of several human malignancies. npm has been identified as the most frequently mutated gene in acute myeloid leukemia (aml) patients, accounting for approximately % of cases ( ). the most frequent human npm mutations lead to variants with altered c-terminal sequences of the c-terminal domain (ctd) that, in its wild form, folds as a three helix bundle. aml modifications lead to (a) an unfolding of the ctd in the mutated protein and (b) its accumulation in the cytoplasm due to the loss of nuclear localization sequences with mutations of trp (mut e) and also of trp (mut a) ( ) . to gain insights into the role of isolated fragments in npm activities we dissected the ctd in its helical fragments. here we describe the unexpected structural behavior of the fragments corresponding to the helices h and h in both wild-type and aml-mutated variants. h region shows a remarkable tendency to form amyloid-like assemblies while only the muta sequence of h region is endowed with and b-sheet structure, under physiological conditions, as shown by circular dichroism, thioflavin t and dynamic light scattering. the aggregates of h , are also toxic to neuroblastoma cells, as determined by using the mtt reduction and ca influx assays ( ) . furthermore the effects of the local context on the different tendencies to aggregate of h and h were investigated and appeared to influence for the aggregation propensity of the entire ctd. since in aml mutants the ctd is not properly folded, we hypothesize that the aggregation propensity of npm regions may be implicated in aml etiology. these findings have implications to elucidate the pathogenesis of aml caused by npm mutants and aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology. we report a resume of our study regarding the effects of microwaves in the range - mhz on a typical protein, myglobin. previous literature have concerned the effects on living and in vitro organic systems induced by high frequencies electromagnetic fields. we have focused our attention on a typical protein, myoglobin, because proteins are the simplest organic systems that are fundamentals in organic functions of livings. myoglobin is a protein found mainly in muscle tissue of vertebrates, consisting of a single protein chain with amino acids and one heme group that stores oxygen in the muscle cells. the physiological importance of myoglobin is mainly related to its ability to bind molecular oxygen. in particular, we focused our attention on the secondary structure of this protein in order to highlight whether exposure to microwaves unfold the protein producing transitions from a-helix component to b-sheet features. to this aim fourier transform infrared (ftir) spectroscopy have been used. the importance of this study is related to previous literature which indicated that transition from a-helix to b-sheet structure in a protein can be responsible for aggregation mechanisms that can lead to neurotoxicity and neurodegenerative disorders that can be considered as the first step to some pathologies [ ] [ ] [ ] . the aggregates consist of fibers containing unfolded proteins with a prevalent b-sheet structure termed amyloid [ ] . in our studies myoglobin in deuterium oxide (d o) solution was exposed for h to mobile phone microwaves at and mhz at a power density of w/m . ftir spectra were recorded by a spectrometer vertex v from bruker optics, following the protocol accurately described in [ ] [ ] [ ] . ftir spectroscopy analysis evidenced an increase in intensity of b-sheet structures and a significant shift to lower frequencies of about . cm- of the amide i vibration after exposure [ , ] . these results led to conclude that mobile phone microwaves induce proteins unfolding and formation of aggregates [ , ] . membrane proteins play a vital role in many biological processes, and yet remain poorly understood as they are frequently unstable in vitro. the goal of this project is to investigate the insertion and folding of membrane proteins into lipid bilayers, using a cell free expression system. we have used both e.colibased cell extracts (s ), and commercial translation systems (purexpress) in combination with synthetic liposomes of defined lipid composition. these studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. model e.coli proteins have been investigated, as they can offer important insights into other proteins, and thus facilitate the further study of more biologically relevant proteins. it has been found that the rhomboid protease glpg spontaneously inserts into liposomes without the aid of an insertase such as secyeg. this spontaneously inserted glpg is functional, and is able to cleave bodipy-labeled casein, yielding a fluorescent product. the major facilitator superfamily (mfs) transport proteins lacy, galp and glpt have also been found to insert spontaneously into liposomes. it has been shown that the lipid composition of the liposomes has an effect on the amount of protein inserted into the bilayer, with all proteins tested to date preferring liposomes containing at least mol% dopg. ongoing and future work will involve the use of rare codons to alter the rate of translation, to investigate the effect this has on the final folded structure of the protein. preliminary work is also currently being done into whether the two domains of the mfs family transporters fold cooperatively or independently, thus aiding understanding into the folding and stability of membrane transport proteins. frederic greco , audrey toinon , nadege moreno , marie claire nicola€ ı rabies remains an important worldwide health problem that causes a fatal encephalomyelitis [ ] . currently, rabies in humans is under control in europe and north america following the use of efficient vaccines for dogs and wild animals. however, it still kills more than , people every year mainly in africa and asia [ ] . human vaccination prevents infection with very high efficacy. the vaccine contains an inactivated rabv produced on vero cells. rabv is an enveloped, negative single stranded rna virus which encodes five proteins, namely the nucleoprotein (n), the phosphoprotein (p), the matrix protein (m), the glycoprotein (g), and the viral rna polymerase (l) [ ] . the viral envelope is covered by trimer spikes of g-glycoprotein which is the most significant surface antigen for generating virus-neutralizing antibodies. here we illustrate the use of dsc (differential scanning calorimetry) to identify structural domains or proteins involved in thermal transitions. the dsc thermogram for intact beta-propiolactone inactivated rabv samples in pbs buffer reveals two major thermal transitions with a tm respectively at c and c. we have initially focused our investigations on one of the major proteins encode in rabv, glycoprotein g [ ] . glycoprotein g contains disulfide bridges on the ectodomain [ ] , is sensitive to bromelain cleavage [ ] and shows reversible conformation changes at low ph [ ] . considering these characteristics, our results provide evidence on the identity of one thermal transition observed by dsc. keywords: rabies virus, differential scanning calorimetry, protein unfolding domain swapping of the dna-binding domain of human foxp is facilitated by its low folding stability exequiel medina, sandro l. valenzuela, crist obal c ordova, c esar a. ram ırez-sarmiento and jorge babul departamento de biolog ıa, facultad de ciencias, universidad de chile, santiago, chile protein folding and dimerization (or oligomerization) are biologically relevant processes when reaching the quaternary structure is required for function. proteins that form dimers by exchanging segments or domains of their tertiary structure with another subunit, the so-called domain swapping phenomenon, are examples where folding and dimerization are tightly concerted processes. previous studies on domain swapping proteins, such as p suc and diphtheria toxin, have shown that, in general, a high kinetic barrier separates monomers and domain swapped dimers, and that this barrier can be lowered by promoting protein unfolding and refolding at high protein concentrations, thus favoring the swapped oligomer. recent crystal structures of the dna-binding domain of several human forkhead box (fox) proteins have shown that the p subfamily of these transcription factors (foxp) can form swapped dimers. the human foxp proteins are interesting models of domain swapping, because mutations of the dna-binding domain of these proteins are linked to diverse inherited disorders in humans, such as ipex and language deficits, and some of these mutations are located in the hinge region that connects the exchanged segment with the rest of the protein. moreover, foxp and foxp have been described to reach monomer-dimer equilibrium in solution after hours of incubation, suggesting that a low kinetic barrier separates both species. using foxp as a model of domain swapping, we analyzed the temperature and protein concentration effects on the dimer dissociation, obtaining the free energy change and enthalpy of the process by van't hoff analysis (dh of . kcal•mol- , ds of . kcal•mol- •k- and dg at c of . kcal•mol- ). these results indicate that the monomer-monomer association is an example of an enthalpy-driven process. to understand how foxp domains swap without protein unfolding, we performed equilibrium unfolding experiments using gndhcl as denaturant, showing that the wild-type protein has a low stability (dgu kcal•mol- , cm . m at c), in contrast to other domain swapping proteins with high kinetic barriers. we further explore the domain swapping mechanism of foxp through biased targeted molecular dynamics simulations, showing that the exchange process can occur by specific local destabilization and unfolding of the hinge region and helix h . to further corroborate that the low stability of wild-type foxp facilitates its domain swapping, we engineered a monomeric version of foxp through a single-point mutation in the hinge region, which has been previously described in the literature, and used this protein to visualize the effect of monomer stability in the dimer formation. comparison of the folding stability of the monomeric mutant a p and wild-type foxp shows that ddgu (mutant-wild-type) is . kcal/mol, concluding that the ability of foxp to domain swap rapidly can be explained through its low monomer stability and local unfolding of the exchange region. funding: fondecyt and . determining the coupled interactions that stabilize the structural framework of the ß-propeller fold loretta au , david green , , department of statistics, the university of chicago, department of applied mathematics and statistics, stony brook university, graduate program in biochemistry and structural biology, stony brook university, laufer center of physical and quantitative biology, stony brook university b-propeller proteins are a highly evolved family of repeat proteins that are involved in several biological pathways, such as signal transduction, cell-cycle modulation and transcription regulation, through interactions with diverse binding partners, despite having a similar fold. as for all repeat protein families, there is a consistent pattern in secondary structure for each repetitive region, in addition to the entire family. typically, four to ten propeller blades (each containing four anti-parallel b-sheets) are arranged in a toroidal shape, thus providing a large binding surface for ligands or other proteins. about % of known proteins adopt this distinctive fold, and although the requirements for tertiary structure and protein function are fundamentally encoded in primary structure, this relationship is not fully understood, and addressing it could provide insight on why the b-propeller fold is common. many techniques in comparative sequence analysis can successfully identify amino-acid conservation between closely related proteins, but molecular interactions between amino acids are often neglected, and further experimentation is still needed to determine the reasons underlying conservation. to explore how primary structure can dictate fold and function, we devised a computational approach to perform large-scale mutagenesis, by adapting the dead-end elimination and a* search algorithms (dee/a*), and also leveraged the structural conservation of each repeating region to understand how sequence variation influences protein fitness, defined here as a combination of stabilizing and binding interactions. dee/a* can evaluate low-energy protein sequences and their corresponding three-dimensional structures, and we used the bsubunit of a g-protein heterotrimer (pdb: gp , gia b g ) as a model system to demonstrate: ( ) how the multiple roles of individual amino acids in protein fitness can be deconvolved, and ( ) how epistatic interactions between them can contribute to structural stability. in doing so, we were able to identify important patterns in sequence complementarity between repeating regions that cannot be found using sequencebased methods alone. these results suggest that computational approaches can be used to determine important protein interactions, and help elucidate the prevalence of b-propeller proteins in biology. temperature induced conformational changes of the villin headpiece miniprotein stanislaw oldziej , wioletta _ zmudzi nska , anna hałabis the c-terminal subdomain of the actin-binding protein villin called hp (villin headpiece) has been used as a model protein in a number of studies of protein folding kinetics and protein folding mechanism [ , ] . the hp is a residue miniprotein with an alpha-helix bundle three-dimensional fold. the goal of our work was to determine conformational ensemble of polypeptide chain of the investigated miniprotein at a wide range of temperatures to get detailed information about how protein structure is influenced by temperature. d nmr spectra of the title miniprotein were registered at , and k. the three-dimensional structure of the hp based on restraints derived from nmr spectra registered at k is almost identical with structure deposited in the pdb database in the record f k [ ] . at higher temperatures ( and k) the general shape of the protein remains unchanged, with well packed hydrophobic core. however, with temperature increase alpha-helices start to melt. at k structure of the protein remains compact and in general shape similar to structure observed at k, but none of the alpha-helices could be observed. results obtained for hp protein are in agreement with previous observation for the trp-cage miniprotein [ ] , that with temperature increase regular secondary structure elements melt first before the break-up of the hydrophobic core of the protein. biological membranes provide a selective and chemically sealed barrier for cells. transport of ions and small molecules across the membrane is mediated by transporter proteins and the breakdown of a cell's ability to produce functionally folded membrane transport proteins can lead to dysfunction and has been implicated in many diseases . however little is known about the processes that govern the misfolding of a-helical integral membrane proteins, taking into account that these proteins fold and maintain functional structures within membranes of various organelles. the neurotransmitter sodium symporter (nss) protein family is an example of a-helical transporter proteins. the nss family encompasses a wide range of prokaryotic and eukaryotic ion-coupled transporters that regulate the transport of neurotransmitter molecules whose dysfunction has been implicated in multiple diseases and disor-ders . we have investigated the folding processes of prokaryotic homologue of the nss family leut responsible for the transport of neurotransmitters and amino acids to the sodium electrochemical gradient. previously folding processes of membrane transporters have mainly been characterised within detergent micelles. however, detergent micelles are not an accurate depiction of the environment of the membrane bilayer, with this in mind we have also attempted to investigate folding processes within a bilayer pd- nmr investigation of ph-induced unfolding of b domain of an escherichia coli mannitol transporter ii mannitol in the bacterial phosphotransferase system kim gowoon , yu taekyung , suh jeongyong the bacterial phosphotransferase system (pts) mediates sugar phosphorylation and translocation across the cytoplasmic membrane. cytoplasmic b domain (iib mtl) of the mannitol transporter enzyme ii mannitol, a pts family protein, delivers a phosphoryl group from a domain to an incoming mannitol that is translocated across the membrane. iib mtl is comprised of a four-stranded ß-sheet and three helices, representing a characteristic rossmann fold. we found that the iib mtl of escherichia coli unfolded at a mildly acidic condition. we made iib mtl mutants to investigate the mechanism of the ph-induced unfolding using nmr spectroscopy. we monitored backbone amide groups and side chain imidazole groups of histidine residues using d hsqc nmr, and pointed out a potential histidine residue that might be responsible for the unfolding. histidine residues may be generally important to the folding stability in response to environmental ph changes. can site-directed mutagenesis shed light on the refolding pattern of human glucose -phosphate dehydrogenase (g pd)? nurriza ab latif , , paul engel conway institute, univerversity college dublin, faculty of biosciences and medical engineering, universiti teknologi malaysia human glucose -phosphate dehydrogenase (g pd) is the first enzyme involved in the pentose phosphate pathway (ppp). this oligomeric enzyme catalyses the reaction of glucose -phosphate to form phosphogluconolactone with concomitant reduction of nadp to nadph. in erythrocytes nadph is important mainly for protection against oxidative stress. in connection with its role as the sole source of nadph, g pd deficiency commonly causes haemolytic disease and is known as the most common human enzyme deficiency globally. protein folding problems and instability are believed to be the major defects in the deficient enzymes. in this study, we employed site directed mutagenesis with hope to give more information on the role of -sh groups in the refolding of human g pd. two mutants were created: ) one in which all cys residues were replaced by ser and ) one in which only c and c were retained. the refolding of recombinant human g pd has been studied primarily by measuring the enzyme activity after refolding. we also used a combination of intrinsic protein fluorescence, ans ( -anilino- -naphthalenesulphonic acid) binding and limited proteolysis to look at the conformational change during the refolding. the results showed that gdnhcl-denatured recombinant human g pd wild type could be refolded and reactivated by rapid dilution technique. even though, as recombinants in e. coli, the mutants were well expressed and active, they remained inactive after attempts were made to refold them in vitro. the methods we applied may have provided some insights on the refolding pattern of this oligomeric protein, albeit qualitatively rather than quantitatively. a single aromatic core mutation converts a designed 'primitive' protein from halophile to mesophile folding connie tenorio , liam longo , ozan s. kumru , c. russell middaugh , michael blaber department of biomedical sciences, florida state university, department of pharmaceutical chemistry, university of kansas experiments in prebiotic protein design suggest that the origin of folded proteins may have favored halophile conditions. these results are consistent with salt induced peptide formation which shows that polymerization of amino acids is also promoted by high salt concentrations. as a result of various origin of life studies, a consensus on which amino acids likely populated early earth has emerged. these residues were synthesized by abiotic chemical and physical processes from molecules present in the surrounding environment. the properties of the consensus set of common prebiotic amino acids (a,d,e,g,i,l,p,s,t,v) are compatible with known features of halophile proteins, meaning these proteins are only stable in the presence of high salt concentrations. the halophile environment, thus, has a number of compelling aspects with regard to the origin of structured polypeptides. consequently, a proposed key step in evolution was, movement out of the halophile regime into a mesophile one commensurate with biosynthesis of "phase " amino acids -including the aromatic and basic amino acids. we tested the effects of aromatic residue addition to the core of a "primitive" designed protein enriched for the prebiotic amino acids (a, d, e, g, i, l, p, s, t, v) that required halophilic conditions for folding. the subsequent results show that the inclusion of just a single aromatic residue was sufficient for movement to a mesophile folding environment. thus, the inclusion of aromatic residues into the codon table could have conferred key stability to early proteins enabling adaptive radiation outside of a halophile environment. contact prediction methods that rely on sequence information alone, such as evfold, can be used for de novo d structure prediction and identification of functionally important residues in proteins. large multiple sequence alignments of protein families consisting of evolutionarily related and plausibly isostructural members reveal co-variation patterns that can be used to identify interactions between pairs of amino acids. we use a global probability model to disambiguate direct and indirect correlations. specifically, we use a maximum entropy approach called pseudo-likelihood maximization (plm) to distinguish causation (residue interactions) from correlation (correlated mutations) and compute evolutionary couplings (ecs). the inferred set of residue interactions can then be interpreted as physical contacts and used in de novo d structure prediction. furthermore, the interactions that are inferred can help guide experiments that measure the phenotypic consequences of protein substitutions, making the method useful for functional studies. the present work can be divided into three areas: (i) methodological improvements related to alignment, folding procedure, structure refinement and ranking; (ii) folding of proteins of known structure for benchmarking and prediction of proteins of unknown structure; and (iii) focused exploration of specific cases of interest. developing shuffle as a platform for expression and engineering of antibodies na ke , alana ali-reynolds , bryce causey , berkmen berkmen shuffle is a genetically engineered e.coli strain that allows disulfide bond formationin its cytoplasm with high fidelity. many proteins containing disulfide bonds have been successfully expressed in shuffle. in this study, we expressed, purified and characterized full-length monoclonal antibody igg in shuffle. for the first time, a fulllength igg can be functionally expressed in the cytoplasm compartment of an e.coli strain. in order to improve the folding and assembly of igg, we have investigated the expression of igg in various formats and vectors; we have co-expressed chaperones and other helper proteins with igg. several-fold increase in the yield of fulllength igg was observed. we characterized the shuffle produced igg and found it comparable to hybridoma produced igg. optimization of fermentation conditions for a large-scale production is in progress. we aim to develop shuffle as an easy, fast, robust platform for antibody engineering, screening and expression. experimental and computational studies of the effects of highly concentrated solutes on proteins: insights into the causes and consequences of quinary protein structure and cytoplasmic organization most studies of protein structure and function focus on pure, diluted samples; however, real-world biochemistry and typical biotechnological applications of proteins take place in complex media with very high concentrations of solutes ( - g/l) of varied size and chemical nature. on one side, this has recently fostered the study of proteins in vivo, in cell, or at least in media mimicking the native conditions. on the other hand, physical chemistry has for a long time studied the general effects of crowded and viscous conditions on proteins, looking mainly at coarse traits like diffusion and stability. but the general effects on traits relevant at atomic/residue resolutions have been less studied, and one fundamental issue remains unsolved: to what extent are proteins forced into interactions with highly concentrated solutes, and with what direct consequences? i will present here our ongoing efforts to dissect the fine effects of high solute concentrations and macromolecular crowding on proteins, based on nmr experiments and md simulations, two complementary techniques of high spatial and temporal resolutions. our results show that smaller solutes are prone to extensive interactions with proteins when at high concentrations while large solutes act chiefly through excluded-volume effects. overall, we observe location-specific perturbations of a protein's surface, its internal dynamics and internal dielectrics, and its hydration, all very dependently on the solute's size and chemical nature. our results support the growing notion that proteins should be studied in native-like media, adding that not only macromolecular crowders but also small molecules should be considered in these studies. last, the fact that high-concentration conditions affect far more than a protein's diffusion rate and stability suggests critical consequences of quinary protein structure and cytoplasmic organization on the regulation of proteins within cellular biochemistry. aldona jeli nska , anna lewandrowska , robert hołyst we developed an analytical technique for the study of interactions of ligands (e.g. cefaclor, etodolac, sulindac) with most abundant blood protein (e.g. bovine serum albumin) using the flow injection method. the experiments were conducted at high flow rates ( cm/s) in a long (> m), thin ( mm) and coiled capillaries. the compound of interest ( ml) was injected into carrier phase, which moved by the poisseule laminar flow. at the detection point we measure the concentration distribution of the analyte. the width of the final profile of the analyte concentration is inversely proportional to the effective diffusion coefficient of the analyte. from the differences between the widths of the concentration distribution of free and bound ligand we can determine value of the association constant. carbohydrate binding modules (cbms), which are defined as contiguous amino acid sequences within a carbohydrate-active enzyme, have been found in both hydrolytic and non-hydrolytic proteins and are classified into families, according to their primary structure similarity. the characterization of cbms by different methods has shown that these modules concentrate enzymes on the surface of polysaccharide substrates. it is thought that maintaining the enzyme in proximity with the substrate leads to more rapid degradation of the polysaccharide. therefore, the study of these kinds of modules or domains is relevant, since they are involved in multiple processes in organisms, like signaling, defense and metabolism; and some of them are involved in allergenic responses. in the present work we studied two different models: the first one is a cbm of the family from lactobacillus amylovorus (lacbm ) that binds starch these domains are present in a a-amylase like a repetitive tandem of five modules that are consecutive and do not present connectors. by means of itc and using a single recombinant lacbm domain we determined a ka . x m- for b-cyclodextrin and a ka . x m- for acyclodextrin. when the number of consecutive recombinant modules increased to three or five tandem modules, the ka values increased to m- ; however, these constants did not show an additive or a synergic effect. for these experiments we fitted the isotherms to different models and used different algorithms. additionally, we used circular dichroism in the uv-far region to determine if there existed conformational changes upon binding of the cyclodextrin molecules to the different tandem modules. we could only observe slight changes in a positive band centered around - nm, which has been explained in terms of p-p; interactions of the aromatic residues at the binding site. these cbms have been used as carriers for in vivo vaccine delivery and affinity tags. the second model is a hevein-like cbm of the family present in a chitinase-like protein from hevea brasiliensis (hbcbm ). hevein is a lectin from h. brasiliensis that shows a % identity with hbcbm . these cmbs are connected to the catalytic domain, in proteins such as chitinases, by a linker of approximately residues. in these experiments we used fluorescence techniques to determine the affinity constants for chitotriose. we previously reported a ka . x m- when using a hbcbm that has a met residue at the nterminal region. besides the aromatic residues at the binding site, the met residue also interacts with the ligand, as determined using crystallographic and docking techniques. the mutant hbcbm -r w that does not have the met residue showed a ka of . x m- with chitotriose, similar to the value reported for hevein using itc (ka . x m- ). interestingly, there exists an isoform of the hbcbm that has a connector between one cbm and a half cbm ( . xhbcbm ). this protein has a ka of . x m- with the same ligand. initiating vesicle formation at the golgi complex: auto-regulation and protein interactions govern the arf-gefs gea and gea margaret gustafson , j. chris fromme molecular decision-makers play critical roles in the effort to maintain efficient and accurate cellular functions. in the case of vesicular traffic at the golgi complex, the decision to initiate vesicle formation is made by a set of guanine nucleotide exchange factors (gefs) that activate the small gtpase arf , which is the master controller for the recruitment of cargos and coat proteins. saccharomyces cerevisiae possess three golgi arf-gefs, gea , gea , and sec , which work at distinct sub-compartments of the golgi to activate arf only when and where appropriate. in the case of sec at the trans-golgi network (tgn), this requires a positive feedback loop in which active arf relieves autoinhibition of sec , as well as recruitment to the golgi membrane and catalytic stimulation by signaling rab gtpases. we know far less about the decisionmaking process for gea and gea , which are responsible for retrograde traffic within the golgi and to the endoplasmic reticulum. i have found that both gea and gea can bind membranes weakly in vitro, an ability which is counteracted by their c-terminal hds domains. in addition, i have discovered membrane recruitment in vitro is aided by the rab gtpase ypt . however, these interactions cannot fully explain the distinct localization patterns of gea , gea , and sec , as all three have been shown to be recruited by ypt , which is found throughout the golgi. my work has revealed that in addition to the well-established distinct localization from sec , gea also occupies different golgi compartments from gea , so specific signals must exist which help the gefs decide where to go. my current efforts focus on understanding the roles of the other domains of gea and gea , identifying the signals which send them to different parts of the golgi, and unraveling the different roles they play in vesicle trafficking pathways. sequence variation in archaea through diversity-generating retroelements sumit handa , blair g paul , kharissa l shaw , david l valentine , partho ghosh department of chemistry and biochemistry, university of california san diego, marine science institute, university of california protein diversification is an essential tool for the survival and evolution for various species. diversitygenerating retroelements (dgr) in bacteria is known to generate massive variation in dna through an error prone reverse transcriptase and retrohoming, which leads to variation in protein sequence. recent discovery of dgrs in intraterrestrial archaeal systems have opened an opportunity to study this massive sequence variation in third domain of life (paul bg, et al. nat. comm.) here, we present the first crystal structure of variable protein from archaea with ligand-binding pocket is surface exposed. also, it has conserved c-type lectin (clec) fold, as shown by previous work on variable proteins, major tropism determinant (mtd) and treponema variable protein a (tvpa) which bind ligands through the clec fold. despite weak sequence identities ( - %) among these variable proteins, clec fold was found to be conserved. this variable ligand-binding site for archaea variable proteins can potentially generate variants. protein synthesis is a dynamic process mediated by a variety of proteins and enzymes. recent studies have shown that hydroxylation is a key post-translational modification involved in translation termination. in particular, the fe(ii)-and -oxoglutarate-dependent oxygenase, jumonji domaincontaining (jmjd ), regulates translation termination via the carbon hydroxylation of an invariant lysine residue, k , of the eukaryotic release factor, erf . in eukaryotes, translation termination is mediated by a release factor complex that includes erf . erf is comprised of three domains, and it is responsible for recognizing stop codons in mrna transcripts before triggering polypeptide release from the ribosome. the lysine residue hydroxylated by jmjd falls within the n-terminal domain and more specifically within the highly conserved niks motif. this motif has been identified by cross-linking and mutagenesis studies to play an essential role in stop codon recognition. while hydroxylation of k by jmjd has been found to increase translational termination efficiency, the exact molecular mechanism by which hydroxylation influences termination remains unclear. this work aims to understand how hydroxylation of erf affects translation termination by exploring the effect of hydroxylation on the structure, dynamics, stability, and binding of the n-terminal domain of erf (erf -n) using mass spectrometry, protein nmr spectroscopy, circular dichroism and differential scanning fluorimetry. in our efforts to understand the effect of hydroxylation, an additional jmjd -catalyzed modification, characterized by a da mass shift on k , was identified in vitro. the effect of this modification on erf was similarly explored. our findings suggest that hydroxylation has no effect on the in-solution nmr structure of erf -n, which experiences chemical shift changes localized to the target lysine residue. correspondingly, there are no significant differences in secondary structure content between wild type and hydroxylated erf -n. hydroxylation was also found to have no effect on protein stability or dynamics. interestingly however, the da modification appears to cause more significant chemical shift changes dispersed beyond the niks motif. this suggests a more global effect on the in-solution nmr structure despite the little differences observed in protein dynamics and secondary structure content. the da modification was also found to have a destabilizing effect on erf -n. neither hydroxylated nor da modified erf -n exhibited differences in rrna binding. while hydroxylation of erf was found to have little effect on protein structure, dynamics, stability, or binding, the da modification has marked effects on protein structure and stability. such differences suggest that this modification has the potential to play an important role in translation. functional and structural analysis of a gh ß-n-acetylglucosaminidase from the marine bacterium vibrio harveyi piyanat meekrathok , arthur t. porfetye , marco b€ urger , ingrid r. vetter , wipa suginta biochemistry-electrochemistry research unit, suranaree university of technology, max planck institute of molecular physiology vibrio harveyi b-n-acetylglucosaminidase (so-called vhglcnacase) is a new member of the gh glycoside hydrolase family responsible for the complete degradation of chitin fragments, with nacetylglucosamine (glcnac) monomers as the final products. however, the d structure of glcnacase is still unknown. in this study, crystal structure and function of glcnacase were investigated based on protein crystallography. size-exclusion chromatography and the native-page were employed to verify the protein state of glcnacase in a native form and the acidic active-site residues were mutated using sitedirected mutagenesis method. the effects of mutations on the binding and hydrolytic activities were studied by enzyme kinetics. to provide a structural basis of glcnacase, the wild-type enzyme was crystalized at k using a solution containing . m sodium acetate ph . and . m sodium malonate and recorded x-ray data. the wild-type enzyme was crystallized within days in the monoclinic crystal form, belonging to space group p , with unit-cell parameters a . , b . , c . Å. the crystal structures of v. harveyi glcnacase were solved and refined to highest resolution of . Å. structural investigation revealed that glcnacase comprises three distinct domains, designated as the n-terminal carbohydrate-binding domain, the a b topology domain and the tim-barrel catalytic domain. the substrate binding groove of glcnacase is a small pocket, which is suitable to accommodate a shortchain chitooligosaccharide. kinetic analysis revealed that a group of the adjacent d -h -e showed a significantly decreased activity as compared with the wild-type enzyme, and these residues might be important for enzyme catalysis. silencing the molecular timekeeper in human cancer alicia michael , stacy harvey , patrick sammons , amanda anderson , hema kopalle , alison banham , carrie partch university of california -santa cruz, university of oxford the circadian clock coordinates temporal control of physiology by regulating the expression of at least % of the genome on a daily basis. disruption of circadian rhythms through environmental stimuli (e.g. light at night) or genetic means can lead to the onset of diseases such as: diabetes, cardiovascular disease, premature aging and cancer. - the circadian clock orchestrates global changes in transcriptional regulation via the bhlh-pas transcription factor clock:bmal . pathways driven by other bhlh-pas transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for clock:bmal . we discovered that the cancer/testis antigen pasd fulfills this role to suppress circadian rhythms. pasd is evolutionarily related to clock and interacts with the clock:bmal complex to repress transcriptional activation. furthermore, deletion of one region, highly conserved with clock exon , alleviates repression by pasd to suggest that it utilizes molecular mimicry to interfere with clock:bmal function. structural and biochemical studies of the direct interaction of pasd with the clock:bmal complex using recombinant protein expression and biophysical techniques are currently underway. as a cancer/testis antigen, expression of pasd is natively restricted to gametogenic tissues but can be upregulated in somatic tissues as a consequence of oncogenic transformation. reducing pasd in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. our work suggests that mechanisms to suppress circadian cycling can be hard-wired in a tissue-specific manner and our data show that they can be co-opted in cancer cells to attenuate clock function. the scaffolding protein iqgap participates in various cellular functions such as cell-cell adhesion, cell polarization and migration, neuronal motility, and tumor cell invasion by binding to target proteins, including rac and cdc , two members of the rho family. to better understand the molecular basis of these interactions, we utilized in this study a novel time-resolved fluorescence spectroscopy to determine individual rate constants for iqgap interaction with fourteen different rho proteins. the results indicated that iqgap binds among rho proteins selectively to rac-and cdc -like proteins only in a gtp-dependent manner. moreover, the interaction of rho proteins with the c-terminal half of iqgap (grd-c), shorter fragment contains grd-gbd, only the grd and also grd-gbd with single and double phosphomimetic mutations s e and s d was performed. obtained results showed that, when both grd and gbd are existing, fluorescence changes is detected but for grd alone or in the case of s d or s e/s d no change was observed, suggesting that gbd and specifically, cysteine is critical for this interaction. furthermore, fluorescence polarization results showed that the grd-c interact with cdc and rac but not with rhoa, and interestingly the grd domain showed similar behavior, but with to folds lower affinity as compared with the grd-c. consistent with this, a gdp-bound form of cdc showed interaction with both grd and the grd-c in quiet comparable affinities. at last, competition experiments utilizing interacting partners of rac , e.g. tiam , p rhogap, plexin-b , p phox, pak and rhogdia, along with structural analysis, revealed two negative charged areas on the surface of rho-and rnd-like proteins, which might explain their inaccessible interaction with iqgap . the overlapping binding site of cdc and rac on the surface of iqgap together with the kinetic details of the selective interaction of iqgap with rac-and cdc -like proteins suggests that these interactions are most likely mediated via the same mechanism. ing dimerizes through its n-terminal domain, with a symmetric antiparallel coiled-coil structure , making it a bivalent reader of the h k me mark. ing is highly homologous with ing , but forms part of a different histone acetyl transferase complex . here, we show that ing is also a dimer and thus a bivalent reader of the h k me mark. however, the crystal structure of the n-terminal domain of ing shows an asymmetric dimer, different from the homologous ing domain. our nmr data (backbone assignment and paramagnetic relaxation effects) and saxs data indicate that the structure of the n-terminal domain of ing in solution is similar to ing , suggesting that the crystal structure of ing is likely a crystallization artifact. three point mutations in the n-terminal domain of ing have been described in oral squamous cell carcinoma: q r, i v, and c r . we have found that the n-terminal domains of the three mutants are dimeric coiled-coils but with different stability, as measured by thermal denaturation. while the q r mutant is as stable as the wild type, the i v and c r mutants are strongly destabilized, suggesting a role in cancer development at least for these two mutants. efforts so far, to combat alzheimer's disease (ad) have focused predominantly on inhibiting the activity of enzyme(s) that are responsible for the production of the main causative beta amyloid forming peptide. however, the inherent complexity associated with the network of pathways leading to the progress of the disease may involve additional targets for designing effective therapies. recent experimental findings have identified abelson's tyrosine kinase (c-abl), a non-receptor kinase involved in a variety of cellular functions as a new target for ad. in the present study we employed energy optimized multiple pharmacophore modeling strategy from multiple c-abl structures bound with ligands in the inactive atp binding conformation. virtual screening followed by docking of molecules from chembridge_cns database, and maybridge databases resulted in the identification of best scoring molecules. based on docking score and selectivity assessment and druggability parameters, four out of the molecules are predicted to show increased specificity for c-abl in comparison to closely related kinases. given the implied role of c-abl not only in ad but in parkinson's disease, the identified compounds may serve as leads to be developed as effective neurotherapeutics. rafael palomino , glenn millhauser , pietro sanna university of california santa cruz, the scripps research institute the central melanocortin system is recognized as a key regulator of energy balance and appetite. the hypothalamic melanocortin receptor, mc r, is a g-protein coupled receptor that is antagonized by the peptide ligand, agouti-related peptide (agrp), leading to increased feeding and weight gain. while much research has gone into how this ligand exerts its effects at the receptor, less is known regarding nonmelanocortin components of the pathway. syndecan- , a heparan sulfate proteoglycan, has previously been implicated in potentiating agrp antagonism, however details of this interaction are unclear. this work aims to investigate the role of syndecans at both a molecular level and in vivo. we hypothesize that agrp binds the glycosaminoglycan (gag) components of syndecans, and that this interaction increases the local concentration of the peptide near mc r. furthermore, we have previously shown that designed positive charge mutations to agrp lead to increased in vivo efficacy that is independent of mc r activity, and we hypothesize that this is due to greater affinity for the negatively charged gags. using isothermal titration calorimetry we have shown tight binding between agrp and heparan sulfate, the major gag component of syndecan- , and this affinity is strengthened by additional peptide positive charge. through nmr, we see that both positively charged and polar residues are necessary for binding various heparan sulfate polymers. these data implicate a specific region of agrp that is not required for mc r binding as being necessary in its role as a heparan sulfate binding protein. expanding on these findings, we are now using a syndecan knockout mouse line to explore the mechanism of differential feeding in our designed mutants. preliminary results indicate a reduction in weight gain in knockouts compared to their wildtype littermates post peptide administration. collectively, these data show that the physiologically relevant form of agrp, previously considered unable to interact with syndecans, is indeed a heparan sulfate binding protein. furthermore, our designed mutants have differential affinities for gags, with increased affinity correlating to increased feeding potency. finally, as the mc r pathway is thought to be a viable target for wasting disorders such as cachexia, we are interested in leveraging this data to improve the potency and stability of our designed agrp mutants. taken together, this work aims to develop new insights and probe the therapeutic potential of a critical metabolic pathway. evidence of a proteolytic phenomenon in the starch binding domain of the a-amylase from lactobacillus amylovorus zaira esmeralda s anchez cuapio , alejandra hern andez santoyo , sergio s anchez esquivel , romina rodr ıguez sanoja instituto de investigaciones biom edicas, universidad nacional aut onoma de m exico, instituto de qu ımica, universidad nacional aut onoma de m exico a-amylases are glycoside-hydrolases that catalyze the hydrolysis of internal a- , glycosidic bonds in starch and glycogen generating smaller oligosaccharides ( ). these multidomain proteins contain a catalytic barrel (b/a) and, in some cases, one or more non-catalytic domains whose function is generally described as carbohydrate binding module (cbm) and particularly as starch-binding domains (sbd). the sbd can bind granular starch increasing the local concentration of substrate at the active site of the enzyme and may also disrupt the structure of the starch surface ( ) . the a-amylase from lactobacillus amylovorus has a structure that consists of a catalytic domain (cd) and an unusual carboxy-terminal starch-binding domain with identical cbms (belonging to family ) in tandem ( ). each repeat acts as an independent fixing module with an additive or synergic effect between the units ( ). when we stored pure sbd from l. amylovorus we found multiple forms of low molecular weight with a constant pattern, which does not correspond to random degradation. interestingly, when the protein is stored at ph close to and edta is added, such proteolysis appears to decrease. so far, there is little information about the proteolytic process of amylases and the nature of it. here we show that divalent ions induce a proteolytic cleavage of the sbd, raising the possibility of an autoproteolytic activity. acknowledgments: this work is supported by grants papiit in - and conacyt . s anchez cuapio z is supported by a personal grant from consejo nacional de ciencia y tecnolog ıa, m exico. unnatural amino acid and related methods provided a special mechanism to implement site-specific spectroscopy active probe incorporation in a specific membrane protein in cells. the site specific incorporation resulted in a single signal during acquisition, resulting in unambiguous signal assignment. the protein specific labeling makes it possible for in situ membrane protein analysis using nmr or fluorescence detection. the f containing unnatural amino acid incorporation has been applied for dynamic studies of transporters in native lipid membrane, and the phosphorylation quantification analysis for tyrosine kinase in native lipid membrane with the aid of lipodisc. the fluorescent unnatural amino acid incorporation enabled the site-specific channel responses analysis upon ligand binding in a single cell. heather wiebe , noham weinberg , department of chemistry, simon fraser university, department of chemistry, university of the fraser valley the mechanism by which conformational changes, particularly folding and unfolding, occur in proteins and other biopolymers has been widely discussed in the literature. molecular dynamics (md) simulations of protein folding present a formidable challenge since these conformational changes occur on a time scale much longer than what can be afforded at the current level of computational technology. transition state (ts) theory offers a more economic description of kinetic properties of a reaction system by relating them to the properties of the ts, or for flexible systems, the ts ensemble (tse). the application of ts theory to protein folding is limited by ambiguity in the definition of the tse, although the experimentally observed first-order kinetics for folding of small single-domain proteins lends itself to interpretation by this theory. the pressure dependences of the folding rate constant can be used to obtain activation energies and activation volumes, which are rationalized as the properties of the folding tse. the large amount of activation volume data in the literature has gone largely uninterpreted at the quantitative level. we propose to utilize this data in conjunction with md-calculated volumetric properties to identify the tse for protein folding. the effect of pressure on reaction rates is expressed in terms of logarithmic pressure derivatives, known as activation volumes. according to ts theory, activation volumes can be identified as the difference in volume between the ts and reactant species: activation volumes dv ‡ have been experimentally determined for the folding of several proteins. the concept of activation volume can be extended to that of a volume profile, dv(y), which describes how the volume of a system changes along reaction coordinate y. if the position y ‡ of the ts along the reaction coordinate is unknown, it can be found by locating dv ‡ on the volume profile: such volume profiles can be built using our recently developed md-based displacement volume method.* using this method, volumes of single molecules can be calculated by taking the difference between the volume of pure solvent and solvent containing the desired solute. this method takes into account the strength and type of solvent-solute interactions as well as the geometrical configuration of the solute. in this work, we present the successful application of this method to several conformationally flexible systems. structure of the p paf/pcna complex and implications for clamp sliding on the dna during replication and repair to the canonical pip-box binding groove on the pcna front face. in contrast to other pcna interacting proteins, however, p paf also contacts the inside of, and passes through, the pcna ring. the mostly disordered p paf chain termini thus emerge at opposite faces of the ring, but remain protected from degradation by the s core proteasome. we also unveil a novel dna binding activity of p paf, both free and bound to pcna, which is mainly mediated by its conserved histone-like n-terminal tail. molecular modeling shows that a ternary complex with a duplex dna inside the pcna ring is energetically feasible and our electron micrographs show increased density inside the ring. we propose that p paf acts as a flexible drag that regulates pcna sliding along the dna, and may facilitate the switch from replicative to translesion synthesis polymerase binding upon dna damage. acknowledgements: this work has been mainly sponsored by mineco grant ctq - and juan de la cierva- contract to alfredo de biasio. metabolic syndrome (mets) is one of the leading causes of the death worldwide; however, exact pathophysiological mechanisms of mets remain largely unknown. growing evidence suggests that the increased availability of glucocorticoids at the tissue level play an important in mets development. one of the major determinants of glucocorticoid local action seems to be the enzyme b-hydroxysteroid dehydrogenase ( b-hsd ). this enzyme is a well-known member of the short-chain dehydrogenase/reductase (sdr) superfamily. it is an important carbonyl reducing enzyme that, besides its role fine-tuning of glucocorticoids actions, is involved in the biotransformation of drugs and in the development of lung cancer through metabolism of the tobacco specific carcinogen nnk. the phylogenetically closest relative of b-hsd is dhrs enzyme from the same superfamily. unlike b-hsd , dhrs is poorly characterized however it can be supposed at least partially overlapping function to b-hsd . moreover its possible association with similar pathological conditions in human as b-hsd has already been indicated by several studies. the aim of this study is the basic biochemical characterization of dhrs . the enzyme is a member of cluster of "classical" sdr; such members are considered to be retinoid and steroid metabolizing enzymes, so characterization the enzyme was based on this assumption. dhrs was prepared in recombinant form in the sf cell line. it was proved that this enzymes is an integral membrane-bound enzyme localized in the endoplasmic reticulum with luminal orientation, similarly to b-hsd . known substrates of b-hsd and related enzymes were tested also as substrates of dhrs . it was proved that dhrs is nadph-dependent reductase with important substrates as steroid hormones cortisone and androstene- , -dione, all-transretinal and also xenobiotics as , -naphtoquinone or carcinogen nnk at least in vitro. for better understanding of the catalytic function of dhrs its structural model was prepared and it is used also for the identification of additional substrates by ligand virtual screening. dhrs enzyme is expressed in several human tissues as adrenals, liver, prostate, small intestine and kidney. these brand new initial results point to the possible involvement of dhrs in important cellular processes that deserve further investigation. these results will lay the foundation for an understanding of dhrs role in human physiology resp. pathophysiology. this project was supported by grant agency of charles university ( /c/ ) and unce / ). structure-based functional identification of helicobacter pylori hp as a nuclease with both dna nicking and rnase activities bong-jin lee , ki-young lee hp is a conserved, uncharacterized protein from helicobacter pylori. here, we determined the solution structure of hp using three-dimensional nuclear magnetic resonance (nmr) spectroscopy, revealing that this protein is structurally most similar to a small muts-related (smr) domain that exhibits nicking endonuclease activity. we also demonstrated for the first time that hp is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. the nuclease activities for dna and rna were maximally increased by mn( ) and mg( ) ions, respectively, and decreased by cu ( ) ions. using nmr chemical shift perturbations, the metal and nucleotide binding sites of hp were determined to be spatially divided but close to each other. the lysine residues (lys , lys and lys ) are clustered and form the nucleotide binding site. moreover, site-directed mutagenesis was used to define the catalytic active site of hp , revealing that this site contains two acidic residues, asp and glu , in the metal binding site. the nucleotide binding and active sites are not conserved in the structural homologues of hp . this study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases. high-fidelity recombinant protein production in a silkworm bioreactor sungjo park , in-wook hwang , tatsuya kato , enoch park , andre terzic center for regenerative medicine, mayo clinic, laboratory of biotechnology, shizuoka university the domesticated silkworm, bombyx mori, is an attractive host naturally equipped with a proficient posttranslational modification machinery adequate to fulfill stringent demands of authentic recombinant protein production. silkworm-based protein expression has originally relied on a prototype baculovirus vector system that employs silkworm as a bioreactor in place of more traditional cell lines. recent development of the silkworm trophic b. mori nucleopolyhedrovirus (bmnpv) bacmid launches a second generation of silkworm-based protein production technology. introducing the recombinant bacmid dna into silkworms expedites heterologous protein expression by eliminating prior virus construction and amplification steps. salient examples of heterologous eukaryotic proteins produced in silkworms are acetyl-coa carboxylase , malonyl-coa decarboxylase, spot /mig heterodimer and a , -sialyltransferase with consistent high levels of protein expression. thus, equipped with a fail-safe post-translational modification machinery, eukaryotic proteins are readily bioengineered using a silkworm-based protein expression platform. studies exploring potential applications of synthetic antifreeze proteins in the frozen food industry ho zee (charles) kong , conrad perera , ivanhoe leung , nazimah hamid , viji sarojini school of chemical sciences, the university of auckland., school of applied sciences, auckland university of technology in nature, certain species of plants, insects and fish produce a group of antifreeze glycoproteins and polypeptides which enable them to survive the freezing temperatures of their natural habitat. these naturally occurring antifreeze proteins (afps) were first discovered in polar fishes such as antarctic notothenioids and winter flounder. these afps have the ability to bind to ice crystals and restrict their size and morphology; decrease the freezing point of water and inhibit the ice recrystallization processes. ice crystal formation is of primary concern to the frozen food industry, as ice crystal formation during freezing can be disruptive to and cause damage to the cellular structures in food. the unique properties of afps can be developed into a potential solution to minimize freeze-thaw damage to frozen food. a number of tailor made synthetic analogues based on the naturally occurring afps were successfully designed and synthesized. antifreeze activity studies of the afps were carried out using the clifton nanoliter osmometer attached with a microscope. the afps exhibited thermal hysteresis as well as modification of ice crystal morphology, confirming their antifreeze activity in vitro. the ability of these synthetic afps in preserving the texture and structure of frozen food was evaluated using the techniques of scanning electron microscopy. the afps showed great potential to preserve the cellular structures of frozen food samples during freeze-thaw process. additionally, secondary structure analysis of the afps was carried out using circular dichroism. this presentation will summarize our current results on the design, synthesis and anti-freeze activity analysis of the synthetic afps. invasive fungal infections remain a leading cause of death in immunocompromised patients. current antifungal agents have a host of issues including limited efficacy, host toxicity and an alarming increase in resistance. current research in our laboratories is focused on targeting the calcineurin signaling pathway that has been shown to be required for fungal pathogenesis. calcineurin is a highly conserved serine-threonine-specific ca -calmodulin-activated phosphatase important in mediating fungal pathogenesis and stress responses. it is a key regulator of a signal transduction network required for survival of the most common pathogenic fungi in humans, making it an ideal target for fungal drug development. calcineurin is a heterodimer of a catalytic (a) and regulatory (b) subunit. phosphatase activity requires association of the two subunits. calcineurin is also the target of the immunosuppressant fk , which functions as an inhibitor by first complexing with the peptidyl-prolyl cis-trans isomerase immunophilin, fkbp . the fkbp -fk complex subsequently binds to calcineurin in a groove between the a and b subunits and inhibits its activity. although fungal calcineurins are targeted by fk , it also targets mammalian calcineurin and is thus immunosuppressive in the host. in order to improve therapeutic efficacy, we have undertaken a unique effort that utilizes both structural biology and molecular mycology in an effort to overcome the fungal versus human specificity barrier. the nmr studies to be presented here have been focused on determining the resonance assignments and solution structures for the fkbp proteins from the pathogenic fungi candida albicans, candida glabrata and aspergillus fumigatus. notably, the x-ray crystallography structures of the wild-type candida albicans and aspergillus fumigatus fkbp proteins revealed an intriguing intermolecular interaction involving four residues in the 's loop including pro (in c. albicans) and pro (in a. fumigatus) which are stabilized in the cis conformation. these data suggest that the protein might use itself as an enzyme substrate. in efforts to establish if this interaction remains in a solution environment, we have determined the nmr structure and measured the t relaxation rates for the wild-type a. fumigatus fkbp protein and for the p g mutant variant that adopts a dramatically different orientation of the 's loop and does not form an intermolecular interaction in the crystal structure. the nmr chemical shift data indicate that, while the remainder of the protein structure remains unchanged, the 's loops in the two variants are indeed different. in addition, the t relaxation rates of the residues in this region are dramatically dissimilar in the two variants, but remain identical throughout the rest of the protein. we have also begun inhibitor binding studies of all of the fkbp proteins from each of the pathogens by titrating the fk inhibitor into native and mutant fkbp proteins in order to examine conformational changes associated in the protein upon complex formation. using this approach we plan to determine the relative kd values for binding of each inhibitor to the fkbp protein from each pathogen for comparison of binding proclivities. lupin (lupinus angustifolius l.) b-conglutin proteins: structure functional features, catalytic mechanism modeling and cross-allergenicity identification using protein threading and molecular docking methods lupin is an important pulse, which displays a wide range of benefits in agriculture, particularly these involved in possible plant pathogen suppression. furthermore, lupin seed proteins promote different positive health aspects, preventing cardiovascular disease, and reduction of glucose and cholesterol blood levels. "sweet lupine" seeds seem to be promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. thus, lupin seeds may be important source of proteins for human and animal consumption. however, and as drawback feature, the number of allergic people to lupin seed proteins is rising, becoming a serious and a growing problem in the western world, because of the rapid introduction of lupin seeds as new ingredients in traditional and novel foods. the goals of this study are the characterization the structure-functional properties of lupinus angustifolius l or narrow leafed lupin (nll) b-conglutin proteins, with a focus in its catalytic mechanism, and its molecular cross-allergenicity with other legumes, i.e. peanut, by extensive analysis using different computer-aided molecular approaches covering (i) physicochemical properties and functional-regulatory motifs, (ii) sequence analysis, -d and d structural (threading) modeling comparative study and molecular docking, (iii) conservational and evolutionary analysis, (iv) catalytic mechanism modeling, and (v) sequence, structure-docking based b-cell epitopes prediction, while t-cell epitopes were predicted by inhibitory concentration and binding score methods. b-conglutins (vicilin-like or s proteins) are seed proteins typically found in reserve tissues (endosperm and cotyledon). they belong to the cupin superfamily of proteins, containing a globular domain constituted by a conserved b-barrel. two barrels were found in all b-conglutin protein isoforms and an additional mobile n-terminal arm constituted bye a-helices. molecular modeling analysis has shown that one of this barrel contain a semi-conserved metal binding motive (hyx. . .r), typically found in oxalate oxidase (oxox) enzymes. interestingly, our results revealed considerable structural differences between b-conglutin isoforms, particularly affecting -d elements (loops and coils), and numerous micro-heterogeneities are present in fundamental residues directly involved in epitopes variability, which might be a major contributor to the observed differences in cross-reactivity among legumes. we also identified multiple forms of b-conglutins polypeptides ranging from - kda, with ige-binding characteristics in atopic patients. thus, b-conglutins might be considered as major allergen in different species of lupin, including the "sweet lupin" group, since several of these polypeptides were recognized by human iges, having the potential to trigger an immune response leading to allergy symptoms. influenza virus is one of the most prevalent pathogens causing respiratory illness which often leads to serious post influenza complications such as pneumonia and myocarditis. some viruses, as the avian influenza h n , are especially dangerous and draw special attention of who. this highly pathogenic virus spreads quickly among domestic poultry and wild birds resulting in high mortality. what is more distressing, the h n virus may be transmitted to humans. because of antigenic drift it is impossible to deliver an effective vaccine against all subtypes of the h n virus. moreover, traditional egg-based production of influenza vaccines is time-and cost-consuming, what makes it inadequate in case of a pandemic. hence, we have developed an efficient production process of influenza vaccine based on a recombinant hemagglutinin antigen (rha). recombinant vaccines underlay strict regulations and quality requirements. the purpose of this work was to develop a battery of analytical methods that allow to evaluate key quality attributes of rha on each stage of production. at first, we have focused on rha structure as a crucial issue for its activity. the primary structure of rha was confirmed by peptide mapping and tof/tof fragmentation (hplc, maldi tof/tof). furthermore, ftir analysis was used to evaluate the secondary structure of the protein. the disulfide bonds, which stabilize the tertiary structure, were assigned by peptide mapping. additionally, free thiols were measured using ellman's reagent. moreover, we have employed rp-hplc, sec-mals and dls to explore oligomerization of rha. these techniques appeared to be useful not only to confirm existence of native oligomers, but also to find and discard misfolded fraction, aggregates and truncated forms. in addition, two analytical methods (rp-hplc and cge) were developed to assess the purity of rha as required by ich guidelines. we also have determined isoelectric point and heterogeneity of rha by cief. afterward, developed methods were applied in the stability studies that provide a valuable insight into a chemical degradation process and conformational changes of rha during storage. this work was supported by innovative economy operational program, grant no. wnd-poig. . . - - / - as a part of project "centre of medicinal product biotechnology. package of innovative biopharmaceuticals for human and animal therapy and prophylactics." muscle cell atrophy via hsp gene silencing was counteracted by celastrol-mediated hsp overexpression molecular chaperone heat shock proteins (hsp) are known to assist protein quality control under various stresses. although overexpression of hsp was found to promote muscle mass retention in an unloading state, it is unclear whether muscle atrophy is induced by suppression of hsp expression and is counteracted by active hsp overexpression. in this study, we pre-treated hsp sirna to rat l cells for the hsp gene-silencing, and determined myotube diameter, hsp expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (cel), the hsp inducer. relative to a negative control (nc), muscle cell diameter was reduced by % in the sirna-treated group, increased . -fold in the cel-treated group and remained at the size of nc in the sirna cel group. hsp expression was decreased % by sirna whereas the level was increased -to -fold in the cel and sirna cel groups. expression of foxo and atrogin- was increased . -to . -fold by sirna, which was abolished by cel treatment. finally, phosphorylation of akt , s k and erk / was not affected by sirna, but was elevated -to -fold in the cel and sirna cel groups. these results suggest that hsp downregulation by hsp gene-silencing led to muscle cell atrophy principally via elevation of catabolic activities. such anti-atrophic effect was counteracted by cel-mediated hsp overexpression. the centers for disease control and prevention report that at least million people in the united states will become ill due to antibiotic resistant pathogens leading to , deaths each year. in order to circumvent these resistance mechanisms, it is essential to quantitatively understand how the function of the protein(s) involved relates directly to resistance. integral membrane efflux pumps are known determinants of single-drug and multi-drug resistance in a wide variety of pathogenic organisms. these transporters are proteins whose characterization typically requires reconstitution in an artificial membrane. subsequently, these important proteins are difficult to characterize by traditional in vitro studies. my project aims to determine the physicochemical parameters of the efflux pump tetb utilizing molecular biology and mathematical modeling. tetb is composed of transmembrane (tm) alpha-helices and is found within the inner membrane of gram-negative bacteria. this protein allows for the efflux of tetracycline (tet), doxycycline (dox), and minocycline (mcn) antibiotics from the cytoplasm into the periplasm. these tetracyclines are a bacteriostatic class of antibiotics that inhibit protein synthesis by binding to the s ribosomal, therefore, blocking the binding of aminoacyl-trna. for cells grown in tetracyclines, the efflux mechanism of tetb decreases the cytosolic antibiotic concentration allowing for the rate of protein translation to increase. i have inserted a tet(b) expression system into the chromosome of an escherichia coli lab strain and have determined its growth profile under various concentrations of tet, mcn, and dox using a high-throughput -well plate format. the growth rate profiles correlate with tetb pumping rates for each drug. tetb more readily pumps out tet compared with dox and mcn and we observe that cells expressing tetb can grow at higher tet concentrations compared with dox and mcn. the shapes of the growth rate profiles produced in the different drugs give insight into the physicochemical mechanism of tetb. we have built a preliminary mathematical model that can simulate these growth profiles and predict efflux pump physicochemical parameters. we are currently working on understanding how efflux expression effects bacterial growth by testing ribosome binding site (rbs) sequences of varying strengths in our tet(b) expression system. future work is geared toward modeling more complex efflux pumps such as the tripartite pumps which traverse both bacterial membranes and cause multi-drug resistance. collectively, this project aims to build an in vivo system which will allow for the characterization of a variety of efflux pumps without the arduous tasks of protein purification and subsequent reconstitution. ( ) identified a small transmembrane region of both kcne and kcne that are essential for their unique modulation of the kcnq channel. by swapping a triplet motif in the transmembrane region of kcne and kcne , we can flip the primary function of these two proteins. while the key for kcne and kcne 's unique modulating is believed to lie in this triplet motif, the mechanism and structural changes involved in this modulation is not fully understood. by using nmr spectroscopy, biochemical studies, and computational docking, we aim to look at the structural and conformational differences between kcne and the triple mutant kcne substituted with the three essential kcne residues. we have expressed and purified n-labled kcne triple-mutant in sufficient quantities for nmr studies in lmpg detergent micelles and other membrane mimetics, and we have collected d nmr spectra using a trosy-based pulse sequence. partial backbone assignments of kcne triple mutant have been determined by aligning and transfer assignments of the wt kcne previous determined in our lab. with the structure of kcne triple mutant determined, we aim to computationally dock the triple mutant into a model of the full-length kcnq channel in the open and closed state. lastly, we will compare the known structure of kcne docked to a model of kcnq to that of the kcne triple mutant to determine key interactions, significant structural and conformational changes, and how the triple motif region gives rise to its specific structural and functional differences. with this information, we can begin to understand the mechanism of the functional diversity of the kcne family on kcnq potassium channel. biochemical characterization of brassica napus diacylglycerol acyltransferase and its regulatory domain .a) expressed in saccharomyces cerevisiae. purified bnadgat in n-dodecyl-b-d-maltopyranoside (ddm) micelles behaves as dimers, which can associate further to form tetramers. the acyl donor preference of the major dimeric form with sn- , -diolein as acceptor follows the following order: a-linolenoyl-coa > oleoyl-coa palmitoyl-coa > linoleoyl-coa > stearoyl-coa. the first residues of bnac.dg-at .a corresponding to a soluble regulatory region was expressed in escherichia coli and purified. truncation of this soluble domain reveals that the dimeric interface is located within residues - , while the first residues allow formation of tetramers. this n-terminal region was implicated as an allosteric exosite for acyl-coas as revealed by previous lipidex- binding studies. in the current study, circular dichroism spectroscopy and isothermal titration calorimetry were used to probe the binding kinetics and thermodynamics. dgat appears to shift between two oligomerization states, a phenomenon that may be related to regulation of enzyme activity and mediated by the n-terminal domain. alteration of lysine and arginine content as a strategy to modify such an interaction was found to increase the activity of rdrp in vitro. further, deletion of c terminal amino acid residues also resulted in increase in the polymerase activity that was comparable to the full length rdrp-p complex. it was proposed that the conserved c terminal disordered domain of rdrp was responsible for interaction with p and modulation of the activity. in the present study, role of the c terminal disordered domain was further investigated by determining the oligomeric status of the complex and the c terminal deletion mutants of rdrp and also by quantitating the rdrp-p interaction using surface plasmon resonance. size exclusion chromatography revealed that rdrp eluted in the void volume of the column whereas a significant fraction of the rdrp-p complex eluted at a position corresponding to the size of the : complex of rdrp and p ( kda). activity measurements indicated that the heterodimeric complex was more active than the aggregate eluting in the void fraction. interestingly, the c terminal deletion mutants of rdrp (c del & c del rdrp) were also found to be less aggregated as compared to full length rdrp and some of the protein eluted at a position corresponding to the respective monomers. these monomers were also more active than the aggregate fractions. these results demonstrate that the increase in activity observed either upon interaction with p or deletion of the c terminal domain could be due to the change in the oligomeric state of rdrp. in order to further analyze the interaction of rdrp with p surface plasmon resonance was used. rdrp and its deletion mutants were immobilized on biacore sensor surface and p protein was used as an analyte. full length rdrp and c del rdrp were shown to interact with p with kd values of . and um respectively. however, c del and c del rdrp did not show any binding with p . these results suggest that the region - from the c terminus of rdrp is essential for the interaction with p . further, the c del rdrp was inactive although c del rdrp continued to be active suggesting that residues - from the c terminus are crucial for rdrp activity. further studies are in progress to identify the residues within these motifs that may be essential for the activity or interaction with p . aggregation of androgen receptor in spinal bulbar muscular atrophy is a multistep process spinal bulbar muscular atrophy (sbma) is a member of the polyglutamine (polyq) expansion diseases, like huntington disease, and it is caused by a genetic expansion of the polycag tract in exon of androgen receptor (ar) that codes for the polyq region. sbma is a late onset disease, which involves a progressive degeneration of the motor neurons and consequent muscular atrophy. there is still no treatment available for this disease. ar is a nuclear receptor that responds to testosterone and that regulates the expression of the masculine phenotype. it is composed of an intrinsically disordered nterminal domain (ntd) that bears the polyq tract, a dna binding domain and a ligand binding domain. aggregates of ar protein with an extended polyq are observed in the motor neurons of sbma patients. in vitro studies showed that aggregation of androgen receptor takes place only in presence of testosterone and that the cleavage of the protein by caspase is a crucial event for cytotoxicity. however, there is no clear knowledge of the mechanism of aggregation, for this protein. an increasing body of evidence supports the hypothesis that the aggregation of these proteins is controlled by regions flanking the polyq tract, by regulating the rate of aggregation depending on their secondary structure. we have applied nuclear magnetic resonance (nmr) and circular dichroism for generating information on the secondary structure of the n-terminal cleavage product of ar by caspase and we have studied its aggregation with a set of biophysical methods, like dynamic light scattering, an hplc sedimentation assay and transmission electron microscopy. we have found that the polyq tract of ar presents a high degree of helicity. we attribute this conformation to the n-terminal flanking region, characterized by high helicity and we have tested this hypothesis by performing mutations. we have also observed that the rate of the first step of oligomerization is not dependent on the number of glutamine repeats, but instead is due to self interactions of a region n-terminal to and far from the polyq. its progression to fibril is dependent to the number of glutamines in the tract. we have therefore identified two steps in the aggregation process of ar, where a motif far from the polyq at its n-terminal drives the early oligomerization, followed by the interaction of the polyq chains that stabilize it and determine the progression to fibrils. these findings shed a light for possible interventions on the ar oligomerization process, thus suggesting a different strategy to study the onset of the disease in sbma patients. destabilizing the transient helical conformation of islet amyloid polypeptide hastens peptide self-assembly and potentiates cytotoxicity carole anne de carufel , phuong trang nguyen , alexandre arnold , isabelle marcotte , steve bourgault amyloidogenic polypeptides can be divided into two different structural classes: those that are intrinsically disordered and those that show a well-defined structure in their monomeric soluble state. natively folded proteins, such as transthyretin, have to unfold (or misfold), at least partially, to form amyloids. in contrast, intrinsically disordered polypeptides, such as the islet amyloid polypeptides (iapp) and abeta peptide, need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate the amyloidogenic process. studies have shown that iapp and abeta adopt an alphahelix conformation in the initial steps of amyloidogenesis. this intermediate is believed to be on-pathway to fibril formation, although this hypothesis is still the matter of debate. in this study, we designed human iapp (hiapp) derivatives in which alpha-helix destabilizing substitutions were incorporated into the putative helical segment of iapp to probe the initial structural event in amyloid formation. using trifluoroethanol titration, we observed by cd spectroscopy that strategic incorporation of d-amino acids at positions and leads to an iapp derivative (diapp) that cannot fold into a helix. in homogeneous solution, hiapp and diapp show similar kinetics of fibrillization, as measured by thioflavin t fluorescence. although their amyloid fibrils display different characteristics by afm, iapp and diapp are able to self-associate to form amyloids when mixed together and when seeded with one another. studies in heterogeneous environment, notably in presence of glycosaminoglycans and model membranes of dopc/dopg ( : ), showed a helical intermediate for hiapp while only a beta-sheet secondary structure was apparent for diapp. while the rate of amyloid fibril formation was increased for both peptides, diapp was drastically affected by these anionic biomolecules with an absence of lag phase. the incapacity of adopting a transient helical conformation accentuates cell toxicity, supported by the caspase / activation level and the increase in intracellular calcium level. overall, this study indicates that the helical intermediate is offpathway to iapp amyloid formation and offers novel mechanistic insights for the development of molecular identities modulating peptide self-assembly and iapp-induced cytotoxicity. for an organism to survive, its proteins must adopt complex conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. the situation is perilous: many diseases arise from improper folding of just a single protein. to cope, cells employ a repertoire of molecular chaperones and remodeling factors that usher unfolded proteins into active conformations, sequester them, or target them for degradation. yet, not all aggregated proteins are the result of mis-folding. yeast prions are self-templating protein-based mechanisms of inheritance that rely upon chaperones for their propagation. the best studied of these is the prion domain (nm) of sup , which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. using genetic, biochemical, spectroscopic, and solid state nmr techniques, we investigated the structural and dynamic underpinnings of sup amyloids and found that prion strains differ in both their atomic structure as well as their dynamic motions. interestingly, these mobility differences correlate with differences in the interaction with molecular chaperones in vivo. limitations on the specificity and sensitivity of biophysical techniques typically restrict structural investigations to purified systems at concentrations that are orders of magnitude above endogenous levels. therefore, i developed an approach to apply a sensitivity-enhancement technique for nmr, dynamic nuclear polarization (dnp), to investigate interactions between sup and molecular chaperones at endogenous concentrations in their native environments. critically, i found that the cellular environment induced structural changes in a region of sup that is intrinsically disordered in purified samples but known genetically to influence prion propagation from one generation to the next. this approach enables structural and mechanistic investigation of proteins in biologically relevant contexts. genetic instability within regions encoding repetitive proteins as a driver of adaptation stephen fuchs more than ten percent of all eukaryotic proteins contain within them a region of repetitive amino acid sequence. these repetitive domains range from short stretches of a single amino acid to multiple copies of longer, heterogeneous amino acid sequences and generally show lack of defined structure. they play diverse roles in cells including acting as structural proteins, promoting cell-cell interactions, and mediating the assembly of molecular machines. tandem repeat proteins are known to be variable in length within cellular populations although the mechanisms dictating this variability have not been elucidated. here we describe work uncovering specific features within the coding sequences of repetitive proteins that contribute to tandem repeat instability in yeast. furthermore, we demonstrate that cells will expand and/or contract repetitive regions in order to adapt to environmental stresses and describe a role for dna repair proteins in this process. lastly, we demonstrate how these mechanisms are likely conserved in higher eukaryotes, including humans. this study uncovers the molecular basis for an important aspect of natural protein evolution and describes a novel mechanism for adaptation in response to environmental changes. a proline-tryptophan turn in the intrinsically disordered domain of ns a protein is essential for hepatitis hepatitis c virus (hcv) nonstructural protein a (ns a) and its interaction with the human chaperone cyclophilin a (cypa), a peptidyl-prolyl cis-trans isomerase (ppiase), are both targets for highly potent and promising antiviral drugs that are in late stage of clinical development [ , ] . despite its high interest in the development of drugs to counteract the worldwide hcv burden, ns a is still an enigmatic multifunctional protein poorly characterized at the molecular level. ns a is required for hcv rna replication and is involved in viral particles formation and regulation of host pathways. thus far, no enzymatic activity or precise molecular function has been ascribed to ns a that is composed of a highly structured domain (-d ), as well as two intrinsically disordered domains (-d ) and (-d ). ns a-d structure has been solved by x-ray crystallography and ns a-d and -d have been characterized by nmr spectroscopy. these two last domains do not adopt a stable d structure but rather exist as an ensemble of highly dynamic conformers. using nmr spectroscopy, hcv ns a-d has been shown to establish a direct interaction with the human cypa and to be a substrate for the enzymatic ppiase activity of cypa [ ] . the cypa interaction site in ns a-d is composed of nearly residues that correspond to the most conserved region of the domain, with proline residues being strictly conserved among all hcv genotypes. whereas ns a-d is mainly disordered, some of its nmr resonances, corresponding to residues in the cypa binding site, display unexpected h and n nmr chemical shifts for an intrinsically disordered domain. thus we have further characterized this region by nmr spectroscopy. a short structural motif in the disordered ns a-d has been identified and we solved its nmr structure. in a cellular assay, we showed that this structural motif, a minimal pro -trp turn, is essential for hcv rna replication. we demonstrated that this pro-trp (pw) turn is required for proper interaction with the host cypa and influenced its enzymatic ppiase activity on residue p of ns a-d . this work provides a molecular basis for further understanding of the function of the intrinsically disordered domain of hcv ns a protein. in addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. [ ] [ ] . this -residue peptide also shows toxicity towards mammalian cells but at higher concentrations, suggesting its possible usefulness as a treatment for trypanosomiasis. here we present the peptide's relative cytotoxicity for bloodstream and procyclic forms of t. brucei and for mammalian cells, the fate of the peptide in t. brucei using fluorescently-labelled bt- , and its three dimensional structure using nmr spectroscopy.minimum inhibitory assays confirmed the peptide's selective toxicity towards both bloodstream and procyclic forms of t. brucei, demonstrating its potential to serve as a starting point for a trypanocidal drug. fluorescence spectrophotometric experiments, carried out using fluorescein labelled bt- , show that the peptide is released from the external surface of the parasite into the suspending medium under de-energized conditions but retained in energized cells. heteronuclear and homonuclear biomolecular nmr experiments (tocsy, noesy, h- c-hsqc, h- n-hsqc, etc) folowed by structural calculations (chemical-shift based as well as simulated annealing techniques) in the free state indicate that this peptide is mostly unstructured in aqueous solution, suggesting that there is a major conformational change upon binding to t. brucei that is required for uptake. we suggest that the evolutionary pressure that selected for the intrinsically disordered structure of this peptide was the advantage it conferred upon the host to bind to many different surface structures throughout the microbiological world. physikalische biologie, heinrich heine university, structural biochemistry (ics- ), research centre j€ ulich, chemistry and biotechnology, swedish university of agricultural sciences (slu) the misfolding and amyloid formation of proteins featuring intrinsically disordered regions is a pathological hallmark of several neurodegenerative diseases, including alzheimer's disease and parkinson's disease. engineered binding proteins targeting amyloidogenic proteins aid in the elucidation of the aggregation mechanism and suggest therapeutic strategies. we have constructed phage display libraries enriched in binders to amyloidogenic intrinsically disordered proteins, using zab , a protein with high affinity for the amyloid-beta peptide, as a scaffold. binding proteins selected from these libraries are termed beta-wrapins (beta-wrap proteins). the beta-wrapins as and hi exhibit nanomolar affinity for monomeric alpha-synuclein or islet amyloid polypeptide, respectively. as and hi potently inhibit in vitro amyloid formation and toxicity at substoichiometric concentration ratios, indicating that they interfere with the nucleation and/or elongation of amyloid fibrils. the nmr structures of the betawrapin:target complexes reveal beta-hairpin motifs in alpha-synuclein and islet amyloid polypeptide which are stabilized by coupled folding and binding. in the case of alpha-synuclein, the beta-hairpin is formed in the sequence region - which contains the beta-strand segments b and b of amyloid fibril models and most disease-related mutations. we show by disulfide engineering, biophysical techniques, and cell viability assays that intramolecular tertiary interactions between the b and b segments of alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of amyloid-beta peptide and islet amyloid polypeptide. our results reveal a common preference of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation. interaction profiling through proteomic peptide phage display cecilia blikstad , moon-hyeong seo , norman davey , roland arnold , sachdev s sidhu , philip m kim , ylva ivarsson department of chemistry -bmc, donnelly centre a considerable part of the human proteome is intrinsically disordered. the disordered regions are enriched in short motifs serving as docking sites for peptide binding domains. domain-motif interactions are crucial for the wiring of signaling pathways. these interactions are typically transient and difficult to capture through most conventional high-throughput methods. we therefore developed a novel approach for the large-scale profiling of domain-motifs interactions called proteomic peptide phage display (prop-pd) ( ). in prop-pd we combine bioinformatics, oligonucleotide arrays, peptide phage display and next-generation sequencing. this allows the interrogation of domain-motif interactions on a proteome-wide scale and the de novo motif discovery.in our pilot experiment we generated two distinct phage libraries, one displaying all human c-terminal sequences and one displaying c-termini of known virus proteins. we used the prop-pd libraries to identify interactions of human postsynaptic density /discs large/zonula occludens- (pdz) domains. we successfully identified novel pdz domain interactions of potential relevance to cellular signaling pathways and validated a subset of interactions with a high success rate. recently, we created a prop-pd library that displays peptides representing the disordered regions of the human proteome. we validate our disorderome library against a range of peptide binding domains, which provides novel insights into their binding preferences and suggest interactions of potential biological relevance as will be presented here. prop-pd can be used to uncover protein-protein interactions of potential biological relevance in high-throughput experiments and provides information that is complementary to other methods. prop-pd is scalable and can be developed to any target proteome of interest. phosducin is a kda phosphoprotein that regulates visual signal transduction by interacting with the gtbg; subunit of the retinal g-protein transducin. the function of pdc is regulated by phosphorylation at ser and ser in a process that involves the binding of phosphorylated pdc to the regulatory - - protein, but the molecular mechanism of the regulation by - - protein is still unknown. pdc was also suggested to be involved in transcriptional control, the regulation of transmission at the photoreceptorto-on-bipolar cell synapse, and the regulation of the sympathetic activity and blood pressure [ ] [ ] [ ] . here, the solution structure of pdc and its interaction with the - - protein were investigated using small angle x-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. we show that the - - protein interacts with and sterically occludes both the n-and c-terminal gtbg binding interfaces of phosphorylated pdc, thus providing a mechanistic explanation for the - - depedent inhibition of pdc function. the - - protein dimer interacts with pdc using surfaces both inside and outside its central channel. the n-terminal domain of pdc, where both phosphorylation sites and the - - binding motifs are located, is intrinsically disordered protein which remains likely highly flexible when bound to - - indicating the fuzzy-like character of this complex. in addition, it has been speculated that the - - protein binding decreases the rate of pdc dephosphorylation after a light stimulus through its interaction with phosphorylated ser and ser , thus lengthening the time that pdc remains phosphorylated after a light exposure. pdc is dephosphorylated in vivo by protein phosphatases known to cause neurodegenerative disease in a polyglutamine-length dependent manner. despite intense study, the molecular basis of polyq toxicity in hd or any of the other diseases has only partially been elucidated and potential routes to therapeutic intervention are sparse. the use of genetically tractable model organisms to identify the cellular pathologies caused by mutant huntingtin expression is essential to our understanding of the disease pathology in humans. in eukaryotes, many of the protein folding homeostasis pathways are highly conserved and yeast cells expressing a glutamine-expanded fragment of huntingtin exon exhibit a polyq length-dependent toxicity that recapitulates many of the basic protein folding defects associated with polyq diseases in neurons. taking an unbiased approach, we screened an overexpression library of the entire yeast genome for suppressors and enhancers of polyq toxicity and identified seven proteins with prion-like, q-rich domains that are strong suppressors in yeast. intriguingly, the q-rich domains of these proteins, and several other q-rich domains, suppress toxicity when expressed in isolation. these suppressors are also efficacious in mammalian cells and, strikingly, one suppressor was independently shown to alleviate polyq-expanded ataxin- toxicity in a drosophila model. in yeast, the suppressors co-aggregated with an otherwise highly toxic glutamine expanded huntingtin exon protein (htt q), resulting in a non-toxic aggregate and eliminating populations of diffusible oligomeric species. using a transcriptional sensor for protein coaggregation, we determined that yeast and human proteins that normally co-aggregated with htt q did not co-aggregate with these hetero-aggregates. thus, these q-rich domains may suppress htt q toxicity by two complementary mechanisms: trapping potentially toxic oligomers in larger aggregates and by limiting the interactome of the larger htt q aggregates. structuring disorder: the case of the intrinsically disordered unique domain of c-src mariano maffei about two thirds of eukaryotic proteins contain large intrinsically disordered regions. they represent a change of paradigm from "structure-function" to "information-function" (uversky, ; babu et al., ). structured proteins are information rich, but the current challenge is to discover how information is stored in disordered protein. regulation of c-src activity, the first discovered oncoprotein, by its intrinsically disordered n-terminal region has been recently demonstrated (perez et al., ). functional studies have revealed that mutations in the ulbr cause strong phenotypes when introduced in fulllength c-src and expressed in xenopus laevis oocytes (perez et al., ) or in human sw colorectal cancer cells (unpublished). however, the connection with the classical regulatory mechanisms is still missing. c-src domain structure consists of four "src-homology" domains: sh , sh , sh and sh , arranged in this order from the n-terminus to the c-terminus, with the intrinsically disordered "unique" domain separating the sh and sh domains. classically, the sh and sh domains are involved in regulation and the sh domain is the membrane anchoring site. we will present our recent results showing that the unique domain is part of a long loop closed by the interaction of the sh and sh domains (maffei et al., ). the conformational freedom of this disordered region is further restricted through direct contacts between the rt-loop of the sh domain and, primarily, residues located within the recently discovered unique lipid binding region (ulbr). the interaction between the unique and sh domains is allosterically modulated by a poly-proline ligand binding to the canonical binding site of the sh domain (maffei et al., ) . these results demonstrate a direct connection between classical c-src regulation involving the sh domain and the new regulation mechanisms involving the intrinsically disordered regions and provide new evidence of the functional importance and the underlying mechanism behind regulation of signalling pathways by intrinsically disordered domains. in mammalian cells, the golgi reassembly and stacking proteins (grasp and grasp ) are involved in the stacking of golgi apparatus cisternae and in the formation of the golgi ribbon. since grasps have been identified in many organisms, other roles for grasps have already been pointed out, such as chaperoning and transport of other proteins, involvement in cell apoptosis, cell migration, unconventional secretion, and in mitosis. in saccharomyces cerevisiae, it is observed that only % of the golgi cisternae are in stacks and do not form ribbon structures. this build yeast contains a single grasp, called grh , that is analogue to grasp . the structural differences of the golgi apparatus and the functional repertoire of grasps suggest a structural dynamic of these proteins. here, we used a combination of biophysical/biochemical methods to investigate the behavior of grh . bioinformatics and circular dichroism (cd) analyses of grh indicated a high percentage of either flexible regions or extended loops. the partial unfolded grh structure in solution folded into more ordered structures under temperature increasing, dehydration onto a surface and nonaqueous solvents as reported also by cd. hydration of the dehydrated folded protein is a reversible process that is accompanied by unfolding. furthermore, grh showed slow migration in sds-page, high susceptibility to proteases and low cooperativity of the chemical-induced unfolding process. fluorescence of trp residues along with cd data showed grh preserves a considerable amount of residual secondary structure, and the unfolding transition monitored by trp presented higher cooperativity. another cooperative transition was also reported by the extrinsic hydrophobic fluorescence probe ans upon chemical denaturation. these set of experiments indicate that grh behaves as a protein containing intrinsically disordered regions (idrs), characterized by unstructured regions of high polypeptide mobility experiencing many conformations. these findings suggest that an idp-like behavior may be the solution found by nature to account for grh functional need for interactions with several different partners in the cell. conformational changes governing dengue virus capsid protein function and its inhibition by pep andr e f. abstract dengue virus (denv) infection affects millions of people and is becoming a major global disease for which there is no specific treatment available. the interaction of denv capsid (c) protein with host lipid droplets (lds) is essential for viral replication. pep - , a peptide designed based on a denv c intrinsically disordered conserved region, inhibits this crucial interaction. combining bioinformatics and biophysics we determined pep - structure and ability to bind different phospholipids, in the context of denv c function. pep - becomes a-helical upon binding to anionic phospholipids. structure prediction of denv c n-terminal intrinsically disordered region reveals orientations that alternatively shield or expose denv c hydrophobic pocket, supporting a novel autoinhibitory role for this region. these findings pave the way for similar studies to understand disordered proteins and improved peptidomimetics drug development strategies against flaviviruses. topics intrinsically disordered proteins protein-lipid interactions pf- developing mechanistic insight into modulators of tau aggregation eri nakatani-webster , hannah baughman , shaylin higgins , abhinav nath the pathological self-association of microtubule-associated protein tau is implicated in a range of neurodegenerative disorders collectively called tauopathies, perhaps the most prominent of which are alzheimer's disease (ad) and chronic traumatic encephalopathy (cte). tau aggregation in vitro shares many features in common with fibril formation by other amyloid-forming proteins: a nucleationdependent polymerization reaction progressing via oligomeric intermediates into b-sheet-rich fibrillar aggregates, characterized by a distinctive sigmoidal kinetic. over the years, many investigators have advanced our understanding of how these time-courses might best be characterized and interpreted. in particular, elegant analytical and numerical approaches have been developed that supersede the empirical sigmoidal equations typically used to fit fibril formation traces. these modern approaches have enabled more rigorous insight into the mechanism of amyloid formation, and into how small molecules, protein chaperones, and other binding partners can modulate the process. an understanding of a modulator's effects on amyloid formation mechanism is necessary in order for us to predict and engineer its effects on amyloid pathology in a biological context. a given modulator may affect rates of primary or secondary nucleation, elongation, or fibril fragmentation to different extents. each of these perturbations, individually or in combination, can alter the kinetics of aggregation, the final state of the amyloid fibrils, and the sampled ensemble of oligomeric intermediates. unfortunately, fitting of mechanistic models to amyloid formation kinetics is an example of an "ill-posed problem", in that dramatically different combinations of elementary parameters can nevertheless generate very similar sigmoidal kinetic traces. this has typically necessitated global analysis of amyloid kinetic traces collected over a broad range of protein concentrations -a substantial expenditure of time, effort and material that must then be repeated in the presence of a modulator in order to gain insight into its effects. we propose an alternative approach: to fit amyloid formation traces to a large distribution of parameter sets, and determine how various aggregation modulators affect the distribution of parameters. this socalled "parameter distribution analysis" enables the inference of mechanistic effects from measurements at a single protein concentration. parameter distribution analysis based on numerical modeling has been made tractable by advances in computer hardware and software, and can be easily extended to include additional mechanisms or phases relevant to a protein or modulator of interest. here, we illustrate how parameter distribution analysis, complemented by fluorescence correlation spectroscopy (fcs), electron microscopy (em) and other biochemical techniques, can shed light on fundamental aspects of tau amyloidogenesis. we examine the disparate effects that natural products, pharmacotherapies and protein chaperones can have on the mechanism of aggregation, and also discuss the effects of heparin (widely used as an inducer of tau aggregation). these insights demonstrate the value of parameter distribution analysis as applied to amyloid formation and other ill-posed biochemical problems. new insights into amyloidogenesis of tau protein induced by enantiomers of polyglutamic acid amyloidogenesis of tau protein leads to the formation of amyloid fibrils (ordered fibrillar protein aggregates) which are accumulated in neurons of central nervous system during the course of neurodegenerative diseases called tauopathies. studying tau (a typical intrinsically disordered protein) amyloidogenesis has been challenging for many reasons. positive charge on the tau molecule must be compensated (e.g. in the presence of polyanions) in order to initiate the process. heparin (glycosaminoglycan) has been the most intensively studied charge-compensating agent in this context. on the other hand induction of tau aggregation by polyglutamic acid is poorly characterized. mechanisms responsible for the propagation of tau conformations has become an interesting research objective. prion-like features of tau amyloid can be studied in vitro also in the seed-induced regime of aggregation. tau amyloid seeds can act as nuclei for amyloidogenesis. such seeds can be obtained by fragmentation of amyloid fibrils by means of sonication. given that amyloidogenesis can proceed through various assembly pathways resulting in distinct amyloid 'strains' (self-propagating structural variants of amyloid) we have used poly-l-glutamic acid (plga) and poly-d-glutamic acid (pdga) to direct tau onto different amyloidogenic pathways. we have hypothesized that the chirality of the inducers could lead to fibril polymorphism. in our studies, we have used a recombinant human n r tau isoform. we have been using transmission electron microscopy (tem), sedimentation and kinetic measurment. firstly, we have characterized unseeded plga-/pdga-induced tau aggregation to find out that corresponding kinetics were significantly different. secondly, we have used sonicated fibrils to characterize the kinetics of seeded processes. both plga-/pdga-induced amyloid seeds were able to efficiently seed tau aggregation in the presence of plga, whereas in the presence of pdga the aggregation was much less effective. surprisingly, we found that pdgainduced amyloid seeds were able to catalyze fibrillogenesis of tau more clearly in the presence of soluble plga than in the presence of pdga -the primary inducer. we could not induce aggregation of tau in the absence of polyglutamic acids which indicates that positive charge on tau molecules must be unconditionally compensated in order to promote amyloidogenesis. thirdly, using tem we have characterized different morphologies of tau amyloid fibrils generated in unseeded and seeded processes. finally, to further characterize properties of the fibrils we have performed sedimentation experiments. fibrils induced by plga, pdga and heparin revealed different sedimentation properties. heparin-induced fibrils underwent sedimentation more readily than pdga-induced fibrils, whereas plga-induced fibrils remained in the supernatant. these results indicate distinct physicochemical properties of these fibrils. we believe that our findings will contribute to the current understanding of the molecular dynamics of tau amyloidogenesis. self-organizing structures of alpha-synulceins and its aggregates by a coarse-grained monte carlo simulation ras pandey , peter mirau , barry farmer alpha-synuclein (asn) consisting of residues, an intrinsically disordered protein, is linked to such neurodegenerative diseases as parkinson's disease (pd) and alzheimer disease via toxic clumping into abstract amyloid fibrils. we investigate the structure and dynamics of an asn chain as a function of temperature by a coarse-grained approach where a residue is represented by a node. in our coarse-grained approach, a residue is represented by a node. the basic idea is borrowed from the 'united atom' approach in polymer chain modeling that has been used extensively where the benefits and pitfalls of the method is explored for decades. such coarse-grained method has also been used protein chain modeling in recent years (e.g. aip advances , ( )). although the atomic scale structural resolution is sacrificed its specificity is captured via a set of unique knowledge-based residue-residue interactions matrix (e.g. classic miyazawa-jernigan matrix, macromolecules , ( )). a number of local and global physical quantities are analyzed such as contact map, neighborhood and mobility profiles, mean square displacement of protein, its radius of gyration and the structure factor. based on the mobility profile, we are able to identify three distinct segment of asn along its contour, i.e. sluggish nterminal ( - ) and c-terminal ( - , least mobile) separated by the central region ( - ), the nonamyloid component (nac) with higher mobility. contact profile shows that the probability of intrachain residue aggregation (clumping) is higher in the n-terminal region than the c-terminal with least aggregation in the nac region. we find that the radius of gyration (rg) decays monotonically with the temperature, consistent with the finding of allison et al. (jacs, , ( ) ). from the detail analysis of the structure factor we are able to predict the variation of the spatial mass distribution with the temperature as the residues in asn chain organize and disperse by evaluating its effective dimension d. we find the protein conforms to a globular structure (d ) at the low temperatures and to a random coil (d ) at high temperatures which is consistent with the estimates of uversky et al. (j. biol. chem. , ( )). in addition, we provide the estimates of d ( d ) for the intermediate structures as the protein chain makes a transition from globular to random coil. questions under-investigation includes what are the effects of mutations (e.g. b-and g-synuclein), how does the structure of an isolated asn chain change in presence of many interacting protein chains, and how do they organize over the multiple length scales? attempts will be made to address some of these issues as the data become available. tear down the wall: dismantling the biofilm scaffold of e.coli cesyen cedeno , nani van gerven , wim jonckheere , imke van den broek , han remaut , peter tompa csga is the major subunit of the so-called curli fiber system. this is an amyloid structure formed in the outer membrane on e.coli and acts as a scaffold for the biochemical machinery/matrix in the extracellular milieu (biofilms). extracellular matrices of this nature are robust platforms helping bacteria colonization; in this context csga becomes a key target in order to break the architecture within bacterial biofilms. chaperones are molecular machines able to stabilize misfolding prone proteins or even retrieve proteins trapped in non-physiological states. here we show how erd acts as a molecular chaperone inhibiting the formation of csga amyloid fibers in vitro. this work illustrates an alternative approach towards biofilm treatment at a molecular level. coupled folding and binding of transcription factors sarah shammas , alexandra travis , jane clarke intrinsic protein disorder is ubiquitous in transcription, particularly within transcription factors, which frequently fold into structures upon binding to partner molecules (dna or protein). the coupled folding and binding reactions that take place between individual transcription factors and the key hub co-activator proteins are crucial in determining the expression profile of the cell, and hence its phenotype. these interactions have been well studied by structural and equilibrium methods. here we present mechanistic insights into the process, gained through complementary kinetics experiments, for the binding of five separate transcription factors to a single prototypical co-activator (cbp kix). the transcription factors investigated belong to cellular (cmyb, mll, creb, e a) and viral (htlv- blz) classes. these reactions are remarkably fast; after removing the effect of long-range electrostatic rate enhancement the association rate constant is still approximately x m- s- , which is just above the typically quoted upper limit for diffusion-limited reactions between pairs of proteins ( - m- s- ), and is also the highest such value we have found reported. this, combined with the apparent insensitivity of the association rate to residual structure within the unbound state, indicates that binding preceeds folding (induced fit mechanism). interactions between kix and its transcription factors are additionally modulated by allostery between its two binding sites. we investigate the basis for this, finding it to be mediated by changes in protein flexibility. alternative hit finding strategies for intrinsically disordered proteins, exemplified by forkheadbox transcription factors harm jan (arjan) snijder , maria saline , tomas jacso , frank janssen , mattias rohman , tyrrell norris astrazeneca r&d, discovery sciences, se- ,pepparedsleden forkhead box o (foxo) proteins are emerging as key transcription factors in insulin and glucose metabolism, regulation of immune responses, and to balance cell proliferation, apoptosis and senescence. foxo proteins are predicted to be intrinsically disordered proteins (idps); idps are largely unstructured and often function as hubs mediating multiple interactions. idps are considered to be largely evasive from classical small molecule interference and lead-generation approaches, as they lack defined binding pockets. the available methods for addressing these targets have been lagging behind and needs to be developed to assess tractability of this target class. here we have evaluated the tractability of fragment screening on various domains of a forkhead box o member. we could confirm the intrinsically disordered character of foxo and used nmr screening to identify fragments that interact with foxo. one of these fragments was subsequently confirmed as a direct foxo binder in d hsqc-nmr spectroscopy and this fragment showed an effect in a foxo reporter gene assay. these results demonstrate that fragment screening may be a valuable approach for intrinsically disordered proteins although challenges remain to expand these fragments into more potent hits in the absence of detailed structural data. the characterization of amyloid-beta peptide (abeta) oligomer samples is critical to advance in the field of alzheime rs disease (ad). here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), extensively used in the field, and electrospray ionization ion mobility coupled to mass spectrometry (esi-im-ms), an emerging technique with great potential for oligomer characterization. to evaluate their performance, we first obtained pure cross-linked abeta and abeta oligomers of specific order. analysis of these samples by sds-page revealed that sds affects the oligomerization state of abeta oligomers, thus providing flawed information on their order and distribution. in contrast, esi-im-ms provided accurate information, while also reported on the chemical modifications and on the structure of the oligomers. our findings have important implications as they challenge scientific paradigms in the ad field built upon the sds-page characterization of abeta oligomer samples. coarse-grained simulation of protein association: application to rate prediction and implication for association mechanisms yinghao wu , the kinetics of protein binding is of paramount importance for understanding cellular functions. for instance, the binding kinetics between membrane receptors and their ligands control the speed of signal transduction after cells are exposed to stimulation. the experimentally measured association rates of protein binding span ten orders of magnitude, a range that was divided into two regimes. it was proposed that a fast association regime is limited by protein diffusion, while the other side of the spectrum is controlled by conformational changes. consequently, all previous simulation methods neglected conformational changes when calculating the association rate of a diffusion-limited regime. however, the most updated theory of protein binding suggests that a protein remains in a pre-existing equilibrium of unbound conformations. binding shifts the equilibrium toward its bound state. this highlights the importance of conformational factors for regulating protein binding. enlightened by this conformational selection model, we hypothesize that the conformational flexibility of protein structures regulates association more widely than previously anticipated. we develop a new coarse-grained model to simulate the process of protein association via the kinetic monte carlo (kmc) algorithm. each residue in this model is represented by its ca atom and a side-chain functional site. a simple physically based potential is used to guide the relative diffusion of two interacting proteins. given the size of the simulation box and the length of the simulation, the association rate constant can be derived by counting the frequency of dimerization among a large number of simulation trajectories. we further designed a prediction strategy that accounts for both the conformational and energetic factors of binding. our method is able to predict rates of protein association that are highly correlated with experimentally measured values. due to the coarse-grained feature, our model was further applied to several special cases of protein association. in one example, we studied the binding kinetics of proteins with flexible linkers. the interaction between thrombin and its functional inhibitor, rhodniin, was used as a testing system. we captured the conformational changes of flexible linkers from the all-atom molecular dynamic simulations. we found that the association with full-length flexible rhodniin was faster than its two individual domains and that their dissociation was more difficult, supporting a "flycasting" mechanism in which partial structures of an intrinsic disordered protein (idp) dock to the target first, while the remaining segments undergo conformational searches and sequentially coalesce around the target. in another example, we studied the binding kinetics of membrane receptors from cellular interfaces. the interaction between membrane proteins cd and cd , cell adhesion molecules known to mediate the activation of t cells and natural killer cells, was used as a testing system. the diffusive properties of these proteins on lipid bilayer were captured from all-atom molecular dynamic simulations. we showed that both d and d association rates could be simulated quantitatively with our method. the calculated values were close to the experimental measurements. we also provided detailed analysis of how molecular diffusions and membrane fluctuations affected d association. pf- (un)structure-function relationships on the ureg enzyme in the nickel-dependent urease system barbara zambelli , francesco musiani , stefano ciurli urease is an essential enzyme for many pathogens and soil microorganisms. its activity relies on the presence of nickel in the active site ( ) . the incorporation of this metal ion into the enzyme requires the formation of a supra-molecular chaperone involving four accessory proteins, named ured, uref, ureg and uree. uree is a metallo-chaperone involved in nickel binding and delivery into the enzyme active site. ureg is a gtpase essential for providing energy to the process of nickel site assembly. uref and ured form a complex that regulates the gtpase activity of ureg. the present work focuses on ureg, which exists in solution as an ensemble of inter-converting conformations ( ) . this observation made this protein the firstly discovered natural enzyme with an intrinsically disordered behavior, possibly allowing it to interact with different protein partners, such as uree ( , ) and uref ( ) and cofactors, such as metal ions ( ), in the urease activation network. ureg folding was studied perturbing protein conformation with temperature and denaturants, and investigating its folding response using circular dichroism, nmr and fluorescence ( ). a combination of light scattering, calorimetry, mass spectrometry, and nmr spectroscopy shed light on the effect of metal ion binding onto the conformational equilibrium of ureg ensemble ( ) . the results suggest that metal binding and solution conditions modulate affect the protein-protein interactions and enzymatic activity of ureg. nuclear inclusion protein a-protease (nia-pro) is a protease involved in processing of pepper vein banding virus (pvbv) encoded polyprotein to generate various intermediates and mature proteins at different stages of the viral life-cycle. nia-pro has two domains-n-terminal viral protein genome linked (vpg) and the c-terminal protease domain (pro).vpg belongs to the group of proteins that are intrinsically disordered, but attain stable structures upon interaction with other globular proteins. such proteinprotein interactions have a regulatory role on the function of the interacting partners. previously, the influence of vpg domain on the activity of pro was studied and it was shown that there was a substantial increase in the protease activity upon interaction with vpg (both in cis and in trans). in the present investigation, several deletion mutants of vpg and nia were constructed with a view to delineate the domain of vpg involved in interaction with pro. it was observed that deletion of residues from nterminus of vpg resulted in a decrease in the activity of pro in cis and in trans probably because of the abrogation of interaction between the two domains. interaction studies using spr (surface plasmon resonance) and elisa confirmed that the n-terminal residues of vpg are important for interaction with pro. the n-terminal residues of vpg are a part of the disordered region of vpg and their deletion resulted in the change in the secondary structure of the vpg and its oligomeric state. the ser and trp residues of pro domain were shown to be important both for the interaction of the two domains and for the activity of protease by mutational analysis earlier. these residues were identified to be a part of wc loop (w -c ) which relay the conformational changes to the active site catalytic triad (his , asp and cys ) leading to activation. however, mutations of these residues did not completely abolish the protease activity as well as the interaction with vpg. therefore, in the present study h and h which are observed to interact with trp and c (via non-covalent interactions) were mutated to alanine and the h a and h a mutants showed a drastic reduction in the activity of protease. molecular dynamics simulations of the wild type pro and the mutants revealed that trp -his -his -cys interaction pathway of the wild type pro was disrupted in the mutants and additional residues were involved in the interaction pathway, such alterations in the network of interactions could be responsible for the loss of activity. however, a change in the oligomeric status of these mutants was also observed as compared to the wild-type pro, suggesting that these residues are important for both the structural and functional integrity of pro and its interaction with vpg. thus, these results provide a molecular insight into the vpg-pro interactions and the modulation of their structure and function upon mutation of residues that are part of the interaction interface. transthyretin (ttr) is one of many proteins that are capable of forming amyloid fibrils in vivo. this protein is associated with two distinct amyloidosis: familial cardiac amyloidosis (fca) that causes a restrictive cardiomyopathy and familial amyloid polyneuropathy (fap) that affect peripheral nerves, they are hereditary and caused by mutations in the ttr gene. the non mutated protein can also aggregate in cardiac tissue in advanced age patients. the diagnosis was established at university hospital since due to a collaborative between our group and the center of amyloidosis antônio rodrigues de mello (ceparm). the only mutation found in brazil was v m in patients diagnosed in france. our group discovered new mutation not described in brazil and a novel mutation not described yet a d. the diagnosed patients are registered in transthyretin amyloidosis outcomes survey (thaos). the novel mutation a d causes a severe restrictive cardiomyopathy that is certainly related to a higher profile of aggregation observed for this mutant if compared to others amyloidogenic mutants of ttr. structural predictions using a bioinformatics tool called foldx showed that the insertion of the mutation cause a electrostatic clash that facilitates the dissociation and aggregation of protein. this mutant was purified heterologously and biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the crystallographic structure indicates that this mutant is structurally identical to wild type. biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the thermodynamic stability of a d is lower than the wild type ttr. the aggregation profile showed us that this protein can aggregate in a higher manner and with a fast kinetic to that observed for others amyloidogenic mutants of ttr, forming fibers in two hours of aggregation. heterotetramers of a d and wt are able to aggregate in the same fiber structure. the analysis of interface interaction of this mutant using the pdbsum showed modifications in the profile of hydrogen bonds and non bonded contacts. in addition the oligomers of a d are toxic for primary culture of cardiomyocytes from murine heart. the amyloidogenic profile displayed by this new mutant can be directly correlated with the aggressiveness observed in the disease developed by the identified patient. furthermore the recent consolidation of ttr diagnosis in our university hospital led to the identification of the rare a d variant in a brazilian patient, suggesting that other new, uncharacterized mutants could be identified in the coming years. multiple cellular proteins interact with ledgf/p through a conserved unstructured consensus motif [ ] . the ledgf/p -mll -menin complex was structurally characterized, but only partially [ ] . using nmr spectroscopy, we identified and mapped a novel mll -ledgf/ p interface. colony forming assays in mll -af leukemic cells expressing mll interactiondefective ledgf/p mutants revealed that this additional interface is essential for leukemic transformation. interestingly, the newly defined interface overlaps with the binding site of known ledgf/p interactor, the hiv integrase [ ] . while the pathophysiological interactions of ledgf/p are intensively studied, its physiological role remains unclear. since ledgf/p contributes to hiv integration and leukemic transformation and has become a new therapeutic target for drug development, it is crucial to study its physiological interactions. in addition to hiv in and mll -menin, the ledgf/p integrase binding domain (ibd) also interacts with several other proteins [ , ] . our recent data (manuscript accepted in nat. commun.) revealed structural details of ledgf/p interactions with physiological binding partners. the interaction with the ledgf/p ibd is maintained by an intrinsically disordered ibd-binding motif (ibm) common to all known cellular partners. based on the knowledge of this motif, we identified and validated iws as a novel ledgf/p interaction partner. naturally occurring single mutants, i t, f i, w r and d h of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including liver, spleen and kidney, resulting in familial systemic amyloidosis. these human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type. here we examined the effects of the aberrant mutant lysozymes i t, f i,w r and d h, each of which possesses a point mutation in its molecule, on a cultured human cell line, hek , in which the genes were individually integrated and overexpressed. western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (er) marker. moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in xbp s and grp /bip, which are downstream agents of the ire _ signaling pathway responding to the unfolded protein response (upr) upon er stress.rnai for the mutant lysozymes' expression greatly suppressed the increases of these agents. next, we addressed the interaction between amyloidogenic lysozyme and grp /bip as the former proteins were obtained by immunoprecipitation with the latter protein as well as colocalization of both proteins in the er. lysozyme composes of a-domain rich in helices and b-domain rich in sheet. two helices of a and a in the n-terminal region arrange in parallel and face to face where hydrophobic amino acids at the f, l , l , l , l and l allocate with equal interval there. in the back of dock, there is a core region of amyloid fibril formation, of which the side chain of i is exposed on the protruding. probably, these hydrophobic amino acids might be crucial for lysozyme folding. although mutated lysozymes undergo folding by grp /bip in such environment, the dissociation of the grp from lysozyme by failure of folding is likely inhibited and both proteins remain bound to, resulting in staying to the er. a part of aberrant lysozymes seem to remain bound to grp /bip during folding and insolubilize with aggregation, thus accumulate in the er accompanied with er stress. lysozyme amyloidosis might be caused by long-term accumulation in the endoplasmic reticulum of the abnormal protein. structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation the accumulation of abnormally aggregated proteins within the body is a common feature of several medical disorders, such as alzheimer's disease, parkinson's disease and diabetes mellitus type . while the specific protein found to be the major component of such deposits varies from one disease to another, the formation of the pathological aggregates seems to occur via a common process of misfolding and self-assembly of a normally soluble polypeptide chain into a series of oligomeric intermediates and, ultimately, into insoluble amyloid fibrils that accumulate within specific organs and tissues. increasing evidence indicates that certain oligomeric protein species generated during the self-assembly of specific proteins into ordered fibrillar aggregates can be highly cytotoxic and are likely to be key players in the initiation and spreading of neurodegenerative diseases. however, little detailed structural information is currently available for these oligomeric species due to their often transient nature and, more importantly, because of their variability in terms of size and structure. we report here the isolation and detailed characterization of an ensemble of stable toxic oligomers of alpha-synuclein, the protein whose deposition is the hallmark of parkinson's disease. by defining and minimizing the degree of heterogeneity of these isolated alpha-synuclein oligomers which have accumulated during the process of amyloid formation, we have identified distinct subgroups of oligomers and determined their structural properties and three-dimensional molecular architectures. this characterization has been achieved by the application of a set of complementary biophysical techniques, including a variety of spectroscopic techniques along with analytical ultracentrifugation, atomic force microscopy, and electron microscopy. although these oligomers exist in a range of sizes, with different extents and nature of beta-sheet content and exposed hydrophobicity, all the oligomeric subgroups possess hollow cylindrical architectures with marked similarities to amyloid fibrils. this suggests that these types of oligomers are kinetically trapped during protein self-assembly and that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their beta-sheet structures. our findings reveal the inherent multiplicity of pathways of protein misfolding and the key role the beta-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the rates of structural conversions, and thus the kinetic stabilities and pathological nature of different amyloid oligomers. the results of this study provide the basis for a more complete understanding of the nature of the self-assembly of polypeptides into beta-sheet rich amyloid aggregates, and potentially contributes to efforts to identify specific targets for drug discovery. fish otoliths and mammalian otoconia, biominerals composed of calcium carbonate and organic matrix, are involved in the functioning of the inner ear, the sensory organ that plays an important role in hearing and balance [ ] . however, their developmental origins, growth, and the role of the matrix, especially the protein component, are still poorly understood. it has been shown that proteins involved in the formation of biominerals are usually very acidic. they often belong to the group of intrinsically disordered proteins (idps), a class of proteins devoid of a rigid tertiary structure [ , ] . the shape and polymorph selection of calcium carbonate otolith in danio rerio is controlled by the starmaker (stm) protein [ ] . recently, a gene was identified encoding the starmaker-like (stm-l) protein from oryzias latipes, a putative homologue of stm. it has been suggested that stm-l has a similar function as stm, although there is no sequence similarity between stm and stm-l [ ] . several methods, such as size exclusion chromatography, cd spectroscopy and analytical ultracentrifugation demonstrated that stm-l is an coil-like idp, with the tendency to form locally ordered structures [ ] . because stm-l was suggested to play a crucial role in calcium carbonate mineralization, it is possible that calcium ions may influence its conformation, as was previously shown for stm [ ] . however, other ions may also be involved in this process. the aim of this study was to investigate the effect of mono and divalent metal ions on the conformation of stm-l. we used single molecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs), which have shown that calcium ions compacts the proteins most efficiently, followed by magnesium and the monovalent ions. the difference in the effect of monovalent and divalent ions on the protein dimensions is likely to result from the different properties of the ions, like charge density and radius. cd experiments have shown that a high excess of calcium ions caused the formation of ordered secondary structure in stm-l, which may be crucial for the formation of calcium carbonate crystals, when the ratio of building ions to protein is high. it has been demonstrated that dmp is proteolytically processed into fragments, including k n-terminal region and k c-terminal region. as many proteins characterized to be engaged in biomineralization, dmp and its fragments belong to the group of intrinsically disordered proteins (idps). it has been suggested that dmp and its fragments can take a part in otoconia mineralization, as the protein is present in mouse otoconia, but the role of dmp and its fragments in the mineralization of calcium carbonate has not been examined until now. to determine the influence of the dmp fragments for otoconia development, k dmp protein was expressed in bacterial expression system, purified and used in in vitro biomineralization test of calcium carbonate. in particular, immobilized metal anion affinity chromatography (imac) was applied as a first step of purification procedure. because of high content of acidic amino acids, ion exchange chromatography with a mono q column was used as a next step. the development of insects is regulated by the combined action of ecdysteroids and juvenile hormones (jh). pulses of -hydroxyecdysone ( e) initiate each step of metamorphosis, while jh modulates its action and prevents precocious differentiation. the biological and molecular mechanism of e action is well described. in contrary, the way of the jh activity is still poorly understood. in wilson and fabian [ ] reported that drosophila melanogaster mutants lacking met are resistant to toxic doses of jh and its analogue methoprene. it has been proved, that met binds jh at physiological conditions. therefore met is believed to be a putative jh receptor. met may also be involved in a cross-talk between two hormonal signalling pathways, involving e and jh. the detailed structure of met is still unknown. therefore our main aim is to characterize structural properties of met. in silico analysis performed on a full-length met suggested, that n-terminal part of met contains three conserved domains characteristic for bhlh-pas transcription factors, whereas c-terminal part is most probably unstructured. )). capitalizing on self-and cross-amyloid interactions, we designed highly effective, peptide-based inhibitors of amyloid self-assembly of abeta and iapp. due to their favourable properties the designed peptides are promising leads for targeting protein aggregation in ad, t d or both diseases while the inhibitor design strategy should be applicable to other amyloidogenic polypeptides and proteins as well. apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular organisms to ensure proper tissue homeostasis, embryonic development and immune system activity. the bcl- family of proteins regulates the activation of apoptosis through the mitochondria pathway. dynamic interactions between pro-and anti-apoptotic members of this family keep each other in check until the proper time to commit to apoptosis. the point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (omm). translocation of the pro apoptotic member, bax, from the cytosol to the mitochondria is the molecular signature of this event. molecular interactions and conformational changes associated with this event have been difficult to obtain due to challenges associated with taking subtle measurements in the complex environment of live cells. to circumvent these challenges, we developed a novel method to reliably detect f€ orster resonance energy transfer (fret) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in bax as this translocation occurs in live cells. in the cytosol, our fret measurements indicated that the c-terminal helix is exposed instead of tucked away in the core of the protein. this coincided with measurements using fluorescence correlation spectroscopy (fcs) that showed that cytosolic bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. we propose that this exposed helix allows for this contact to occur. cross-linking the c-terminal helix (a ) to helix a reduced the instances of these interactions while at the same time yielded fret measurements that are consistent with the a helix tucked into the core of the protein. after translocation, our fret measurements showed that bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the bh domain (helix a ) and the c-terminal helix. these findings provide insight into the molecular architecture that may involve possible contacts with other bcl- proteins to permeabilize the omm, which would also be necessary for the regulation of apoptosis. abstract spatial resolution is especially advantageous for bacterial cells because of their small sizes. in the past few years the spatial organization and dynamics of a variety of bacterial cellular structures and protein macromachineries have been revealed with unprecedented details. as the field matures, it is now time to focus on the functional aspect of the observed spatial organizations and dynamics. are they essential in carrying out a specific cellular function? do they play a regulatory role in controlling the on and off of a certain cellular process? in this work i will present a few examples from our laboratory that examine the spatial and functional organization of macromolecules involved in bacterial cell division. transcription factors (tf) exert their function by interacting with other proteins and binding to dna. the nucleus is a compartmentalized space, and the spatial organization of tfs and their partners represents other step of gene expression regulation. we used the glucocorticoid receptor (gr) as a model of tf's mechanism of action. gr is a ligand-activated tf with a relevant role in physiology and a great variety of effects. it can be recruited to specific response elements on dna or interact with other tfs. also, the activity of gr is modulated by different co-regulators, e.g. tif /grip . gr and tif do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. the functional role of this particular intranuclear organization remains unknown. we used advanced fluorescence microscopy techniques to study the dynamics of gr and tif in the nucleus of living cells with high spatial and time resolution. gr and tif fused to fluorescent tags were transiently expressed in newborn hamster kidney (bhk) cells and visualized by a confocal microscope. fluorescence correlation spectroscopy (fcs) experiments were carried on to measure the intranuclear mobility of both proteins. the method is based on the analysis of fluorescence intensity fluctuations due to the movement of fluorescent molecules in and out the confocal volume. the data could be fitted with a model that considers a free diffusion of tif and gr in the nucleus and their binding to fixed targets. we also studied the dynamics of different gr mutants in the presence of different ligands and our results suggest that the binding depends on dna. both gr and tif autocorrelation curves reveal an increase in the bound population upon gr activation by its agonist dexamethasone (dex). a cross-correlation analysis showed that, as expected, dex-stimulus increases the population of gr-tif complexes. without hormone, gr shows a homogeneous distribution and tif forms large clusters in the nucleus. upon dex-binding, gr accumulates in the nucleus, is rapidly recruited to tif foci and there is an important re-distribution of both proteins, that co-localize in the same pattern of small intranuclear clusters. the dynamics of gr and tif molecules at these clusters were studied by performing orbital-scanning measurements, tracking the clusters position in silico and analyzing the intensity fluctuations of the clusters along time. a positive cross-correlation between both channels indicates that dex-bound gr and tif interact at these foci and dissociate from them forming tif -gr hetero-complexes. in conclusion, advanced fluorescence microscopy methods allowed obtaining a dynamical map of gr distribution and function in the nucleus of mammalian living cells. assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial prionoid repa-wh cristina fern andez , rafael n uñez-ramirez , mercedes jimenez , germ an rivas , rafael giraldo amyloid fibril formation is associated with human neurodegenerative diseases. prefibrillar oligomers formed during the fibril assembly process, rather than mature fibrils are known to be central to disease abstract and may be responsible for cell damage. a commonly proposed mechanism for the toxicity of small oligomers is their interaction with the lipid bilayer of cell membranes, leading to loss of membrane integrity [ ] . recent studies from our laboratory have shown that repa-wh , a winged-helix domain from a bacterial plasmid replication protein, can assemble into amyloid fibrils in vitro. when expressed in escherichia coli repa-wh functions as a cytotoxic protein that shares features with the mammalian amyloid proteinopathies. these features have proved repa-wh to be a suitable synthetic model system to study protein amyloidosis [ , , ] . in this work, using the repa-wh bacterial model system, we have studied the interaction between the protein and model membranes (large and giant unilamellar lipid vesicles, luvs, and guvs respectively). repa-wh shows association and aggregation to membranes composed of anionic phospholipids. protein association in guvs did not result in lysis of the vesicles, suggesting the assembly of discrete protein pores as the mechanism for repa-wh membrane damage. to investigate the formation of pores we analyzed by electron microscopy the aggregation of repa-wh in the presence of a pre-formed e. coli lipid monolayer. the em images show the presence of pore-like particles on the monolayer. amyloid pores formation explains the permeabilization effect of repa-wh in vesicle models and is in agreement with observations for human amyloidogenic proteins. the approaches presented here provide a deeper insight into amyloid cytotoxicity towards membranes and will make possible the assay of inhibitors and effectors of amyloidosis under controlled conditions. references: b -adrenergic receptor (b ar) is a member of g protein-coupled receptors, which represent the single largest family of cell surface receptors involved in signal transduction. b ar recognizes a variety of ligands and communicates with cytoplasmic g-proteins by transmitting signals through the cellular membrane. thus, investigation of communication pathways for b ar may give important insights for understanding its allosteric mechanisms and identifying new target sites for more specific and efficient drug molecules to be used in the treatment of pulmonary and cardiovascular disease. in this study, various conformations from ms molecular dynamics (md) simulations and available crystal structures of human b ar were investigated to reveal alternative signaling pathways between its extra and intracellular regions. specifically, shortest communication paths connecting key residues (more than Å apart) at the orthosteric ligand binding site (d , s , t , f , n ) to either l or s located near the g-protein binding site were investigated. the conformers from previous md simulations [ ] include the intracellular loop (icl ), which especially affects the transmembrane collective dynamics but is lacking in x-ray structures. the protein was described as a graph composed of nodes linked by edges. nodes were placed at the alpha-carbon atoms and the edges were calculated based on the number of atom-atom interactions within a cut-off distance . Å for each residue pair. twenty shortest pathways were revealed using k-shortest path algorithm [ ] on the coarse-grained network. our results indicated that distinct signaling paths progressed most frequently on tm but alternative paths were also present, which passed partially through tm , tm , tm or tm depending on the conformation. among the critical residues that transmitted the signal between distant sites, f and n were detected, whose functional roles were reported in previous experimental studies. pathway shifting was observed depending on the open-to-closed transition of icl during md simulations. the sulfonylurea receptor (sur ) is an atp binding cassette (abc) protein that forms the regulatory subunit in katp channels found in the pancreas and the brain. mgatp binding and hydrolysis at the two cytosolic nucleotide binding domains (nbd and nbd ) in sur control gating of the katp channel pore. , proper regulation of katp channel gating by sur is critical. over mutations that lead to diabetes, hyperinsulinism and developmental delay have been identified in different domains of sur , including the nbds. therefore, molecular-level understanding of the structure and function of the nbds is essential for designing improved treatments for sur-related diseases. here we present biophysical and biochemical studies aimed at understanding the effect of disease-causing mutations on the conformation and nucleotide binding of sur nbd . specifically, we are investigating sur nbd mutations that cause neonatal diabetes (r w and h t) or congenital hyperinsulinism (c d, g v, r g, r d and k t). our nuclear magnetic resonance (nmr) data shows that the hyperinsulinism mutation k t causes chemical shift changes throughout the spectrum of nbd , implying overall changes in protein conformation that may affect mgatp binding and inter-domain interactions with other domains in the sur protein. size-exclusion data show that the other hyperinsulinism mutations (c d, g v, r g, r d) produce mostly aggregated protein, likely as a result of misfolding of nbd . misfolding of nbd may be the underlying cause of reduced katp trafficking seen with these mutations and hence decreased katp channel gating observed in hyperinsulinism. in contrast to the k t mutations, the congenital diabetes-causing mutations (r w and h t) cause few nbd nmr spectral changes. however, the congenital diabetes mutation r w decreases the affinity of nbd for mgatp, which is unexpected for congenital diabetes mutations. our fluorescence, circular dichroism and microscale thermophoresis data corroborate the results that we have obtained by nmr spectroscopy. our data provide molecular-level details on the effects of disease causing mutations in human sur . egfr increased stability: rmsf of the ca atoms during the md simulations suggest that glycosylation is associated with dampened motions, suggesting that the glycans stabilize the structure. subdomain iii is the most stabilized while subdomain i is stabilized largely in the proximity of the ligand. both dimer interfaces including the dimerization arm from domain ii and the tip of domain iv fluctuate less upon glycosylation. hydrogen bonding; persistent interactions seen for protein-glycan: in the disaccharide-containing system, we observed three highly occupied hydrogen bonds between the glycans and domain iii and iv of egfr. hydrogen bonds of domain iii involve the residue asp in which a sidechain oxygen interacts with oxygen atoms of the n-acetylglucoseamine linked to asn . in domain iv a hydrogen bond is seen between the cys backbone amide and the oxygen atom of n-acetylglucosamine linked to asn . in the oligosaccharide-containing system hydrogen bonds observed between the glycan attached to asn and domain ii. these hydrogen bonds form between the gln sidechain oxygen atom and cys backbone oxygen atom and the mannose linked to asn . the reduction in the mobility of these amino acids suggests that hydrogen bonds impart stability to both the sugars and to the interacting egfr. insects possess a complement-like immune response utilizing thioester-containing proteins, or teps. the only arthropod tep of known structure is anopheles gambiae tep , which is a key component in the natural immunity of this mosquito to malaria parasites (genus plasmodium). unlike vertebrate complement factors, agtep does not contain an anaphylatoxin domain which acts to regulate a massive conformational change accompanying activation of the protein. the mechanism of agtep must therefore involve an alternative mechanism for allosteric regulation of thioester activation. in place of a small internal domain, a large, heterodimeric complex of two leucine-rich repeat (lrr) proteins, lrim and apl c, have been shown to specifically bind and stabilize the active conformation of agtep . i will present my group's most recent work in this area. we have shown that different alleles of tep , which are known to influence the vectoral capacity of wild mosquitoes, differ significantly in their susceptibility to thioester hydrolysis. allelic variation is centered on residues at the protein-protein interface within tep containing the thioester bond. the lrim /apl c heterodimer is shown to form an extended and flexible ensemble in solution. two closely-related genes to apl c, apl a and apl b, can also form a complex with lrim , and apl b lrr domain can form a homodimer. we propose that a flexible and heterogeneous group ensemble of lrim /apl dimers interact with the active conformation of tep , thereby producing an array of immune complexes to protect mosquitoes from a diverse set of pathogens. human flap endonuclease- (hfen ) is an essential metallo-nuclease involved in okazaki fragment maturation and long-patch base excision repair. during these processes, bifurcated nucleic acid intermediates with ssdna '-flaps are generated by polymerase strand displacement synthesis and then cleaved one nucleotide into the downstream duplex by fen to create a nicked-dna that is a suitable substrate for ligase. until recently, how hfen achieves tremendous catalytic power (rate enhancements > exp ) and exquisite selectivity for the scissile phosphate had been understood poorly ( ) . in , the grasby and tainer labs solved the structures of hfen in complex with product and substrate. this study revealed that scissile phosphate selectivity is largely due to the substrate dna undergoing a novel di-nucleotide unpairing (dnu), which places the scissile phosphate diester in contact with the requisite divalent metal ions. in addition, by comparing the structures of hfen alone ( ) and in complex with substrate and product dnas ( ), grasby and tainer proposed a model, whereby protein conformational changes occur upon binding substrate resulting in placement of key basic residues that position and/or electrophillically catalyse hydrolysis of the scissile phosphate diester. further work using a cd-based assay showed that metals are absolutely required for dnu, whereas the key basic residues in the active site are not. surprisingly, perturbations to the protein structure that are much more distant from the fen active site (i.e., helical cap) prevent dna unpairing, implying that the fen protein actively participates in the unpairing process ( , ); however, how it does remains a mystery. the maximal multiple turnover rate of hfen reaction is rate-limited by enzyme product release, whereas hfen kinetics under substrate-limiting conditions ([e]<[s]<km) suggest that the enzyme is approaching catalytic perfection (i.e., almost diffusion controlled - exp m- s- ) ( ) . previous work has shown that a paralogue of hfen (i.e., t fen ) is not ratelimited by chemistry under single turnover (st) conditions, but rather some physical limitation such as conformational change ( ) . to ascertain whether the protein and/or dna conformational changes of hfen mentioned above are rate limiting under st conditions, we have initiated kinetic and nmr studies to determine the role of conformational dynamics in the catalytic cycle of hfen . owing to the latest advance in the computational technologies of microprocessor, high-speed inter-processor connection, and parallelization algorithm, all-atom molecular dynamics (md) simulations of microsecond time scale are getting popular to study the pharmaceutical target proteins. an antibody binds to its antigen with structure changes. a small molecule moves around the target protein and finds an entrance to get into the binding site. these phenomena can be observed in microsecond simulations, but the vital issue is whether the adopted force field is enough accurate to correctly describe the phenomena. we are developing a force field (fuji) based on general amber atom types and amber van der waals parameters in order to describe arbitrary organic molecules in a unified manner including proteins and nucleic acids. we use the first principle theoretical method to refine the force field in contrast to common empirical fittings to experimental data. the dihedral torsion parameters of protein backbone were determined to agree with the torsion energy profiles calculated by high-level quantum mechanical theory for the model systems of protein backbone. conformational preferences of dipeptides in water were measured by vibrational spectroscopy. comparing with the experimental distribution of ramachandran angles of dipeptides, how accurately molecular calculations predict the conformation distribution of dipeptides were investigated for various force fields and semiempirical quantum methods. fuji force field gave the best prediction score among the various methods (tzanov et al, ) . in this work we examine dynamical structure behaviors of pharmaceutical target proteins by microsecond md simulations with fuji force field. epiregulin (epr) is a transmembrane protein with residues belonging to the epidermal growth factor family. mature epr ( residues) binds to epidermal growth factor receptor (egfr), which stimulates the proliferative signaling in cancer cells. our epr antibody has a proline at the residue in the third complementarity-determining region (cdr) of the heavy chain, which has cis peptide bond in free state and trans one in the bound state with epr according to x-ray crystallography analysis. in order to clarify the structure changes in binding we performed extensive md simulations of our antibody (fab; residues) and epr. the total simulation time was about microseconds. we also performed md simulations for a protein kinase and a small molecule. we show how the complex system reaches thermal equilibrium states in simulations from the stiff x-ray crystal structure. the calculations are compared with experiments such as x-ray crystal structures and thermodynamic quantities measured by isothermal titration calorimetry (itc) and surface plasmon resonance (spr). this research has been supported by mext spire supercomputational life science (hp , hp ) and first kodama project in japan. structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes our understanding of the contribution of protein dynamics to function is still emergent. in a protein engineering context, do we need to take into account the dynamics in order to maximize the fitness and function of the resulting proteins? using high resolution crystal structures, nmr relaxation dispersion and ms molecular dynamics simulations, we compare two naturally evolved homologous class a b-lactamases, tem- and pse- which share a high degree of structural and functional conservation. we observed a conservation of dynamics on a catalytically relevant timescale. this is consistent with dynamics being an evolutionarily conserved feature. however, laboratory-engineered chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic differences, despite the function and structure being conserved. the laboratory-engineered chimeras are thus functionally and structurally tolerant to modified dynamics on the timescale of the catalytic turnover. this tolerance of blactamases to dynamic changes could be linked to the high fitness of the naturally evolved proteins and implies that maintenance of native-like protein dynamics may not be essential when engineering functional proteins. conformations solution. an immense advantage of our recombinant system is the possibility to perturb it by introducing unnatural amino acids for subsequent labelling with fluorescent dyes or to omit subunits from the multisubunit rnap. comparing free, nucleic acid-and factor-bound rnap complexes we found that the position of the clamp is modulated during the transcription cycle in a fashion exceeding the simplified closed-open picture drawn from crystallographic studies. we show that the clamp adopts at least two distinct conformations in both initiation and elongation complexes. moreover, we were able to link conformational states to the catalytic activity of the rnap. transcription factors tfe and spt / , which both bind to the rnap clamp domain and stimulate the activity of the enzyme, shift the equilibria towards one of the main conformations suggesting that both prompt an allosteric switch that influences the structure of the rnap. hence, our data provide a mechanistic rationale for the function of these factors and provide new insights into the complex dynamic behaviour of the transcriptional machinery. solvent models for protein simulations -the good, the bad and the applications duy hua , amitava roy , he huang , carol post the solvent environment plays a crucial role in determining the structure, dynamics and function of a biomolecule. in order to utilize molecular dynamics (md) simulations to examine the structural changes abstract of biomolecules, it is imperative that the solvent environment be accurately modeled. implicit solvent models (isms), in which water molecules are absent and the solvent effect is estimated using an energy function, offer a low-cost approach to describe the solvent environment in md simulations. isms have been used for a variety of biological studies; yet, the performance of implicit solvation methods in capturing the structural characteristics of proteins has not been rigorously demonstrated. in this work, three isms, namely gbmv ii, facts, and scpism, were evaluated for their abilities to emulate the solvent environment and its effect on the structures, dynamics and electrostatic interac- thioredoxins (trx) are ubiquitous proteins that play a key role in redox state regulation of the cell. they interact with multiple targets in the cell. our group solved the structure of s. cerevisiae (ytrx ) and measured its dynamics in different timescales (pico to seconds). it is well established that the asp is the proton acceptor in the reduction process of the target protein. we proposed that asp also works to couple hydration and motion of the interacting loops. we studied conformational motions of the residues of water cavity and interacting loops. the water molecules in water cavity of the protein play a vital role in the redox activity of thioredoxin. the degree of mutational frustration explains in a great deal the multiple timescales observed in the protein dynamics in ytrx and the mutant d a. the ytrx displays a complex equilibrium between the ground state and several excited hydration states. this excited states are more permeable to water and it is key to understand the proton transfer and activation of cys . in this current study, we examined the mutant d a, and observed that this small hydrophobic residue induces conformational exchange in residues exposed to the water cavity, such as cys . along with molecular dynamics simulations and calculation of mutational frustration levels, we were able to describe conformational details of the water cavity. it is formed by three independent contiguous lobes which we called lobes a, b and c. lobe b is the central lobe that contains the catalytic important residues cys and asp . it closed upon mutation d a. lobe a and c remains open upon mutation, meaning that their hydration are independent. the nmr structures for the oxidized and reduced state of the mutant d a has been studied. in-vitro and in-silico studies of ligand binding to the nuclear receptor ppargamma using fret and md narutoshi kamiya , gert-jan bekker , takuma shiraki , haruki nakamura ipr, osaka university, kinki university the nuclear receptor ppargamma is a ligand-dependent transcription factor which regulates gene expression related to glucose homeostasis and insulin sensitization. it is a potential therapeutic target for metabolic syndrome, cancer, and inflammatory diseases. crystal structures have revealed that its ligands bind deep inside the pocket. however, how the ligand recognizes ppargamma and penetrates inside the pocket remains unclear. we have measured the binding kinetics using the fret method and have calculated potential dissociation paths via molecular dynamics (md) simulations. the binding kinetics were measured using fret between the ligand, c -bodipy, which is a fluorescent pigment structurally similar to one of its agonists -oxo-eicosatetraenoic acid, and cyan fluorescent protein (cfp) which is fused to ppargamma. fluorescence of c -bodipy is observed when it is close to cfp when cfp is excited by light at nm. the ligand was simultaneously added to the ppargamma-cfp solution using the stopped-flow method. time-resolved fluorescence signals of c -bodipy and cfp were also monitored. two time constants were observed by the fret experiment, which correspond with the landing and docking processes. we used our new md program, psygene-g [ ] , which utilizes the gpu for acceleration of the non-bonded interactions, such as electrostatic interaction which uses our original zero-dipole summation method [ , ] . previously we were able to attain similar thermodynamics with respect to the particle mesh ewald method in membrane proteins and dna-water-ion systems [ , ] and have applied it to the thermodynamic study of dynein [ ] . we have introduced random accelerated md (ramd) into the psygene-g program to predict a ligand's dissociation path from the initial crystal structure (pdb id: zk ). in ramd, an external random-directional constant force is added to the ligand. we executed -ramd simulations with different random seeds for the initial pull directions. twelve ramd trajectories managed to dissociate within ns, while the remaining simulations did not manage to disassociate within this time-frame. in all twelve cases the ligand went past r , which is located at the surface of ppargamma. interestingly, a somatic mutant (r h) is related to colon cancer. we concluded that r plays a role of a gate keeper to assist ligand binding. sumoylation is a post-translational modification involved in various cellular processes, such as nuclearcytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. sumoylation is a conjugation of glysine residue of sumo protein with lysine residue in target(rangap ) protein through the influence of enzyme e (ubc ) and e (ranbp ). sumo proteins modify the function of target protein in the cell. but the kinetic mechanism of sumoylation is not well-understand. we traced out the full kinetic process of sumoylation of the sumo -ubc -rangap -ranbp complex in atomic detail via molecular dynamic simulation. we uncovered the kinetics of sumoylation mechanism and verified the important residues which play a key role in the sumoylation process. the comparison of our results with the experimental one are also discussed. human potassium channel kcnq is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. to tune its output current, kcnq complexes with several accessory proteins from the kcne family, each kcne family member modulating kcnq distinctly: kcne causes the channel to delay opening and become more conductive in the open state, kcne causes the channel to be constitutively conductive, and kcne closes the channel. the purpose of this study is to uncover the atomic-scale, mechanical mechanism of how kcne modulates kcnq . to do this we modeled the interaction between kcnq and kcne with a hybrid experimental-computational approach. our strategy is to determine the nmr structure of kcne alone in bilayer-mimicking bicelles, build a homology model of the open-state kcnq , and dock the structure of kcne onto the model of kcnq using electrophysiology-based restraints to validate and refine the resulting model complexes. paramagnetic relaxation enhancement, residual dipolar coupling and sequence analysis suggest the single span transmembrane helix of kcne is significantly curved. hydrogen-deuterium exchange, nuclear overhauser effect cross peaks, and amber simulation suggest kcne carries a significant amount of water in the c-terminal side of the transmembrane helix. the h- n two dimensional nuclear magnetic resonance spectra of a known phenotype-changing mutation in the center of the transmembrane helix, v t, suggests the helix adopts multiple conformations when the extra side-chain hydroxyl group is present. a docking funnel corroborates a binding pocket suggested by the in vivo electrophysiology data where kcne wedges between a helix near the potassium pore and two helices in the voltage sensitive element of the channel. within this pocket, we believe the conformational sampling and structural rigidity-or flexibility, in the context of the v t mutant-of the accessory protein kcne directly influence the modulation profile. watching conformational changes in proteins by molecular dynamics simulations kresten lindorff-larsen proteins are dynamical molecules and their ability to adopt alternative conformations is central to their biological function. examples include motions that underlie allosteric regulation or ligand binding, or protein dynamics in enzymes that can modulate the overall catalytic efficiency. protein motions can often be described as an exchange between a dominant, ground state structure and one or more minor states. the structural and biophysical properties of these transiently and sparsely populated states are, however, difficult to study, and an atomic-level description of those states is challenging. in an attempt to determine how well molecular dynamics simulations can capture slow, conformational changes in protein molecules we have studied two different protein systems which are known to undergo conformational exchange on the millisecond timescale, and for which structural information is available for both major and minor states. using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from x-ray crystallography and nmr, we show that current force fields and sampling methods allow us to sample experimentally-determined alternative conformations with surprisingly high accuracy. in particular, we find that we can reversible sample both the ground state and minor state, and that the simulations capture the structure of the minor states also. our simulations enable us to calculate the conformational free energy between the two states, and comparison with relaxation dispersion nmr experiments demonstrates a high accuracy. thus, we show for two distinct proteins that we can map the free energy landscapes and that both the structure and energetics are in excellent agreement with nmr experiments. our simulations provide insight into the structural and biophysical properties of transiently populated minor states, and help reinterpret previous experimental measurements. further, our results demonstrate that, at least in the two cases we have studied, modern simulation methods enable us to examine these otherwise "invisible" states of proteins and describe their structural, functional and thermodynamic properties. our results in this way help demonstrate how simulations can now add to our knowledge of transiently formed, "hidden" states of proteins. cell signaling is a fundamental process for all living organisms. g protein-coupled receptors (gpcrs) are a large and diverse group of transmembrane receptors which convert extracellular signals into intracellular responses primarily via coupling to heterotrimeric g proteins. in order to integrate the range of very diverse extracellular signals into a message the cell can recognize and respond to, conformational changes occur that rewire the interactions between the receptor and heterotrimer in a specific and coordinated manner. by interrogating the energetics of these interactions within the individual proteins and across protein-protein interfaces, a communication network between amino acids involved in conformational changes for signaling, is created. to construct this mapping of pairwise interaction energies in silico, we analyzed rhodopsin gpcr coupled to a gai b g heterotrimer. the structure of this g protein complex was modeled in the receptor-bound and unbound heterotrimeric states as well as the activated, monomeric ga(gtp) state. from these tertiary structural models, we computed the average pairwise residue-residue interactions and interface energies across ten models of each state using the rosetta modeling software suite. here we disseminate a comprehensive analysis of all critical interactions and create intra-protein network communication maps. these networks represent nodes of interaction necessary for g protein activation. structure and dynamics of the polymyxin-resistance-associated response regulator pmra in complex with the promoter dna yuan-chao lou , yi-fen kao , tsi-hsuan weng , yi-chuan li , chwan-deng hsiao , chinpan chen institute of biomedical sciences, academia sinica, institute of molecular biology, academia sinica pmra, an ompr/phob family response regulator (rr), takes part in the two-component system that manages genes for polymyxin resistance through a phosphorylation-dependent regulation. phosphorylation of ompr/phob rr induces the formation of a two-fold symmetric dimer in the n-terminal receive domain (rec), promoting c-terminal dna-binding domains (dbds) to recognize tandem repeat dna sequences on the promoter to elicit adaptive responses. recently, narayanan et al. presented the complex structure of active-like kdpe in complex with dna, which reveals a unique asymmetric rec-dbd interface that is necessary to form stable complexes for transcription activation. in this study, we report the . Å resolution crystal structure of bef -activated pmra in complex with promoter dna as well as its dynamics in solution by nmr. unlike the case of kdpe, pmra-dna complex structure reveals a different asymmetric rec-dbd interaction. interestingly, nmr assignments and dynamics study suggest that rec and dbd do not form stable contacts in solution; instead, domains tumble separately in the absence or presence of dna. relaxation dispersion experiments on methyl groups further show that several rec-dbd interfacial residues exhibit similar slow dynamics in the presence of dna. it is hence highly possible that the slow dynamics observed on these interfacial methyl groups are related to the formation of asymmetric rec-dbd interaction, which reduces the mobility of pmra-dna complex and promotes crystallization. in solution, two domains tumble independently and have diverse orientations, which together with the dbd-dbd interface may facilitate searching best interactions with rna polymerase holoenzyme for transcription activation. time-resolved x-ray observations of nano-scale protein assembly networks yufuku matsushita , hiroshi sekiguchi , noboru ohta , keigo ikezaki , yuji goto , yuji sasaki , the university of tokyo, graduate school of frontier science, advanced materials science, spring- , osaka university, institute for protein research protein aggregation and network formation in diverse protein species had been studying in various experimental approaches such as absorbance spectrophotometry, dynamical light scattering and x-ray scattering. however, these conventional methods are only able to observe averaged information on bulk conditions and also these techniques have a limitation of time resolved observations. recently, development of single molecular observation techniques are remarkable. diffracted x-ray tracking (dxt) is one of the notable single molecular observation methods to capture detailed intramolecular dynamics of an individual single protein molecule by the labeling x-ray method. in this study, we present an innovative method for observing the protein assembly networks of nano-scale size on the bulk solution using a dxt that possess pico-meter scale positional accuracy and micro-second time resolution. this method is founded by detecting angular rotational displacement of a coexisted and dispersed single gold nanocrystal (approximately nm) on target solution. for target sample, we choose a crystal precursor metastable state of mg/ml lysozyme solution (hen egg white lysozyme: . g/ mole on . m sodium acetate - w/v nacl, ph . buffer) and mg/ml of lysozyme solution such as stable condition. for reference sample, we prepare mg/ml and mg/ml of ribonuclease a solution (ribonuclease a (bovine): . g/mol, . m sodium acetate - w/v nacl, ph . buffer). the experiment was carried out at spring- bl xu. from dxt analysis, we obtained logarithmic rotational velocity distribution of mg/ml, mg/ml lysozyme solution and mg/ml, mg/ml of ribonuclease a solution during ms and we processed regression analysis of gaussian fitting in each distribution. from this result, mg/ml of lysozyme solution (stable) and mg/ml, mg/ml (reference) have definitely regressed by single peak normal gaussian distribution that are positional peaks at . mrad and . mrad and . mrad, respectively. in contrast, mg/ml of lysozyme solution consisted of double peak logarithmic distribution at . and . mrad. this peak separation tendency from dxt are typically observed in a supersaturated condition of the sodium acetate solution which coexisting nano-scale solute networks (dxt and small angle x-ray scattering (saxs) experiment). from this study, dxt measurement results and detailed analysis process for a protein solution such as crystal precursor metastable state of lysozyme solution are concerned that this technique is a powerful tool for observing nano-scale protein network's existence and its local dynamics in bulk solution. at the present time, we will demonstrate the detailed analysis and processing from dxt raw data and the conformity of the results from another experiment confirmation such as saxs and darkfield microscopy. finally, we present a detailed protein network model of lysozyme metastable solution from dxt result. increasing evidence suggests that conformational fluctuations experienced by proteins play a key role in promoting function. however, the experimentally derived dynamic behavior of discrete protein systems has yet to provide clear evidence on the evolutionary conservation of motional events between structural and functional protein homologues. in this work, we used a statistical coupling analysis (sca) approach to analyze the role of co-evolving amino acids in protein function and flexibility. sca facilitates the identification of distinct residue sectors and provides an understanding of the correlation between amino acid co-evolution and biological function. as a model system, we used the ribonuclease a (rnase a) family, which includes canonical members that are structurally similar, but perform diverse activities such as angiogenesis, anti-pathogenicity and neurotoxicity, while preserving the common ribonucleolytic function. analysis of sequences with sca . allowed the determination of five independent components (ics) corresponding to amino-acid sectors with distinct functional, structural and/ or dynamical roles within the family. while most ics correspond to residues critical for protein stability and catalytic function, ic correlates with residues experimentally determined to be highly dynamic on the catalytic timescale in several rnase a homologues, as we confirmed by nmr n-cpmg experiments. additionally, ic residues form a hydrogen-bonding network shown to allosterically modulate and coordinate catalytically productive motions. these results provide a direct observation between the role of residue sectors in stability and motions relevant to function in the rnase a family. this study highlights the role of concerted action of multiple residues towards a common function, which can guide experiments for engineering proteins to enhance function such as catalysis. halophilic archea are a type of extremophiles that thrive in hypersaline conditions. in order to avoid the osmotic shock, their cytoplasm is maintained with higher ionic strength up to - m. such a high ionic strength of intracellular environment protects the cells from desiccation or salting out. however, one wonders how such high salt concentrations keep the cell functional and active throughout all the cellular reactions (i.e. enzyme catalysis). the answer lies in the evolution of the halophiles that has left the halophilic proteins with a certain preference for amino acids composition [ ] (prefers more acidic residues and overall decrease in hydrophobic side-chains). it's also known that the salt concentration can affect the activity and the underlying mechanism [ ] of the halophilic enzymes. in order to further understand the haloadaptation, we have chosen to study a model system, dihydrofolate reductase (dhfr) from haloferax volcanii (hvdhfr). dhfr is a very well understood enzyme and known to bound variety of ligands that can modulate the dynamics and the energy landscapes of the enzyme [ ] . we have probed the ls-ms dynamics of hvdhfr bound to the cofactor nadph (holoenzyme) and other substrates (dhf, thf) using relaxation dispersion nmr. in order to understand the salt contribution to the dynamics and catalytic mechanism, the experiments were done in the increasing order of salt concentration, shedding light on the conformational ensembles involved in the catalytic mechanism. the nmr relaxation dispersion analysis of different enzyme complexes in the presence of m and m kcl has shown more conformational fluctuations than compared to the enzyme with m kcl. this result suggests that the enzyme preferably is highly active & samples more flexible conformers in the hypersaline environment. further, quantification of these conformers/invisible-states and their salt-dependence will provide new insights into the mechanism of the enzyme-catalysis. also, it will facilitate designing novel enzymes that can be used in the bioprocess industrial set-up under extreme conditions. . tadeo we have identified the extracellular protein hbps in the cellulose degrader streptomyces reticuli and shown that it specifically binds iron ions, heme and aquo-cobalamin. based on d crystal structures, structural alignments, sequence comparisons, mutagenesis, and comparative biochemical investigations, we identified the coordination sites for iron, heme and aquo-cobalamin, and binding kinetics were elucidated [ , ] . hbps interacts with the membrane-embedded sensor kinase sens. while under nonstressed conditions hbps inhibits sens autophosphorylation, under oxidative-stressing conditions activates it. sens in turn phosphorylates the response regulator senr that activates the transcription of anti-oxidative genes [ ] . we crystallized hbps and solved its d crystal structure that revealed an octomeric assembly which is required for interaction with sens [ ] . using mutagenesis, fret, cd spectroscopy, fluorescence spectroscopy and site-directed spin labelling combined with pulse electron paramagnetic resonance spectroscopy, we demonstrated that ironmediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within hbps. we additionally showed that hbps is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds [ ] . we concluded that these molecular events are responsible for the hbps-mediated activation of the sensor kinase sens. this presentation will focus on the hbps structure and dynamics within the protein. indian institute of technology bombay for (fop-related protein of kda), a centrosomal protein, is conserved across all ciliated eukaryotes. it has been shown to be involved in ciliogenesis in rpe cells and in the s-phase progression in hela cells. it localizes to the pericentriolar satellite and at the centrosome. the localization of for at the centrosome has been found to be throughout the cell cycle. in paramecium, for is involved in docking of the basal body to the cell membrane and in the assembly of the transition zone. we found that for colocalizes with ciliated microtubules in nih t cells. since, the localization of for to the pericentriolar satellite is dependent on microtubules and the cilium itself is a microtubule-based structure; we sought to investigate the role of for in microtubule assembly. the over-expression of gfp-for in hela cells led to the depolymerization of microtubules while only the gfp expressing hela cells showed typical microtubule network. in addition, the microtubules of gfp-for expressing hela cells were found to be more sensitive towards nocodazole, a microtubule-depolymerizing agent, suggesting that for destabilizes microtubules. the effect of for on the polymerization of tubulin was monitored by light scattering, sedimentation assay and electron microscopy. in vitro, for inhibited the rate and extent of polymerization of tubulin. for example, , , and mm for inhibited the polymerization of purified tubulin by , , and %, respectively. further, the sedimentation assay suggested that for inhibited the polymerization of tubulin. in addition, electron microscopic analysis of the assembly mixture showed that for inhibited microtubule formation in vitro. the binding of for to purified tubulin was monitored by several complimentary techniques. using size exclusion chromatography, for was found to be co-eluted with tubulin indicating that for binds to tubulin. further, the interaction between for and tubulin was analyzed by surface plasma resonance technique and the result showed that for binds to tubulin with a modest affinity. the data together suggested that for regulates microtubule assembly through a direct interaction with tubulin. amide hydrogen exchange (hx) is widely used in protein biophysics even though our ignorance about the hx mechanism makes data interpretation imprecise. notably, the open exchange-competent conformational state has not been identified. based on analysis of an ultra-long molecular dynamics trajectory of the protein bpti, we propose that the open (o) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the n-h group. the hx protection factors computed from the relative o-state populations agree well with experiment. the o states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. the mean residence time of the o state is ps for all examined amides, so the large variation in measured hx rate must be attributed to the opening frequency. a few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. in either case, we argue that an over-coordinated n-h group is necessary for efficient proton transfer by grotthuss-type structural diffusion. differences in redox reactions with nadp /h between ferredoxin-nadp oxidoreductases from bacillus subtilis and rhodopseudomonas palustris daisuke seo , hidehiro sakurai , pierre s etif , takeshi sakurai graduate school of natural science and technology, kanazawa univ., research institute for photobiological hydrogen production, kanagawa university, ferredoxin-nad(p) oxidoreductase (fnr) is a ubiquitous enzyme that catalyze the redox reaction between soluble small iron-sulfur protein ferredoxin and nadp /h. fnrs from photosynthetic green sulfur bacterium chlorobaculum tepidum (ctfnr), low-gc content gram-positive bacterium bacillus subtilis (bsfnr) and purple non-sulfur bacterium rhodopseudomonas palustris (rpfnr) are homodimeric proteins containing one fad prosthetic group per subunit. crystal structure analyses of ct-and bsfnrs have revealed their significant structural homology to nadph-thioredoxin reductase from eschelicia coli, which is distinct from the monomeric fnrs from plastids of higher plants, cyanobacteria, g-proteobacteria and putidaredoxin reductase. previous studies on steady-state reaction of bs-and rpfnrs with nadp /h by diaphorase assay have demonstrated that nadph oxidation rates of bsfnr and rpfnr were comparable to those of the fnrs from plastid and cyanobacteria. but affinities toward nadp /h and rates for oxidation of nadph differed significantly between bsfnr and rpfnr, despite their high amino acid sequence homology. in this work, we report pre-steady state reactions of bsfnr and rpfnr with nadp /h by a stopped-flow spectrophotometry. mixing oxidized bsfnr with nadph yielded a rapid formation of charge transfer complexes (ctcs) followed by a reduction of the enzyme. bsfnr was almost fully reduced at equilibrium. mixing photochemically reduced bsfnr with nadp also rapidly provided an absorption spectrum of ctc but followed reoxidation of reduced bsfnr was very slow. the amount of oxidized bsfnr after equilibrium depended on nadp concentration. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was much faster than that for the reverse direction. mixing oxidized rpfnr with nadph exhibited a rapid ctcs formation followed by a slower reduction of the enzyme. increase in nadph concentration reduced the observed rate, suggesting redox potential of rpfnr was similar to that of nadp /h couple in the presence of excess nadph. mixing photochemically reduced rpfnr with nadp rapidly provided ctcs. the decay corresponding to the reoxidation of reduced rpfnr with nadp involved two distinctive kinetic phases, which would be due to the similarity in hydride transfer rates of forward and reverse directions, and the presence of excess amount of nadp . kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was close to that for the reverse direction. obtained data suggested bsfnr and rpfnr are optimized for different direction of the reaction and may play different physiological roles. stephan k€ ohler , , friederike schmid , giovanni settanni , institute of physics, johannes gutenberg university mainz, germany, graduate school materials science in mainz, fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. this picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. here, we present extensive molecular dynamics simulations of the fibrinogen complex which reveal large bending motions centered at a hinge point in the coiled-coil region of the complex. this feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. it also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. in addition the simulations suggest how the dynamics of the d region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a-and b-holes, important for fibrin polymerization, and the integrin binding site p . membrane curvature -the assembler of proteins mijo simunovic , , gregory voth , patricia bassereau chemistry department, the university of chicago, physico-chimie curie, institut curie many biological phenomena require the membrane to change its shape. this process is often mediated by curvature-generating proteins, most notably by those containing one of many bar domains. at the same time, membrane curvature controls the way proteins interact with one another and so it acts as a vital signaling mechanism in the cell. we combine theoretical modeling with microscopy imaging techniques to study the driving force underlying the reshaping of biological membranes induced by n-bar proteins. in particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical simulation methods to study the assembly of proteins on the membrane at molecular resolution. this approach allowed us to elucidate the precise mechanism by which cell membranes rapidly and from large distances recruit proteins to membrane-reshaping sites. it also let us identify a surprising role of membrane tension in directing the strength and geometry of protein-protein interactions. by complementing our simulations with fluorescence and atomic force microscopies, we study the way molecular assembly of proteins affects the membrane morphology at a more macroscopic level. we demonstrated how largescale ordering of the proteins on the membrane is mediated by a strikingly long-range interaction that is driven by membrane fluctuations. in sum, our combined theoretical and experimental approach gives vital clues on how the mechanical properties of the membrane may regulate protein dynamics in living cells. adnan sljoka , alexandr bezginov kyoto university, university of toronto understanding how a protein functions depends in critical ways on predicting which parts are rigid and which are flexible. using rigidity-theoretical techniques, a number or programs such as first, proflex or abstract kinari can rapidly decompose a protein into flexible and rigid regions. in this study we extend this technique and develop a novel computational approach for detecting protein allosteric interactions. it is widely believed that the binding of a ligand at the allosteric site triggers a conformational change that is transmitted through the protein to cause a rearrangement and alteration of the shape of the active site. allostery was first described more than years ago; however, the underlying allosteric mechanism is still not well understood. we will introduce a rigidity-based allosteric mode of communication together with an algorithm which can detect transmission of rigidity and shape changes between two (or more) distant binding sites in allosteric proteins. our algorithm is also used to predict and identify regions in the protein that are critical for the coupled communication between distant sites (i.e. allosteric pathways). starting with a set of known gpcr -dimensional structures, we apply our methods and show how binding of an activating ligand (i.e. agonist) triggers small rigidity changes which propagate to the critical g-protein binding regions. in contrast, in the inactive gpcr structures no such rigidity allosteric communication is observed. detailed predictions and analysis on activated (agonist-bound) and inactive adenosine receptors is discussed and results are compared with experimental evidence. these results show that rigidity-based allosteric model and algorithm is a powerful new tool for detecting allostery in gpcrs. comparing the intrinsic dynamics of multiple proteins using elastic network models reveals global similarities based on their overall shape sandhya tiwari , , nathalie reuter , with increasing protein structural data, we find ourselves with the opportunity to study the dynamical similarities within large ensembles in order to understand the role of protein dynamics at various levels. is there evolutionary pressure to conserve dynamics? is this necessary to retain functionality, as it has been suggested? we performed strategic comparative analysis using the elastic network model, which has proven to be highly efficient and informative (fuglebakk et al., bmc bioinformatics, ) to investigate how the dynamics of related proteins change when their functional or oligomeric state shifts, and if it is conserved within protein families. in our work, we have found that proteins with different ligandbinding states, as in adenylate kinase (tiwari et al., bmc bioinformatics, ), or oligomeric states, as in the pyrr family (perica et al., science, ), can be classified based on the global similarity of their intrinsic dynamics. moreover, our recent analysis on functionally distinct protein families with the tim barrel fold indicates that the overall similarity in shape dictates similarity in intrinsic dynamics regardless of sequence and functional conservation or evolutionary links. we suggest that since shape dictates a large part of dynamical similarity in proteins, the changes in local flexibility play a strong role in differentiating various functional states. oxygen-affinity and cooperativity of hemoglobin introduction: the widely-held mechanism of allostery of hb has been that the changes from the r-quaternary/tertiary structures of oxy-hb to the t-quaternary/tertiary structures of deoxy-hb exert certain constraints on the coordination structure of the heme group, leading to a lower o -affinity of the hemes and thus that of hb [ ] . however, we found that there is no causal correlation between the static t/r-quaternary structures and the low/high o -affinities of hb, respectively [ ] . we explore an alternative mechanism of the regulation of the ligand-affinity and cooperativity in hb. results and discussion: the o -affinities of free fe[ii]-protoporphyrin ix-nitrogenous base complexes in organic solvents are very low (p > torr), whereas the apparent o -affinities of these metalloporphyrins, which are incorporated in apo-myoglobin, apo-hb, serum albumin, etc., increase substantially to p < - torr, though their coordination structures are apparently unchanged [ ] . such substantial increases in the apparent ligand-affinities of metalloporphyrin-containing proteins are accomplished by preventing/inteferring with the dissociation of the ligand by protein matrix, since the interior of globin is nearly fully packed by protein matrix. in hb, the dissociation process of the ligand proceeds through the "caged" state [ ] [ ] [ ] , which can be produced by cryogenic photolysis of the ligated-states at . k and in which the metal-ligand bond is broken and the un-bonded ligand is trapped near the bonding site within the globin moietiy. this "caged" state has spectral features distinct from those of either deoxyor ligated states of the respective hemoproteins. the apparent ligand-affinities of hb are regulated by heterotropic effectors without detectable changes in either static quaternary/tertiary structures of the globin moiety or the coordination/electronic structures of the metalloporphyrin moiety and thus the ligand-affinity of the metalloporphyrins themselves [ ] [ ] [ ] . the reduction of the apparent ligand-affinities of hb may be caused by increases in the migration rate of ligands through globin matrix from the "caged" state to solvent, resulting from the effector-linked, enhanced high-frequency thermal fluctuations which increase the transparency of the globin matrix toward small diatomic ligands [ ] [ ] [ ] . conclusion: the ligand-affinity of hb is regulated through protein dynamics by heterotropic effectors, rather than static quaternary/tertiary structural changes. thus, the "caged" state of hb acts as a critical transition state in regulation of the affinity for small diatomic ligands in hb [ ] . the role of metal ions in the regulation of life processes is extremely important. they act as signal transducers, protein configuration stabilizers, enzymatic cofactors, oxygen transport supporters and many others. for example, subtle perturbations in calcium homeostasis may lead to mental disabilities and are linked to diseases such as autism spectrum disorders (asd). in this study we focus on complex protein systems, mainly those present in the brain. we search for dimers mediated by the presence of metal ions, and determine the impact of the presence or absence of the latter on the structure and energetic properties of the complex in the protein-protein interface. we investigate ions' influence on the interface stability using classic molecular dynamics methods (md), including steered md. moreover, we apply a novel suite of enhanced md-based methods recently developed by our team (rydzewski & nowak) to explore ion diffusion pathways in protein fragments of the synapses. finally, we describe specific inter-protein ion binding motifs with the most important interactions, collating them with various structures deposited in the protein data bank [ ] . the binding of integrins to collagen plays a critical role in numerous cellular adhesion processes including platelet activation and aggregation, a key process in clot formation. collagen is an unusually shaped ligand, and its mechanism of recognition and role in selectivity and affinity are unique, and at this stage not well understood. the i-domain of the integrin protein binds to collagen specifically at multiple sites with variable affinities, however the molecular mechanism of integrin i-domain (ai) regulation remains unknown. using nmr, along with isothermal titration calorimetry, mutagenesis, and binding assays we are developing a novel integrated picture of the full recognition process of the integrin a i binding to collagen. the adhesion of the a b integrin receptors to collagen is cation-dependent with collagen binding a mg(ii) ion that is located at the top of the extracellular integrin a i-domain (a i). our results show evidence for a regulatory effect of the mg(ii) ion on a i affinity, by inducing allosteric ms-ms motions of residues distant from the binding site. we propose a novel model of a i recognition to collagen, comprising a two-step mechanism: a conformational selection step, induced by mg(ii) coordination, and an induced-fit step caused by collagen binding. hydrogen-deuterium exchange experiments show that the induced-fit step is facilitated by the reduced local stability of the c-terminus. we propose that the conformational selection step is the key factor that allows discrimination between high and low affinity collagen sequences. cytochromes p (cyp) are heme containing enzymes involved in the metabolism of endobiotics and xenobiotics, such as drugs or pollutants. [ ] in humans, cyps are attached to the biological membranes of endoplasmic reticulum or mitochondria by n-terminal transmembrane anchor and they are partially immersed by their catalytic domain to different level. [ ] generally, the composition of lipid membrane may significantly affect behavior of protein embedded in respective membrane e.g. the cholesterol in membrane alters membrane properties such as: thickening of the membrane, changing the stiffness or enhancing ordering of the membrane. furthermore, the increasing amount of cholesterol in membrane may also alter interaction with membrane proteins and affect solute partitioning between membrane and water molecules. [ ] cholesterol is also known to noncompetitively inhibit the most typical drugmetabolizing cyp -cyp a , [ ] however the mechanism was unknown. for this reason, we prepared the set of simulations of cyp a embedded in dopc lipid bilayers with various cholesterol concentrations ( , , , and % wt; figure ) and the ns long md simulations were carried out. md simulations showed the formation of funnel-like shape of the lipids close to the catalytic domain of cyp. in addition, the cholesterol molecules have tendency to accumulate in the vicinity of membrane-attached f/g loop. the catalytic domain sunk deeper into the membrane with cholesterol and also the number of amino acids in contact with membrane was bigger than in the pure dopc bilayer. in contrast, the presence of higher amount of cholesterol affected the pattern of channel opening effectively blocking the access to the active site from the membrane, which in turn may affect the substrate preferences and catalytic efficiency. [ ] finally, we study the effect of different lipid types on membrane-attached cyp a . anti-( -hydroxy- -nitrophenyl)acetyl (np) antibodies are one of the most widely analyzed type of antibodies, especially with respect to affinity maturation [ ] [ ] [ ] . affinity maturation is a process in which b cells produce antibodies with increased affinity for the antigen during the course of an immune response, and is like "evolution" in term of increasing antigen-binding affinity. during the course of affinity maturation, the structural dynamics of antibodies, which are closely correlated with the binding function, can change. to analyze the structural dynamics at atomic resolution and the single-molecule level, we tried to express and purify single-chain fv (scfv) antibodies against np. using scfv antibodies, we can also analyze the effects of key residues on affinity maturation via site-directed mutagenesis. as the first step, we have succeeded in generating a sufficient quantity and good quality of scfv of affinity-mature anti-np antibody, c , with a linker composed of four repeats of gggs. the scfv protein was expressed in the insoluble fraction of e. coli, and solubilized using m urea, followed by refolding by step-wise dialysis to decrease the urea concentration. the final step of purification using an antigen column indicated that approximately % of the solubilized protein was correctly refolded and possessed antigen-binding ability. the analytical ultracentrifugation (auc) analysis showed that the purified c scfv exists in the monomeric state with little oligomeric contamination. the secondary structure and thermal stability of c scfv were analyzed using circular dichroism (cd). the far-uv cd spectra of c scfv indicated typical b-sheet-rich structures. upon antigen binding, the far-uv cd spectrum remained unchanged, but the thermal stability increased by approximately oc. the antigen-binding function of c scfv was analyzed using a surface plasmon resonance (spr) biosensor, biacore. the binding affinity and kinetics of c scfv for np conjugated to bovine serum albumin immobilized on the sensor chip were similar to those of intact c . taken together, the results of auc, cd, and spr indicated that c scfv could be refolded successfully and would possess its functional structure. next, to analyze the structural dynamics of c scfv in the absence or presence of antigen, experiments involving diffracted x-ray tracking (dxt) were performed [ ] . c scfv with an n-terminal his-tag was immobilized on substrate surfaces using tag chemistry, and au-nanocrystals were labeled on the surface of scfv as tracers. the motions of c scfv were analyzed in two rotational directions representing tilting (u) and twisting (v) mean square displacement (msd) analysis from more than trajectories showed that the slope for c scfv without antigen, especially in the u direction, was greater than that for c scfv with antigen, suggesting that the motion of scfv was suppressed on antigen binding. the antibiotic resistance enzyme aph( '')-ia confers antimicrobial resistance to aminoglycoside antibiotics in staphylococci and enterococci. this kinase phosphorylates aminoglycosides such as gentamicin and kanamycin, chemically inactivating the compounds. we have determined multiple structures of the enzyme in complex with nucleoside and aminoglycoside substrates and cofactor magnesium. introduction of aminoglycoside to crystals of aph( '')-ia induce gross conformational changes in crystallo, illustrating several important stages of the catalytic cycle of the enzyme. an interaction between nucleoside triphosphate and an amino acid residue on a conserved loop has also been identified that appears to govern a conformational selectivity and modulates the enzyme activity when no substrate is present. comparisons between multiple protein molecules both within and between crystal structures allow us to infer functional states of the enzyme as it carries out catalysis. these structures collectively highlight an enzymatic flexibility that not only allows the binding of diverse aminoglycosides, but also appears to transition from a stabilized, inactive enzymatic state to a catalytically active enzyme with an active site geometry identical to distantly-related eukaryotic protein kinases. mechanistic insight gained from these studies begin to demystify a widespread staphylococcal resistance factor, and provide a starting point for the development of anti-infectives toward this important antimicrobial resistance machine. ryan godwin , william gmeiner , freddie salsbury wake forest university -department of physics, wake forest university health sciences -department of cancer biology the zinc-finger of the nf-jb essential modulator (nemo) is a ubiquitin binding domain, and an important regulator of various physiological processes including immune/inflammatory responses, apoptosis, and oncogenesis. the nominally functioning residue monomer ( jvx) is represented by a bba motif, with a cchc active site coordinating the zinc ion. here, we investigate the effects of a single point mutation that has been linked to the disease states associated with ectodermal dysplasia. the single mutation of the last binding cysteine (residue ) to a phenylalanine ( jvy) distorts the available conformation and dynamics of the protein, as shown via microsecond, gpuaccelerated molecular dynamics simulations. we examine these two proteins in various states of zinc-binding and coordinating cysteine protonation. in addition to destabilization of the alphahelix induced by the cysteine to phenylalanine mutation, prominent conformations show the bsheets turned perpendicular to the alpha-helix, providing a possible mechanism for the induced disease state. , catalytic ( - aa) and c-terminal ( - aa)) were expressed in e. coli. several truncated in variants containing amino acids - , - , - and - were also prepared. a full-size ku with a gst-tag on its n-terminus was purified from e. coli. all the experiments performed showed that neither n-terminal nor c-terminal domains of hiv- in are essential for its binding with ku despite a weak binding capacity retaining to the c-terminal domain. the catalytic core ( - aa) as well as the mutant lacking c-terminal domain ( - ) both demonstrated affinity to ku comparable to the affinity of the full-size in, whereas its truncated variant ( - aa) bound to ku protein only weakly. we also expressed a c-terminal ha-tagged full-length in and its - variant in hek t cells together with a wt ku - flag and showed that both in variants are stabilized by co-expression with ku by approx. twofold. we hypothesize that the binding surface within in lies in the region from to a.a. that is a long a-helix. we have shown that a homologous integrase from prototype foamy virus that lacks this structural element does not bind to ku . it is worth noting that ku does not affect the interaction of in with its major cellular partner -ledgf/p as well as its interaction with the dna substrate. this work was supported by an rfbr grant - - and by an rscf grant - - . the nadph-dependent cytochrome p oxidoreductase (cypor) is large amino-acid long microsomal multidomain enzyme responsible for electron donation to its redox partner cytochrome p (cyp) involved in drug metabolism. electron transfer (et) chain is mediated by two riboflavin-based cofactors -flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad) within their respective domains and nicotinamide adenine dinucleotide phosphate (nadph). during this electron transfer cypor undergoes several structural changes in open and closed state of both domains in different degree of contact. in spite of the fact that cyp-cypor complexes play a key role in drug metabolism, the atomistic mechanism of structural rearrangements during complex electron transfers is still lacking. here, we present the results of our study on structural changes during cypor multidomain complex movement between individual electron transfers using classical molecular dynamics (md) and metadynamics (mtd) simulations with cofactors of nadph, fad and fmn in resting state. homology model of human cypor in both forms (opened and closed) were embedded into pure dioleoylphosphatidylcholine (dopc) bilayer. after system equilibration (figure ), structural changes of protein, anchor and cofactor movement were studied. we were able to select possible cypor-membrane orientation which would allow interaction with cytochrome p . in addition, spontaneous closing of open cypor was observed. however structural changes between crystal structures and structures obtain from md simulations lead us to the use of metadynamics in order to speed up the process. fmn and fad cofactor remained in close van der waals contact during the -ns long simulation stabilized by p stack interaction of fad with trp , whereas continual movement of nadph continually weakens its p stack interaction with fad. after ns of classical md additional metadynamics simulations were performed in order to investigate internal motion of cofactors during electron transfer. atoms c n (nadph) and n (fad) which are responsible for et were able to move closer to the distance of Å after adding biasing potential. this distance is more than sufficient for electron transfer to occur. after switching back to classical md cofactors got into resting positions ( Å) again. our results show that cypor undergo several structural changes and internal motions of cofactors in order to transfer electrons to its redox partner -cyp. research & utilization div., jasri/spring- , grad. school frontier sci., univ. tokyo, grad. sch. sci., univ. hyogo, japan, national institute of advanced industrial science and technology, japan, pentameric ligand-gated ion channels (plgics) are a major family of membrane receptors that open to allow ions to pass through the membrane upon binding of specific ligands. plgics are made up of five identical (homopentamers) or homologous (heteropentamers) subunits surrounding a central pore. structural information about their multiple allosteric states, carrying either an open or a closed channel, has become available by recent studies by x-ray crystallography. however, dynamic information are needed to understand their mechanism of gating, notably the long-range allosteric coupling between the agonist binding site and the ion channel gate. here we used the diffracted x-ray tracking (dxt) method ( ) to detect the motion of the extracellular and transmembrane domain two plgics: the nicotinic acetylcholine receptor (nachr) and a proton-gated bacterial ion channel from gloeobacter called glic. dxt is a powerful technique in biological science for detecting atomic-scale dynamic motion of allosteric proteins at the single molecular level and at tens of micro seconds timescale resolution. the dynamics of a single protein can be monitored through trajectory of a laue spot from a nanocrystal which is attached to the target protein immobilized on the substrate surface ( , ). dxt detects two kinds of rotational motions of nanocrystal, tilting and twisting, based on x-ray incident beam axis. dxt analysis with . ms/f time resolution showed that tilting motion of the transmembrane domain of glic and both tilting and twisting motions of the extracellular domain of glic and nachr were enhanced upon application of agonists (lowering the ph for glic, and binding of acetylcholine for nachr). the detailed dynamic information, including size effect of gold nanocrystal to the motion of them, is discussed. [ proteins possess unique structure-encoded dynamics that underlie their biological functions. here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. crystallographic structures were determined for several ancestral gfp-like proteins that were reconstructed based on posterior sequence predictions, using members of the stony coral suborder faviina as a model system. the ancestral proteins belong to the kaede-type class of gfps, a group of proteins that undergoes irreversible green-to-red photoconversion and is therefore frequently employed in superresolution microscopy. surprisingly, we find that the structures of reconstructed common green ancestors and evolved green-to-red photoconvertible proteins are very similar. therefore, we analyzed their chain flexibility using molecular dynamics and perturbation response scanning. we find that the minimal number of residue replacements both necessary and sufficient to support lightinduced color conversion provide for increased fold stiffness at a region remote from the active site. at the same time, the allosterically coupled mutational sites appear to increase active site conformational mobility via epistasis. these data suggest that during evolution, the locations of fold-anchoring and breathing regions have been reversed by allosteric means. therefore, we conclude that the green-tored photoconvertible phenotype has arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. based on titration experiments, we estimate that at ph , . % of the protein population harbors neutral side chains for his and glu , residues that form an internal salt bridge near the chromophore. we propose that this reverse-protonated subpopulation constitutes the catalytically competent state. in the electronically excited state, light-induced chromophore twisting may be enhanced, activating internal acid-base chemistry that facilitates backbone cleavage to enlarge the chromophore. in this way, a softer active site appears to be coupled to a mechanism involving concerted carbon acid deprotonation and betaelimination. dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities by tuning motions in the active site. the binding of an agonist to a gpcr causes a conformational change in the receptor that leads to its activated functional state. rhodopsin, the membrane receptor responsible for photoreception in the vertebrate retina, is a prototypical gpcr and has been extensively used in structural, biochemical and biophysical studies of this class of receptors. different small molecules have been described to be capable of binding to rhodopsin. in addition, mutations in rhodopsin have been associated with retinal diseases and efforts have been carried out in order to find potential ligands that can offset the effect of these mutations. cyanidins, a group of flavonoids within the larger family of polyphenols, have been reported to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration intermediates. the aim of the current study was to evaluate the effect of the flavonoid quercetin on the conformational properties of both native bovine rhodopsin and heterologously expressed recombinant rhodopsin. rhodopsin was purified from bovine retinas by immunoaffinity chromatography, and photobleaching, thermal stability, metarhodopsin ii decay and chromophore regeneration assays were carried out in the absence or in the presence of mm quercetin. for recombinant rhodopsin, a plasmid encoding wild-type opsin was transfected into mammalian cos- cells, in the absence or in the presence of mm quercetin, harvested, regenerated with -cis-retinal, or -cis-retinal, and subsequently purified in dodecyl maltoside solution. no differences in photobleaching behavior, upon illumination, could be detected in the purified quercetin-containing samples compared to those in the absence of this flavonoid. in the case of rhodopsin, and the recombinant wild-type protein regenerated with -cis-retinal, quercetin did not significantly alter the thermal stability and rate of regeneration of the purified proteins under our experimental conditions. however, a two-fold increase in the thermal stability and a % increase in chromophore regeneration were observed for the recombinant wild-type protein regenerated with -cis-retinal in the presence of quercetin. in contrast, the presence of quercetin did not alter the electrophoretic and basic spectroscopic properties of rhodopsin, or those of the recombinant wild-type protein, suggesting no important structural alterations as a result of quercetin binding to the receptor. the positive effect of quercetin on the stability, and chromophore regeneration of rhodopsin, could be potentially used to counteract the effect of naturally-occurring misfolding mutations in rhodopsin. thus, quercetin could help stabilizing rhodopsin mutants associated with retinal diseases such as retinitis pigmentosa. furthermore, docking of the ligand, carried out on the crystallographic structure of rhodopsin (entry gzm), reveals several favorable sites for quercetin binding. one of this would be compatible with -cis-retinal suggesting a complementary binding to the receptor of this isomer which would not be compatible with -cis-retinal binding. identification of prospective allosteric sites of p by computational methods protein function is intrinsically associated with structural flexibility, so that understanding the functional properties of proteins requires going beyond the static picture produced by x-ray diffraction studies. structural flexibility can also be interpreted as a dynamic exchange between different conformational states with low energy barriers at room temperature. allosterism is a mechanism to regulate protein function associated with the plasticity exhibited by proteins. allosteric sites can be considered transient cavities that can be occupied by a small molecule with the subsequent modulation of the protein plasticity. occupation of these sites may modify the affinity of the protein for its native substrate that can be positive when the affinity increases or negative when the affinity decreases. allosterism can be used for the design of non-competitive ligands as new therapeutic agents. this mechanism of activity modulation is particularly interesting for those targets that use a common substrate for activation, like in the case of kinases to search for selective compounds. proteins can be viewed in solution as an ensemble of diverse energy accessible conformations. binding of an allosteric ligand produces a redistribution of the population of the diverse conformational states, which at the end modulate the affinity of the native substrate. allosteric sites can be characterized using computational methods by ensemble docking. it consist of characterize a set of structures that represent the accessible conformations of a protein that can then be used to perform virtual screening. in the present work we have studied prospective allosteric sites of p using computational methods. the protein is a member of the mitogen-activated protein kinases (mapks), a highly regulated group of enzymes that control a variety of physiological processes, including mitosis, gene expression, apoptosis and metabolism movement among others. the conformational profile of p was assessed using a us trajectory of accelerated molecular dynamics as sampling technique in explicit solvent. we used as starting structure the apoform of p in its inactive conformation (entry p ). the conformational features of the protein were assessed through the analysis of the variance of the most flexible regions of the protein using principal component analysis. the snapshots of the trajectory were projected onto the two principal components. subsequent cluster analysis permitted us to select a few structures for further studies. specifically, prospective biding sites were identified using a hydrophobic probe as implemented in the sitemap program. the results show previously described regulatory sites and some new prospective ones. hydrogen/deuterium exchange-mass spectrometry provides clues on the mechanism of action of min e maria t. villar , kyung-tae park , joe lutkenhaus , antonio artigues cell division in most bacteria is initiated by the formation of the z ring, an essential cytoskeletal element that serves as a scaffold for the cytokinesis machinery, at the mid body of the cell. in e coli the spatial location of the z ring is regulated by the min protein system, comprised by three major proteins: minc, mind and mine. the dynamic interaction between these proteins results in the formation of an oscillating protein gradient between the poles of the cell. this oscillation determines the position of the formation of the z ring. many aspects of this simple mechanism are beginning to be understood. in particular, the conformational changes associated with the interaction of the three min proteins between them and with the cell membrane, are of especial interest. hydrogen/deuterium exchange mass spectrometry (hdx ms) is a sensitive technique for the detection of changes in protein conformation and dynamics. the main advantages of this methodology are the ability to study native proteins in solution, the requirement for low protein concentrations, the potential to discriminate multiple coexisting conformations, and the lack of an upper limit to the size of protein to be analyzed. here we use hdx ms to analyze the dynamics of the wild type mine and of its inactive double mutant d a d a. our results show significant differences in the rates of exchange and in the total amount of deuterium exchanged at the end of the reaction between these two forms of mine. the wild type protein exchanges most of the amide hydrogen during the first few seconds of initiation of the exchange reaction. on the other hand, the mutant protein exchanges only % of the total amide hydrogen atoms during the first seconds of initiation of the exchange, and the remaining % amide hydrogen atoms are exchanged more slowly during the next few minutes of the reaction. our data are consistent with the existence of a highly flexible structure for the wild type protein and the coexistence of at least two rigid conformations for the double mutant that are undergoing a cooperative transition. interestingly, the central b-sheet forming the interface between the two subunits is protected against exchange on both proteins. these results provide insights into the conformational changes that mine undergoes during its interaction with mind. biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease pablo herrera nieto , james dalton _ , jes us giraldo _ universidad aut onoma de barcelona biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease as a significant component of dopamine signalling in the brain, the dopamine d receptor (d r), a member of the class a gpcr family, is an important target in the treatment of neurological conditions such as schizophrenia and parkinson's disease. d r shows a variety of signalling pathways through g proteins, including adenylyl cyclase inhibition, gbgpotentiation of adenylyl cyclase , and erk kinase activation, in addition to b-arrestin recruitment,. these pathways are differentially activated by some agonists and it has been suggested that d r ligands with gai/o antagonist and b-arrestin agonist activity may have anti-psychotic behavioural activity with reduced extra-pyramidal side effects. d r has also been found to form homodimers or higher-order hetero-oligomers with other gpcrs, which may modulate d r conformation and activity, thus constituting an additional form of allosteric receptor regulation. based on these findings, we have computationally modelled the full-length structure of d r, including its long intracellular loop (icl ) that is residues in length and absent in all homologous gpcr crystal structures. using state-of-the-art tools, such as rosetta for ab initio protein folding and acemd for micro-second molecular dynamics (md) simulations we have successfully de novo folded icl , which primarily consists of extensions to transmembrane helices (tmh) and and an intervening disordered histidine/proline-rich region, which is highly flexible. the latter is observed to interact with other receptor intracellular loops (icl and icl ) and appears to restrict access to the g-protein binding-site. in addition, we have docked a structurally diverse collection of ligands (biased agonists, antagonists and allosteric modulators) into our d r model and observed characteristic binding patterns suggestive of different biased signalling mechanisms. finally, through protein-protein docking with rosettadock, we have generated a complete heterodimer model of d r with the adenosine a a receptor (aa ar), where a mutual interface is formed between their respective tmhs and , as well as an association between the c-terminus of aa ar and icl of d r. this may be a particularly relevant biological complex in the treatment of parkinson's disease where antagonists of aa ar have been shown to ameliorate disease effects, potentially through direct interaction with d r. bis-ans as a tool to monitor conformational changes upon assembly of binary and ternary complexes of eif e, e-bp inhibitory protein, and the mrna 'cap specific recognition of the mrna ' terminal cap structure by the eukaryotic initiation factor eif e is the first and rate-limiting step in the cap-dependent translation. small e-binding proteins, e-bp , e-bp , and e-bp , inhibit the translation initiation by competing with eif g initiation factor for the same binding site, and by blocking the assembly of the translation machinery [ ] . our recent studies revealed intricate cooperativity between the cap and e-bp binding sites of eif e [ ] . here, we applied a fluorescent dye, , '-dianilino- , '-binaphthyl- , '-disulfonate (bis-ans) to investigate conformational changes upon assembly of binary and ternary complexes composed of human eif e, e-bp , and the mrna 'cap analogue, m gtp. the fluorescence quantum yield of bis-ans increases significantly upon binding to hydrophobic sites of proteins, making the probe a convenient tool to determine the accessibility to hydrophobic surfaces, and to monitor structural reorganisation of macromolecules [ ] . we characterised the interaction of bis-ans with eif e and e-bp by fluorescence titration. the association processes takes up to several hours until the saturation of the fluorescence signal is achieved, reflecting high flexibility of the protein structures. the association constants kas of eif e/bis-ans complexes are very high for the non-specific interaction. the kas values for eif e/bis-ans and eif e/ e-bp /bis-ans are similar ( m ), whereas the presence of m gtp results in ca. -fold weaker binding of the probe to eif e. the affinity of bis-ans for e-bp is -fold lower than that for eif e. we found no effect of either m gtp or e-bp on the fluorescence of bis-ans in complex with eif e, thus indicating lack of conformational changes around the probe on eif e/m gtp or eif e/ e-bp complex formation. it also testifies that bis-ans does not bind to the cap-binding site, despite the hydrophobic nature of this eif e region. on the contrary, addition of m gtp to the eif e/ e-bp /bis-ans complex causes an increase of the probe fluorescence, which indicates differences in the structural reorganisation in the binary, m gtp/eif e, compared with the ternary, m gtp/eif e/ e-bp , complexes, and confirms the spatial cooperation between the cap and e-bp binding sites. we also observed an increase of fluorescence for bis-ans bound to e-bp in the presence of eif e, pointing out that e-bp partially folds upon association with eif e. in summary, our results provide a deeper insight into the structural aspects of the molecular interaction at early stages of the cap-dependent translation. acknowledgements: this work was supported by the bst /bf project from university of warsaw background: beta -glycoprotein (b gpi) is a protein abundantly present in human plasma and highly conserved in all mammals. b gpi has been identified as the major antigen in the antiphospholipid syndrome (aps), a severe thrombotic autoimmune disease. despite its importance in the pathogenesis of aps, the physiological role of b gpi is still elusive. in a previous work we have demonstrated that b gpi significantly prolongs the clotting time in fibrin generation assays, and inhibits aggregation of gel-filtered platelets (ic . um), either isolated or in whole blood, by inhibiting cleavage of par on intact platelets (ic . um) and in solution. importantly, b gpi does not alter the ability of thrombin (fiia) to generate the anticoagulant protein c, with or without thrombomodulin added. hence, we concluded that b gpi inhibits the key procoagulant properties of fiia, without affecting its unique anticoagulant function. we also proposed that b gpi, together with other more efficient anticoagulant pathways such as thrombomodulin-fiia -protein c and antithrombin iii-fiia, may function as a mild anticoagulant in vivo especially in those compartments were the efficacy of thrombomodulin is limited, as in the large vessels, or is even absent, as in the brain vasculature. aims: lacking the threedimensional structure of b gpi-thrombin complex, the aim of this work is to identify the peptide regions either on thrombin and b gpi involved in complex formation. results: data obtained by fluorescence and surface plasmon resonance (spr) indicated that b gpi interacts whit fiia whit physiological affinity (kd nm). kd values calculated by reverting the interacting systems are very similar to each other (kd nm), suggesting that b gpi in the mobile phase has a conformation which is competent for the binding to immobilized fiia. the affinity of fiia for immobilized b gpi is markedly decreased by increased ionic strength (i.e. kd increases by -fold going from . m to . m), suggesting the electrostatic interactions play a key role in fiia -b gpi recognition. filling/inactivation or perturbation of fiia active site does not alter the affinity of fiia for immobilized b gpi, confirming that the active site is not involved in the interaction. mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, gpibalpha, hd aptamer) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of fiia plays a key role in b gpi binding. from the docking model of the bb gpi-thrombin complex, we identified a highly negatively charged segment - in domain v of b gpi interacting with positively charged pathes in thrombin exosite ii. the synthetic peptide b gpi( - ) was able to bind to fiia with an affinity (kd nm) comparable to that of full-length b gpi, deduced from fluorescence or spr measurements and to compete in spr measueremnts with the binding of full-length b gpi to thrombin. hence, combining experimental and theoretical data, we obtained a reliable model of the b gpi-thrombin complex. metalloproteases are one of the most diverse types of proteases, presenting a wide range of folds and catalytic metal ions. in the case of the merops ma clan, where most of the known metalloproteases are grouped based on the consensus hexxh sequence motif, a single catalytic zinc ion and common fold architecture [ ] . despite these common features, members from distinct families present distinct domain composition and topology. given our interest in developing new tailor-made metalloproteases for bioengineering applications, an in-depth understanding of the factors governing their function is required. protein internal dynamics includes the space of functionally-relevant structural changes occurring during an enzymatic reaction, and there is an increasing understanding on how it relates with protein sequence and structure evolution. therefore, we have recently assessed how the structural heterogeneity of metalloproteases relates with the similarity of their dynamical profiles [ ] . first, the dynamical profile of the clan ma type protein thermolysin, derived from the anisotropic network model, was evaluated and compared with those obtained from principal component (pc) analysis of a set of crystallographic structures and essential dynamics (ed) analysis of a ns molecular dynamics simulation trajectory [ ] . a close correspondence was obtained between normal modes (nm) derived from the coarse-grained model and experimentally-observed conformational changes (rmsip between nm -nm and pc of . ), corresponding to functionally-relevant hinge bending motions that were shown to be encoded in the internal dynamics of the protein (cumulative overlap of ed -ed and pc of . ). next, dynamics-based comparison methods that employ a related coarse-grained model (b-gaussian elastic network model) was made for a representative set of ma clan members [ ] , allowing for a quantitative description of its structural and dynamical variability. although members are structurally similar ( % pairs with dalilite z-score > . ), they nonetheless present distinct dynamical profiles ( % of pairs with aladyn p-value > . ), with no identified correlation between structural and dynamical similarity. for cases where high dynamical similarity was observed, the respective modes corresponded to hinge-bending motions encompassing regions close to the active site. further inspection of the produced alignments indicates that for ma clan metalloproteases, conservation of internal dynamics has a functional basis, namely the need for maintaining proper intermolecular interactions between the protein and respective substrate. previously unnoticed dynamical similarity between clan members botulinum neurotoxin type a, leishmanolysin and carboxypeptidase pfu was also found. together, these results suggest that distinct selective pressure mechanisms acted on metalloprotease structure and dynamics through the course of evolution. this work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate additional information of protein dynamics. glucokinase from antarctic psychrotroph pseudoalteromonas sp. as- (psgk) has a higher specific activity at low temperatures and a higher thermal stability than its mesophilic counterpart from e. coli (ecgk). in order to elucidate the structural basis for cold-adaptation and thermal stabilization of psgk, we have determined the crystal structure of psgk at . Å and compared it with the ecgk structure. psgk is a homodimer of the subunit of amino acid residues. each subunit consists of two domains, a small a/b domain (residues - and - ) and a large a b domain (residues - ). the active site is located in a cleft formed between the two domains. the identity of amino acid sequence between psgk and ecgk was %, but three dimensional structures of them are very similar to each other, having the conserved catalytic residues and substrate-binding residues. the analysis of the mainchain temperature factors revealed that the regions of small domain and the hinge region connecting two domains of psgk showed higher temperature factors with a lower number of intramolecular hydrogen bonds and ionic interactions than the corresponding regions of ecgk. however, the large domain regions of psgk showed lower temperature factors with a higher number of intramolecular hydrogen bonds than ecgk. furthermore, the atomic temperature factors of catalytic asp on the small domain were higher, but those of glucose-binding glu , his , and glu on the large domain were lower than ecgk. these results suggest that highly flexible hinge region and the catalytic residue on the small domain of psgk may contribute to its cold-adaptation, namely higher activity at low temperatures, whereas a more rigid structure of the large domain of psgk stabilizes its overall structure more strongly than ecgk. nowadays non-waste technologies in synthetic chemistry become more and more popular. such processes are often carried out using different enzymes. dehydrogenases represent the large group of enzymes, which are widely used in synthesis of chiral compounds and other useful molecules. such enzymes need nadh or nadph as a cofactor and due to high cost of reduced coenzymes a cofactor regeneration system is an obligate part in such kind of processes. it was shown that formate dehydrogenase (fdh, ec . . . .) is one of the best enzymes for nad(p)h regeneration. fdh catalyses the reaction of formate oxidation to carbon dioxide coupled with reduction of nad(p) to nad(p)h. the main advantages of fdh are the irreversibility of catalyzed reaction, low price of formate ion and wide ph optimum of activity. our laboratory has the largest collection of formate dehydrogenases from different sources. many fdh genes from bacteria, yeasts and plants were cloned and enzymes were expressed in active and soluble forms. mutant formate dehydrogenases from bacterium pseudomonas sp. show the highest thermal stability as well as activity in comparison with other reported formate dehydrogenases. now we have focused on eukaryotic genes. the recombinant enzymes from soya glycine max (soyfdh), arabidopsis thaliana (athfdh), moss physcomitrella patens (ppafdh) and yeast ogataea parapolymorpha (opafdh) were obtained by genetic engineering methods. it was revealed, that soyfdh has the best michaelis constants among all known fdhs, but it's less thermally stable compared to other fdhs. new mutant forms of soyfdh with excellent catalytic characteristics and high thermal stability were obtained by protein engineering. other enzymes (athfdh, ppafdh and opafdh) are comparable in their stability with majority of bacterial enzymes (but not with psefdh), so all the new obtained fdhs can be successfully used for cofactor regeneration. marmara university, wellesley college, antibiotics are essential therapeutic drugs widely used in the treatment of bacterial infections. unfortunately, misuse of these drugs resulted in the development of bacterial defense mechanisms. blactamase synthesis is among these mechanisms that renders b-lactam antibiotics ineffective. understanding the dynamic behavior of this enzyme is an important step in controlling its activity. in a former study, the importance of highly conserved w in modulating the hinge type h motion was reported. in the light of this information, mutant tem- b-lactamase enzymes with w a, w f and w y substitutions were constructed. wild-type and mutant tem- b-lactamases purified with ni affinity chromatography were subjected to enzyme assay using centa as the substrate. with w f and w y mutations, the remaining activity was approximately % of the initial activity. however with the w a mutation, activity was totally lost. structural studies of the w a mutant with cd and florescence spectroscopy indicated that there was no major change in the overall structure. however this mutation disrupted the interactions of w which resulted in an increase in the flexibility of this region of the protein. this project was supported by t € ub _ itak project no m . light-switchable zn binding proteins to study the role of intracellular zn signaling stijn aper , maarten merkx zn plays an important catalytic and structural role in many fundamental cellular processes and its homeostasis is tightly controlled. recently, free zn has also been suggested to act as an intracellular signaling molecule. to get increased understanding of the signaling role of zn we are developing light-switchable zn binding proteins to perturb the intracellular zn concentration using light. these protein switches consist of two light-responsive vivid domains and the zn binding domains atox and wd , linked together with flexible peptide linkers. in the dark, zn is tightly bound in between the two zn binding proteins. light-induced dimerization of the vivid proteins disrupts this interaction and thus results in zn release. the fluorescent proteins cerulean and citrine were attached to the vivid domains to allow the different conformational states of the protein switch to be monitored using fret. zn titrations revealed a -fold decrease in zn affinity going from dark-to light-state for the initial design, which was further improved to -fold by optimizing the linkers between the protein domains. in addition, the zn affinities of both states were tuned to be optimal for intracellular applications. switching between the high affinity dark-state and the low affinity lightstate was found to be reversible for at least two light-dark cycles. following the in vitro characterization, we are currently assessing the performance of this genetically encoded 'caged' zn in mammalian cells. proteins as supramolecular building blocks: engineering nanoscale structures school of biological sciences, university of auckland, school of biological sciences, victoria university proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics and pharmaceuticals. the potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. we are exploring self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the n-terminal domain of a dna binding protein (nterm-lsr ) and a typical -cys peroxiredoxin (hsprx ). non-native forms of these proteins have been designed undergo selfassembly into supramolecular structures in a controllable manner. self-assembly of nterm-lsr is initiated via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. we will show that the degree of oligomerisation can be controlled by variations in environmental conditions such as ph and protein concentration. furthermore, via protein engineering, we have introduced a new "switch" for oligomerisation via enteropeptidase cleavage. the new construct of nterm-lsr can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. hsprx has been shown to oligomerise into dimers, toroids, stacks and tubes in response to specific triggers such as ph and redox state. in this work we have utilised the histidine tag to further control the assembly of this versatile protein tecton. we will show that minute variations in ph can induce oligomersation of hsprx toroids into stacks and tubes. furthermore, by utilising the histidine tag as a ligand we can bind divalent metals to these supramolecular structures. this not only drives the formation of higher ordered oligomers but also provides a facile route which may facilitate the functionalisation of these protein nanoscale structures after they have been assembled. danielle basore , , rajesh naz , scott michael , sharon isern , benjamin wright , katie saporita , donna crone , christopher bystroff , , biological sciences, rensselaer polytechnic institute, cbis, rensselaer polytechnic institute, chemical and biological engineering, rensselaer polytechnic institute, computer science, rensselaer polytechnic institute, obstetrics and gynecology, west virginia university, unintended pregnancy is a worldwide public health concern, with million pregnancies being classed as unintended in . the magnitude of this number clearly indicates an unmet need in terms of contraception. methods that are currently available are effective, but exhibit many problems. side effects, ease of use, cost, and availability are all concerns. we propose a contraceptive vaccine that would be safe, effective, long-lasting, cheap, and reversible. our vaccine would prevent pregnancy by targeting sperm with antibodies raised in the woman's body. several approaches have been taken to developing a contraceptive vaccine in recent years. the most successful so far has been using human chorionic gonadotropin (hcg), a hormone produced during pregnancy, as an antigen . the hcg vaccine progressed to phase clinical trials, but only displayed an % efficacy, which is insufficient for a contraceptive. our lab uses a structure based approach to the design of an anti-sperm antigenic protein. we believe this will raise a more vigorous immune response that will produce a longer lasting titer. the catsper complex is a heterotetrameric calcium channel found in the tail region of sperm . each subunit of the complex contains an exposed loop known as the p-loop. the p-loop is unique on the surface of sperm because it is not glycosylated, allowing antibodies to potentially recognize and bind it. ylp is a twelve residue peptide that mimics the glycans in the glycocalyx of sperm . ylp is a member of the flitrx library, and in mice, produced protective titers that were reversible both voluntarily and involuntarily. our designs will introduce these two potential antigens into a loop of the l protein of human papilloma virus. l spontaneously assembles into virus like particles, and will aid in the production of a robust immune response. protein carriers for passage of the blood-brain barrier sinisa bjelic medical solutions that help protein therapeutics accumulate into the brain are crucial for future treatment of neurological disorders. biodrugs have a tremendous potential to treat disorders of the nervous system, but their efficiency has been severely restricted. to reach the brain all drugs must traverse the blood-brain barrier (bbb) -a permeable wall that separates blood from the brain -whose main function is to protect the nervous system from environmental influences of bacteria and toxins. unfortunately the bbb is also the culprit that effectively blocks access to therapeutics required for treatment of neurological diseases. a way to boost exposure of therapeutics across the bbb is to piggyback onto the transferrin receptor, a multidomain protein anchored in the membrane, which is involved in the physiological facilitation of iron uptake. here i present research that aims at successfully developing potent protein carriers for transferrin receptor-mediated passage of the bbb by using computational protein design in combination with yeast display methodology for hit validation and optimization. the longterm goal is to couple therapeutics -as for example drugs against alzheimer's -to the designed carriers to increase the brain uptake and cure neurological disorders. medium-throughput multistep purification of coagulation factor viia jais r. bjelke , gorm andersen , henrik Østergaard , laust b. johnsen , anette a. pedersen , tina h. glue there is a need of medium-to-high throughput purification of low-titre recombinant protein variants for screening to identify the final biopharmaceutical lead. such proteins include coagulation factors to be used for treatment of haemophilia and other bleeding disorders. at novo nordisk we have established a platform for production of recombinant coagulation factor viia variants, which include a spectrum of single-point mutations to large domain insertions. the variants were produced using transiently transfected hek f, hkb or choebnalt (qmcf technology) suspension cells. harvest cultivations were typical in the range of . -to l. a -step continuous, multistep purification method was implemented on € aktaxpress systems (ge healthcare). the interlinked process steps include capture using an immunoaffinity column, polish, concentration and buffer exchange using an anion-exchange column and proteolytic activation of the zymogen variant forms using a coagulation factor xaimmobilized column. buffers were designed such that elution from the capture column was aligned with binding conditions on the polish column to avoid a desalting step in-between. the following and final enzymatic activation was optimized with regards to flow rate to ensure full conversion while minimizing unwanted secondary cleavages in factor viia. the final products were fractionated in sharp chromatographic peaks ready for characterization. hplc and sds-page analyses showed a solid quality of the produced variants and more than variants have been produced in sub mg scale using the outlined method. biomimetic sequestration of co : reprogramming the b domain of protein g through a combined computational and experimental approach esra bozkurt , ruud hovius , thereza a. soares , ursula rothlisberger ecole polytechnique f ed erale de lausanne, federal university of pernambuco protein engineering is a powerful tool to generate highly specific enzymes for biomimetic production of chemicals. among many applications, the development of enzymes to accelerate carbon dioxide fixation is a possible route to limit co emission. in this project, we are inspired by the ancient enzyme carbonic anhydrase which efficiently catalyzes the reversible hydration of carbon dioxide in the presence of a zinc ion active site. to create an efficient biocatalyst, the engineered gb domain containing a his cys zn (ii) binding site was used as a starting point. in subsequent work, b domains comprising of his wat zn (ii) binding sites have been rationally designed to produce carbonic anhydrase mimics. the re-engineering was accomplished through a series of mutations to orient the zinc bound reactive species to form a hydrogen bond network in the active site while retaining the native secondary structure. we performed classical molecular dynamics (md), quantum mechanics/molecular mechanics (qm/ mm) simulations and metadynamics, with the aim to explore potential catalytic roles of the reengineered b domains and to elaborate the reaction mechanism. briefly, we introduced novel zn (ii) binding sites into thermostable b domain. in parallel, experiments are underway. wild-type protein was expressed and purified. structural and mutagenesis studies are ongoing. the results emphasize the power of theoretical work to enable the mimicking of nature's enzymes for desired catalytic functions. the roles of entropy and packing efficiency in determining protein-peptide interaction affinities diego caballero , , corey o'hern , , , , lynne regan , , physics, yale university, integrated graduate program in physical and engineering biology, yale university, mechanical engineering and materials science, yale university, applied physics, yale university, molecular biophysics and biochemistry, yale university, chemistry, yale university despite many recent improvements in computational methods for protein design, we still lack a quantitative and predictive understanding of the driving forces that control protein stability, for example, we do not know the relative magnitudes of the side-chain entropy, van der waals contact interactions, and other enthalpic contributions to the free energy of folded proteins. in addition, we cannot reliably predict the effects of point mutations on enzyme specificity or sequence tolerance in ligand binding sites. the tetratricopeptide repeat (tpr) motif is a common and versatile protein system that has been used as a model to study protein-protein interactions. for example, recent studies have experimentally measured the binding affinity and specificity for different tpr binding pockets and peptide ligands and generated a ranking of the protein-peptide pairs with the highest affinity. to gain a fundamental understanding of the interplay between atomic close packing and fluctuations of side-chain conformations in protein-peptide binding pairs, we performed all-atom langevin dynamics simulations of key residues near the binding interface of tpr proteins and their cognate peptides. the langevin dynamics simulations enabled us to calculate the entropy and potential energy of side chain conformations in the presence of backbone fluctuations for each protein-peptide pair. we compile rankings of the stability and affinity of mutant tpr-peptide structures to those obtained from experimental studies. this research has enhanced our ability to rationally manipulate protein-peptide interfaces. advances from this research will enable the design of tpr modules that specifically recognize biologically important proteins. monitoring protein-protein interactions using tripartite split-gfp complementation assays protein-fragment complementation assay (or pca) is a powerful strategy for visualizing protein-protein interactions in living cells. previously described split-gfp based sensors suffer from the poor solubility of individual pca fragments in addition to background signal originating from their spontaneous selfassembly ( ). we developed a new encoded genetic reporter called "tripartite split-gfp" for visualizing protein-protein interactions in vitro and in living cells. the assay is based on tripartite association between two twenty amino-acids long split-gfp tags, gfp and gfp , fused to interacting protein partners, and the complementary gfp - detector. when proteins interact, gfp and gfp selfassociate with gfp - to reconstitute a functional gfp ( ). using coiled-coils and frb/fkbp model systems we characterize the sensor in vitro and in escherichia coli. we extended our studies to mammalian cells and examine the fk- inhibition of the rapamycin-induced association of frb/fkbp . the small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence and for screening modulators of complex formation in cell-based assays. aldehyde dehydrogenases (aldhs) catalyze the oxidation of aldehydes to their corresponding acids using nad(p) as coenzyme. these enzymes are responsible for the detoxification of lipid peroxidation products, which have been involved in the etiology and pathogenesis of different diseases involving increments in oxidative stress. recent data from our group, showed that aldh a is resistant to inactivation by lipid peroxidation products, even at concentrations - times higher than those required to inactivate aldh a and aldh . the amino acids sequence of the aldehyde-binding site of the three enzymes was analyzed, and it was found that the enzymes susceptible to the effect of lipid peroxidation products (aldh a and aldh ), have cys residues flanking the reactive cys (position ), based on this criteria and considering that these aldehydes react preferentially with cysteine, a mutant of aldh was generated changing the cys residues adjacent to cys . the mutant aldh -cys thr-cys val, was resistant to the inactivation by acrolein and -hne, even at concentrations -fold higher than those required to inactivate aldh . however, the mutant presented values of km , and -fold higher for acrolein, propionaldehyde and acetaldehyde, respectively, compared to the wild type enzyme, but showed a catalytic efficiency similar to the parent enzyme. these data revealed that cys residues near to the reactive cys in aldh are important in the inactivation process induced by lipid aldehydes, but also participate in determining the specificity for the substrates in this enzyme. small molecule-assisted shutoff: a widely applicable method for tunable and reversible control of protein production h. kay chung , conor jacobs , yunwen huo , jin yang , stefanie krumm , richard plemper , , roger tsien , michael lin department of biology, stanford university, department of pediatrics, stanford university, department of pharmacology, university of california san diego, department of pediatrics, emory university, institute for biomedical sciences, georgia state university, department of chemistry and biochemistry, university of california san diego, howard hughes medical institute, university of california san diego, the ability to quickly control the production of specific proteins would be useful in biomedical research and biotechnology. we describe small molecule-assisted shutoff (smash), a technique in which proteins are fused to a self-excising degron and thereby expressed in a minimally modified form by default. degron removal is performed by a cis-encoded hepatitis c virus (hcv) protease, so that applying clinically available hcv protease inhibitors causes degron retention on subsequently synthesized protein copies and suppresses further protein production. we find that smash allows reversible and dosedependent shutoff of various proteins with high dynamic range in multiple cell types, including yeast. we also successfully use smash to confer drug responsiveness onto a rna virus for which no licensed drug inhibitors exist. as smash does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. furthermore, as smash only uses a single tag and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts. top, a protein of interest is fused to the smash tag via a hcv ns protease recognition site. after protein folding, the smash tag is removed by its internal ns protease activity, and is degraded due to an internal degron activity. bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. vaccine development has emerged, epitope-focused immunogens, but in the past these have failed to deliver the expected outcome. here, we employed a new computational design methodology (rosetta fold from loops or ffl) to design epitope-focused immunogens. ffl was devised to insert structurally defined functional sites into protein scaffolds. throughout the ffl stages the structure of the scaffold is folded and its sequence designed to stabilize the desired functional conformation of the inserted site. we used ffl to design epitope-focused immunogens for the respiratory syncytial virus (rsv), for which despite the intense research we are still lacking an approved vaccine. we designed three-helix bundles harboring an rsv epitope, that was previously co-crystallized with the neutralizing antibody motavizumab. the designs were thermodynamically stable (tm > ˚c) and showed extremely high affinities to motavizumab (kd pm). structural characterization through x-ray crystallography of antibodybound and unbound scaffolds showed good agreement to the computational models in the overall structure (rmsd - . Å) and exquisite mimicry of the epitope region (rmsd - . Å), when compared to the peptide-epitope in complex with motavizumab. the designed immunogens were used to immunize non-human primates (nhp), and approximately % of the cohort developed rsv neutralizing activity, in some instances with high potency. to evaluate the therapeutic relevance of the elicited neutralization activity, we compared the nhp neutralization titers to those of human sera after natural rsv infection, which generally yields protective levels of antibodies. the neutralization potency of the best nhp responders was comparable to that of the human sera. to better understand the features of the antibodies elicited, we isolated several rhesus monoclonal antibodies (rhmabs) from the animal that exhibited the most potent neutralization. two of the rhmabs bound to the immunogen with very high affinity (kd pm) and were potent rsv neutralizers. interestingly, these rhmabs were approximately fold more potent than the fda-approved prophylactic antibody palivizumab. our results provide the first proof-of-principle for epitope-focused vaccine design, and demonstrate the power of the ffl figure . schematic of nucleotide binding, exchange and hydrolysis in tubulin, and its coupling to mt assembly. exchange of gdp (orange) for gtp (magenta) at the e-site in b-tubulin (blue) happens in the unpolymerized dimer (left). the active, gtpbound tubulin dimer adds to a growing mt (right). interaction of the incoming a-tubulin (green) with the e-site nucleotide at the plus end of a mt (with b-tubulin exposed) results in gtp hydrolysis. the mt cartoon (bottom right) shows an oversimplified representation of a gtp cap as it first grows by tubulin addition and then shrinks by polymerization-coupled gtp hydrolysis (here b-tubulin that is bound to gtp is shown in red and that bound to gdp is shown in blue). cryo-em density map (emdb- ) and atomic model (pdb: jak) for an eb -decorated mt bound to gtpgs. a-tubulin, b-tubulin and eb are colored green, blue, and orange, respectively. computational methodology. we anticipate that ffl will be useful for a variety of other challenges in the computational design of functional proteins. designed repeat proteins as templates for photoactive molecules and fluorescent nanoclusters sara h. mejias , , antonio aires , , javier l opez-andarias , pierre couleaud , , begoña sot , , carmen atienza , nazario mart ın , , aitziber l. cortajarena , imdea nanoscience, c/faraday, , ciudad universitaria de cantoblanco , cnb-csic-imdea nanociencia associated unit "unidad de nanobiotecnolog ıa", departamento de qu ımica org anica i, facultad de qu ımica, universidad complutense self-assembly of biological molecules into defined functional structures has a tremendous potential in nanopatterning, and the design of novel bionanomaterials and functional devices. molecular selfassembly is a process by which complex three-dimensional structures with specified functions are constructed from simple molecular building blocks. we present first the study and characterization of the assembly properties of modular repeat proteins, in particular designed consensus tetratricopeptide repeats (ctprs), and their application as building blocks in order to generate functional nanostructures and biomaterials. ctpr proteins can be assembled into self-standing thin films, and thin nanometer fibers in solution. in this work, we show the use of the designed consensus repeat proteins as scaffolds to template: ( ) photoactive organic molecules, and ( ) fluorescent nanoclusters. .we explore the potential of ctpr proteins to arrange donor-acceptor pairs for electro-active materials. in particular, porphyrin rings arranged by ctprs in a defined distance and orientation for favoring face-to-face orientation which should lead to an improvement in the optoelectronic properties. our results confirm the successful ability of ctpr proteins to be used as scaffold for ordering organic chromophores, while preserving their structure. the unique self assembly properties of ctpr scaffolds have been exploited to generate ordered conductive films of the protein-porphyrin conjugates. these results open the door to fabricate hybrid protein-based solid devices. .we show results on the ability of ctpr to encapsulate and stabilize fluorescent gold nanoclusters. we investigated the influence of the protein sequence in the final properties of the nanoclusters. the structural and functional integrity of the protein template is critical for future applications of the protein-cluster complexes. therefore synthetic protocols that retain the protein structure and function have been developed. as a proof of concept, a ctpr module with specific binding capabilities has been successfully used to stabilize nano clusters. biohybrid photoelectrochemical cells have been developed by functionalizing the hematite photoanode with the light-harvesting cyanobacterial protein c-phycocyanin (pc) yielding a substantial enhancement of the photocurrent density. photoelectrochemical cells combining light-harvesting proteins and inorganic semiconductors have potential for the use in artificial photosynthesis. in this work we present processing routes for the functionalization of hematite photoanodes with pc, including in situ co-polymerization of pc with enzymatically-produced melanin and using a recombinantly produced pc . moreover, recombinant forms of the light-harvesting protein c-phycocyanin from synechocystis sp. pcc were engineered to carry a peptide with affinity for hematite. similarly, a bacterial laccase was engineered to acquire affinity for hematite. results obtained from the different approaches to hematite functionalization and the advantages offered by protein engineering will be presented. minimizing a suitable free energy expression is arguably the most common approach in (ab initio) protein structure prediction. the achieved accuracy depends crucially on the quality of the free energy expression in use. here, we present corrections to existing free energy expressions which arise from the thermal motion of the protein. we (i) devise a term accounting for the vibrational entropy of the protein, and (ii) correct existing potentials for 'thermal smoothing'. (i) vibrational entropy is almost always neglected in free energy expressions as its consideration is difficult. this practice, however, may lead to incorrect output because distinct conformations of a protein can contain very different amount of vibrational entropy, as we show for the chicken villin headpiece explicitly [ ] . for considering vibrational entropy, we suggest a knowledge based approach where typical fluctuation and correlation patterns are extracted from known proteins and then applied to new targets. (ii) at ambient conditions, timeaveraged potentials of proteins are considerably smoothened due to thermal motion where the strength of this effect varies strongly between atoms. distinguishing these inhomogeneities by introducing new atom species regarding their locale environment can therefore increase the precision of time-averaged potentials [ ] . extraction of general principles from the continually growing protein data bank (pdb) has been a significant driving force in our understanding of protein structure. atomistic or residue-level statistical potentials, secondary-structural propensities, and geometric preferences for hydrogen bonding are among the classical insights that arose from observations in the pdb. given the magnitude of structural data available today, it is likely that many quantitative generalizations remain to be made. here we hypothesize that the pdb contains valuable quantitative information on the level of local tertiary structural motifs (terms), with term statistics reflecting fundamental relationships between sequence and structure. we define a term to be the structural fragment that captures the local secondary and tertiary environments of a given residue, and put our hypothesis through a series of rigorous tests. first, we show that by breaking a protein structure into its constituent terms, and querying the pdb to characterize the natural ensemble around each, we can estimate the compatibility of the structure with a given amino-acid sequence through a metric we term "structure score." considering submissions from recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy, with poorly predicted regions readily identifiable. this performance exceeds that of leading atomistic statistical energy functions. next, we show that by considering the terms of a structure that are affected by a given mutation, and mining the pdb to characterize sequence statistics associated with each, we are able to predict mutational free energies on par with or better than far more sophisticated atomistic energy functions. finally, we ask whether term statistics are sufficient to enable the design of proteins de-novo. we demonstrate that given a native backbone conformation, term considerations alone with no input from molecular mechanics correctly predict roughly the same fraction of amino acids from the corresponding native sequence as state-ofthe-art computational protein design methods. knowledge-based energy functions have already put pdb statistics to good use by parsing structural environments into geometric descriptors, generally assuming their conditional independence. our results suggest that it may now be possible to instead consider local structural environments in their entirety, asking questions about them directly. if this is the case, then the pdb is an even larger treasure trove of information than it has been generally known to be, and methods of mining it for term-based statistics should present opportunities for advances in structure prediction and protein design. comprehensive understanding of a protein fold is intertwined with successful design. recent advances in designing de novo structures have shown that proteins can be designed for a few globular and helical folds. however, designing all-b structures and barrels remains challenging because loops and intricate long range interactions that are important in these topologies are difficult to control. for designing novel catalysts, the (a/b) -barrel (or tim-barrel) fold is one of the most important examples, for it is the most common topology for enzymes. for almost year, attempts in designing de novo tim barrel structures have all resulted in poorly folded proteins. here we describe the successful design of a -fold symmetrical (a/b) barrel directly from geometrical and chemical principles. designed variants with a wide range of stabilities from being molten globules to cooperatively folded proteins were experimentally characterized, and the results revealed the importance of sidechain-backbone hydrogen bonding for defining the characteristic a/b-barrel. the residue tim barrel structure is among the smallest tim-barrels and has a fully-reversible melting temperature of c. the x-ray crystal structure shows atomic-level agreement with the design model. despite this structural similarity, psi-blast searches do not identify sequence similarities to known tim-barrel proteins. more sensitive profile-profile searches suggest that the design is sufficiently distant from other native tim-barrel superfamilies to be in a superfamily of its own, further implying that nature has only sampled a subset of the sequence space available to the tim-barrel fold. the ability to de novo design tim-barrels opens new possibilities for custom-made enzymes. university of texas southwestern medical center, biofrontiers institute, university of colorado creation of new molecular sensors and actuators based on fluorescent proteins relies on methods for identifying complex photophysical phenotypes and subsequently performing separations on cell populations. we developed a microfluidic flow cytometry approach tailored to interrogating the performance of genetically-encoded fluorophores and present the results of studies employing this technology. the system screens cell-based libraries on the basis of multiple photophysical parameters relevant to imaging, including brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence quantum yield) at a rate of up to cells/sec. in a first generation of experiments, molecular dynamics-guided design was used to create a library of mcherry mutants that was screened with this system, resulting in the identification of a variant with a higher stability b-barrel and improved photostability but with a decreased brightness due to reduction in the fluorescence quantum yield. to avoid inadvertent decreases in this important performance criterion, subsequent rounds of selection were performed on the basis of both photostability and excited-state lifetime as sorting criteria. in these second generation selections, mutations were designed to target pathways of oxygen access through the bottom of the bbarrel in addition to a position that directly interacts with the chromophore. furthermore, subsequent rounds of screening were used to improve folding and maturation. the multiparameter sort identified multiple clones with up to -fold improved photostability and up to double the excited-state lifetime of the parent mcherry fluorescent protein. the best mutant we identified produces one order of magnitude more photons before photobleaching compared to mcherry, at excitation conditions characteristic of confocal fluorescence microscopy. our results demonstrate the utility of combining moleculardynamics-guided library design with technology for photophysics-based selections. we anticipate that the new fluorescent proteins obtained in this work will find use in low-copy-number and long-duration imaging live cell imaging applications in cell-lines created by genomic editing techniques. targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase rushikesh joshi , ratna prabha c. the maharaja sayajirao university of baroda targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase targeting the over accumulated protein in the cell for degradation using specific degrons is an emerging research area. the degradation of the vast majority of cellular proteins is targeted by the ubiquitin-proteasome pathway. but in the case of ubiquitin independent protein degradation, odc/az system is more effective in achieving targeted protein degradation than other types of degradation . ornithine decarboxylase (odc) is key regulatory enzyme in the biosynthesis of polyamines. the protein has two domains namely, n terminal a/b barrel domain and c-terminal b-sheet domain. degradation of odc is mediated by polyamine inducible protein, antizyme (az). antizyme interacts with odc on n-terminal region, which results in degradation of odc by proteasomes. in mammalian odc the c-terminal has an unstructured tail of residues, which pulls odc into proteasome for degradation. it was reported earlier by coffino's group that the unstructured tail acts as a degron in chimeric fusion with gfp . in yeast, same function is achieved by n-terminal residues . present study focuses on accomplishing targeted protein degradation in saccharomyces cerevisiae by adding these two degradation signals or degrons of yeast odc and mammalian odc as tags to a reporter protein. we have selected two degrons namely, n terminal a/b barrel domain of yeast odc and c-terminal residues of mouse odc and grafted them to n and c-terminus of the reporter protein yegfp. degradation of yegfp and yegfp fusion with degrons of odc (degron-yegfp) were monitored by western blot using anti-gfp antibody and fluorescence spectroscopy. initially, the amount of degron-yegfp fusion protein was very low compared to control yegfp. it means that the chimeric protein underwent rapid degradation in the cells. after inhibition of proteasome, increase in the level of degron-yegfp was observed, confirming that the degrons cause rapid degradation of reporter protein through proteasome. earlier, we have also tagged ubiquitin from yeast with last residues of modc and observed enhanced degradation of ubiquitin in saccharomyces cerevisiae. therefore, both the degrons of odc alone and in combination are capable of decreasing stability of reporter protein in the cells. however, the combination of degrons is more effective than either of them in isolation. enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. the requirement that they be stably folded is a likely factor that contributes to their relatively large size (> , dalton). however, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. to test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of -residue peptides that act as zn dependent esterases. zn helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. the fibril activity is on par with the most active to date zinc-protein complex. such remarkable efficiency is due to the small size of the active unit (likely a dimer of -residue peptides), while the protein is at least -fold larger in molecular weight. the observed catalytic activity is not limited to ester hydrolysis. we have designed copper binding peptides that are capable oxygen activation. these results indicate that prion-like fibrils are able to not only catalyze their own formation -they also can catalyze chemical reactions. thus, they might have served as intermediates in the evolution of modern-day metalloenzymes. these results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions. rational design of the cold active subtilisin-like serine protease vpr with improved catalytic properties and thermal stability abstract proteinase vpr, from a psychrophilic vibrio species and its thermophilic structural homologue, aqualysin i (aqui) from thermus aquaticus, we set out to design a mutant of vpr which would be more thermostable, but would retain the high catalytic activity of the wild type enzyme. our starting protein template was a previously stabilized mutant containing two inserted proline residues close to the nterminus of vpr (n p/i p). this vpr_n p/i p mutant was shown to have a significantly increased thermal stability but displayed a concomitant tenfold loss of catalytic efficiency. from our previous studies we selected two mutations, one which increased catalytic activity (q k) of the enzyme significantly and another which stabilized the protein against thermal denaturation (n d). the n d mutation had been shown to introduce a salt bridge into the structure of the cold adapted proteinase, yielding higher stability but without negative effects on activity. the q k exchange had been shown to double the turnover number (kcat) to that of the wild type enzyme. insertions of these selected mutations into the vpr_n p/i p mutant were according to predictions; the q k increased the kcat tenfold, and the n d mutation increased the thermal stability. in the combination mutant, vpr_n p/i p/n d/q k, thermal stability was increased by c and c, in terms of tm and t %, respectively. furthermore, the catalytic activity of the mutant was somewhat higher than that of the wild type enzyme. critical peptide stretches may not serve as faithful experimental mimics for protein amyloidogenesis bishwajit kundu , dushyant garg certain amino acid stretches are considered critical to trigger the amyloidogenesis in a protein. these peptide stretches are often synthetically produced to serve as experimental mimics for studying amyloidogenesis of the parent protein. here we provide evidence that such simple extrapolation may be misleading. we studied the amyloidogenesis of full length bovine carbonic anhydrase ii (bcaii) and compared it with those formed by its critical amyloidogenic peptide stretch - (pepb). under similar solution conditions and initial monomeric concentrations, we found that while amyloid formation by bcaii followed aggregation kinetics dominated by surface-catalyzed secondary nucleation, pepb followed classical nucleation-dependent pathway. the afm images showed that bcaii forms short, thick and branched fibrils, whereas pepb formed thin, long and unbranched fibrils. atr-ftir revealed parallel arrangement of cross b sheet in bcaii amyloids, while pepb arranged into antiparallel b sheets. amyloids formed by bcaii were unable to seed the fibrillation of pepb and vice versa. even the intermediates formed during lag phase revealed contrasting ftir, far uv cd signature, hydrophobicity and morphology. we propose that for any polypeptide, the sequences flanking a critical region are equally effective in modulating the initial nucleation events, generating prefibrillar and finally fibrillar species with contrasting characteristic. the results have been discussed in light of amyloid polymorphism and its importance in the design of therapeutic strategies targeting such toxic regions. aksana labokha , ralph minter all approved biological drugs target extracellular proteins and not the majority of the expressed human genome, which resides within intracellular compartments. included in the latter category are many important, disease-relevant targets which cannot be easily addressed by small molecule approaches, such as the oncology targets c-myc and k-ras. although bacteria and viruses have evolved strategies to deliver biological material to the cell cytoplasm and nucleus, our ability to engineer recombinant proteins to replicate this is somewhat limited by (i) our nascent understanding of protein uptake and trafficking pathways and (ii) the ability to easily quantify cell delivery to the cytoplasm and cellular organelles. the aim of my project is to address these challenges by developing an effective assay for cytoplasmic uptake and then using it to measure the delivery efficiency of recombinant proteins which mimic natural delivery strategies e.g. cell penetrating peptides fusion, exotoxin mimics, and supercharged proteins (proteins with high surface charge which can enter cells). i also intend to explore the influence of the rab superfamily, which are the master regulators of protein trafficking, to influence and control both the kinetics and final subcellular destination of exogenous proteins. protein engineering: what's next? with the growing industrial need for engineering enzymes for the deconstruction and transformation of plant biomass in biorefineries, there is a want for the development of new approaches for designing special purpose biocatalysts. techniques, such as directed evolution, which mimic the natural selection process by evolving proteins towards the improvement of a given property, have unquestionably demonstrated their value and are routinely used in large industrial companies. nevertheless, the brute force employed in these methods, could significantly gain from an all-atom description of the underlying catalytic mechanisms, to center the efforts on more limited areas of the protein. in the last years, we have developed computational tools, which combine the electronic structure description of qm/mm methods with the potential to model long time scale processes of pele, to study the details of a variety of reactions. examples, which will be discussed, include rationalizing the selective oxyfunctionalization of steroids using fungal enzymes and the study of the effect of point mutations on the oxidation efficiency of laccases. these methods have shown their potential not only at the descriptive level but, more importantly, through their high predictive capability that opens many opportunities for their use in biotechnology. in this talk, we will show how recent advances in in silico approaches are setting new grounds for future computer guided directed evolution. several orthogonal bioreactions take place simultaneously within membrane bound organelles in eukaryotes and proteinaceous microcompartments in bacteria. these subcellular structures contain sets of enzymes co-involved in metabolic pathways. towards the goal of creating artificial protein microreactors, we seek to develop an artificial organelle that emulates the metabolic activity of the carbon fixating organelle of autotrophic bacteria, the carboxysome. here, we show that the two key carboxysomal enzymes, ribulose- , -bisphosphate carboxylase/oxygenase (rubisco) and carbonic anhydrase (ca), can be efficiently co-encapsulated using our previously reported encapsulation system which is based on a bacterial capsid formed from the protein lumazine synthase (aals- ). our preliminary results suggest that the enzymes can act in tandem and that the co-encapsulation of ca with rubisco in the capsid is necessary for enhanced rubisco activity in vitro. we attribute this observation to the high local concentrations of the rubisco substrate, co , produced by ca within the capsid. we are developing a theoretical model of a minimal carboxysome using the kinetic rate constants of our rubisco and ca variants and aals- as the shell to complement these experiments. next, we will incorporate our minimal carboxysome within an expression host such as e.coli, opening up the possibility of further optimization through directed evolution. in the past targeting and engineering of chemokines has led to several interesting drug candidates. [ ] amongst them, met-rantes, a met-ccl with high g protein-coupled receptor (gpcr) affinity but no subsequent signal transduction, as well as mutants addressing the interaction with the so-called glycosaminoglycans (gags) seem to be the most promising candidates. both, gag knockout as well as gag affinity matured chemokine isoforms have been considered as anti-inflammatory drug candidates, out of which an il- mutant with modifications reached clinical phase where it was profiled for acute neutrophil-related exacerbation in copd. [ ] cxcl (ip- ) is a proinflammatory chemokine released by various cells following stimulation by interferon g (ifn-g) . it is therefore considered as a late chemokine being responsible for the attraction of different lymphocytes. [ ] any therapeutic indication is consequently related to chronic and multiple applications. we have therefore engineered cxcl very conservatively at positions to ultimately generate dominant-negative mutants with a mildly improved gagbinding affinity and an entire knock off gpcr activity. the first steps of our engineering approach were in silico modelling of the mutants and the establishment of a suitable upstream-and downstreamprocessing protocol. next we generated a fluorescently engineered cxcl variant for our fluorescence-based affinity studies which was subjected to biocomparability investigations relative to the native, non-fluorescent protein. compared to the wild type, the fluorescently engineered mutant exhibited similar biological, chemotactic and gag-binding properties. next we started to produce sufficient amounts of the members of our nascent mutant library which were tested with respect to their biophysically behavior as well as to their knocked out chemotactic potency on cells. these experiments included gel electrophoresis and western blot analysis to determine identity and purity; circular dichroism (cd) and chaotrope-induced unfolding to approximate structure; isothermal fluorescence titration (ift); surface plasmon resonance (spr) and isothermal titration calorimetry (itc) to quantify gagbinding affinity and boyden chamber experiments to determine the chemotactic activity. our results show that we are able to tune the gag binding strength along with the gpcr activity of human cxcl which could lead to therapeutic applications in the future. nanodiscs are composed of a nanometer-sized phospholipid bilayer encircled by two a helical, amphipathic membrane scaffold proteins (msps). these particles provide a unique detergent free lipid bilayer model enabling biochemical and biophysical characterization of membrane proteins in a physiologically relevant medium. previously, the largest diameter reported of a nanodisc assembled using msps was about - nm. here we present a method to create large nanodiscs (up to nm in diameter) assembled with covalently circularized msps (cmsp). we can observe the homogeneity in nanodiscs diameter as a narrow distribution using negative-stain em. using our method, we have created nm nanodiscs and used them to study poliovirus ( nm diameter) entry and rna translocation. a nm nanodisc is sufficiently large to accommodate multiple copies of the cd receptor (also known as the poliovirus receptor), and has enough surface area to act as a surrogate membrane for the rna translocation complex during viral uncoating. the nm nanodiscs functionalized with the his-tagged ectodomain of poliovirus receptor, cd , were generated by adding lipids derivatized with a nta nickel- chelating head group to the lipid mixture during nanodisc assembly. cd receptor was added to the already assembled nanodiscs and incubated for minutes at room temperature. the receptordecorated nanodisc complex was purified by size exclusion chromatography. the purified complex was then incubated with poliovirus for minute at c, and then heated to c for minutes to initiate receptor-mediated viral uncoating. virus binding to nanodisc-cd complex and subsequent insertion of viral components into and across the membrane were confirmed by negative-stain electron microscopy (figure c) . to obtain a high-resolution structure for the rna translocation complex we conducted single-particle cryo-em studies using a polara f microscope. unlike liposomes, generating a reconstruction of samples containing nanodiscs is less complicated since the nanodiscs are more homogenous in size, and allow for thinner ice. also, the viral rna can be visualized more easily. the method for making large nanodiscs as well as the negative stain and cryo-em data will be will be presented and discussed. parametric design of alpha-helical barrels and pore-like assemblies with very high thermodynamic stabilities computational design of novel protein structures and enzymes with new functions is a promising tool to create superior biological materials with tailor-made properties, new pharmaceuticals, complex fine chemicals or renewable fuels. it also challenges our understanding of protein folding, protein evolution, molecular recognition and catalysis. here we present a procedure for designing proteins with backbones produced by varying the parameters in the crick coiled-coil generating equations [ ] . combinatorial design calculations using the software suite rosetta identify low energy sequences for alternative helix supercoil arrangements. after that, loop modeling is applied to connect the designs with lowest energy. the extent to which the designed sequences encode the designed structures is evaluated using large-scale structure prediction calculations, as well as symmetric and asymmetric protein-protein docking calculations. subsequently, synthetic genes are generated for sequences that converge strongly on the designed structure for experimental characterization. we applied this approach to monomeric three and four helical bundle structures as well as a pentameric five-helix bundle structure using idealized coiled-coil geometries [ ] . recently we expanded this approach to higher complexity backbones, which resulted in the de-novo design of monomeric, antiparallel six-helix bundles with untwisted, left-and right-handed geometries. circular dichroism (cd), size-exclusion coupled multi-angle light scattering measurements (sec-mals), negative stain electron micrographs (em) and small angle x-ray scattering (saxs) of these designs suggest that they indeed form the designed structures. in addition, we used rosetta protein-protein interface design functionality to computationally design oligomers out of our previously published three and four helix bundle structures to generate self-assembling pore-like structures with the potential use as channels or transporters. again, experimental validation of these designs by cd, sec-mals, em and saxs show that the designs are correct. we are currently undertaking further structural investigation of all these designs by x-ray crystallography. the designs described above can act as templates for protein or small molecule binding, holding a catalytic machinery or for scaffolding enzymes in reaction cascades. some of these applications are currently under investigation, including a self-sufficient redox system employing two copper-centers, binding of heme-moieties as a prosthetic group and tailoring the pore-like geometries to be used in nanopore sequencing. university of washington, university of california, san francisco, repeat proteins are an example of how evolution proceeds by building on existing structures and functions, but also a source of modular protein scaffolds for molecular recognition and biomaterials. however, it is unclear whether the limited number of folds and families that we know today is the result of the intrinsic limitations of polypeptide chains or the consequence of the path followed by evolution. we explored this hypothesis by computational design of repeat proteins based on modular units formed by two alpha helices and two loops of variable lengths, without relying on information from available repeat protein families. the automated sampling of the conformational space resulted in a large number of architectures from which de novo designs were selected for experimental characterization. % of the proteins were stable up to c and monodisperse and designs were structurally validated by small angle x-ray scattering. crystal structures were solved for of them, with root mean square deviation from the models between . Å and . Å. the designs differ from known proteins both at the sequence and structure levels and cover a broader range of geometries than observed in naturally occurring repeat protein families, indicating that existing architectures represent only a small fraction of what can be achieved. our results show that it is possible to expand the range of repeat protein architectures beyond the naturally occurring families, and that computational design can provide new scaffolds and enable the design of proteins tailored for specific applications. the serpin family of proteins consists of over members, all with a highly conserved native structure that is metastable ( ). serpins use this metastability to control the activity of proteases, via a specific inhibitory process. the serpin binds to its target protease through specific residues within the reactive centre loop, the protease cleaves the loop and results in a large conformational change causing the protease to become distorted and catalytically inactive whilst the serpin becomes much more stable ( , , ) . the metastable nature of aat is therefore required to facilitate the rapid and gross conformational changes required for its inhibitory function ( , ) . several disease-causing mutants of aat have been identified, the most common of them being the z-variant ( ). the z-variant has an increased propensity to polymerize in the endoplasmic reticulum of hepatocytes leading to cell death and liver damage ( ) . during the past fifteen years, many groups have unsuccessfully screened a number of serpins and a vast range of solution conditions to identify a combination of serpin and conditions that will enable the folding reaction of a serpin to be characterized. we have now taken an alternative approach and designed a synthetic "model" serpin that folds reversibly to its native state. in order to do this, we used a consensus design approach, analysing a sequence alignment of serpin sequences and determining the prevalent amino acid residue at each position, we termed this serpin conserpin (consensus serpin). here we present the structural, biophysical and functional characterisation of conserpin. combined crystallographic and folding studies reveal the characteristics of conserpin that likely dictate its unique stability and folding behaviour, whilst retaining activity as a serine protease inhibitor. the development of enhanced protein binding scaffolds is a key for engineering protein inhibitors and biosensors with advanced characteristics. utilizing the structural variability and designability of repeat proteins offers a means for designing protein binders where the overall shape is customized to optimally match a target molecule. we developed a computational protocol for the design of repeat proteins with a predefined geometry. by combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeat (lrr) proteins where the building blocks assemble into a novel structure. the suggested design procedure was validated by engineering an artificial donut-like ring structure, which is constructed from ten self-compatible repeats. characterization of several designed constructs further suggests that buried cysteines play a central role for stability and folding cooperativity in certain lrr proteins. this effect could provide a means for selectively stabilizing or destabilizing specific parts of an lrr-based protein binder. the computational procedure may now be employed to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired. engineering apobec g enzymes for altered specificity and processivity louis scott , muhammad razif , aleksandra filipovska , , oliver rackham , harry perkins institute of medical research, school of chemistry and biochemistry, the university of western australia apobec g (a g) is a host-encoded protein involved in the defense against hiv- and other retroviral infections. a g is a cytidine deaminase with a ' to ' processive nature, causing targeted c to t mutations along a dna strand. the catalytic and processive activity of a g leads to the hypermutation of nascent retroviral cdna, resulting in premature termination codons and dysfunctional proteins. ultimately, the action of a g inhibits viral replication. the ability of a g to jump and slide along a dna strand, deaminating at targeted sequences, makes it an interesting candidate for protein engineering. engineered a g enzymes for increased activity, altered specificity, and altered processivity are attractive options for expanding the dna modifying enzyme toolbox. mutation of catalytic residues, residues thought to affect its processive nature and those thought to be involved in target recognition, can create novel a g enzymes. using structure guided selection, residues in key functional sites that are amiable to mutation will be chosen. individuals from the resulting libraries of mutants will be selected by directed evolution for desired characteristics. the resulting a g enzymes will be examined for the relationship between their structure and function. such engineered a g enzymes could be targeted to catalyse the reversion of deleterious genetic mutations. furthermore, engineered a g enzymes could be used in mutational studies that call for targeted deamination along a dna strand, or mutational studies that call for unspecific and high throughput dna deamination. engineering porous protein crystals as scaffolds for programmed assembly thaddaus huber , luke hartje , christopher snow a key motivation for nano-biotechnology efforts is the creation of designer materials in which the assembly acts to organize functional domains in three dimensions. crystalline materials are ideal from the validation perspective because x-ray diffraction can elucidate the atomic structure. relatively little work has focused on engineering protein crystals as scaffolds for nanotechnology, due to the technical challenges of coaxing typical proteins into crystallizing, and the likelihood of disrupting the crystallization process if changes are made to the monomers. we have circumvented these limitations by installing guest protein domains within engineered porous crystals ( nm pore diameter) that have been rendered robust using covalent crosslinks. the retention of the scaffold structure despite changes to the solution conditions and macromolecule uptake can be validated through x-ray diffraction. we have engineered scaffold crystals for the non-covalent and covalent capture of guest macromolecules. by controlling the reversible loading and release, we can prepare "integrated" crystals with spatially segregated guest loading patterns. as assessed using confocal microscopy, such host-guest crystals are highly stable. ultimately, the resulting crystals may serve as a robust alternative to dna assemblies for the programmed placement of macromolecules within materials. engineering ultrasensitive protein probes of voltage dynamics for imaging neural activity in vivo francois st-pierre , , michael pan , , helen yang , xiaozhe ding , , ying yang , , thomas clandinin , michael lin , department of bioengineering, stanford university, department of pediatrics, stanford university, nervous systems encode information as spatiotemporal patterns of membrane voltage transients, so accurate measurement of electrical activity has been of long-standing interest. recent engineering efforts have improved our ability to monitor membrane voltage dynamics using genetically encoded voltage indicators. in comparison with electrophysiological approaches, such protein-based indicators can monitor many genetically defined neurons simultaneously; they can also more easily measure voltage changes from subcellular compartments such as axons and dendrites. compared with genetically encoded calcium indicators, voltage sensors enable a more direct, accurate, and rapid readout of membrane potential changes. however, several challenges remain for in vivo voltage imaging with genetically encoded indicators. in particular, current voltage sensors are characterized by insufficient sensitivity, kinetics, and/or brightness to be true optical replacements for electrodes in vivo. as a first step towards addressing these challenges, we sought to develop new voltage indicators that further improve upon the performance of the fast voltage sensor accelerated sensor of action potentials (asap ). in asap , voltage-induced conformational changes in a natural voltage-sensing domain perturb the fluorescence emission of a covalently linked green fluorescent protein (gfp). using a structurebased approach to guide mutagenesis, we discovered several amino acids that tune the kinetics and voltage sensitivity of asap . these residues are not only located in the voltage-sensing domain, but also in the fluorescent protein and in the linkers bridging sensing domain and gfp. our most improved variant, asap , exhibits improved sensitivity to voltage transients such as neuronal action potentials and subthreshold depolarizations. we sought to characterize the ability of these new voltage sensors to monitor neural activity in vivo using laser-scanning two-photon microscopy, a technique that allows imaging with lower autofluorescence and deeper tissue penetration. we report that asap sensors were able report stimulus-evoked voltage responses in axonal termini of the fly visual interneuron l . asap sensors enabled voltage imaging with dramatically improved temporal resolution compared to three recently reported calcium and voltage sensors. overall, our study reports novel voltage indicators with improved performance and highlights how specific amino acids can tune the performance of a proteinbased fluorescent sensor. we anticipate that these results will pave the way for further engineering of voltage sensing proteins, and that our new sensor asap will facilitate current and future efforts to understand how neural circuits represent and transform information. assembly of armadillo repeat proteins from complementary fragments erich michel , randall watson , martin christen , fabian bumback , andreas pl€ uckthun , oliver zerbe demonstrated that complementary fragments of a designed consensus armadillo repeat protein (armrp) recognize each other [ ] . the two fragments ym : ma, in which y, m and a denote the n-cap, internal repeats and the c-cap, respectively, form a : complex with a nanomolar dissociation constant, which is essentially identical to the crystal structure of the continuous ym a protein. we further demonstrate that structurally intact armadillo repeat protein complexes can be reconstituted from fragments obtained at various split sites -essentially after every repeat but also within repeats. the fragments display variable affinities towards each other, depending on the split site. the low affinity of some complementary pairs can be dramatically increased upon addition of peptide ligands. while a number of proteins are known that can be reconstituted from fragments we believe that the fact that armadillo repeat proteins can be reconstituted from various complementary fragments is novel and opens new interesting perspectives and applications in biochemistry. a reliable method for generating optically controllable proteins would enable researchers to interrogate protein functions with high spatiotemporal specificity. we recently engineered a tetrameric fluorescent protein, dronpa n, that undergoes light-induced monomerization, then developed a general architecture for lightinducible proteins based on this light-induced transition. we created proteins whose active sites were blocked by fused dronpa n domains in the dark, but would become unblocked by light. here we present further two extensions to this concept that together enabled the generalization of this method to additional classes of proteins. first, we engineered a photodissociable dimeric dronpa (pddronpa) with tunable affinity, faster photoswitching speed, and decreased level of protein aggregation, enabling better performance of fusion proteins. second, we introduce the concept of caging a protein active site by insertion of dronpa domains into loops rather than strictly at the protein termini. we use the pddronpa system to impose optical control on kinases and the cas endonuclease. the resulting light-inducible mek kinase, raf kinase, and cas endonuclease showed high caging efficiency of protein activities in the dark, and robust protein activation upon light illumination. we believe that our efforts on further improving and generalizing this method would bring the power and benefits of light control to a broad community of biologists. exploring the evolution of folds and its application for the design of functional hybrid proteins saacnicteh toledo patiño , birte h€ ocker the structural diversity of proteins may appear endless, nevertheless even large protein complexes can be decomposed into protein domains and smaller sub-domain sized fragments. only recently, we could identify such fragments employing sequence-based comparisons of different folds, as the tim-barrel and the flavodoxin-like fold (farias-rico et al., ) . as an extension of this work, we compared all a/b proteins and identified several fragments shared by different folds illustrating how nature may have achieved structural and functional diversity from a reduced set of building blocks. inspired by this combinatorial concept, we searched for homologous fragments bearing active sites to engineer a functional fold-chimera. we extracted the vitamin-b binding part from methylmalonyl coa mutase, which belongs to the flavodoxin-like fold (fl) and used it to replace the corresponding fragment in uroporphyrinogen iii synthase, which belongs to the hemd-like fold (hdl). the new hybrid resulted in a stable and well-folded protein whose structure was determined by x-ray crystallography. moreover, cobalamin-binding function was successfully transferred to the new protein from the fl parent, which shows the advantage of using this approach for the design of new functional proteins. in addition, profile alignments revealed sequence and structural evidence that suggested an evolutionary path for hdl from fl by gene duplication. to test this hypothesis, we expressed a modified c-terminal half of uroporphyrinogen iii synthase and solved its structure by nmr spectroscopy, thereby confirming the predicted fl architecture. altogether, our approach facilitates the detection of common ancestry among different folds contributing to our understanding of protein development. furthermore, our results show how new complex proteins can be designed using fragments of existing proteins that serve as building blocks in a lego-like manner. we believe that combining fragments containing existing properties will provide a successful method for the design of novel functionalities in the future. [ ] . the active site cysteine plays a key role in the reaction mechanism and we investigated this residue in more detail by exchanging this moiety with selenocysteine (sec) and homocysteine (hcy). the sortase mutants were generated by semisynthesis using expressed protein ligation (epl). the resulting cys-, sec-and hcy-sortase enzymes were characterized and showed a moderate - -fold reduction of activity for sec-sortase. the activity of hcysortase was barely detectable with less than % of wildtype activity. the alkylation efficiency of the active site nucleophiles correlated with the expected pka values of sec, cys and hcy. analysis of the ph dependency of the transpeptidation reactions showed that the activity optimum of sec-sortase was shifted towards more acidic conditions. these investigations provide further insights into the reaction mechanism of sortase a and the semisynthetic enzymes may provide new tool for further biochemical studies. propanediol oxidoreductase from escherichia coli (fuco) uses nadh/nad as cofactors to catalyze the conversion of s-lactaldehyde to s- , -propanediol and vice versa. fuco is an attractive enzyme in the search for possible biocatalysts producing a-hydroxy aldehydes, which are important for the synthesis of natural products and synthetic drugs. enzymes catalyzing these types of reactions are unique in catalytic power and stereoselectivity. the usage of fuco in synthetic industry is limited by the restricted substrate scope, which makes fuco inactive with larger phenyl-substituted alcohols. we used reengineering and directed evolution to enable fuco to catalyze the regio-and enantioselective oxidation of arylsubstituted vicinal diols, such as phenylpropanediols, into a-hydroxy aldehyde products. we mutated amino acids considered to restrict the entry into the active site, and modeled the mutants that were most active with the substrates phenylacetaldehyde and s- -phenyl- , -propanediol and performed docking studies with them. as expected, our experimental and in silico results show that the mutations enlarge the active site cavity and enable the mutant enzymes to accommodate the new substrates. we also found specific amino acids in the active site, which need to be conserved to allow the substrates to make stabilizing interactions. interestingly, an asparagine residue makes the mutant enzymes able to discriminate between phenylacetaldehyde and s- -phenyl- , -propanediol. in conclusion, we successfully re-engineered the specialist enzyme fuco to accept also bulkier molecules as substrates, thereby making it more useful for industrial purposes. one way to gain insight into the sequence-structure-function relationship in proteins is to de novo design artificial proteins. despite impressive successes in de novo protein design, designing a folded protein of more than amino acids still remains a challenge. using this approach, an idealized (beta/ alpha) fold protein was designed leading to the production of a protein of amino acids (octarellin v). this protein showed a low solubility and stability. through directed evolution we produced a soluble variant, octarellin v. . the biophysical characterization of octarellin v. shows a well folded monomeric and thermostable protein with a tm over c. however, after several screenings, we could not find crystallization conditions for this protein. as an alternative, we decided to co-crystallize octarellin v. with a protein partner that helps the crystallization process. we used protein partners: alpha-reps and nanobodies. the first one is characterized to interact through a large surface contact, whereas the second is characterized to recognize an specific small epitope. crystallization of both complexes was performed successfully by vapor diffusion and the structures were solved. the experimental structures correspond to the first for an artificial protein of this size and it will allow to criticize the computational design of the octarellin v. generation of synthetic antibodies against membrane proteins in nanodiscs for use in structural biology methods. here, we describe a robust strategy for generating a class of high performance antibodybased affinity reagents that have proven useful in determining the structures of relevant functional states of membrane proteins. these reagents are fab fragments that are generated by phage display from fully synthetic libraries and are called synthetic antibody fragments, or sabs. we have developed phage display sorting strategies that can trap a desired conformational state, making it accessible to structural analysis, or target a particular epitope on the protein surface. however, to maximize this technology for membrane proteins, several limitations of phage display sorting in detergent formats had to be overcome, the greatest being that using detergents can produce non-native conformational biases. we sought to address these limitations by embedding membrane proteins into nanodiscs, soluble lipidfilled discoidal particles, to better mimic the native membrane environment. nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing stimuli such as ligands, ions and ph during phage display selections. we have established and validated an improved protocol using two membrane protein systems: ) mj , an archaeal membrane protein of unknown function, and ) cora, a pentameric magnesium ion channel. using mj , we compared the nanodisc protocol with the standard method performed in detergent, and as an important byproduct, we characterized the influence of the membrane protein environment on the apparent affinity of sabs to their cognate antigen. using cora, we developed a more sophisticated sorting strategy resulting in a variety of sabs specific to either the open or closed conformation of the channel. finally, using sabs as crystallization chaperones we obtained the structure of mj at . Å resolution, and crystallized cora in several new conditions. lipocalin-type prostaglandin d synthase (l-pgds) is a member of the lipocalin superfamily, and binds a large variety of small hydrophobic molecules. using this function of l-pgds, we have already reported the feasibility of l-pgds as a novel drug delivery vehicle for the poorly water-soluble drugs [ ] . sn- , -ethyl- -hydroxy-camptothecin, is a semi-synthetic analogue of anti-cancer alkaloid camptothecin that targets dna topoisomerase i. despite of the potent anti-tumor activity, however, sn- was not used directly in a clinical practice due to its poor water solubility. thus, irinotecan hydrochloride (cpt- ), which is the water-soluble prodrug of sn- , is used for the cancer treatment. however, cpt- shows approximately . % cytotoxic activity of sn- against the various cancer cell lines in vitro, and its metabolic conversion rate is % of the original volume of cpt- . here, we show the development of the drug delivery system utilizing l-pgds, which enables a direct clinical usage of sn- . first, we investigated the effect of l-pgds on the solubility of sn- . in the presence of mm l-pgds, the concentration of sn- was . mm, which was , -fold as compared with that in pbs. then, we carried out isothermal titration calorimetry measurements to investigate the detailed binding mode of sn- to l-pgds. as a result, it was revealed that l-pgds binds three molecules of sn- , and the dissocia- control over the sensitivity with which artificial biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. such control could likewise be highly useful in artificial biotechnologies in which highly responsive behavior is of value, such as biosensors, genetic logic gates, and "smart" materials and delivery devices. in nature, the control of molecular responsiveness is often achieved using "hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. here we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. to do so we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. the loop spatially separates the two sets of the two halves of the binding sites, preventing a complete binding site that enables target molecule binding without prior closure of the loop. thus, the first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a pre-formed site. using this approach we have rationally introduced cooperativity into three unrelated aptamers, achieving in the best of these a hill coefficient experimentally indistinguishable from the theoretically expected maximum. the extent of cooperativity, and thus the steepness of the binding transition, are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. essential and non-essential amino acid species for an ancestral protein satoshi akanuma the translation system is an essential element for life because it links genetic information embedded in genes to functional molecules, proteins. the modern genetic code, which encodes the standard amino acids (and three terminations) using triplet codons, is shared by most of the extant organisms on the earth. a number of theories have been proposed for the origin and evolution of the genetic code, and these theories suggest that only a fewer amino acids were used in primitive proteins and later the amino acid repertoire gradually increased up to through the course of evolution. if so, one would wonder how many number of and which types of amino acids were involved in the primitive proteins. i have begun to address this issue experimentally. i first resurrected several ancestral proteins and then restricted the amino acid usage of one of the resurrected proteins. i targeted nucleoside diphosphate kinase (ndk) that catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate. ndk may have arisen early because at least one gene that encodes ndk is present in most extant organisms. the first step in the reconstruction of ancestral ndk sequences is to prepare multiple amino acid sequence alignments using homologous sequences of ndk from extant species. then, phylogenetic trees were built. ancestral sequences of ndk that represent the last common ancestors of archaea and of bacteria were reconstructed using the information contained in the predictive phylogenetic trees. the reconstructed ancestral kinases are extremely thermally stable [akanuma et al., ] . then, using the most thermally stable ancestral ndk, arc , as the starting molecule, i restricted its amino acid usage. arc does not contain any cysteine residue and therefore consists of amino acid species. i completely replaced one of the amino acid species by other amino acid species and thus created proteins each of which consisted of amino acid species. then, i evaluated the stabilities and activities of the resulting arc variants to assess the individual contributions of the amino acid species. as the result, i found that the amino acid species do not equally contribute to the stability and activity of arc and that some amino acid species can be easily lacked but others are important or essential for its stability and function. the result clearly shows that the full amino acid species are not necessarily essential and supports the hypothesis that proteins in the early stage of evolution were made from a reduced amino acid set. the protein surface recognition for protein-protein interactions (ppi) is involved in signal transduction, immune reaction, and creation of the nanostructures in living cells. the methods for rational designing of ppi that could provide non-antibody scaffolds and nanostructured materials are required for the therapeutic and nanotechnological applications. although there have been some successful rational designs with computational methods, it is still difficult to design freely the ppi onto arbitrary proteins. the reason for this limitation is decreased solubility in the designed protein due to the additional hydrophobic residues in order to drive ppi. another reason is a limited set of design modes by which proteins can interact, because the target proteins have individual surface structures. therefore, many methods of constructing an interface for numerous target scaffold proteins without loss of their solubility are necessary. surface exposed a-helices are often observed in natural globular proteins. moreover, there are many examples for naturally occurring oligomeric proteins where an a-helix from each subunit interacts to form an intermolecule coiled coil. further, the works related to designing of artificial helical bundle reported by the several other groups have provided information about how to generate and tune the interaction between a-helices. therefore, a surface exposed a-helix would be a good target for designing a de novo interface onto the scaffold protein. here we engineered two different proteins, sulerythrin and cys-larfh, to form the cys-larfh-sulerythrin dimer-cys-larfh heterotetramer via an intermolecular helix-helix interaction. wild-type sulerythrin forms a dimeric eight-helix bundle. cys-larfh is a designed monomeric protein that forms four-helix bundle containing interhelical s-s bonds. both sulerythrin and cys-larfh are extremely thermostable. to design protein-protein interfaces onto the individual proteins, we first introduced six leucines to the two a-helices of sulerythrin and three leucines to a a-helix of cys-larfh. as expected, the introduction of the hydrophobic amino acids reduced their solubilities. to recover the solubility, we then introduced six aspartates or glutamates around the hydrophobic surface of the sulerythrin (hereafter referred to as l d or l e). similarly, three arginines were introduced around the artificial hydrophobic surface of the cys-larfh (hereafter referred as iv- l r). the solubilities of the mutants with the hydrophobic interface and additional charged residues were recovered their solubility. in addition, the sulerythrin mutants l d and l e exist mainly as dimer. the cys-larfh mutants iv- l r, also exists as monomer. we then examined the interaction between l e or l d and iv- l r. a pull-down experiment, in which co beads bound to either his-tagged cys-larfh and iv- l r were used to pull down wild-type sulerythrin, l d, or l e, demonstrates that l d or l e specifically interacts to iv- l r. furthermore, when analysed by size exclusion chromatography, the dominant peaks of the mixture of l d and iv- l r appeared at the volume expected for the heterotetrameric complex. thus we successfully created the de novo ppi by using a very simple concept involving hydrophobic interaction in combination with charge interactions. in vitro selection of liposome anchoring peptide by cdna display naoto nemoto , ryoya okawa , yuki yoshikawa , toshiki miyajima , shota kobayashi a liposome-anchoring peptide (la peptide) was selected against liposomes composed of dioleoyl-snglycero- -phosphocholine (dopc) by in vitro selection using cdna display method. the selected peptide la peptide consists of the n-terminal region (hydrophobic) and the c-terminal region (basic) in a characteristic manner. thus, la peptide was synthesized chemically and the interactions between la peptide and particular types of liposomes were investigated and confirmed by confocal laser scanning microscopy. designing of a novel platinum-binding amino acid sequence on a protein surface asumi kaji , hiroya niiro , satoshi akanuma , tetsuya uchida , akihiko yamagishi designing of a novel interaction between a metal and a protein is a key to create hybrid materials between organic and inorganic materials. for example, in a glucose biosensor, which is widely used for measuring glucose concentration in blood, glucose oxidoreductase molecules are immobilized on a platinum electrode by polyacrylamide gel. a metal-binding tags that is added to the n-or cterminus of a protein is also used for fix the protein to a metal. however, a technique to create a metal binding site on a desired position of a protein has not been invent. if such a technique would be established, the technique would contribute to developing and improving biosensors and to producing new bionanoelectronic materials. in this study, we created a platinum-binding site on a loop located at a protein surface. we used an artificial protein, larfh, that had been synthesized by connecting four identical alpha helices originated from the c-terminal segment of the escherichia coli lac repressor with three identical loops. we randomized the ser, gly, gln, gly, gly, ser sequence within one of the inter-helical loops and then selected for binding to platinum by a t phage display system. most of the selected larfh variants contained the tyr, lys, arg, gly, tyr, lys (ykrgyk) sequence in the randomized segment. we then evaluated the affinity of the larfh variant to platinum by means of quartz crystal microbalance analysis. we found that the variant binds to platinum more strongly than does the original larfh. in the annual symposium, we will also report about the affinity of the isolated ykrgyk sequence to platinum and about the crucial role of the first tyrosine in binding to platinum. engineering of an isolated p a subunit of pi ka permits crystallization and provides a platform for structure-based drug design pi ka remains an attractive target for development of anticancer targeted therapy. a number of p a crystal structures in complex with the nsh -ish fragment of p regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the atp binding site. taking advantage of recent data on the functional characterization of the lipid binding properties of p a, we designed a set of novel constructs allowing production of isolated stable p a subunit missing the adapter binding domain (abd) and lacking or featuring a modified c-terminal lipid binding motif. while this protein is not catalytically competent to phosphorylate its substrate pip , it retains ligand binding properties as indicated by direct binding studies with a pan-pi ka inhibitor. additionally, we determined apo and pf- bound crystal structures of the p a ( - ) subunit at . Å and . Å respectively. comparison of isolated p a ( - ) with the p a/p complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p a for iterative sbdd. importantly, this crystal form of p a readily accommodates the binding of non-covalent inhibitor by means of a fully accessible atp site. the strategy presented here can be also applied to structural studies of other members of pi kia family. identification of structural determinants involved in the differential conformational changes of ef-hand modules emma liliana arevalo salina , joel osuna quintero , humberto flores soto , gloria saab rinc on instituto de biotecnolog ıa, universidad nacional aut onoma de m exico identification of structural determinants involved in the differential conformational changes of ef-hand modules calcium signals are regulated by several proteins, most of which belong to the ef-hand superfamily. the ef-hand motif is formed by a helix-loop-helix that binds calcium through its loop . these motifs occur in adjacent pairs, forming a single globular domain which is the basic structural and functional ca binding unit. the proteins in this family can be classified as calcium sensors or modulators, according with their function. the first group undergoes a major conformational change upon calcium binding, while the second one remains practically unchanged , . to explain the biophysics behind the different behavior of these proteins upon ca binding, we have sought to identify structural determinants that could account for these features, especially for the difference in the conformational change. we examined the primary structure from two ef-hand motifs: a sensor ef-hand from chicken troponin c (sciii) and a modulator ef-hand from bovine calbindin d k (clbn). the main differences were in the binding ca loop and a group of charged residues in the h helix of the modulator ef-hand. then, we constructed chimeric clbn motifs containing the loop or the loop and h from sciii motif (h clbnsciii and h h clbnsciii). these constructs were analyzed using a reporter system that discriminates ef-hand-sensor motifs from signal-modulators at the single-motif level. this reporter is based on the fusion of genes codifying for the ef-hand and the prephenate dehydrogenase from e. coli (tyra), a protein which is active only as a dimer. isolated ef-hand motifs have the ability to homo-dimerize and in the fusion can stabilize and activate tyra. the sensor motif exhibits a conformational change by binding calcium and in doing so, destabilizes the dimeric conformation of tyra and virtually eliminates its activity. in the modulators, on the other hand, the rather small conformational change only gives rise to a decreased tyra activity. both constructed chimeric ef-hand fusions showed a loss of activity upon ca binding, indicating that the residues connector of the sensor ef-hand from sciii is sufficient to confer the conformational change. in addition we used cd and extrinsic fluorescence spectroscopies to analyze any conformational change in the h h clbnsciii and h clbnsciii isolated modules, not finding any difference between the ca free and ca bound chimeras, suggesting that the change in activity of the reporter protein is due to a change in the orientation of the helices in the ef-hands induced by calcium. the effect of ca binding of the chimeras in the context of the entire calbindin d k protein is under investigation. mapping side chain interactions at the n-and c-termini of protein helices nicholas e newell, independent researcher interactions involving one or more amino acid side chains near the ends of protein helices stabilize helix termini and shape the geometry of the adjacent loops, contributing to supersecondary structure. side chain structures that have been identified at the helical n-terminus include the asx/st n-caps, the capping box, and hydrophobic and electrostatic interactions. at the cterminus, capping is often achieved with main-chain polar groups, (e.g. the schellman loop), but here also particular side chain motifs clearly favor specific loop geometries. key questions that remain concerning side chain interactions at helix termini include: ) to what extent are helix-terminal motifs that include multiple amino acids likely to represent genuine cooperative interactions between side chains, rather than chance alignments? ) which particular helix-terminal loop geometries are favored by each side chain interaction? ) can an exhaustive statistical scan of a large, recent dataset identify new side chain interactions at helix termini? in this work, three analytical tools are applied to answer the above questions for both n-and c-termini. first, a new perturbative least-squares d clustering algorithm is applied to partition the helix terminal structures in a large ( , example), low-redundancy pdb dataset by loop backbone geometry. the clustering algorithm also generates a set of structural exemplars, one for each cluster, that is used to represent the most important loop geometries at each terminus. next, cascade detection (newell, bioinformatics, ), an algorithm that detects multi-amino acid cooperativities by identifying overrepresented sequence motifs, is applied to each cluster separately to determine which motifs are most important in each loop geometry. finally, the results for each motif are displayed in a capmap, a d conformational heatmap that depicts the distribution of motif abundance and overrepresentation across all loop geometries by projecting these quantities onto the structural exemplars generated by clustering. the capmap reveals the loop conformations most favored by a motif. actual structures from the clusters corresponding to these favored conformations are then examined in a structure browser to characterize the side chain interaction associated with the motif. this work identifies a 'toolkit' of side chain motifs which are good candidates for use in the design of synthetic helix-terminal loops with specific desired geometries, because they are used in nature to support these geometries. highlights of the analysis include determinations of the favored loop geometries for the asx/st motifs, capping boxes, big boxes, and other previously known and unknown hydrophobic, electrostatic, h-bond, and pi-stacking interactions. a goal of future work is to make these results available in a structurally-addressable database that would enable researchers to immediately retrieve the side chain interactions most compatible with a desired loop geometry. generation of fluorescent protein-tagged gp mutants to analyze the intracellular distribution of hiv- envelope protein shuhei nakane , zene matsuda green earth research center, green earth institute co., ltd., res ctr for asian infect dis, inst of med sci, the univ of tokyo, lab of struct virol and immunol, institute of biophysics, cas hiv- is a causative enveloped virus of aids. its envelope protein (env) has two non-covalently associated subunits, gp and gp , which are proteolytically processed from a gp precursor. the gp subunit is a surface protein and gp is a transmembrane protein. the gp and gp subunits are responsible for the receptor recognition and membrane fusion, respectively. the cytoplasmic tail (ct) of gp is about amino acids long and is believed to play a critical role in intracellular trafficking of env. to visualize dynamic trafficking, the c-terminus of gp has been tagged with fluorescent proteins such as gfp. however, tagging of ct may cause a concern to affect the interactions between the ct and cellular proteins that are involved in intracellular trafficking. to avoid this problem, here we tried to insert gfpopt, a gfp variant, into five variable regions of gp . we have analyzed the phenotypes of env mutants, such as the cell surface expression, processing of gp , membrane fusion activity, and virion incorporation. among variable regions of gp , the v region was most sensitive to insertion. v /v region was less sensitive than v . consistent with the recently revealed structure, exteriorly located v and v were highly tolerant to insertion. we used the mutant with the gfp insertion in the v region to analyze the intracellular distribution of env with and without ct. we found that deletion of ct increased the presence of vesicles colocalized with late endosome markers. this is consistent with the hypothesis that the ct region contains a motif regulating intracellular trafficking. our results showed that env with gfpopt insertion in its gp subunit is a useful tool for the study of intracellular dynamics of hiv- env. these mutants would also be useful to trace the fate of virus particles during infection. pi- ngs-guided phage panning: comparison to conventional panning strategy buyung santoso , dorain thompson , john nuss , john dwyer phage display is a powerful tool for generating binders to a target protein. multiple rounds of panning with conventional phage display strategies typically result in a number of hits, which are then individually screened using in vitro assays. clones screened at this stage are a combination of specific binders, sequences that are selected due to amplification bias, and non-specific binders. if the number of specific clones is low relative to the non-specific sequences, a larger number of clones have to be screened to ensure sufficient diversity of early leads. with the advent of next generation sequencing (ngs) technology, we aim to test whether we can increase the diversity of specific hits and decrease the number of non-specific sequences. in our experiment, four rounds of conventional panning produced ten peptide binders to target protein. ngs analysis after two rounds of panning was done in parallel, yielding more than ten thousand sequences, ranked by abundance. all ten binders from conventional panning were found in the top most abundant ngs hits. more importantly, additional hits were found in ngs analysis but not in conventional panning, highlighting this strategy as a promising alternative for hit discovery with the significant upside of more diverse and higher affinity leads. numerous processes in pharmaceutical development, including construct screening, structural genomics, protein engineering and expression optimization among others, require the use of higher throughput plasmid dna purification. the majority of issues encountered in mini, midi, and maxiprep purification kits involve flocculate removal following alkaline lysis, and there is currently no easy way to produce large amounts of plasmid dna without the addition of complicated and time consuming clarification steps. the existence of a hassle-free automated system that is not restricted by sample size would significantly help in cutting time and costs during the initial processing steps of plasmid purification. the autoplasmid mea instrument provides a fully automated solution to traditional problems faced in plasmid purifications, allowing mini, midi, and maxiprep plasmid purifications to be performed on a single instrument. the data presented here on plasmid yield, purity, and suitability for sequencing and transfection/transformation illustrate a new strategy for automated plasmid preps. by eliminating traditional clarification methods, cell culture volumes between - ml can be processed leading to yields ranging from - lg. this flexible system was developed in order to satisfy a wide variety of concentration and yield requirement, while eliminating the time consuming steps previously needed to obtain similar results. the ability to perform fully automated mini, midi, and maxi plasmid preps on one instrument allows for a customized all-in-one purification system that is not restricted by traditional clarification methods, eliminating manual intervention, and streamlining the purification process. the modular nature of protein architectures suggests that proteins have evolved through duplication and fusion to give rise to modular, often symmetric forms, which later diversified under the influence of evolutionary pressure. we have developed a computational protein design method termed reverse engineer evolution (re volution) to create symmetrically self-assembling protein building blocks. we have used this method to design a perfectly symmetric b-propeller protein called pizza. subsequently, we have engineered a metal binding site into this pizza protein. this new pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. the designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. two copies of the trimer associate in the presence of cadmium chloride in solution, and high resolution x-ray crystallographic analysis reveals a nano-crystal of cadmium chloride, sandwiched between two trimers of the protein. this nano-crystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. our results indicate the feasibility of using rationally-designed symmetrical proteins to biomineralize nano-crystals with applications in bionanotechnology. bacillus licheniformis trehalose- -phosphate hydrolase structures suggest keys to substrate specificity chwan-deng hsiao , min-guan lin , long-liu lin , yuh-ju sun institute of molecular biology, academia sinica, department of applied chemistry, national chiayi university, depaertment of life science, national tsing hua university trehalose- -phosphate hydrolase (trea) of the glycoside hydrolase family (gh ) catalyzes the hydrolysis of trehalose- -phosphate (t p) to yield glucose and glucose- -phosphate. products of this reaction can be further metabolized by the energy-generating glycolytic pathway. here we present the crystal structures of bacillus licheniformis trea (bltrea) and its r q mutant complexed with p-nitrophenyl-a-d-glucopyranoside (r q/ ppng) at . Å and . Å resolution, respectively. the overall structure of bltrea is similar to other gh family enzymes. however, detailed structural comparisons revealed that the catalytic groove of bltrea contains a long loop adopting a different conformation from those of gh family members. unlike the homologous regions of bacillus cereus oligo- , -glucosidase (bcogl) and erwinia rhapontici isomaltulose synthase (nx- ), the active site surface potential of bltrea exhibits a largely positive charge, contributed by the four basic residues his , his , lys and lys . mutations at these residues resulted in significant decreases of bltrea enzymatic activity. strikingly, a hhlk motif and the lys residue played critical roles in bltrea substrate discrimination. crystal structure of engineered lrrtm synaptic adhesion molecule and a model for neurexin binding anja paatero , katja rosti , alexander shkumatov , cecilia brunello , kai kysenius , prosanta singha , henri huttunen , tommi kajander institute of biotechnology, university of helsinki, helsinki, finland, dept of pharmaceutical and pharmacological sciences, ku leuven, leuven, belgium, neuroscience center, university of helsinki synaptic adhesion molecules are key components in the development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of the chemical synapses. several families of neuronal adhesion molecules have been identified such as ncams, neurexins and neuroligins, and in particular recently several leucine rich repeat protein families, e.g. netrin g-ligands, slitrks and lrrtms. the lrrtms form a family of four proteins. they have been implicated in excitatory glutamatergic synapse function, and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. in addition, lrrtm and lrrtm have been found to be ligands for heparan sulphate proteoglycans. we report here the crystal structure of a stability-engineered mouse lrrtm , with a tm c higher than the wild type protein, while retaining its function. we localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation and prior information on the ligand interaction with neurexins, allowing us to propose a tentative model for lrrtm:neurexin interaction compex. cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapse-like contacts. small angle x-ray scattering data suggests that the wild type protein forms transient dimers, which may have importance for the function. the structural and functional data presented here provide the first structure of an lrrtm protein, and a model for molecular mechanism of lrrtm function in adhesion. computational design of phenylalanine binder olga khersonsky , gil benezer , sarel fleishman recently, abdesign algorithm was developed in our lab for de novo design of antibodies ( ). it is guided by natural conformations and sequences, and exploits the modular nature of antibodies to abstract generate an immense space of conformations, which can be used as scaffolds for design of stable highaffinity binders. we have used abdesign to design a binder of phenylalanine. , antibody scaffolds were obtained by splicing h and l fragments into a template (pdb id brr), and subsequent optimization of vh and vl orientation. phenylalanine binding site, based on native phenylalanine binders, was introduced into the scaffolds with rosettamatch ( ), and the sequences were subsequently optimized by rosetta enzyme design protocol ( ) . designs were experimentally tested by yeast display for binding of biotinylated phenylalanine ligand. several designs were found to bind the ligand, and we plan to further characterize this affinity and improve it using directed evolution techniques. in collaboration with the group of prof. johnsonn, the resulting phenylalanine binder will be incorporated in a bio-luminescent (lucid) sensor for phenylalanine ( ) . phenylalanine monitoring device would be of primary importance for patients with phenylketonuria, a genetic disease with phenylalanine metabolism problem. cold-adapted enzymes are interesting because of their higher catalytic activity compared to mesophilic and thermophilic homologues. alkaline phosphatase (ap) from a psychrophilic vibrio marine bacteria (vap) has an unusual large surface loop that extends from each of its monomers to stabilize a homodimeric structure ( ). in many cold-adapted enzymes, the loop regions are longer compared to proteins of mesophilic organisms and our aim was to study the functional and structural role of this loop. three substitutions (r l, y f and f y) were introduced within the large surface loop as directed by microsecond molecular dynamics (md) simulations. with the r l mutation, two hydrogen bonds were broken that connect the loop to residues on the adjacent subunit, and further two hydrogen bonds broken with the adjacent q . as a consequence, r l displayed a % higher kcat compared with wild-type and a slight decrease in the km value. overall, the catalytic efficient improved by %. the global heat stability (tm) and the active site sensitivity to heat (t %) were reduced by c and c, respectively. md simulations showed that hydrogen bonds to arg are important for longrange communication to the active site. certain rotamers of two important residues in the catalytic site, ser and arg , were favored, presumably toward states more competent for catalysis upon the replacement of arg with leu. in the y f variant, removal of one hydrogen bond between the loop and the other subunit caused a small drop in stability parameters, whereas both kcat and km were reduced by about half, giving similar kinetic efficiency (kcat/km) to that of wild-type. finally, we changed a residue at the root of the large loop (f y) such that one new intersubunit hydrogen bond could form. this variant maintained the wild-type characteristics. in conclusion, removing hydrogen bonds connecting the major loop of one subunit to the protein surface of the other subunit in vap produced higher catalytic activity and this shows functional connections between loop mobility and the active site. our study also demonstrates that interactions between residues in the large disordered loop and the opposite subunit in the dimeric vap are determinants of its stability. thus, we managed to show that loosening of interface contacts between the two vap subunits by replacement of crucial residues provides a way to orchestrate structural and kinetic dynamics in a productive way. the de novo design of artificial proteins arises as a stringent test of our understanding of the relationship between sequence, structure, and function. examples include the design of a four a-helix bundle, a new protein topology called top , and a series of artificial (ba) -barrels called octarellins. however, de novo design has proven difficult for larger proteins with more than amino acids. here we present two methods to generate the backbone and to perform the de novo design of (ba) -barrel proteins through the use of the software rosetta; both have different advantages and limitations. the first method for generating the backbone is knowledge-based, with a first analysis of a non-redundant database of natural (ba) -barrel proteins in order to obtain statistical analysis on preferred secondary structure element length and amino acidic propensities. with this information we use the rosetta cm software to create more than models which are then ranked in term of rosetta energy. the second method is performed with the parametricdesign package of rosetta, in which only geometrical information are requested (number of strands and helices, radius of the b-and a-barrels, degree of inclination, orientation of the side chains, among others). both methods contain a step of loop refinement and multiple steps of sequence design with the package rosetta design, in order to find low scoring amino acid sequences for each of the starting backbone conformations. thousands of models will be generated by both methods and then analyzed in term of sequence similarity, secondary and tertiary structure prediction, and stability by molecular dynamics simulations. the best candidate sequences will be selected for the experimental verification. in order to identify a putative successfully design, we added a metal binding site during the design step. all the proteins will be expressed in e. coli. the solubility of the designed proteins inside bacteria will be determined thanks to the fusion to green fluorescence protein (gfp). solubility, stability, secondary structure, and cooperativity of folding will be assessed for each protein before determination of their three-dimensional structure. construction of protein capsule possessing drugs controlled release ability shota shimizu , masatoshi nakatsuji , keisuke yamaguchi , yuya sano , yuya miyamoto , takashi inui most compounds that exhibit anti-tumor activities are water-insoluble, thus limiting their clinical use. chemical modification of these compounds and the use of solubilizing agents such as organic solvents, surfactants and ph modifiers improve their solubility. however, chemical modification of compounds decreases their potency, and the use of solubilizing agents causes toxicity in many cases. thus, drug delivery systems (dds) for poorly water-soluble anti-tumor drugs which exploit liposomes, cyclodextrins, and lipid nanoparticles have been studied intensely. in these dds, the controlled release of drugs from the delivery vehicle is one of the most important functions. selective release in target cells leads to adequate therapeutic efficacy with few side effects. in our laboratory, we have already demonstrated that lipocalin-type prostaglandin d synthase (l-pgds), an intravital transporter protein, is a novel and valid drug delivery vehicle for sn- , a poorly water-soluble anti-tumor drug. in this study, we generated l-pgds-based protein capsules with a controlled-release function by introducing a disulfide bond into the upper part of the drug-binding cavity of l-pgds. the intracellular concentration of glutathione ( . mm) is known to be substantially higher than the extracellular concentration ( mm). therefore, it is expected that in the extracellular oxidative environment the disulfide bonds in the protein capsule remain stable, avoiding premature release of the internal drugs during circulation of blood, after reaching the target cells, the disulfide bonds are cleaved in the intracellular redox-environment, and then the internal drugs are released. we generated three kinds of protein capsules which have disulfide bonds in different positions, w c/w c, k c/h c, k c/w c, based on tertiary structure information of human l-pgds (pdb id: o y). firstly, we performed circular dichroism (cd) measurements to confirm the structure of each capsule. the cd spectra of three protein capsules were similar to that of wild-type l-pgds in the far-uv region. therefore, the secondary structures of three protein capsules were not changed from wild-type l-pgds by introducing the mutations. quantitative analysis of the free thiol group in the protein capsule by dtnb assay revealed that the intermolecular disulfide bond was formed by h o -induced oxidation and cleaved by dithiothreitol-induced reduction. in addition, to investigate the solubility of sn- in the presence of protein capsules, we mixed the protein capsule of reduced-form with sn- suspension, and stirred at c for hours. the resulting concentrations of sn- in pbs with mm w c/w c, k c/h c, and k c/w c were mm, mm, and mm, respectively. these values were approximately -fold higher than without protein capsules. sds-page analysis showed that the bond formation decreased in a time-dependent manner, and that new intermolecular disulfide bond was not formed in the protein capsules after hours' incubation. from the above, we succeeded in generating drug delivery vehicles possessing openable and closable lids that are responsive in an oxidation-reduction environment. takaaki miyamoto , mai kuribayashi , satoshi nagao , yasuhito shomura , yoshiki higuchi , , shun hirota graduate school of materials science, nara institutte of science and technology, graduate school of science and engineering, ibaraki university, department of life science, graduate school of life science, university of hyogo, domain swapping has been of interest as a mechanism of protein oligomerization, where a secondary structural region or a domain of one protein molecule is replaced with the corresponding region or domain of another protein molecule. we have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. , in this study, we show that a four-helix bundle protein cyt cb , in which the heme of cyt b is attached to the protein moiety by insertion of two cys residues, forms a domain-swapped dimer. dimeric cyt cb was more stable than dimeric cyt b at c, showing that attachment of the heme to the protein moiety stabilizes the domain-swapped structure. absorption and cd spectra of dimeric cyt cb were similar to the corresponding spectra of the monomer, showing that the active site and secondary structures were similar between the dimer and monomer. the redox potential of dimeric cyt cb was also similar to that of its monomer. the dissociation temperature of dimeric cyt cb was c, and its dh on dissociation to monomers was . kcal/mol (per dimer). according to x-ray crystallographic analysis, dimeric cyt cb exhibited a domain-swapped structure, where the two helices in the n-terminal region (helices and ) in a protomer and the other two helices in the c-terminal region (helices and ) of the other protomer interacted between each other. the heme coordination structure of the dimer was similar to that of the monomer. we have previously shown that domain-swapped oligomers of horse cyt c form through intermolecular hydrophobic interaction between the n-and c-terminal a-helices at the early stage of folding. it has been suggested that helices and form first at the initial stage of folding in wild-type apo cyt b . therefore, we propose that cyt cb forms a domain-swapped dimer when helices and interact intermolecularly at the initial stage of folding, whereas the intramolecular interaction of helices and results in formation of a monomer. a highly buried and conserved tryptophan residue close to the dimer interface in a cold-adapted phosphatase is phosphorescent and important for activity. jens g. hj€ orleifsson and bjarni asgeirsson. department of biochemistry, science institute, university of iceland, dunhagi , reykjavik, iceland. alkaline phosphatase (ap) from vibrio g - is a cold-adapted dimeric enzyme with one of the highest catalytic efficiency reported for known aps. it contains five intrinsic tryptophan (trp) residues and one additional trp located on the c-terminal streptag used for expression and purification. in this study, we made several single trp-substitutions to determine the role of each of the trp in the fluorescence emission spectrum. we also determined their solvent exposure by acrylamide fluorescence quenching. the results indicate that trp , trp and trp are mostly responsible for the fluorescence emission. quenching experiments with acrylamide indicated that all the trp residues were about equally accessible for quenching, except trp which was shown to be highly buried in the core of the protein. interestingly, the enzyme was found to be highly phosphorescent at c, having two phosphorescence lifetimes. the longer lifetime is due to trp . trp is located close to the dimer interface and points towards a helix in the active site where his binds an active-site zinc ion. in other aps, an aromatic amino acid is conserved in the location occupied by the trp residue. in most cases for cold-adapted aps it is indeed a trp. interestingly, the mutation of the trp to a phenylalanine affected both stability and activity of the enzyme. kcat/km was -fold lower than for wild-type. overall, this study reveals that trp can be used as a phosphorescent probe of local dynamics and could possibly also serve to study the dimer-monomer equilibrium due to proximity to the dimer interface, an area clearly crucial for enzyme activity and stability. modulating protein-protein interaction with a molecular tether helen farrants , oliver hantschel , kai johnsson ecole polytechnique f ed erale de lausanne (epfl) high-affinity scaffolds for protein-protein interactions, such as monobodies and darpins can be engineered in vitro to bind to protein targets. we speculate that the affinity for the target protein can be modulated by incorporating these evolved scaffolds and a synthetic intramolecular tether into protein switches, in a protein construct of composed of snap-tag, a monobody and a circular permutated dihydrofolate reductase. the tether, attached to the construct via snap-tag, was composed of a linker and trimethoprim, which interacts reversibly with the circular permutated dihydrofolate reductase. we have investigated the affinity between the n-sh domain of the phosphatase shp and an evolved monobody in such a protein construct using a fret assay. when the intramolecular tether was bound the circular permutated dihydrofolate reductase ("closed" conformation), there was an increase in the affinity of the construct to the target n-sh . in the presence of a small molecule competitor ("open" conformation) the affinity of the monobody construct to its target was reverted to the value reported in the literature. the intramolecular tether in these protein constructs combined with engineered scaffolds for protein-protein interactions may be a general approach towards protein switches. because most proteins are long polymers of amino acids with twenty or more chemically-distinct sidechains, there are an enormous number of potential protein sequences. here, we report the construction of biologically active proteins with minimal chemical diversity. transmembrane domains of proteins can specifically interact with other transmembrane domains to modulate the folding, oligomerization, and function of transmembrane proteins. for example, the bovine papillomavirus e protein is a -amino acid transmembrane protein that transforms fibroblasts to tumorigenicity by binding directly to the transmembrane domain of the platelet-derived growth factor b receptor (pdgfbr), resulting in ligandindependent receptor activation and cell transformation. these studies showed that a free-standing transmembrane domain could fold properly in cells and act in trans to modulate the activity of a larger transmembrane protein target. because of the relative chemical simplicity of transmembrane domains and this ability to act even when not linked to more complex soluble protein domains, we reasoned that short transmembrane proteins could be used to define the minimal chemical diversity sufficient to construct biologically active proteins. to accomplish this, we infected cultured mouse cells with a retroviral library expressing -amino acid proteins consisting of an initiating methionine followed by a randomized sequence of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a single methyl group, and selected rare proteins with transforming activity. we isolated numerous proteins consisting of diverse sequences of leucine and isoleucine that cause morphologic transformation, escape from contact inhibition and focus formation, and growth factor independence. genetic and biochemical analysis of these proteins indicate that like e they interact with the transmembrane domain of the pdgfbr to specifically activate the receptor and transform cells. mutational analysis of individual proteins identified specific leucines and isoleucines required for transforming activity, and insertion of a single isoleucine at a particular position in a stretch of leucines is sufficient for activity. these proteins identify the minimal chemical diversity required to generate a biologically active protein and have important implications for biochemistry, protein evolution, protein engineering and synthetic biology. yusuke azuma , donald hilvert virus-like particles that are precisely loaded with functional cargo are an important tool to study the effect of spatial confinement and create novel entities with application in biotechnology and medicine. by genetic fusion to a positively supercharged green fluorescent protein (gfp( )), an enzyme retroaldolase (ra) was efficiently targeted to the negatively charged lumen of an engineered protein cage, aquifex aeolicus lumazine synthase variant (aals- ). the encapsulation is quantitative under mild aqueous condition up to a mixing ratio of guest enzymes per host cages. the chromophoric tag is used for precisely quantifying the enzyme concentration, which allows detailed characterization of the effect of encapsulation on the enzyme activity. the generality of the encapsulation system was examined with structurally different enzymes. introduction and purpose: in the immune system, high affinity antibodies are generated by selection of b cells activated by antigen-stimulation followed by additional optimization through somatic hyper mutation of antibody genes. in the artificial antibody libraries, such as phage libraries, selection of specific antibody clones from the library is performed by in vitro selection process called biopanning and the subsequent binding screening. however, in spite of high efficiency of enrichment in biopanning, there is a possibility that we overlook the minor antigen-specific clones in the screening because of the limitation of the number of clones employed for screening. in recent years, high-throughput analysis of dna sequences by the next-generation sequencer (ngs) has become available not only for genomic analysis of organisms but also repertoire analysis of antibodies. in this presentation, we report the successful isolation of a variety of antigen specific antibodies from patients-derived antibody phage library by a combination method of high throughput sequence analysis on ngs and biopanning. method: we constructed two kinds of human single chain fv (scfv) antibody libraries from pooled mrna of five cancer patients and of a wheat allergy patient, respectively. after biopanning against a cancer antigen or wheat allergy antigen "gluten", the phagemid vector dna prepared from the pooled phages before or after biopanning was used for pcr amplifications of vh genes, adding the index and adapter sequences for ngs analysis. the high throughput sequencing was performed on miseq (illumina) using miseq reagent kits v . after discarding the short sequences and low quality data, '-and '-reading sequences were unified by a merge program. the frequencies (%) of all vh sequences were evaluated using a program based on usearch . clustering software and the changes of the frequency (%) of each sequence between before and after panning were assigned as amplification rate. results and discussion: vh sequences at each round of pooled phages after biopanning against cancer antigen were analyzed on ngs. after three rounds of biopanning, three clusters of antibody sequences were specifically enriched suggesting these are specific binders. to check this, scfv gene were regenerated by pcr using h-cdr specific primers and scfv-displaying phages reconstructed were subjected to binding analysis. all three phages showed a clear specific binding to cancer antigen in elisa. subsequently, to test the usefulness of this method, we applied it to identify allergen-specific scfv from allergy patient-derived antibody phage library. the phylogenetic tree analysis of vh sequences which showed the amplification rate higher than . by a single round of biopanning elucidated total eleven clusters of vh sequences. the vh sequences in the two clusters with the highest amplification factor were selected and the regenerated scfv-displayed phages were tested for binding analysis. the prepared scfvdisplayed phages and also scfv proteins showed a clear binding ability to allergen. thus, it is suggested that the analytical method of vh sequences on ngs before and after biopanning is very useful to isolate a variety of disease related antigen-specific novel antibodies quickly with high degree of certainty. biochemical analysis of the recognition helix of z-dna binding proteins: roles in conformational specificity yang-gyun kim , xu zheng , so-young park conversion of right-handed b-dna into left-handed z-dna is one of the dramatic structural transitions in biological processes including gene regulation and chromatin remodeling. z-dna binding motif, zalpha (za), was first discovered from human adar . subsequently, with sequence and structure similarity to the hzaadar , families of proteins including viral e l, interferon-induced protein dai (zbp ) and pkz has been identified to have za domain(s). interestingly, the za domain of the e l protein from vaccinia virus (vvzae l) was confirmed to have the ability of z-dna-binding, but it does not have the b-to-z conversion activity. here, we showed that the replacement of the a -helix of vvzae l (vvzae l-a ) with that of hzaadar results in acquiring the ability to converting b-dna to z-dna. the detailed biochemical analysis of the a -helix mutants of vvzae l further suggested that the contribution of positively charged residues in the c-terminal part of the a -helix is crucial during the b-to-z transition. in addition, hydrophobic residues of the n-terminal part of the vvzae l-a also influence on the b-to-z conversion activity, possibly through forming a tightly-packed structure. in conclusion, our results revealed the previously-unknown contribution of amino acid residues existed in the a -helix of the za domains to the b-to-z conversion. moreover, it strongly implies that such residues may play important roles in initiating conformational changes of dna structure during the b-to-z conversion event. the ability of switching the activity of proteins at will is of great interest from an application point of view. one promising approach utilizes a protein modification with an organic photochromic molecule. linking two protein side chains with the photochrome that undergoes a light induced conformational change, protein secondary and tertiary structure can be stabilized or destabilized and thus the structure dependent activity can be switched "on" and "off" by light irradiation. for this the photochrome must fulfil several requirements. foremost, it must possess two states of comparable stability that differ significantly in their geometry. it must further be water soluble and non-toxic, and should not experience fatigue phenomena upon multiple irradiations. there are two classes of molecules that fulfil those requirements: azobenzenes and spiropyrans. we are pursuing two different strategies for the design of photoswitchable proteins. in the first approach we attach an azobenzene compounds to side chains of the alpha-helical antifreeze protein type i. the end to end distance of the photochromic molecule is sterically compatible with the folded helix only in one form, photoisomerization therefore switches the folding state between an active helical state and an inactive unfolded form. in a second, more general approach we use the trp-cage domain as a switching unit. the trp-cage is the smallest known folded protein ( amino acids). its folding is induced by hydrophobic interactions of a tryptophan side chain in a short helical segment. after modification with a photochromic molecule in appropriate positions, its structure is rendered sensitive to the state of the chromophore. by creating protein chimera of such a trp-cage and biologically active peptides with helical propensity, we aim at conferring the light-dependent fold of the cage to the attached peptide moiety. salt-bridges are electrostatic interactions between groups of opposite charges. net interaction energy (ddgnet) of a salt-bridge is partitioned into bridge (ddgbrd), desolvation (ddgdsolv) and protein (ddgprot) energy-terms of which estimation of ddgdsolv and ddgprot are only possible by computational means. thus, general purpose poisson-boltzmann equation solver: "delphi" (in commercial package of insight-ii) and "apbs" (open-source) are popularly used to determine these energy-terms. nevertheless, the computation-method is highly involved one than other uses of these solvers. moreover, protein-specific saltbridges, grid-points, center, hydrophobic-isosteres-mediated mutation-files of original charge-radius file and others are to be worked out prior to the computation. this might answer as to why only limited numbers of structure files ( % of crystal-structure-database) are worked out till date. at this juncture, an efficient fully automated all-in-one-procedure that could analyze large dataset in a single run would be useful. to the best of our knowledge, such procedure is truly lacking in public domain. at this end, our fully automated all-in-one procedure: adsetmeas (available freely at http://sourceforge.net/projects/adsetmeas/along with detailed documentation) uses "apbs" method to compute component as well as net energy-terms of salt-bridges and redirect compact output in excelformat. further, micro-environments of salt-bridges are also been reported based on the presence of polar, dipolar, acidic, basic and hydrophobic side-chains in their proximity. the procedure provides versatility to users in choosing a] model for computation of energy-terms to-date available in the literature and b] method (default or advanced) for parametric optimization in "apbs" calculations. it works in unix like environment including cygwin. it processes all proteins present in the working directory with any number of salt-bridges in them. a pre-released version of the procedure was successfully applied for energy-terms on salt-bridges from halophilic proteins. overall, our adsetmeas provides intricate details on salt-bridge energetic from crystal structures and find application in the field of computational structural biology. these and other results will be discussed in the conference. next generation analgesics -targeting ion channels with antibody-drug conjugates (adcs) anna wojciechowska-bason , clare jones , chris lloyd postdoctoral fellow, adpe, medimmune, cambridge, ria, medimmune, cambridge, adpe ion channels are common targets for chronic pain therapies. small molecule analgesics are widely used therapeutically, but due to poor specificity they often cause a wide range of side effects. as a result, efficacy of existing treatments is very limited. we believe that to achieve the required specificity and efficacy, a novel and innovative approach is required that would combine the potency of the small molecule with the selectivity of an antibody. therefore, we propose to apply antibody-drug conjugates (adcs) to deliver small molecules or peptides to ion channels in order to specifically modulate pain signalling pathways. voltage-gated sodium channel nav . has a well characterised role in the perception of pain. here we present the activity of the peptide huwentoxin-iv (hwtx-iv) and small molecule inhibitors ptc-a, ptc-b and ptc-c on voltage gated sodium channels nav . and nav . . in novel findings, we report that these inhibitors show little selectivity between the voltage-gated sodium channel family members, nav . and nav . , and that the ic values and the impact on channel biophysics (voltage-dependence of activation and fast inactivation) of the inhibitors are largely similar for both channel types. therefore, the use of hwtx-iv and other small molecule inhibitors of nav . for pain therapy could be dose-limited due to side effects mediated by the inhibition of channel nav . . in conclusion, we propose that hwtx-iv and the investigated small molecule inhibitors could be used for the treatment of pain as part of a nav . antibody-drug conjugate (nav . -adc), establishing nav . specificity and minimising side effects. maria antonietta carillo , daniel varon silva malaria is one of the most infectious diseases caused by plasmodium species parasites. the merozoite surface protein (msp ) is the most abundant protein on the surface of the plasmodium species merozoite stage, which plays an important role during the erythrocytes invasion process [ ] . msp is synthesized as a -kda glycosylphosphatidylinositol (gpi) anchored protein precursor which is processed at the end of the schizogony into four different fragments. the primary processing step produces a complex of four fragments that are present on the merozoite surface. the secondary processing step at erythrocytes invasion results in the detaching of the complex from the surface, except for the cterminal -kda domain (msp ), which remains anchored to the parasite surface by the gpi moiety. in human malarial infections, the gpi is considered to be a toxin that causes the expression of various host genes and induces a pro-inflammatory immune response, making it a valuable candidate for the development of anti-malarial drugs. in order to study the function of the gpi and evaluate the effects, msp fragment has been expressed, purified and anchored to the synthetic gpi molecule using protein trans-splicing strategy based on the split intein method [ ] . the role of the gpi moiety will be studied through protein folding experiments and the effect of the anchored protein will be evaluated in vitro in order to understand the function of the gpis. assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, john love san diego state university, san diego state university, assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, and john j. love san diego state university the ubiquitin proteasome system (ups) is a complex system composed of multiple structural and functional elements that play key roles in cellular processes such as signal transduction, cell cycle regulation, apoptosis, and protein degradation. proteins destined for degradation are first tagged with the protein, ubiquitin, which is covalently attached to internal lysine residues. once the target has be degraded by the proteasome; the enzyme ubiquitin carboxy hydrolase l (uch-l ) is believed to prepare ubiquitin for additional rounds of ubiquitination by cleaving small peptides and chemical adducts from the ubiquitin c-terminus. previously in our laboratory, protein substrates of uch-l were engineered and used to characterize uch-l substrate selectivity. the engineered substrates consisted of n-terminal monoubiquitinated test variants derived from streptococcal protein g (protein gb ) and staphylococcal protein a (spab). the thermal denaturation temperatures (tm) of the fusion proteins were measured using circular dichroism and span a range of over c. more importantly, the rate of hydrolysis for the fusion proteins is demonstrated to be directly correlated to the tm of the test variant fused to the c-terminus of ubiquitin. previously, the engineered substrates were designed to emulate natural ubiquitin fusions and thus did not contain any 'linker' residues between the c-terminus of ubiquitin and the n-terminus of the test protein. to explore the effects of linker length on uch-l hydrolysis we are engineering new uch-l substrates that contain an unstructured amino acid linker between ubiquitin and the test protein. to further explore the catalytic efficiency of uch-l we will revisit diubiquitin (ub-ub), which is not hydrolyzed by uch-l , and will make mutations in the hopes of generating a hydrolysable substrate. using rational design, the new variants will be engineered to destabilize the c-terminal ubiquitin to determine if this results in hydrolysis of the new ub-ub construct. the thermal stability of these new fusion protein substrates will be measured using circular dichroism spectroscopy (cd) and uch-l hydrolysis rates will be characterized using existing assays. our goal is to continue the use of engineered substrates to further explore the catalytic properties of uch-l activity and the potential role in protein trafficking and degradation within living cells. we present a biophysical study of a suite of helical proteins that have been modified to contain and -amino acid additions on their termini that impart increased resistance to degradation in e. coli abstract recombinant expression systems. the b domain of staphylococcal protein a (ab) and the homeobox dna-binding domain from d. melanogaster engrailed (en) are small -helix bundles. these domains do not appreciably accumulate in the e. coli bl (de ) cytoplasm when expression in a pet vector is chemically induced. this is likely due to host protein degradation/recycling factors that function to efficiently degrade these two proteins. addition of sequences encoding either of two amino-terminal beta-hairpins to either the n-or c-terminus of ab and en results in the accumulation of large amounts of these new chimeric proteins. additionally, destabilization of the ab or en sequence does not abolish the expression enhancement effect of the beta-hairpin addition. we have investigated the biophysical origins and effects of the beta-hairpin additions using circular dichroism (cd) spectroscopy, and have determined that the added sequence does not significantly perturb the secondary structure of ab or en, nor does it significantly influence the unfolding temperature (tm). while investigation into the origin of the accumulation effect is ongoing, we hypothesize that the addition of the sequence is disruptive to recognition events in the native protein degradation machinery in e. coli. thus, this approach represents both a biotechnological tool for expressing helical peptides recalcitrant to expression, as well as a system well-suited to probing mechanisms of protein recycling and homeostasis. a special class of these proteins are lipidated proteins containing a glycosylphosphatidylinositol (gpi) glycolipid moiety at the c-terminus. the lipid chains of the gpi anchor molecule are responsible for the membrane association of the attached protein. a unique feature of gpi-anchored proteins is that after isolation they can be reinserted into the membrane of recipient cells with the retention of the biological function. accordingly, the exogenous introduction of fluorescent gpi-anchored protein analogues into cell membranes is a useful method for visualizing the cellular traffic of membrane associated proteins and for engineering cell surfaces. we have recently shown that cholesterol can be applied for anchoring proteins to the plasma membrane of live cells without perturbing the membrane. in order to introduce proteins containing covalent modifications that are not genetically encoded, an enzymatic method was considered and fused with the c-terminal cholesterylation method. the usefulness of the method is demonstrated via the preparation of multimeric model proteins of kda monomers, that is an appropriate representation of the ligation of domain size proteins. transmembrane domain dimerization drives p ntr partitioning to lipid rafts irmina garc ıa carpio , marc¸al vilar sociedad de biof ısica de españa. sbe p neurotrophin receptor (p ntr), is best known for its role in mediating neuron cell death during development or after injury but it also regulates cell proliferation, axon guidance or survival. the key to understand its signaling could rely in its structure and conformational states. it has been described that p forms disulfide-linked dimmers through the cys in the transmembrane domain which are essential for its ngf mediated signaling. previous studies have shown that p is present in lipid rafts, where it interacts with intracellular adaptors to activate different signaling pathways. we design several p mutants in the tm domain that impairs dimerization and study the role of tm domain dimerization in lipid rafts recruitment. our analysis suggests that p tm domain dimerization influences lipid raft partitioning. these results could be a key role to understand its signaling and processing pi- bioluminescent sensor proteins for therapeutic drug monitoring of the monoclonal antibody cetuximab martijn van rosmalen , remco arts , brian janssen , natalie hendrikse , dave wanders , maarten merkx therapeutic drug monitoring (tdm) -adapting the drug dosage scheme to the individual patient's pharmacokinetic and pharmacodynamic characteristics -is still uncommon for therapeutic monoclonal antibodies, despite preliminary studies showing its potential benefits. one of the factors impairing tdm implementation is the lack of equipment and trained personnel to regularly measure drug concentrations in patients receiving treatment. point-of-care diagnostic devices which could be used by patients themselves or by their general practitioners would greatly advance the feasibility of tdm. here we present a biosensor for the therapeutic monoclonal antibody cetuximab. we developed a series of cyclic peptides that specifically recognize cetuximab, covering a fourfold range of affinities, and incorporated these cyclic peptide sequences into a set of luminescent sensor proteins. the sensors translate cetuximab concentrations into a change in emission color that can be read out using a mobile phone camera. together, these sensors can quantify cetuximab levels within the relevant therapeutic concentration range and we propose that they can be used for therapeutic drug monitoring applications. genetically encoded biosensor for cell permeability of inhibitors of the p -hdm interaction silvia scarabelli , thomas vorherr , kai johnsson ecole polytechnique f ed erale de lausanne, the evaluation of the permeability across the cellular membrane is a key step in the development of therapeutics, since it affects the distribution and the efficacy of the latters. reliable and versatile techniques for the determination of structural permeability determinants of molecules and information about the entry kinetics are still missing. we introduced in the past a class of semi-synthetic ratiometric sensor proteins (snifits) that has been shown to be suitable for the measurement of intracellular metabolites concentrations. here we describe a totally genetically encoded sensor based on the snifits modular design for the assessment of the cell permeability of small molecules and peptides inhibitors of the protein-protein interaction between p and hdm . we show that our sensor detects the presence of hdm -binding stapled peptides in vitro, and, when expressed in mammalian cells, it responds to the perfusion of the known small molecule hdm inhibitor nutlin- a. moreover, experiments made with an automated microscope show that the sensor is suitable for measuring and comparing the kinetics of entry of different kinds of inhibitors in the cytosol of living cells. in parallel, we are developing an hcaii-based sensor protein for the sensing of sulfonamides and eventually their peptide derivatives. we show that the sensor responds to the presence of different kinds of hca-inhibitors in vitro and in perfusion experiments. this second sensor would broaden the range of molecules and peptides whose permeability can be studied with our tools beyond the family of the hdm -binders. our sensors overcome the limitations of the already existing techniques for measurements of permeability while offering a simultaneous measurement of the cell permeability and of the binding efficiency of small molecules and peptides of interest. archer: predicting protein function using local structural features. a helpful tool for protein redesign. jaume bonet , javier garcia-garcia , joan planas-iglesias , narcis fernandez-fuentes , baldo oliva structural bioinformatics lab, grib, upf, division of metabolic and vascular health, university of warwick, the advance of high-throughput sequencing methodologies has led to an exponential increase of new protein sequences, a large proportion of which remain unannotated. the gap between the number of known proteins and those with assigned function is increasing. in light of this situation, computational methods to predict the function of proteins have become a valid and necessary strategy. here we present archer, a server that exploits archdb's hierarchy of super-secondary structures to map go and enzyme functions upon protein regions and, thus, infer the function of a protein. the server relies on either the sequence or structure of the protein of interest and returns the mapping of functional subclasses extracted from archdb. moreover, it computes the functional enrichment and significance of each subclass, combines the functional descriptors and predicts the function of the query-protein. combining the functional enrichment analysis of the super-secondary structures with the structural classification of archdb, users can select variants of the target sequence that swap the region of a supersecondary structure by another that putatively fits in the same scaffold minimizing the effect on the global tertiary structure. only variants that modify the predicted function are offered for selection, thus providing a rational, knowledge-based, approach for protein design and functionalization. the archer server is accessible at http://sbi.imim.es/archer. phytochromes are natural photoreceptors known to regulate photosynthesis in plants, fungi and bacteria. phytochromes found in bacteria share common architecture and consist of a pas-gaf-phy photosensory core and a c-terminal output module, responsible for biological function. a bacterial phytochrome, bphp , from rhodopseudomonas palustris undergoes reversible conversion from the farred absorbing state (pfr) to the red-absorbing state (pr) followed by the conformational change upon nm light irradiation. as most of bacterial phytochromes, bphp forms a dimer. it was shown that nm light causes a protomer swapping between the bphp dimers; and likely, the output module is involved in this process. however, the mechanism of the light-induced swapping is poorly studied. we tested an ability of the protomer swapping between bphp dimers using pull-down biochemical assay. for this, strep-tagged bphp was immobilized on strep-tactin sepharose beads in the presence of untagged bphp fused to mruby at different concentrations. after incubation, the proteins were eluted and visualized in sds-gel using a zinc-induced fluorescence assay. an amount of the bound to beads protein was estimated by densitometry. it was found that more than % of heterodimers (streptagged-bphp and bphp -mruby ) form within . h of incubation under nm light at -fold excess of one of the interacting partners. in darkness, the swapping was much slower. in the similar setup we checked the amount of heterodimers after , and min of incubation. no difference was observed for different time points, suggesting that the protomer swapping is relatively fast process. next, a role of the c-terminal effector domain of bphp in the light-induced interaction was studied. for this, kinetics of the pfr-to-pr transition was analysed by measuring of absorbance at nm and nm for full-length bphp and a bphp mutant with the deleted c-terminal domain. while full-length bphp showed the normal pfr-to-pr transition, absorbance of the mutated bphp at nm did not raise. however, nm absorbance changes were similar for both proteins; and surprisingly, the similar dark relaxation kinetics was observed. we propose that the impaired pfr-to-pr transition is caused by restricted pr conformation in the mutant rather than by fast pr-to-pfr relaxation. understanding the mechanisms of the bphp light-induced structural changes and the protomer interaction should advance engineering of bacterial phytochromes into fluorescent probes and optogenetic tools. antibody detection is an integral part of many diagnostic strategies, most crucially so when infectious diseases are involved. currently used assays, such as elisa or spr, enable detection of antibodies in the laboratory with high sensitivity, yet a translation of these technologies to an application outside of the laboratory setting is far from trivial. problematically, the burden of disease for many infectious diseases is carried precisely by those countries where access to laboratory facilities is severely limited. we therefore developed a novel, one-step assay that allows the detection of antibodies directly in solution using a luminescent sensor protein. our strategy is based on the use of a bright luciferase, nanoluc, tethered to a green fluorescent protein (mneongreen) via a semi-flexible linker containing two epitope sequences. crucially, two small helper domains were fused to the protein termini. these domains keep nano-luc and mneongreen in close proximity in the absence of antibody, enabling efficient bioluminescence resonance energy transfer (bret). binding of antibody to the epitopes in the sensor proteins linker domain pulls the bret partners apart, effectively changing the color of emission from green to blue. the assay allowed the detection of picomolar amounts of anti hiv -p antibodies directly in solution, both under optimized buffer conditions and in blood plasma. in principle. the modular sensor architecture should allow detection of any antibody with a well-defined epitope of sufficient affinity. to demonstrate this, the hiv-epitopes were substituted for two ha-tag epitopes, yielding a sensor that enabled the detection of picomolar amounts of anti-ha antibodies. the simple optical readout provided by the sensor system allowed us to record the emitted signal with a conventional mobile phone camera. a simple software application that analyzes the image based on rgb values sufficed to interpret the recorded image vis-a-vis the presence of antibody. bearing in mind the eventually envisioned application in a point-of-care diagnostic setting, this combination of sensor recording and interpretation using nothing more than a mobile phone and a software application holds considerable diagnostic potential. beyond point-of-care diagnosis of infectious diseases, a simple assay to detect and quantify antibodies directly in solution could also have a substantial impact in other fields. antibodies are ubiquitous in biotechnology, and this is reflected by the plethora of potential sensor applications, which range from a role in microfluidic circuits or monitoring the biotechnological production of antibodies, including validation of bispecificity, to veterinary applications, diagnosis of autoimmune diseases and monitoring the success of vaccination campaigns. the continually growing protein data bank (pdb) has been a key resource for general principles of protein structure. for example, parsing structural observations in the pdb into simple geometric descriptors has given rise to statistical energy functions. here we present a novel strategy for mining the pdb on the basis of local tertiary structural motifs (term). we define a term to be the structural fragment that captures all local secondary and tertiary structural environments of a given residue, and query the pdb to obtain quantitative information for each terms. first, we show that by breaking a protein structure into its constituent terms, we can describe its sequence-structure relationship via a new metric we call "structure score." using submissions in recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy -a performance that exceeds leading atomistic statistical energy functions. next, we show that querying terms affected by point mutations enables the quantitative prediction of mutational free energies. our simple approach performs on par with state-of-the-art methods fold-x and popmusic on mutations, and provides superior predictions in certain cases where other methods tend to fail. in all, our results suggest that the data available in the pdb are now sufficient to enable the quantification of much more sophisticated structural observations, such as those associated with entire terms, which should present opportunities for advances in computational structural biology techniques, including structure prediction and design. exploiting natural sequence diversity for protein crystallization sergio mart ınez-rodr ıguez , valeria risso , jos e m sanchez-ruiz , jos e a. gavira , departamento de qu ımica-f ısica, universidad de granada, laboratorio de estudios cristalogr aficos, iact-csic-ugr granada during the last decade, different rational and high-throughput approaches have been successfully applied in the protein crystallography field to widen thejjso-called "protein crystallization bottleneck" [ , ] . despite the enormous efforts carried out by our community, the statistics presented by structural biology consortiums [ ] suggest that so far only the easy-to-pick fruit has been attained; thus, new approaches are necessary to further expand the crystallization limiting step to relevant targets. on the basis of previous hypothesis suggesting that the difficulties found in protein crystallization might be a result of evolutionary negative design [ ] , we have used two different protein engineering approaches exploiting natural sequence diversity using beta-lactamase as toolbox: i) ancestral reconstruction and ii) consensus approach [ ] . both approaches resulted in hyperstable and promiscuous ancestral derivatives. furthermore, our initial crystallization results also suggest that both approaches increased the crystallizability of the resulting enzymes when compared to the extant tem- beta-lactamase. the adipocyte-derived hormone adiponectin has become a key player for the understanding of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. one of its abstract major functions are the insulin sensitizing effects, which are mediated by the activation of ampk, p -mapk and ppara ( ). furthermore adiponectin is involved into glucose regulation and fatty acid oxidation. recently, three adiponectin receptors adipor , adipor and t-cadherin have been described while an unknown fourth receptor is hypothesized ( ) . for only two of them (adipor and adipor ) the signaling transduction via adiponectin has been confirmed ( ). in order to find new binding partners or co-receptors, we cloned and expressed full length adiponectin as a fusion protein with a c-terminal intein and a chitin binding domain (cbd) as well as an n-terminal his -tag. by using the impactsystem, the fusion protein was cleaved to form the corresponding thioester. to separate the starting materials as well as the cleaved intein chitin binding domain, the purification was performed with chitin beads. furthermore, the product was concentrated by ni-nta-affinity chromatography. accordingly, the obtained adiponectin thioester was reacted with a tamra-or a biotin labeled peptide, respectively, to receive the corresponding ligation product. finally the functionalized adiponectin was purified by size exclusion chromatography. further studies will allow screening for interacting molecules in cell and tissue derived samples. departamento de quimica fisica, facultad de ciencias university of granada, dpto. de quimica fisica biologica. instituto de quimica fisica rocasolano, departamento de quimica organica, facultad de ciencias university of granada, rational design of non-natural enzyme activities has proved challenging. here, we report the introduction of catalysis of the kemp elimination (a model of proton abstraction from carbon) in scaffolds corresponding to precambrian nodes in the evolution of the antibiotic resistance protein b-lactamase. we used a single-mutation, minimalist approach based on chemical intuition, and obtained catalysis levels similar to those reported in the literature for computational kemp-eliminase designs involving multiple mutations. remarkably, the approach was unsuccessful when performed on modern b-lactamases. we provide experimental evidence that enhanced conformational flexibility contributes to the success of the minimalist design in the ancestral scaffolds. this work has implications for the understanding of function emergence in protein evolution and demonstrates the potential of ancestral protein resurrection in enzyme engineering and design. exploring the importance of dimerization for dj- function through engineered domain fusions sierra hansen , jiusheng lin , mark wilson parkinson's disease is a progressive neurodegenerative disease that affects approximately . million people worldwide and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. dj- (park ) is one of several genes that are mutated in rare forms of familial parkinsonism. dj- is a dimeric cytoprotective protein that defends against oxidative stress and preserves mitochondrial function. dimerization of dj- is thought to be essential for this function, as some diseaseassociated mutations cause poor folding and disrupt the dj- dimer. however, recent reports suggest that dj- may be functional as a monomer. to test this, we have engineered a non-dissociable dj- dimer that is a fusion of two human dj- domains. this construct cannot dissociate into monomers and thus will provide a stringent test of the importance of monomeric dj- . our engineered construct is modeled on plant dj- homologs, which feature naturally occurring duplicate dj- domains separated by a small ( amino acid) linker region. using x-ray crystallography, we confirmed that this engineered non-dissociable human dj- dimer has identical structure to the naturally occurring dimeric protein. we have investigated the influence of enforced dimerization of the pathogenic effects of the parkinsonian l p and l p mutations. cd spectroscopic analysis reveals that single and double l p mutations in the non-dissociable dj- dimer maintain a higher degree of structure than l p mutations in the native protein. additional characterization of the protective capacity and subcellular trafficking of this non-dissociable dj- dimer is underway. the purification, crystallization and preliminary characterization of sdre from s. aureus the purification, crystallization and preliminary characterization of sdre from s. aureus staphylococcus aureus (s.aureus) is an important human opportunistic pathogen which colonizes about % of the human population persistently [ ] . surface proteins of s.aureus can excretion a kind of sortase, which represents a surface organelle responsible during the pathogenesis of bacterial infection the host circulation [ ] . sdr proteins were a component of cell wall anchored family proteins, including sdrc, sdrd and sdre [ ] . sdre could combine with the complement regulatory protein factor h to escape the alternative pathway of complement [ ] . to further investigate the functions of sdre, we have expressed and purified the adhesive domain (residues -' : ), and crystallized the recombinant protein. in addition, we also constructed the mutant s.aureus, and the cell experiments confirmed that sdre gene participate in the bacteria invasion. bacterial microcompartments (bmcs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency or to prevent the loss of volatile or toxic intermediates. there is an increasing desire to engineer bmcs for non-native enzymatic processes. it is thought this will increase multi-enzyme pathway efficiency and allow the expression pathways that may produce toxic or volatile intermediates in bacteria. the mechanisms of small molecule transport and retention of toxic intermediates by bmcs remain poorly understood. better understanding of the bmcs pores critical to engineer bmcs for these non-native pathways. in order to better understand the bmc pore we have undertaken structure-guided modifications of the the hexameric pdua shell protein of the , -propanediol utilization microcompartment (pdu mcp). these modifications include pore mutations in an attempt to alter substrate specificity and permutations of pdua to allow more drastic alterations to the structure of the protein. crystal structures of pdua pore mutants, solved to atomic resolution ( - . Å) provide evidence of the pore residues that confer specificity. further, a pdua permutation (pduap) has resulted in a closed icosahedral cage. this novel pduap cage shows a ph and salt dependent assembly and may serve as a reaction vessel or be utilized for cargo delivery. ( , ) . anm-mc is used to identify targeted transition pathways and intermediates between open and closed states of proteins. at each step of this iterative technique, the protein is deformed along the collective anm mode showing the best overlap with the target direction and its energy is minimized via short mc run. in this work, optimization of simulation parameters (number of mc moves and their perturbation strength, anm deformation factor in each cycle and force constant for backbone bonds) was performed in order to increase the efficiency of this technique. as a result, this technique can now be applied to much larger systems and conformational changes. the transition pathway between apo and dna-bound conformations of the yeast rna polymerase, which is a hetero- -mer with more than residues, will be presented here. moreover, the pathway intermediates for more than diverse proteins were analyzed in terms of changes in local strain energy and backbone torsional angles during apo-to-complex transitions. certain residues interacting with the ligand are detected to exhibit large changes with respect to any of these two parameters for more than half of the proteins in our dataset. department of chemistry and chemical biology, harvard university, howard hughes medical institute, harvard university, transgenic crops have radically reshaped the agricultural landscape. since their introduction in the late s, transgenic crops have affected economic gains greater than us$ billion globally due to reduced production costs and increased yield gains. crops modified to produce biological insecticides derived from the soil bacterium bacillus thuringiensis (bt) are among the most robust methods of pest control. bt toxins offer many advantages over traditional insecticides, chiefly their inability to affect human biology and exquisite selectivity for defined pest species. however, the evolution of resistance to bacillus thuringiensis oendotoxins (bt toxins) in insects has been widely observed in the field, and greatly threatens the use of this mechanism of pest control in the future. we developed a phage-assisted continuous evolution (pace) platform for the rapid generation of high-affinity protein-protein interactions and validated the system by evolving known high affinity antibody mimetics in < days of pace. we applied this system to the evolution of the bt toxin protein cry ac to recognize a non-cognate cadherin-like receptor from trichoplusia ni, a pest for which bt toxin resistance has been observed in both the laboratory and the field. the resulting evolved cry ac variants exhibits high affinity for the target receptor, and kill insect cells more potently than wild-type cry ac. our findings establish that the directed evolution of novel receptor recognition in bt toxins can be used to target resistant pests, and has far-reaching implications for biological reagents and therapeutics. optimization of a designed protein-protein interface brian maniaci , collin lipper , john j. love san diego state university, university of california protein-protein interactions play key roles in practically every biological process. protein-protein interactions vary with composition, affinity, and lifetime of the complex. studying designed protein-protein interactions will provide insight into the underlying principles of complex assembly and formation. computational protein docking and amino acid sequence design were used previously to generate protein dimers from monomeric proteins. the normally monomeric b domain of streptococcal protein-g (gb ) was computational docked to itself, followed by optimization of the interfacial side chains. two variants, monomera and monomerb, were computationally derived as a result of a designed protein-protein interface. these designed proteins were characterized using analytical ultracentrifugation and heteronuclear nmr techniques. this design resulted in a pair of protein monomers that formed a heterodimer of modest binding affinity. a tetrahedral metal-templated interface design strategy was implemented in an attempt to strengthen the monomera-monomerb complex by introducing cross-monomer metal coordination. another advantage of using the metal-templated interface is the ability to control the protein-protein interaction both temporarily and spatially. a number of newly engineered variants of monomer a and monomer b with metal coordination sites were designed, produced, and tested for increased affinity of the protein-protein complex. while the generation of a metal-templated monomera-monomerb complex was unsuccessful, we were able to obtain monomera variants that form a homodimer assembly only in the presence of zinc (ii) ions. the crystal structures of metal-templated monomera variants in the presence of zinc provide an explanation for the observed dimer formation. the crystal structure indicates that the protein-protein interaction is not driven by the designed protein interface, but rather non-specific association via edge-strand interactions. new variants were designed with the goal of engineering a high affinity homodimer in a helix-to-helix orientation as the originally designed protein-protein interface. current evaluation of monomera variants for self-association via metal coordination are being evaluated using size exclusion chromatography with a multi-angle light scattering detector for oligomerization state quantification. the results of this protein design project should lead to a greater understanding of the biophysical parameters that drive natural protein-protein interactions. continuous evolution of site-specific recombinases with highly reprogrammed dna specificities the ability to precisely modify the genome of human cells has enormous potential as a novel therapy and a powerful research tool. in contrast to reprogrammable nucleases, such as talens or a cas / sgrna pair -which specifically cleave dna but then rely on stochastic host cells processes to effect gene insertion -site specific recombinases directly catalyze genomic integration with high efficiency. a major limitation of this approach is that recombinases, such as cre, natively bind with high specificity to long dna target sequences (loxp in the case of cre) that do not exist in the human genome. previous attempts at evolving cre resulted in modest changes to its specificity, or required hundreds of rounds of manual protein evolution. we developed and validated a phage assisted continuous evoluiton (pace) selection for rapidly altering the dna specificity of cre recombinase towards a site present in a human genomic safe harbor locus. the pace experiments resulted in cre variants capable of recombining a substrate with nearly % of the nucleotides altered compared to loxp. we successfully used one of these variants to integrate exogenous dna into the genome of unmodified human cells. we are currently using sequencing methods to determine the specificity of the new recombinase clones. aleardo morelli , burckhard seelig generation of comprehensive deletion libraries mediated by in vitro transposition analysis of protein enzymes and ribozymes from nature, and from in vitro evolution, revealed that deletions of up to dozens of amino acids (or nucleotides) can be structurally tolerated. furthermore, shortened variants can exhibit better stability and increased catalytic activity. in order to investigate the effects of deletions, we developed a new procedure based on in vitro transposition to build libraries of more than , deletion mutants in three to four days. we tested our procedure on dna sequences coding for an artificial rna ligase called ligase c. we used the generated library for an mrna display selection, and isolated two active mutants containing and amino acids n-terminal deletions. structural characterization of ppsc, a multi-domain polyketide synthase from mycobacterium tuberculosis using a fragment-based approach alexandre faille , nawel slama , anna grabowska , david ricard , annaik qu emard , lionel mourey , jean-denis pedelacq polyketide synthases are of great interest in numerous scientific fields. they are composed by multiple domains, each having a different role to play in the catalysis of sequential reactions including condensation, reduction and esterification. their reaction products, named polyketides, represent a large variety of chemical compounds, from antibiotics to immunosuppressors or even anticancer drugs. ppsc is a kda polyketide synthase, organised into six catalytic domains (ks-at-dh-er-kr-acp) with singular functions. along with other type i polyketide synthases, ppsc is responsible for the biosynthesis of an essential polyketide for the virulence of mycobacterium tuberculosis (mtb) and thus is a target of choice for the design of inhibitors. to date, no structural information of any type i polyketide synthase in its entire form has been described. main reasons are the length of these large size enzymes and the flexibility imposed by the linkers between domains, thus making them very difficult to crystallize. numerous questions about domain-domain interactions, spatial arrangement of this complex machinery, substrate specificity and stereochemistry are still unanswered. addressing the structural and functional characterization of ppsc would then help answering these questions and provide valuable information for drug design. to overcome the length-and flexible-dependent problem originating from the presence of multiple domains and linkers, we decided to study domains expressed alone. for this purpose, we used our domain trapping strategy to identify soluble fragments representing a single domain from ppsc [ ] . it has the advantage of not relying on the bioinformatically designed domain boundaries and can even sometimes include parts of linkers to obtain more soluble fragments. using this strategy, we were able to identify relatively small and highly soluble fragments representing each domain of ppsc, thus facilitating the downstream structural and functional characterization. more than fragments have been submitted to crystallization trials. among these, gave crystals and allowed us to determine the x-ray structure of ppsc at, er, in addition to the dh domain in complex with a substrate analog for which activity was confirmed in vitro. the computational design of proteins that bind small molecules remains a difficult challenge in protein engineering. the ability to computationally design native-like interactions with high accuracy and efficiency would be an asset towards therapeutic development, enzyme design, and engineering functional proteins. we have developed a systematic approach to designing interfaces. we first identify ligands with naive binding affinity to our protein scaffold, then use rosettaligand to computationally dock the ligand while designing the interface for a tighter interaction. this way, we are taking a 'shot in dim light' for design as opposed to a 'shot in the dark', allowing us to more thoroughly investigate the successful and not-so-successful designs, and improve the computational methods. of ligands screened, we identified weakly-binding hits in the range of - mm. thus far, rosettaligand has successfully designed one tighter protein-ligand interface, from mm to mm. in progress experiments include designing and experimentally validating more designed interfaces. structural studies of human acidic fibroblast-growth factor (fgf ) mutants with a probable anticancer activity lectins are carbohydrate-binding proteins ubiquitously present in nature. they play a role in biological recognition phenomena involving cells and proteins. the interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface lectins specifically directed to carbohydrate residues present only on malignant cells and absent on healthy ones ( ) . lectins have been found to possess anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the t-antigen (gal b - galnac) found specifically on the surface of tumor cells ( ) . the main problem is that their use as a detection agent for the t-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response. previous studies with x-ray crystallography made in our laboratory have characterized a lectin found in mushrooms called bel b-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the t-antigen. unlike other lectins with this property, bel b-trefoil shows structural homology with a human protein, acidic fibroblast growth factor (fgf ) ( ). superposition of their structures suggests that the human protein could be mutated to contain at least one of the binding sites for the t-antigen. such mutations should create in fgf the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein. fgf is mitogenic and chemotactic, and mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor. to reach our purpose, the fgf cdna was cloned into a bacterial plasmid and then mutated in five different positions to eliminate its mitogenic activity and to engineer in the protein the t-antigen binding capacity. attempts to crystalize the mutants of fgf were made using the hanging drop technique with the final aim to carry out their structural characterization by x-ray diffraction analysis of the crystals. the de novo synthesis of proteins in response to the activation of cellular signaling pathways is a crucial element of many high-level biological processes, including the synaptic plasticity underpinning memory formation in the brain. while of fundamental biological importance, there has been a shortage of tools with which to specifically target pools of newly synthesized proteins of interest for study. thus, we have developed timestamp and smash, methods for drug-dependent tagging, or destruction, respectively, of newly synthesized copies of proteins of interest. both methods rely on protein tags that remove themselves by default via an internal hepatitis c virus (hcv) ns protease, but which are retained in the presence of cell-permeable small molecule protease inhibitors. the timestamp tag contains split yfp halves and epitope tags which are reconstituted and preserved, respectively, on proteins of interest following drug application, whereas the smash tag contains a strong degron which remains attached to proteins of interest following drug application, resulting in their clearance. one limitation of time-stamp and smash is that they can only be used to independently manipulate one protein of interest at a time. furthermore, the application of timestamp and smash to study endogenous protein pools in mammals has not yet been explored. here, we report on efforts to extend these techniques by reengineering ns proteases which can be inhibited by two different drugs orthogonally to one another. by incorporating different drug resistance mutations into two ns protease variants, we engineered ns protease domains that are inhibitable either by asunaprevir only, or by telaprevir only. we found that these tags permit simultaneous and independent control over the newly synthesized pools of two proteins of interest within the same population of cells. we also report the development of transgenic knock-in mouse strains incorporating timestamp and smash tags, which allow the interrogation of newly synthesized pools of specific endogenous synaptic proteins in the context of their endogenous regulatory elements, and without relying on overexpression. infectious diseases are often diagnosed by the presence of specific antibodies that are produced in response to the invading pathogen. one example are antibodies that are present in patient blood after infection with the dengue virus serotype and that are directed against an epitope on the virus' nonstructural protein (ns- ). traditional antibody diagnosis relies on time-consuming multi-step assays that require sophisticated equipment in a laboratory environment. a promising alternative are protein switches that are based on bioluminescence resonance energy transfer (bret). these switches comprise a luciferase (nanoluc) and a green fluorescent protein (mneongreen), which are connected via a semiflexible linker. the linker contains two epitope sequences of ns- to which the antibodies bind specifically. if no antibodies are present nanoluc and mneongreen are held in close proximity via two helper domains and bret can occur; thus green light originating from mneongreen is visible. if antibodies are present, they bind to the specific epitopes in the linker of the switch and cause stretching of the linker and therewith break the interaction of the helper domains. as a result, nanoluc and mneongreen are separated in such a way that bret cannot occur anymore; thus only blue light originating from nano-luc remains visible. using this principle, monoclonal anti-ns- antibodies were detectable in a controlled buffer system and in spiked plasma samples. furthermore, the developed antibody switch was applied to plasma samples of macaques after a primary infection with dengue virus serotype . signal readout was possible using a laboratory-based plate reader as well as the camera of a standard smartphone. we demonstrate that this bret-based protein switch can quickly detect antibodies in solution in a single-step assay format using simple equipment for signal readout, such as a standard smartphone. this simplified antibody detection platform has the potential to be carried out outside of a laboratory, thus in areas with limited laboratory infrastructure and a high number of diverse infectious diseases. proteins expressed from more than two-thirds of the human genome reside within intracellular compartments. of these proteins many are important disease-related targets such as kras and c-myc which cannot be easily addressed by conventional small molecule approaches. some of the weaknesses of small molecules can be addressed by biologic drugs, for example high target specificity and inhibition of protein-protein interactions. the challenge for biologics is how to engineer recombinant proteins to access the intracellular space. one strategy is to use systems evolved by bacteria and viruses to deliver material inside the cells. an example of such pathway is used by pseudomonas exotoxin a (pe). the modularity of pe allows the catalytic domain to be replaced with a biologic payload against desired intracellular target. an additional benefit of pe-based delivery is a possibility of targeting the drugs only to relevant cells in the body by modifying the cell-targeting domain of the pe. the aim of this project is to deliver functional payloads against k-ras and c-myc into the cell using a pseudomonas exotoxin a translocation domain. we used phage and ribosome display to select antibody mimetics that bind k-ras and c-myc. here, we present their activity in biochemical assays and the initial results on generation of pe-based constructs. ( ) . hemagglutinin is synthesized as ha molecule assembled as noncovalently bound homotrimers on the viral surface. this precursor protein is cleaved by trypsin-like proteases to yield two subunits ha and ha linked by a single disulphide bond ( ) . ha is also post-translationally modified by n-glycosylation ( ). it is well established that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies. in the attempt towards developing influenza vaccine production (the egg-based manufacturing lasts several months) that would be faster and safer the utilization of recombinant antigen alone is currently being observed. recently we demonstrated that yeast produced influenza h protein although cleaved into two subunits induced strong immunological response in mice ( ) . in this report, we describe the biochemical and immunological characterization of the h antigen, based on hydrolytic domain of the h n gene, with deletion of multibasic cleavage site and expressed in yeast system. the ha encoding gene from h n virus with deletion of nucleotides was cloned into ppiczac vector. rha fusion protein with his -tag was secreted into the culture medium and was purified to homogeneity in one step using ni-nta agarose. the efficiency of the antigen purification was mg/l. glycosylation sites of rha were determined using lc-ms-ms/ms. analysis of the n-linked glycans revealed that the rha is glycosylated at the same sites as the native ha in the vaccine strain. next we investigated if the hemagglutinin with deletion of the cleavage site oligomerize into higher molecular forms. to determine the oligomeric forms of the recombinant antigen various approaches were applied e.g. native-page, size exclusion chromatography or dynamic light scattering. as a final experiment to measure the size of oligomers in a protein sample a combined technology sec-mals was conducted, using multi angle light scattering (mals) as a detector. the immunological activity of rha was tested in chicken and mice, where antigen elicited high immune response. the data presented here demonstrate that new influenza antigen produced in p. pastoris is highly immunogenic and might be consider as a candidate for subunit vaccine. structural motifs capture redundant patterns that frequently occur in proteins. motifs associated with contiguous fragments of structure (i.e., secondary structural motifs) are well studied and have been successfully used to capture "rules" describing sequence-structure relationships in protein design and structure prediction. we have extended this concept to motifs that capture tertiary information-(i.e., tertiary structural motifs or terms. we have discovered that a relatively small alphabet of terms describes the known structural universe (all secondary, tertiary and quaternary information in the pdb) at sub-angstrom resolution. this alphabet of universal motifs reveals the remarkable degeneracy of the protein structure space, with just a few hundred terms sufficient to accurately capture half of the known structural universe. we have begun to demonstrate the considerable promise this structural alphabet has for applications such as protein design, structure prediction, and docking. we have developed a novel protein design framework that selects amino acid sequences, given a desired structure, using solely information from the universal terms. we show that given a native backbone, this framework recovers the native sequences to a level on par with state-of-the-art atomistic protein design methods, indicating that the motifs capture the salient structural rules governing native proteins. further, predicted sequence distributions agree closely with observed evolutionary variation. given the apparently high degeneracy among even complex features of protein structure, methods based on mining the pdb for tertiary information should provide ample opportunities for advancement in problems of computational structural biology. sortase-mediated synthesis of protein-dna conjugates for sensitive biosensing bedabrata saha , marieke op de beeck , remco arts , maarten merkx in recent years, semisynthetic protein-dna conjugates have emerged as attractive biomacromolecules for different applications in bio-nanotechnology, biosensing, diagnostics and therapeutics. in protein-dna conjugates, synthetic oligonucleotides allow the construction of desired molecular architecture with high specificity, while maintaining the original functionality of the protein molecules for desired application. however, the synthesis of site-specific and stoichiometric protein-dna conjugates can be challenging. due to the diversity in composition and physico-chemical properties of the proteins, few generic strategies are available for conjugation of protein molecules to a dna scaffold. a common approach is to use thiol-based covalent conjugation, but the introduction of additional cysteines can lead to the formation of intermolecular disulfides or interfere with the formation of native disulfide bonds. as an alternative, here we have developed a site-directed protein-dna conjugation strategy based on sortase mediated trans-peptidation reaction. the sortase recognizes a 'sorting motif' (i.e. lpxtg, x any amino acid), which is recombinantly introduced by site-directed mutagenesis at the cterminal end of the protein molecule. the sortase cleaves the t-g peptide bond and catalyzed the formation of a new amide bond between the lpxt peptide and the n-terminal amine of any molecule bearing an n-terminal oligoglycine motif. for this purpose, a triglycine motif was introduced at the 'end of single-stranded dna (ssdna). on-column synthesis of triglycine modified ssdna, protected on a controlled pore glass beads, simplified the purification process and enhanced the yield of triglycinemodified ssdna (> %). we used this conjugation strategy in several biosensing applications. for example, we used the method to conjugate ssdna linkers at the c-termini of a range of single-chain antibody fragments (scfv) and applied these constructs to allow oriented display of capture molecules on biosensor surfaces. ssdna-scfv were using an excess of triglycine modified ssdna, we achieved % conversion scfv-ssdna conjugate, which can be further purified by in two step purification process consisting of ni-nta affinity column and ion-exchange chromatography. we also extended this sortase-based conjugation strategy to develop a bioluminescence based assay for sensitive target oligonucleotide detection. in this regard, the ' and ' end triglycine-modified ssdna molecules were successfully conjugated with a bret protein pairs, nanoluc luciferase and mneongreen fluorescent protein. the introduction of a c-terminal sortase-his tag and and n-terminal strep-tag allowed efficient purification of theseprotein-ssdna conjugates from excess oligonucleotides and unreacted protein. mass spectrometry based proteomics to identify the protein differences in human breast milk from breast cancer patients and controls devika channaveerappa , roshanak aslebagh , kathleen f. arcaro , costel c. darie breast cancer is the second leading cause of cancer death in women. about % women in the us develop breast cancer. death rates due to breast cancer have been declined over the years due to advancements in mammography and treatment. although, mammography helps in the early detection of breast cancer, it has few limitations. dense breast tissue makes mammogram less accurate. breast milk can be assessed to evaluate the risk of one getting breast cancer by comparing the proteomes of breast milk from healthy and breast cancer suffering individual. this study makes use of mass spectrometry based proteomics to identify the differences between the control and cancerous samples which would further help in identifying potential biomarkers for breast cancer. firstly, sds-page was used to separate the proteins from the whole milk sample. the gel bands for each sample was then excised and cut into small pieces. the gel pieces were washed and trypsin digested in order to extract the peptides. peptide mixtures in the solution were cleaned using c zip-tipp and then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms). minutes and minutes gradient were used for lc-ms/ms analysis. raw data obtained were converted to pkl files using proteinlynx global served (plgs version . ). raw data were then submitted to mascot database search for protein identification. the mascot results were then exported as .dat files and further analyzed using scaffold version . software. three breast cancer milk samples were investigated against healthy control milk samples. in the sds-page gel, after coomassie staining, the protein patterns did show minor differences. after lc-ms/ms analysis, the proteins identified by mascot database search were imported into the scaffold software and compared for the relative ratio between the proteins from the milk sampled from control donors and the donors with breast cancer. there were significant differences identified in the proteomes of the two sets of samples. some of the proteins were upregulated in the breast cancer samples and some were down regulated when compared with the controls. additional investigation of more breast milk samples is ongoing. this study focuses on identifying biomarkers directly in the milk of donors with breast cancer. leukolike vectors: leukocyte-inspired nanoparticles claudia corbo , , alessandro parodi , , roberto palomba , , roberto molinaro , michael evangelopoulos , francesco salvatore , , ennio tasciotti the houston methodist research institute, fondazione irccs sdn, nanomedicine aims to improve drug efficiency by enhancing targeting and biocompatibility, and reducing side effects. multiple surface modifications have been proposed to provide nanocarriers with these features, based on complex synthesis processes and very often inefficient in contemporary providing biological tolerance and targeting properties [ ] . bio-inspired approaches based on surface coatings developed from the purified cell membrane of immune cells represents a new paradigm shift for the development of carrier enable of prolong circulation and proper tumoritropic capabilities. we showed that nanoporous silicon (nps) particles coated with leukocyte cellular membranes -leukolike vectors (llvs) -possess cell-like properties [ ] . llvs can escape macrophage uptake, delay sequestration by the reticulo-endothelial system, target tumor inflamed vasculature and accumulate within the cancer parenchyma [ ] . llvs were fully characterized for their shape, size, surface charge and coating through dynamic light scattering and scanning electron microscopy. in addition we characterized the content and function of the leukocyte's proteins transferred onto the llvs coating through high-throughput proteomic analysis and the results revealed the presence and the correct orientation of several important markers of leukocytes: cd , cd and mhc-i were identified as key players in determining llvs biocompatibility, while leukocyte associated function- (lfa- ) and mac- contributed to the llvs targeting ability and bioactivity towards inflamed endothelium [ ] . recent investigation showed that the coating induced the formation of a singular protein corona (i.e. the protein adsorption layer) on the surface of the nanoparticles compared to negative control following in vivo injection. in addition, the proteolipid coating favored active extravasation of the llvs in the tumor vasculature by molecular mechanisms similar to those used by tumor infiltrating leukocytes. this work shows that is possible to transfer biologically active leukocyte membrane proteins onto synthetic nanoparticles, thus creating biomimetic carriers retaining cell-like functions that are not affected by the protein corona effect that occurs in vivo. the targeting of the inflamed endothelium can be applied to a broad range of diseases and the approach used to formulate the system could open new avenues for the fabrication of the next generation of personalized treatments by using as cell membrane source the immune cells of patients. references: [ ] alessandro parodi, claudia corbo, armando cevenini, roberto molinaro, roberto palomba, laura pandolfi, marco agostini, francesco salvatore, ennio tasciotti. enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers. nanomedicine uk. accepted. steroid hormone receptors are intracellular receptors that initiate signal transduction in response to steroid hormones, including oestrogen and androgens. generally, the binding of the steroid to the nuclear receptor induces the protein to form a dimer and relocate onto the chromatin, although the order of these events may vary. the location of receptor binding on the chromatin is defined by specific hormone response elements (hre). once located, the receptor promotes gene activation by the recruitment of other co-factors. it is this process that makes the complex of receptor protein and co-factors play a pivotal role in the regulation and activation of genes. the failure to regulate this process correctly is a key step in the development of several endocrine-driven cancers. for example: oestrogen receptor positive (er ) breast cancer is one of the most common forms of cancer and accounts for % of all breast cancer cases. in er tumours, the oestrogen receptor (er) drives the tumour growth and cell proliferation. understanding the interactions of the er with other proteins, either directly or indirectly, can provide vital insight to the regulation of the system that drives this cancer. the progesterone receptor (pr) has also been implicated in breast cancer, and the androgen receptor (ar) is a known driver in the majority of prostate cancers. to meet the challenges of elucidating these systems, we have developed methods to purify and analyse cross-linked regulatory complexes bound to dna by mass spectrometry (chip-ms). this allows for the enrichment of proteins involved in gene regulation. chip-ms, combined with tandem mass tags (tmt), makes it possible to realise a quantitative method to investigate the dynamic network of interactions between proteins within complexes that undertake the regulation of biological systems. chip-seq is a well-established method for identifying where these protein complexes are bound to the genome. this work focuses on how to combine these technologies with my previous development of cross-linking coupled mass spectrometry techniques (xcms) to provide a strategy for visualising the dynamic organisation of the proteins on the chromatin. global kinetic analysis of caspase protein substrates in cell lysate reveals selective roles and target specificity olivier julien , min zhuang , arun wiita , james wells caspases are cysteine proteases that play important roles in development, cell differentiation and cell death. however, the limited number of known caspase substrates hinders our understanding of caspase function. here we performed a non-biased identification and kinetic analysis of caspase- and caspase- proteolytic substrates in cell lysate, using an enzymatic n-termini enrichment approach followed by mass spectrometry. we identified and potential substrates for the initiator caspase- and putative executioner caspase- , respectively. our results not only confirm known substrates but also identify many more new substrates with the precise location of proteolysis. given the emerging roles of caspases- and in inflammation and neurodegeneration, these new substrates may provide molecular insight into the progression of related diseases. the sequence consensus logo of caspase- targets was very similar to a classical executioner caspase motif (devd), while caspase- revealed a vevd motif. using selected reaction monitoring (srm), we quantified the kinetics of proteolysis of a large subset of these substrates by measuring the appearance of the caspase cleavage product over time. in the end, we measured and kcat/km values for individual substrates cut by caspase- and caspase- , respectively. by comparing these data with our previous analysis of caspase- , , and , we found that substrates that are shared between caspases are often cleaved at rates that differ by orders of magnitude. thus, despite having nearly identical primary sequence motifs, the caspases exhibit remarkable substrate specificity that may reflect their specialized roles within the cell. the rockefeller university, new york university school of medicine, johns hopkins university school of medicine line- (l ) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. despite tremendous influence on genome composition, l rnas only encode two proteins. consequently, l particles include a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against l 's mutagenic activity. we previously characterized host proteins associated with synthetic and natural human l retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. to build on these analyses, we have implemented a series of d separations and post-purification treatments to produce a multi-dimensional interactomic characterization of affinity isolated l s. these studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. we report a comprehensive, quantitative survey of the proteins partitioning within these distinct l populations and their associated in vitro activity. our observations provide a basis for the classification of l interactors with respect to their physical and functional links, facilitating hypotheses to direct in vivo experimentation. polyubiquitin recognition by continuous ubiquitin binding domains of rad probed by modeling, small-angle x-ray scattering and mutagenesis sangho lee , trung thanh thach , namsoo lee , donghyuk shin , seungsu han , gyuhee kim , hongtae kim rad is a key protein in double-strand break dna damage response (ddr) pathways by recognizing k -linked polyubiquitylated chromatin proteins through its bipartite ubiquitin binding domains ubz and lrm with extra residues in between. rad binds k -linked polyubiquitin chains as well as k linked ones and mono-ubiquitin. however, the detailed molecular basis of polyubiquitin recognition by ubz and lrm remains unclear. here, we examined the interaction of rad ( - ), including ubz and lrm, with linear polyubiquitin chains that are structurally similar to the k -linked ones. rad ( - ) binds linear polyubiquitin chains (ub , ub , ub ) with similar affinity to a k -linked one for diubiquitin. ab initio modeling suggests that lrm and the extra residues at the c-terminus of ubz (residues - ) likely form a continuous helix, termed 'extended lr motif' (elrm). we obtained a molecular envelope for rad ubz-elrm:linear ub by small-angle x-ray scattering and derived a structural model for the complex. the rad :linear ub model indicates that elrm enhances the binding of rad with linear polyubiquitin by contacting the proximal ubiquitin moiety. consistent with the structural analysis, mutational studies showed that residues in elrm affect binding with linear ub , not monoubiquitin. in cell data support that elrm is crucial in rad localization to dna damage sites. specifically e seems to be the most critical in polyubiquitin binding and localization to nuclear foci. finally, we reveal that the ubiquitin-binding domains of rad bind linear ub more tightly than those of rap , providing a quantitative basis for blockage of rap at dsb sites. taken together, our data demonstrate that rad ( - ) forms continuous ubiquitin binding domains, comprising ubz and elrm, and provides a structural framework for polyubiquitin recognition by rad in the ddr pathway at a molecular level. optimization of a protein extraction method for the proteomic study of pozol cynthia teresa leyva-arguelles , carmen wacher , rosario vera , romina rodr ıguez-sanoja instituto de investigaciones biom edicas, unam., facultad de qu ımica, unam., instituto de biotecnolog ıa, unam key words: proteomics, fermentation, pozol pozol is a mexican traditional no alcoholic beverage elaborated by various ethnic groups in the southeastern of mexico. pozol is obtained from the natural fermentation of nixtamal (heat-and alkali-treated maize) dough. the main carbohydrate in maize dough is starch ( - %), because others such as sucrose, glucose and fructose are mostly lost during nixtamalization; so, the starch remains as the major carbohydrate available for fermentation [ ] . a wide variety of microorganisms have already been isolated from the fermentation of pozol; these microorganisms include fungi, yeasts, lactic acid bacteria, and non-lactic acid bacteria [ ] . however, only few bacteria are amylolytic in this fermentation and all of them are weakly amylolytic [ ] . in an attempt to explain how a very low content of soluble sugars can support a diverse and abundant microbiota, a proteomic approach was designed to understand the fermentation of pozol [ ] . nevertheless, the extraction of proteins from pozol remains a limiting step in proteomic analysis mainly due to the complexity of the sample. on the basis of the aforementioned reasons, the aim of this work was to obtain a suitable extraction method of proteins for proteomic analysis. therefore, the fermentation of pozol was continued for h and samples were taken at , , and h. for each sample, the total sugar content was determined by the dubois et al. method [ ] and protein extraction was performed by two methods: a) direct extraction from the dough [ ] and b) initial extraction of microorganisms and soluble proteins (this work). comparison between the two protein methods was performed on two-dimensional gels with silver stain. then, gels underwent to image analysis by the image master d platinum software. comparing the d-gels, more proteins spots were obtained with method b than that with method a, indicating a more efficient protein extraction with method b. although, using method a higher concentration of total proteins was observed, they were mostly maize proteins, that in turn overlap and reduce the efficiently extraction of the microbial low abundant proteins. then, method b allows a better extraction of those low abundant proteins and removes sample components that may interfere with the determination. these results could help us to find the proteins involved in carbohydrate metabolism of the microbiota and finally elucidate the dynamics of pozol fermentation. proteomics has been applied to the enology field for numerous purposes including fermentation control, improvement of fermentation processes, ensuring wine quality, etc. according to rodriguez et al., ( ), the information provided by wine proteomics is not only useful for these intentions, but also offers excellent prospects for innovation and diversification of winemaking processes in the near future. in this context, our group has focused research on the identification of proteins that might be important for yeast survival under typical wine elaboration conditions (standard fermentation, sherry wine biological aging and sparkling wine second fermentation) as well as proteins that configure the content of metabolites which are ultimately responsible for wine quality. by using novel proteomic (offgel fractionator and ltq orbitrap xl ms) and metabolomic techniques (sbse-td-gc-ms) we have identified a high amount of up-regulated proteins involved in processes like oxidative stress response (in biological aging) or protein biosynthesis (in second fermentation) as well as thirty-three proteins directly involved in the metabolism of glycerol, ethanol and seventeen aroma compounds excreted by the yeast under biological aging conditions. further, in order to validate proteome data; null mutants of genes codifying proteins up-regulated in the biological aging condition were constructed. analyses of correlated phenotypes are in progress. this technique and its combination with metabolomics within the enology context will provide enough knowledge to design or choose yeasts or conditions that satisfy wine production and/or wine characteristics such as color/aroma/texture/flavour profile demands of winemakers and consumers. additional binding sites for cytochrome c on its redox membrane partners facilitate its turnover and sliding mechanisms within respiratory supercomplexes blas moreno-beltr an , antonio d ıaz-quintana , katiuska gonz alez-arzola , alejandra guerra-castellano , adri an vel azquez-campoy , miguel a. de la rosa , irene d ıaz-moreno ibvf, ciccartuja, universidad de sevilla -csic, bifi -iqfr (csic), universidad de zaragoza, departamento de bioqu ımica y biolog ıa molecular celular, universidad de zaragoza, gliding mechanisms of cytochrome c (cc) molecules have been proposed to shuttle electrons between respiratory complexes iii and iv within plant and mammalian mitochondrial supercomplexes, instead of carrying electrons by random diffusion across the intermembrane bulk phase [ ] [ ] . in this work, the binding molecular mechanisms of the plant and human cc with mitochondrial complexes iii and iv have been analyzed by nuclear magnetic resonance and isothermal titration calorimetry. our data reveal that both cc-involving adducts possess a : stoichiometry -that is, two cc molecules per adduct -. the presence of extra binding sites for cc at the surfaces of complexes iii and iv opens new perspectives on the mitochondrial electron transport chain, where membrane respiratory complexes can be either in independent, free diffusional motion or forming macromolecular assemblies. in the latter context, such new binding sites for cc facilitate the turnover and sliding mechanisms of cc molecules within supercomplexes. indeed, the accommodation of several cc molecules between complexes iii and iv in supercomplexes provide a path for cc diffusion from complex iii to iv. such path could have physiological significance in the electron flow, which is controlled in supercomplexes to optimize the use of available substrates [ ] [ ] [ ] . can bio-functionalities be deciphered from protein sequence information using computational approaches? background: the processes of uncovering bio-functionalities such as pharmacological activities, disease processes, physiological and structural properties by means of clinical approaches are irrational. this is because they are resource and time consuming. sometimes, they involve sophisticated and expensive equipments, reagents and animal tissues. contrarily, sequence information-based computerized approaches are rational and have become relevant in assessing bio-functionalities. they include geno pheno [coreceptor] [ ] , position-specific scoring matrix (pssmsi/nsi and pssmcxcr /ccr ) [ ] , and informational spectrum method (ism)-based phylogenetic analysis (istree) [ ] . aim: this presentation demonstrates how bio-functionalities could be deciphered from sequence information using computational approaches. method: ism procedure and peptides, vipmfsals and capagfail are engaged. results: protein sequences of the peptides are converted into bio-functionality (affinity). affinity between the two peptides is demonstrated as significant amplitudes at the point of common interaction also referred to as consensus frequency, signifying remarkable affinity. discussions: bio-functionalities of bio-molecules are known to be expressed in one or two genes, which have been found to provide as much biological information as the bio-molecules. this indicates that biological characteristics, represented in these genes and proteins can now be extracted from their sequence information. for example, multi-drug resistances arising from a variety anti-microbial agent from several classes including alkaloids, flavonoids, etc can be retrieved from the sequence information of their encoding genes (mdr and mdr ). similarly, translation of hiv infection to aids disease can be extracted from the protein sequence alterations in the hiv gp . similarly, effectiveness of anti-retroviral agent, maraviroc on the hiv isolate h bx and ndk can be deciphered from the sequence information of their v observed at the predicted sequences. these positions are important as they surround the cleavage site in the three-dimensional structure, and are probably less tolerant to change. moreover in previous studies, cys at p position has been shown to be the dominant determinant for cleavage efficiency, while cys, pro and glu at p position have also been shown to be correlated with increased cleavage efficiency of ns / a protease. for adv cysteine protease, on the other hand, bsst produces similar significant results for both type (xgx-g) and type (xgg-x) consensus cleavage sites, where p and p ' positions have gly with highest percentage in type (xgx-g) while p and p positions have gly in type (xgg-x). these indicate that the bsst seems to provide a powerful methodology for predicting the substrate specificity for the hcv ns / a serine protease and adv cysteine protease, which are targets in drug discovery studies. protein plasticity improves protein-protein binding description chiara pallara , juan fern andez-recio an accurate description of protein-protein interactions at atomic level is fundamental to understand cellular processes. however the current structural coverage of protein-protein interactions (i.e. available experimental structures plus potential models based on homologous complex structures) is below % of the estimated number of possible complexes formed between human proteins. , for these reasons, computational docking methods aim to become a complementary approach not only to solve the structural interactome but also to elucidate the basis of the protein-protein association mechanism. in spite of the advances in protein-protein binding description by docking, dealing with molecular flexibility is a major bottle-neck, as shown by the recent outcomes of the capri (critical assessment of prediction of interactions) experiment. this data clearly confirms that the protein dynamics plays a key role in protein-protein association. the use of conformational ensembles generated from unbound protein structures in combination with computational docking simulations might represent a more realistic description of protein-protein association. here, we present the first systematic study about the use of precomputed unbound ensembles in docking, as performed on a set of cases of the protein-protein docking benchmark . . the primary aim of our work is to understand the role of the protein conformational heterogeneity in protein-protein recognition. to do this, small conformational ensembles were automatically generated starting from the unbound docking partners, and then an extensive analysis of their binding properties was performed in the context of pydock docking scheme. the results show that considering conformational heterogeneity of interacting proteins can improve docking description in cases that involve intermediate conformational changes in the unbound-to-bound transition. more interestingly, we found that protein plasticity increases chances of finding conformations with better binding energy, not necessarily related to bound geometries. the relevance for future docking methodology development and for understanding protein association mechanism will be discussed. purpose of the research: there is increasing interest in the development of protein scaffolds that can be used to develop affinity reagents that are alternatives to antibodies. the affimer scaffold is based on the cystatin protein fold. the affimer scaffold is biologically inert, biophysically stable and capable of presenting a range of designed or random binding surfaces defined by peptides inserted at different loops. the result is highly specific, high affinity interactions with a wide range of targets including ones that are inaccessible to antibodies. affimers are designed to work in the same way as the very best antibodies, but with a number of key advantages. affimers are quick to develop (typically weeks) without using animals. they contain no disulphide bonds, are expressed easily in e. coli and have no batch to batch variability. affimers are small molecules ( aa, kda), robust and stable (resistant to ph range, thermally stable and not sensitive to edta). affimers can be a direct replacement for antibodies -no process or workflow change required -and perform identically to antibodies in assays such as elisa, facs, ihc, western blots, affinity purification, microarray and potentially therapeutics. we describe some applications of the technology in regards of affimer development for custom targets on one hand and for the biomarker discovery workflow using affimer microarrays on the other. main results: by screening of our very large ( x ) library against yeast sumo protein we identified affimers with high affinity allowing their use for elisa. moreover, no cross-reactivity was observed when affimers were used on western blots leading to a unique band specific to yeast sumo when compared to human proteins. a library of , random affimers, expressed in e. coli, was printed on glass microscope slides and challenged with plasma from children (n ) with sepsis and from healthy children (n ). unsupervised hierarchical clustering based on the , affimers allowed differentiation between the control and patient samples. affimers were found to differentially bind proteins between the groups with a > fold change. the affimer arrays identified a strong signature of sepsis and roc curve analysis allowed confident prediction of disease (auroc of . ). affinity purification and preliminary mass spectrometry analysis identified known biomarkers of sepsis and also potentially novel biomarkers not previously associated with this disease. major conclusions: this work demonstrates the scope of affimer affinity reagents to develop alternative binders to antibodies, where affimers perform identically in most assays without the disadvantages associated with antibodies. moreover, affimers enable a new protein microarray-based biomarker-discovery workflow and we predict that array-based validation of signatures identified using discovery arrays prior to affinity purification and mass spectrometry will offer a cost-and time-effective methodology compared to purely mass spec-driven workflows. tau pathologies, called 'tauopathies', are related to several neurodegenerative diseases including alzheimer disease (ad). in ad, tau protein is observed hyper-phosphorylated and aggregated as paired helical filament (phf). the neuronal tau protein is an intrinsically disordered proteins (idps). nuclear magnetic resonance spectroscopy (nmr) is here used to study the tau protein phosphorylations and protein-protein interactions (ppis). in in vitro assays, tau phosphorylation by rat brain extract is considered as an hyperphosphorylation model that was furthermore pointed out to enable tau aggregation [ ] . in a first step, we have identified all the phosphorylation sites of rat brain extract phosphorylated-tau, using the analytical capacity of nmr. we showed that the protein is modified at ser/thr sites. among the kinases that we have characterized so far using tau as substrate, only the extracellular signal-regulated kinase (erk ) shows an ability to modify in vitro tau protein on so many sites. we have indeed identified phosphorylated ser/thr-pro motifs out of potential phosphorylation sites in the sequence of full length -residue tau. in addition, we showed using transmission electron microscope (tem) a similar in vitro aggregation capacity of erk-phosphorylated tau protein compared to that of rat brain extract phosphorylated-tau. this shows that phosphorylation by the erk kinase generates an hyperphosphorylated tau. given the high efficiency of erk towards tau, we have next looked into the mechanism of tau recognition. erk kinase possesses two well-characterized docking domains: d recruitment sites (drs) and f recruitment sites (frs), which recruit complementary docking sites and increase the specificity and efficiency of the interaction with both its upstream regulators and downstream substrates [ ] . as the interaction between tau protein and erk kinase is analyzed by nmr spectroscopy, multiple sites of interaction are observed along the tau sequence, similar to drs docking sites, all located in the so-called microtubule binding domain of tau. these sites are short sequences loosely matching the reported consensus for d sites w - uxu (w, u, and x refer to positively charged, hydrophobic, or any intervening residues, respectively) [ ] , and also the reverse sequence uxuw - .to confirm the mapping of the interaction, two tau recognition sites were produced as recombinant peptides of about amino-acid in fusion with an n-terminal his-tag sumo. interaction assays using d [ h, n] hsqc spectra of the peptides confirm their binding to erk kinase. the potential of these peptides to inhibit erk activity with tau as substrate is now being investigated. while rigid-body docking has become quite successful for predicting the correct conformations of binary protein complexes, determining whether two given proteins interact remains a difficult problem. successful docking procedures often give equally good scores for pairs of proteins for which there is no evidence of interaction. studies investigating what we define as the 'pre-docking' problem via in silico approaches have only recently become feasible with the help of supercomputers and gridcomputing systems. in a previous work, on a restricted set of protein complexes, we showed how predictions of interacting partners could be greatly improved if the location of the correct binding interface on each protein was known. experimentally identified complexes are found to be much more likely to bring these two interfaces into contact, at the same time as yielding good interaction energies. we present data from a complete cross-docking (cc-d) study of a database of proteins, including the treatment of more than , potential binary interactions. the performance of the interaction index we developed to predict binding probability compares well with other methods. by studying the interaction of all potential protein pairs within a dataset, cc-d calculations can also help to identify correct protein interaction interfaces. the present large-scale study also reveals the influence of various protein families (enzyme-inhibitor, antibody-antigen, antigen-bound antibody, etc.) on binding specificity, showing, in particular, the distinctive behavior of antigenic interfaces compared to enzymes, inhibitors or antibodies. the performance of our approach is encouraging. although identifying interaction interfaces significantly helps in the identification of interacting proteins, further refinements will be necessary to make in silico cross-docking a viable alternative to high-throughput experimental methods. whole-protein mass spectrometry reveals global changes to histone modification patterns in hypoxia sarah wilkins , kuo-feng hsu , christopher schofield chemistry research laboratory, oxford university cells respond to limiting oxygen availability (hypoxia) by altering the gene expression profile. this primarily involves changes at the level of transcription via the activity of hypoxia-responsive transcription factors, although increasing evidence suggests that changes in chromatin structure (i.e. from a condensed 'silent' state to a more open or 'active' state) are required in order for transcription to take place. in particular, post-translational modifications (ptms) to histones have an important regulatory function in gene expression under hypoxic conditions. the n-terminal tails of histone proteins are accessible to a set of enzymes capable of 'writing' and 'erasing' ptms including acetylation, methylation, ubiquitylation, sumoylation and phosphorylation. to date, studies in hypoxia have employed antibody-based methods to investigate changes in histone modifications, and so have focused on individual marks in isolation. the interplay between coexisting ptms is thought to be much more important than the effect of any single mark. therefore, a global view of the histone modification profile is essential to gain a complete understanding of the function of histone ptms and their roles in gene regulation. in this study, we apply whole protein mass spectrometry to investigate hypoxia-induced changes in histone marks. this 'top-down' approach provides insight into combinational modification patterns that are difficult to establish by antibody-based methods or peptide ms analysis. we investigated changes in the global ptm profiles of histones from a range of human cell-lines and tissues under severe hypoxia (< . % o ). we find that hypoxia causes a shift in the overall profile towards a more highly modified state, with significant changes in methylation and phosphorylation. marked changes in histone ptms were also observed following treatment of cells with epigenetic inhibitors and commonly used hypoxia mimetics, including several iron chelators currently in clinical trials for the treatment of anaemia. finally, we show that this method can be used to identify the histone variant h ax, whose phosphorylation at serine is an indicator of double-stranded dna breaks in cancer. overall, these data provide important insights into the epigenetic changes associated with hypoxia in normal and disease contexts. we hope to further develop this method in combination with different labelling strategies to enable quantitative analysis of histonemodifications in cells. mass spectrometry-based protein biomarker discovery in neurodevelopmental disorders interactions. there is currently no biological diagnosis or known cause of asd. slos is characterized by a cholesterol deficiency due to a mutation on the dhcr gene. approximately / , babies are born with slos. diagnosis is achieved by measuring cholesterol and -dehydrocholesterol ( dhc) levels in the blood, however, there is currently no proven treatment for slos. because of this, research is increasing to determine biomarkers for these disorders. here, samples from people with asd (sera and saliva) and slos (saliva), and matched controls were analyzed using a combination of gel electrophoresis (tricine-page, sds-page and blue native page), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanolc-ms/ms) to investigate differences between the proteomes of people with these neurodevelopmental disorders and matched controls. several alterations in protein expression were identified. these differences may lead to potential biomarkers for diagnosis, possible therapeutic targets and an altogether better understanding of the disorders. understanding protein recognition using structural features protein-protein interactions (ppis) play a crucial role in virtually all cell processes. thus, understanding the molecular mechanism of protein recognition is a critical challenge in molecular biology. previous works in this field show that not only the binding region but also the rest of the protein is involved in the interaction, suggesting a funnel-like recognition model as responsible of facilitating the interacting process. further more, we have previously shown that three-dimensional local structural features (groups of protein loops) define characteristic patterns (interaction signatures) that can be used to predict whether two proteins will interact or not. a notable trait of this prediction system is that interaction signatures can be denoted as favouring or disfavouring depending on their role on the promotion of the molecular binding. here, we use such features in order to determine differences between the binding interface and the rest of the protein surface in known ppis. particularly, we study computationally three different groups of protein-protein interfaces: i) native interfaces (the actual binding patches of the interacting pairs), ii) partial interfaces (the docking between a binding patch and a non-interacting patch), and iii) back-to-back interfaces (the docking between non-interacting patches for both of the interacting proteins). our results show that the interaction signatures in partial interfaces are much less favoured than the ones observed in native and back-to-back interfaces. we hypothesise that this phenomenon is related to the dynamics of the molecular association process. back-to-back interfaces preserve the exposure of the real interacting patches (thus, allowing the formation of a native interface), while in a partial interface one interacting patch is sequestered and becomes unavailable to form a native interaction. structural characterization of the cytoplasmic mrna export platform laboratory of cellular and structural biology, the rockefeller university., laboratory of mass spectrometry and gaseous ion chemistry, the rockefeller univ., university of california, san francisco, the new york structural biology center, department of biochemistry, faculty of medicine, university of montreal mrna biogenesis is an intricate process that begins within the nucleus and culminates with the remodeling and nuclear export of the mrnp particles through the nuclear pore complex (npc). defects in this conserved mechanism have been shown to cause serious human diseases. the protein assembly that performs the last steps in mrnp biogenesis and export is located at the cytoplasmic face of the npc and is formed by different proteins, organized into several subcomplexes whose arrangement and molecular architecture are poorly understood. in this study we applied an integrative approach, combining cross-linking and mass spectrometry (cx-ms), electron microscopy and available high-resolution structures, to describe the molecular architecture of the endogenous npc cytoplasmic mrnp export machinery. we generate a hybrid, close-to-atomic structure of the yeast native nup complex, the core of the assembly. our map also reveals how the nup complex organizes the entire cytoplasmic mrnp export machinery, and how this in turn docks into the architectural core of the npc. mapping of phenotypic profiles into our structures allows us to generate a first functional map of the ensemble. we expect that our map will serve as a framework to understand the molecular mechanisms underlying this key step of mrnp biogenesis. study of candidate proteins to pore associated with p x receptor in different cell types carla oliveira , anael alberto , mônica freitas , luiz alves laborat orio de comunicac¸ão celular -fiocruz, centro nacional de ressonância magn etica nuclear -ufrj aim: the p x r is a purinergic receptor, which differs from others subtypes due to its structural and pharmacological characteristics. when exposed for extended time or to high concentrations of its agonist (atp), promotes an increase in membrane permeability, allowing the passage of molecules up to da. there is a controversy among several authors that leave in doubt if this receptor needs a second protein for the pore formation and which protein could be. we select five pore-forming proteins: trpv , trpa , connexins- (cx- ), pannexin- (panx- ) and vdac. we believe that different mechanisms and proteins could be associated with p x r, depending on the cell type and their microenvironment stimuli. in this context, our main goal is identify possible proteins that could be associated with the p x r pore in different cells and species. methods and results: we started with rt-pcr technique of cell lines: j .g , n a, u , u , hek- and primary cells from wistar mouse and swiss mice. we used different primers and pcr cycle for each target at different species. we observed that the p x , panx- and cx- are the most abundant and are present in all cell types except the absence of p x in u cells and panx- in mice macrophages and u cells (n> ). however, trpv was seen at n a and u cells and trpa in and primary cells from mouse and mice and in j .g cells (n> ). regarding to the vdac, it is present in mouse macrophages, j .g and hek- cells (n> ). the further steps, we verified if those proteins could be physically associated with the p x r. we coimmunoprecipitated the p x r of j .g (with or without atp), mice macrophages, hek- and u cells. the samples were applied in two separated . % bis/acrylamide gels: one destined to mass spectrometry (ms) and the other to western blot. at this point, we confirmed the presence of p x r, and observed several others proteins associated to p x r at different cell conditions, mainly when we exposed, j .g cell, to mm atp (n ). at this condition, we found by ms, hsp , , and ; alpha and b tubulin; myosin va; alpha, b and g actin; malate and lactate dehydrogenase (n ). although u and hek- had not received atp treatment, we found several proteins associated to p x . the next step was to immunoprecipitated those proteins in j .g (treated or not with atp) and use it to verify if p x are physically associated to them. as result we saw the p x associated to panx- in j .g cells. conclusion: we conclude that the p x r activated by extracellular atp triggers the recruitment of variety different proteins. at this condition, we can suggest that maybe there is a conformational change, regardless of the numerous recruitment structural proteins. in addition, apparently, the pore-forming protein pannexin- is associated with p x r, and the others pore forming proteins (vdac, cx- , trpv , trpa ) seems not be linked to p x r at j . recently, we developed a series of molecular modeling tools for structure-based studies of protein functions and interactions. these tools are publicly available as web servers that are easily operated even by non-specialists: cabs-fold server for protein structure prediction [ ] ; cabs-flex server for modeling of protein structure flexibility [ ] ; aggrescan d server for prediction of protein aggregation propensities and rational design of protein solubility [ ] ; and cabs-dock server for prediction of peptide binding sites and peptide docking [ ] . the web servers are freely available from the laboratory website: http://biocomp.chem.uw.edu.pl/tools sandy on , pinghui feng university of southern california, keck school of medicine, developing a technique to detect deamidated proteins and peptides using rig-i sandy on, pinghui feng university of southern california, norris comprehensive cancer center, department of microbiology, and molecular biology, los angeles ca perhaps the most notable type of post-translational modification of proteins and peptides into a higher order structure is deamidation of asparagine and glutamine. deamidation occurs when an amine group is removed, degrading the molecule for purpose of regulating intracellular levels. previous studies have demonstrated that this notable post translational modification has been uncovered over time for use in dna recombinant technology as well as use as a biological clock to facilitate the rapid turnover of biologically important components of the cell. while the effects of this non-enzymatic chemical reaction have been widely studied, the method to uncover modification sites over a large quantity of proteins remains an issue. one of the most common types of deamidation is of asparagine and glutamine residues. at this time, most researchers will depend on mass spectrometric based proteomic techniques for identification of these post-translational sites. the issue is that mass spectral analysis of deamidated proteins and peptides is complication and can lead to misassigned identification attributed by an overlapping of c peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by . mda. while these issues can be mediated by using a mass spectrometer with a high mass measurement accuracy, and high resolving power, it is essential to establish simpler methods for identifying substrates that have undergone deamidation. if deamidation is present, different protein bands will be exhibited in the western blot, which will be compared to a triple mutant rig-i, which resists deamidation, to observe the location of this modification on the protein. with enough testing, i will determine specific sites of digestion and use this information to make conclusions of unknown proteins. i will make results regarding whether the protein has been modified based on the digestion sites. i will use mass spectrophotometry analysis to compare the proteins on a wider scale and double check my results. i have narrowed it down to a couple of different digestion sites that indicate deamidation. though the analysis work can be tedious, it is crucial to ensure the sites we isolate are accurate in order to establish this technique. from my research, we can apply this method for wider scale use such as in clinical settings. in areas of inflammation of parkinson's' patients, we can review specifically the infected cells versus uninfected and isolate the proteins, usually deamidated, responsible often smaller in size and more specific. in addition, research articles have already shown that suppressing modification of certain cells such as bcl-xl playing a major in leading the regulation of cancer cell death by apoptosis. by leading the discovery of a simpler methods to uncovering deamidation in cells, researchers will more easily and quickly be able to scan through various proteins, some of which discovered eventually may play pivotal roles in cancer research. influenza virus (iv) hemagglutinin (ha) is a homotrimeric integral membrane glycoprotein that mediates receptor-binding and membrane fusion. it constitutes the prominent viral surface antigen and a main target for neutralizing antibodies. bacterial, recombinant ha-based vaccines indicate high potential to confer protection against highly pathogenic (hp) avian iv (aiv) h n and arise as alternative for the traditional egg-or cell culture-based manufacturing. relatively short time of bacterial has production can be of great importance in case of a pandemic. escherichia coli produced protein, based on the ha sequence of a/swan/poland/ - v / (h n ) hpaiv*, has been successfully expressed in the form of inclusion bodies at institute of biotechnology and antibiotics. refolded and purified antigen was obtained in a soluble form, isolated by reversed phase hplc and identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi tof/tof ms). the performed research in a great extent allowed to confirm the amino acid sequence of the recombinant ha (rha) assumed based on the cdna and allowed to establish the location of a total of six disulfide bridges. however, during purification and storage of the rha, apart from desired higher order rosette-like structures of the protein, other non-native species resulting from posttranslational modifications, misfolding, aggregation and degradation may occur what results in reduced vaccine potency. here, besides the properly folded monomers, we indicate non-native aggregates induced by disulfide crosslinking. moreover, several free cysteine residues and unexpected intrachain s-s were identified in rha tryptic peptide maps. cys was found most susceptible to formation of disulfide bridges between the distinct chains of rha. the above findings allow to assume that not all rha particles fold to form the native structure. reduced cys residues exhibit tendency to undergo oxidation and uncon- new strategies and approaches to understand how antibodies recognize and neutralize snake toxins represent a challenge to improve the antivenoms. the neurotoxic activity of micrurus venom is carried majority by two distinct proteins families, ftx and pla . the conserved structural folding of these toxins can be appreciated as model to generate inhibitors against them. in this regard, monoclonal antibodies (mabs) can be used as tool to find hot spots for inhibit the toxins and represent the first step in order to develop recombinant neutralizing molecules. in this work our goals were analyse a set of monoclonal antibodies against the most toxic components of m. altirostris venom by proteomics approaches. the venom was fractionated; its major toxic proteins identified by in vivo tests based on murine lethal toxicity analyses (approved by the ethical committee for animal experimentation from center of health and science of the federal university of rio de janeiro -no. . / - ). the toxic components were used to generate a panel of five monoclonal antibodies. elisa and antivenomics results allowed us identify the specificity of all mab and their neutralizing efficacy was measured by in vitro tests. three mabs showed reactivity towards ftx and two against pla . all monoclonal antibodies against ftx lack a broad recognition. however, we identified a pair of monoclonal antibodies able to recognize all pla molecules of m. altirostris venom and showed a synergism to inhibit the catalytic activity of them. moreover, we challenge monoclonal antibodies against to micrurus venom for inhibit the pla activity of naja naja, specie taxonomically out of micrurus cluster. our results showed that pla of m. altirostris venom share a pair of conserved antigenic regions and draw attention to use these epitopes to miming antigen to generate antibodies for antivenom production. moreover, face to the cross reactivity and the pla activity inhibition capability by mabs towards the naja naja venom, our results highlight the conservation of neutralizing epitopes across the elapidae family. protein-protein interactions are known to play key roles in the most important cellular and biological processes such as signaling, metabolism, and trafficking. one major goal of structural biology is the structural characterization of all protein complexes in human and other organisms. these efforts can be complemented by computational approaches. in this context, computational docking attempts to predict the structure of complexes from their monomeric constituents. the docking problem presents two main challenges: the generation of structural poses or sampling, and the identification of the correct structures with a scoring function (sf). docking methods can be successful if the interacting partners undergo small conformational changes. however, in a general situation, these algorithms generate a large number of incorrect predictions, and therefore the predictive success strongly depends on the accuracy of the sf used to evaluate the docked conformations. a variety of strategies have been developed to score putative protein-protein docked complexes. they are usually based on atomic level potentials, residue level potentials, or a combination of both. in current work, we have evaluated different sf, taken from cchappi server, on the results of different rigid body docking methods, ftdock, zdock, and sdock, using the docking benchmark . and a docking set built from capri scorers experiment. our results show sf that showed better or similar success rate than the in-built sf. some of these sf increase the docking success rates especially for flexible or weak-binding cases, which are the most challenging for docking. of them are residue level sf robust enough to detected solutions in cases with large conformation change. in particular we found two sf that shows outstanding robustness, one designed for protein modeling and shared among docking methods, and the other is for protein docking which is also the best success rate in the top ranking in the capri scorers set. the other atomic level sf display high success rate to find a solution within weak binding proteins. the most successful sf are shared between the docking methods and display high success rate in the hard cases of the benchmark . and in the capri scorers set. the difference between them in the resolution level at which they work, one being atomistic the other residue-based. we found that they success rate vary according to the docking method chosen, allowing them to explode different properties of the sampling used. this way to characterize a protein complex can help to develop new combined scoring functions in protein docking or a new ranking strategy to enhance the success rate. multi-ptk antibody: a powerful tool to detect a wide variety of protein tyrosine kinases (ptks) isamu kameshita , noriyuki sueyoshi , yasunori sugiyama the eukaryotic protein kinases consist of large families of homologous proteins and play pivotal roles in various cellular functions. these enzymes are classified into two major groups; protein serine/threonine kinases and protein tyrosine kinases (ptks). ptks are believed to be involved in various cellular events such as cell cycle, proliferation, differentiation, apoptosis, and cell adhesion in multicellular eukaryotes. as many as ptk genes have been identified in the human genome and many of these ptks are known to be closely correlated with various diseases such as cancer. therefore, it is important to elucidate the expression profiles of the entire ptk family in cells and tissues. to investigate the expression profiles of the cellular ptks, we produced an antibody that detects a wide variety of ptks. for production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain vib) of ptks were synthesized and immunized to balb/c mice. among various antigens, a peptide with amino acids, cyvhrdlraan, efficiently produced a polyclonal antibody with a broad reactivity to ptks. we established a hybridoma cell line producing a monoclonal antibody, yk , which appeared to cross-react with various ptks. at least ptks could be detected by yk antibody, as evidenced by its reactivity with the recombinant src tyrosine kinases whose subdomain vib had been replaced by those of the other ptks. when differentiated hl- cells were analyzed by western blotting after two-dimensional electrophoresis with yk antibody, we observed significant changes in the immunoreactive spots in hl- cell extracts along with the changes in the morphology of the cells. these results suggest that the multi-ptk antibody, yk , will be a powerful tool for the analysis of a variety of cellular ptks. analysis of the siglec- and hvap- interactions leonor carvalho , vimal parkash , heli elovaara , sirpa jalkanen , xiang-guo li , tiina salminen structural bionformatics laboratory, department of biosciences, medicity research laboratory, department of pharmacology, drug development and therapeutics, sialic acid-binding immunoglobulin (ig)-like lectins (siglec) are type i transmembrane proteins. siglec- has an n-terminal v-set domain followed by two c -set domains in the extracellular region. it contains an immunoreceptor tyrosinebased inhibitory motif (itims) in its cytoplasmic tail and can function as an inhibitory receptor by dampening the tyrosine kinase-driven signaling pathways. these proteins are expressed primarily on leukocyte subsets and, thus, are thought to be involved in regulation of leukocyte functions during inflammatory and immune responses. recently, phage display screening experiments identified siglec- as leukocyte surface ligand for human vascular adhesion protein (hvap- ; aoc gene product) and their interaction was confirmed by cell adhesion and enzymatic assays (kivi et al., ; aalto et al., ) . based on our preliminary data, hvap- sugar units with sialic acid (sa) might mediate interactions with the v-set domain in siglec- . furthermore, it is known that the siglec peptides binding to hvap- are located in the ce loop of the second c -set of domain (siglec- _c ). based on current hypothesis an arginine in siglec- _c interacts with the tpq residue in the active site of hvap- . the ce loop of siglec- _c has two arginines (r and r ) and, therefore, the interacting arginine is unclear. we will now study the interaction mode of hvap- and siglec- in silico to predict the role of the arginines in the c domain and the role of sa-binding using the d model of the full-length ectodomain of siglec- and the hvap- crystal structure. the in silico analysis will be conducted in parallel with experimental site-specific mutational studies and the result will be combined to elucidate the mechanism of hvap- -siglec- interaction. adam middleton , catherine day attachment of ubiquitin to substrate proteins regulates almost all cellular processes, including protein degradation and cell division. ubiquitylation involves a cascade of three families of proteins: ubiquitin activating (e ), ubiquitin conjugating (e ) and ubiquitin ligase (e ) enzymes. the . kda protein can be attached as a monomeric moiety or as a polyubiquitin chain, and the type of modification spells out the 'ubiquitin code' that directs the fate of the substrate. polyubiquitin chains can be formed via eight different linkage types, and the arrangement of chain formation is typically directed by the e enzymes. forming a polyubiquitin chain involves binding of two molecules to the e : the donor (ubd) and acceptor (uba) ubiquitin. ubd is linked to the e via a thioester bond between its c-terminal gly and the active site cys of the e , and when primed for catalysis it interacts with a particular face of the e . in contrast, coordination of uba by e s is transient and cannot be easily measured; however, uba binding defines the linkage type of polyubiquitin chains. the e , ube k, directs lys chain synthesis, which results in modified proteins being degraded by the proteasome. we generated a stable form of the ube kub conjugate and crystallized it, and showed that both ube k and its ubiquitin conjugate are monomeric. using molecular docking, we modelled the position of both ubd and uba and investigated the interfaces with site-directed mutagenesis. these experiments led to a molecular model that revealed how ube k can synthesise lys -linked ubiquitin chains. this molecular explanation provides a foundation for understanding how other e s generate lys -linked polyubiquitin chains. the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis marta bunster, francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedl fac de cs biol., universidad de concepci on the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis. francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedel, marta bunster. departamento de bioqu ımica y biolog ıa molecular, facultad de ciencias biol ogicas, universidad de concepci on. phycoerythrin is a phycobiliprotein present in phycobilisomes in gracilaria chilensis as a complex with chromophorylated linker proteins. our interest is to discover the role of these linkers in the function of phycobilisomes. phycobilisomes(pbp) are auxiliary light harvesting protein complexes in charge of channeling energy towards photosystem ii in alga, cyanobacteria and cryptophyta. this is possible thanks to fluorescent proteins called phycobiliproteins (pbp) and the chromophores (phycobilins, open-chain tetrapyrrols) attached to specific cysteines. phycobiliproteins share a common general structure; they are organized as (alfab) heterodimers which themselves assemble as trimers(alfab) or hexamers (alfab) ; this complexes are organized in high order structures to form the core and the rods. besides pbps, pbs have linker proteins in charge of the assembly and stabilization of the complex, and also it has been proposed that they collaborate in the fine tuning of the energy transfer steps between chromophores. these linkers are located within the rods, the rod-core interface, the core and the core-membrane interface. although most linker proteins are colorless, chromophore bearing linkers have been described, which suggest its participation in the energy transfer process. two of them, g and g are associated to r-phycoerythrin in gracilaria chilensis, nevertheless the information available on these linkers in eukaryots is still limited. to understand how these linkers collaborate with the function of the phycobilisome, we need structural information, especially the coordinates of all the chromophores present in the complex; we have sequenced both linkers from the genomic dna, performed sequence analysis and also we have purified the linkers by anion exchange, molecular sieve and hpl chromatography. the characterization was performed by denaturant electrophoresis, absorption and emission spectroscopy and by mass spectrometry. the results show that they have molecular masses as predicted, with a peptide signal for chloroplasts, an internal sequence repeat; residues - with residues - for g and residues - with residues - for g , and the presence of conserved cysteine residues putative sites of chromophorylation. the spectroscopy shows that they have different composition of phycobilins and a very short t / . a preliminary model for both linkers shows that they belong to aa structural class and that they share a common fold (heat like motifs) frequently involved in protein-protein interactions. dept. of phys., chuo univ., grad. sch. of inform. sci. and eng., tokyo tech, rigid-body docking algorithms are useful for predicting tertiary structures of near-native protein complexes. however, this algorithms generate many protein complex poses including false positives. then, near-native poses are searched in a post-docking process. there are many computational softwares with rigid-body docking algorithms, for example, zdock. we developed a high-performance protein-protein interaction prediction software, megadock, which is basically used on supercomputing environments for a large scale and network level in this work, we then tried to use these docking softwares and the profile method for understanding mechanisms of protein-protein interactions. we focused on some physicochemical properties, electrostatic and hydrophobicity, of a set of protein complex poses generated by a rigid-body docking process. from these poses, we obtained sets of possible interacting amino acid pairs. a set of interaction profiles has some information of docking spaces. from the view of a network prediction, the docking spaces of a set of protein complex poses are one of the properties for discriminating native protein-protein pairs from non-native pairs. in this work, ensemble docking process is performed by megadock ver. . and zdock ver. . . . cluster analysis is used with profiles of physicochemical properties. we used a dataset composed of typical monomer-monomer protein pairs and will discuss mainly differences between native and non-native protein pairs. the structural studies of the two thermostable laccases from the white-rot fungi pycnoporus sanguineus marta orlikowska , grzegorz bujacz institute of technical biochemistry, lodz university of technology, poland laccases (ec . . . , benzenodiol oxygen oxidoreductases) are enzymes that have the ability to catalyze the oxidation a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water [ ] . it has been found that the active site is well conserved in between laccases from different organisms. it contains four copper atoms: one paramagnetic type cooper (t ) that is responsible for their characteristic blue color and where the oxidation of the reducing substrate occurs, one type cooper (t ) and two type coopers (t ) that conform a trinuclear cluster in which molecular oxygen is reduced to two molecules of water [ ] . laccases are present in many different species and they have been isolated from plants, fungi, prokaryotes, and arthropods in most cases laccases are monomeric glycoproteins of around amino acids with molecular weights in the range of - kda. the various functions carried out by those enzymes include the antagonistic ones such as their involvement in lignin biosynthesis (in plants), lignin degradation, pigment production, fruiting body formation, pathogenesis (in fungi) and spore protection against uv light (in bacteria) [ , ] . the diversified functions of laccases make them an interesting enzyme for study from the point of view of their structure, function and application. laccases of white-rot fungi (wrf) are of special interest because one of its role is to degrade lignin and most of them are extracellular enzymes helping purification procedures [ ] . during the last two decades, there has been an increasing interest in the genus pycnoporus for its ability to overproduce high redox potential laccases as the ligninolytic enzymes. we present the crystal structures of two thermostable lacasses produced by strain pycnoporus sanguineus cs (laci and lacii). the molecular weights of laci and lacii, determined by sds-electrophoresis, is and kda, respectively [ ] . both isoforms shows high amino acids sequence similarity ( %) between them and high thermal stability, at c and c. they remained active at high concentration of organic solvent (acetonitrile, ethanol or acetone). the unique properties make them promising candidates for industrial applications in wasterwater treatment. laci exerted a higher thermal and ph stability, tolerance against inhibitors and was a more efficient catalyst for abts and dmp (laccases substrate) then lacii [ ] . based on the structures we would like to understand the isoforms differences that confers laci a markedly better performance than lacii in ph and thermal stability as well as better resistance to inhibitors. analysis of liver proteome in cystathionine ß-synthase deficient mice using d ief/sds-page gel electrophoresis, maldi-tof mass spectrometry, and label-free based relative quantitative proteomics izabela bieli nska , Łukasz marczak , hieronim jakubowski , institute of bioorganic chemistry, polish academy of sciences, rutgers university, new jersey medical school homocysteine (hcy) arises from the metabolism of the essential dietary protein amino acid methionine. levels of hcy are regulated by remethylation to met and transsulfuration to cys. cystathionine bsynthase (cbs) catalyzes the conversion of homocysteine to cystathionine (first step of transsulfuration reaction). human cbs deficiency is a recessive inborn error of homocysteine metabolism that casues severe hyperhomocysteinemia (hhcy) and diverse clinical manifestations, including fatty liver disease [ ] . although the causes of fatty liver disease in cbs deficiency have been studied the underlying mechanism is not understood. we hypothesize that cbs deficiency induces changes in gene expression that could impair liver homeostasis. to test this hypothesis and gain insight into hepatic functions of cbs we analyzed the liver proteome of cbs -/-and cbs / mice [ , ] using d ief/sds-page gel electrophoresis and maldi-tof mass spectrometry (n ) we identified twelve liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of three proteins was upregulated and of nine down-regulated by the cbs-/-genotype. two up-regulated liver proteins are involved in iron metabolism (ftl and fth). those proteins are associated with oxidation stress and inflammation. third up-regulated liver protein (cbr ) is related to oxidation-reduction process. the downregulated protein are involved in the hydrolysis of n-acylated or n-acetylated amino acids (acy ), regulation of endopeptidase activity (a at ), cholesterol biosynthetic process (fpps), amino acid degradation (huth), cellular calcium ion homeostasis and l-ascorbic acid biosynthetic process (rgn). using label-free based relative quantitative proteomics (n ) we identified fourteen liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of four proteins was up-regulated and of ten proteins was down-regulated. the down-regulated liver proteins are linked with regulation of bone mineralization and inflammatory response (ahsg) or regulation of mrna splicing (roa ). the up-regulated liver proteins are involved in tricarboxylic acid cycle (suca), oxidation-reduction process (cy ), cholesterol metabolic process, iron ion homeostasis (fech), fatty acid metabolic process (ssdh; eci ) and response to oxidative stress (lonm). our findings suggests that cbs interacts with diverse cellular processes, including lipid metabolism, that are essential for normal liver homeostasis. deregulation of genes involved in lipid metabolism provides a possible explanation for fatty liver disease associated with cbs deficiency. transcription factors play central roles in coordinating developmental processes, as evidenced by the increasing number of transcription factor-related developmental disorders being uncovered by nextgeneration sequencing and genome-wide studies of copy number variation. the action of a transcription factor in regulating gene expression depends on interactions with other transcription factors, coactivators/co-repressors and chromatin modifying and remodeling complexes. transcription factors are commonly regulated by post-translational modifications. however the study of protein-protein interactions and post-translational modifications of transcription factors by common techniques such as coimmunoprecipitation and mass spectrometry is hampered by the difficulty in preserving interactions and modifications through cell lysis. to circumvent this issue, we developed a bioluminescence resonance energy transfer (bret) assay, which allows protein-protein interactions to be observed in live cells. in this assay, a protein of interest is expressed as a fusion with luciferase from renilla reniformis, and its putative interaction partner as a fusion with yellow fluorescent protein (yfp). upon addition of a cell-permeable substrate, the distance-dependent non-radiative transfer of energy from luciferase to yfp is quantified by measurement of light emission at two wavelengths to assess the interaction between the two fusion proteins. to validate the utility of this assay for investigating transcription factor interactions, we confirmed homodimerization of the foxp transcription factor, haploinsufficiency of which causes a rare and severe speech and language disorder, as well as interaction of foxp with other members of the foxp family. we also confirmed the interaction between foxp and multiple candidate interactors identified through yeast two-hybrid assays, including the autism-related transcription factor tbr , the co-repressors ctbp and ctbp , and post-translational modification enzymes of the pias family. the role of pias enzymes in sumoylation -the covalent modification of proteins with small ubiquitin-like modifier (sumo) proteins -led us to further explore this process, which is notably difficult to investigate because of the dynamic and labile nature of the modification, which is also typically present on only a minor fraction of molecules of a given protein. combining the bret assay with gel-shift techniques we demonstrated that foxp is sumoylated. finally, we used the bret assay to examine the effects of etiological foxp variants in speech and language disorder on protein-protein interactions and post-translational modification. in summary, the bret assay is a sensitive, reliable and potentially high-throughput technique for exploring protein biology in the context of live cells. we have demonstrated applications of the assay in validating putative protein-protein interactions, assessing posttranslational modifications, and investigating functional effects of protein variants identified in patient cohorts. these investigations have provided novel insights into the function of the foxp transcription factor in neurodevelopment and into the etiology of foxp -related speech and language disorder. the directly interaction between pres of human virus b and human heat shock protein (hsp ) deqiang wang , chen ke , jun zhang key laboratory of molecular biology on infectious disease, the department of cell biology and genetics the directly interaction between pres of human virus b and human heat shock protein (hsp ). hepatitis b virus (hbv) has infected billion people worldwide, and million of them are chronically infected. the chronic virus infection, a major public health problem worldwide, leads to bout two-thirds of hepatocellular carcinoma (hcc). the hbv envelope consists of the large (l), middle (m) and small (s) envelope proteins, which contain pres -pres -s, pres -s, and s domain alone, respectively [ ] . the pres domain is believed to mediate virus attachment to the high-affinity receptor. yan et al employed a novel technique to propose sodium taurocholate co-transporting polypeptide (ntcp) as the candidate hbv receptor, and consequently, ntcp is a target for a new family of anti-hbv agents [ ] . whereas, it remains a query to clarify that ntcp is the only or major hbv receptor in vivo. to illuminate if other host proteins cooperatively participate the hbv infection, we detect the interaction between pres and many candidate host proteins. fortunately, we have found that the human heat shocking protein (hsp ) could directly interact with the pres domain of the hbv virus protein. both the pull down and the size exclusion chromatography experiments verify that the grp have the ability binding to pres . whereas, whether the interaction between hsp and pres relates to the hbv infection need further experiments to clarify. the member sponsorship in the vast world of naturally occurring peptides, where more than peptides are known and approximately peptide therapeutics are currently being evaluated in clinical trials (fosgerau & hoffmann, ), the rapid and accurate determination of their physicochemical properties is key in peptide drug discovery. among these properties, hydrophobicity is crucial for understanding molecular recognition and biomolecular aggregation. hence, there is a great interest in determining hydrophobicity scales for amino acid structures. in this work, octanol/water partition (log p) and octanol/water distribution (log dph, fig. ) of n-acetyl-l-amino-acid methyl amides were determined by means of quantum mechanical ief-mst solvation calculations taking into account the intrinsic conformational preferences of each amino acid according to dunbrack's libraries (dunbrack & karplus, ; ) . the results reveal log d . differences for a-helical and b-sheet conformations in arg, lys, hid, asn, gln, met, cys, leu and ile. furthermore, by decomposing the octanol/water transfer free energy into electrostatic and non-electrostatic components, we estimated that the non-electrostatic cost of transferring the amino acid side chain amounts to . . cal/mol.Å , in agreement with previous estimates reported in the literature. comparison of our scale with other theoretical and experimental hydrophobicity scales yields satisfactory results, leading to correlation coefficients ranging from . to . . additionally, the mstderived hydrophobicity scale led to significant correlations with the rp-hplc retention factors measured for eight decapeptides (r . ) and for influenza virus hemagglutinin -mer (ac-ypydvp-dyaslrs-amide) peptides (r . ). finally, the hydrophobicity scale was able to reproduce the experimental log p for random neutral peptides (r . ) and log d . for ' : random charged peptides (r . ), fig. . future studies will address the application of this methodology to nonproteogenic amino acids, the prediction of peptide hydrophobicity at global and atomic level in peptides, and the scoring of peptide-protein interactions. docking-based tools for discovery of protein-protein modulators docking-based tools for discovery of protein-protein modulators. protein-protein interactions (ppis) play an essential role in many biological processes, including disease conditions. strategies to modulate ppis with small molecules have therefore attracted increasing interest over the last few years. although protein-protein interfaces (ppifs) are considered difficult to target with small molecules given its lack of well defined cavities. successful ppi inhibitors have been reported into transient cavities from previously flat ppifs. recent studies emphasize on hotspots (those residues contribute for most of the energy of binding) as promising targets for the modulation of ppi. pydock algorithm is one of the few computational methods that use energy of solvation to predict protein-protein interfaces and hotspots residues. we present an approach aimed at identifying hotspots and transient pockets from predicted proteinprotein interfaces in order to find potential small molecules capable of modulating ppis. the method uses pydock to identify ppifs and hotspots and molecular dynamics (md) techniques to propose putative transient cavities. we benchmarked the protocol in a small set of protein-protein complexes for which both structural data and ppi inhibitors are known. the method applies to the unbound proteins of the complexes the fast fourier transform algorithm, followed by the energy-based scoring from pydock to calculate the normalized interface propensity (nip) values derived from rigid-body protein docking simulations to predict the ppifs and hotspots residues without any prior structural knowledge of the complex. then we used md to describe the possible fluctuations of the interacting proteins in order to suggest transient pockets that could be useful as targets of small molecules for the modulation of ppis. finally, we evaluated by ligand docking, the validity of predicted hotspots and pockets for in silico drug design. we found that the nip-based method from pydock protein-protein docking identifies hotspots residues that are located within the binding site of known inhibitors of ppis. predicting ppifs from a three dimensional structure is a key task for the modulation of ppis. the use of the nip-based hotspots prediction method improve the identification of transient cavities from md simulation when compared to known binding cavities. this approach can be extremely useful in a realistic scenario of drug discovery targeting ppifs, when there is no information at all about the protein-protein complex structure. protein complexes are the fundamental molecular organizations that assemble multiple proteins to achieve various biological processes. identification of protein complex membership should provide a genotype-phenotype map to elucidate human gene-disease associations. it has been routinely assumed that network clusters with dense connections inside and sparse connections outside would form functional protein complexes. therefore, searching highly modular subgraphs in protein-protein interaction networks was explicitly or implicitly implemented in the algorithms to find protein complexes. however, to our surprise, we found a large portion of complexes with a medium-to-low modularity from the analysis of experimentally confirmed protein complexes. we also discovered that these complexes have cellular functions enriched in highly time-and space-dependent expression, such as signal transduction or subcellular localization. we further developed an algorithm to find such complexes by weighing network connections to capture transient interactions with intrinsically disordered regions. we confirmed that our method improved the identification of biologically relevant members of protein complexes and covered more complexes with a medium-to-low modularity. furthermore, newly discovered subunits in protein complexes could explain more disease-gene associations, indicating its utility to expand current genotype-phenotype map of human diseases. expanding template-based protein-protein complex prediction using ab-initio docking sergio mares-s amano , luis angel rodr ıguez-lumbreras , juan fern andez-recio structural characterization of protein-protein interaction (ppi) networks is crucial for understanding the underlying molecular mechanisms whereby life processes and disease arise. however, due to inherent limitations of experimental techniques, such characterization only covers an extremely reduced fraction of the human ppi network (interactome). recent studies have shown that although available structural templates may suffice to model a significant proportion of the interactome, model accuracy and binding specificity remain unsolved problems. consequently, improving the ability to predict ppis structurally will help to provide a better d profile of the known interactome, which may ultimately lead to the development of new therapeutic applications. here we show a novel approach that combines templatebased modeling with protein-protein computational docking to the structure-based prediction of ppis. our approach samples different protein-protein structural models derived from docking simulations. models are subsequently ranked using a function that incorporates an energy-based scoring term and a structural template similarity score. the energy-based scoring function includes electrostatics, van de waals and desolvation calculations, whilst the template similarity score accounts for the degree of structural similarity of models against a high-resolution and diverse dataset of structural templates. our approach highly improved the predictive success rate over individual ab-initio docking and templatebased techniques across a large benchmark dataset, including protein-protein complexes. when compared to the performance of the ab-initio docking algorithm, we found that the approach increased consistently the success rate, by approximately %, for the top , top and top solutions. the success rate improvement was even more notorious when the comparison was performed against the predictions from the traditional template-based docking. though incorporating ab-initio docking expands considerably the scope of the template-based docking method, challenges remain for interacting proteins in which high conformational changes occur upon binding and also the size and diversity of the repertoire of structural templates needs to be increased. is essential for the development of multicellular organisms. in mammalian cells, early events in pcd involve the release of cytochrome c (cc) from mitochondria to the cytoplasm, so letting cc play a key role in assembling the apoptosome and triggering apoptosis. in plants, pcd is part of a general process -the so-called hypersensitive response -in which mitochondrial cc is likewise released into the cytosol but its further role and cytoplasmic partners remain veiled. such a coincidence in cc release made us think of a common link for pcd in such evolutionarily distant species along evolution. to go deeper in understanding the pcd-dependent role of cc, a proteomic approach based on affinity chromatography with cc as bait was run using human and plant cell extracts. upon combining this approach with bimolecular fluorescence complementation (bifc), a total of eight and nine unknown proteins interacting with cc under pcd conditions were identified in human and plant cells, respectively [ , ] . such novel cc-partners -which are located in the cytoplasm and even in the nucleus -are involved in protein folding, translational regulation, oxidative stress, dna damage, energetic and mrna metabolism [ ] . strikingly, some of the novel human cc-partners are closely related to those for plant cc, so indicating that the evolutionarily well-conserved event of cc release from mitochondria could involve a common signalosome consisting of a wide range of common targets [ ] . to also understand such a promiscuity of cc from a structural point of view, the cc surface residues involved in complex formation with each one of its counterparts were mapped by using nmr spectroscopy. the resulting data shows that the heme crevice of cc is at the cc-partner interface in most of the complexes, which is in agreement with the vast majority of known redox adducts of cc. in contrast, however, to the high turnover number of the redox cc adducts inside the mitochondria, the complexes formed by cc under pcd conditions lead to the formation of rather stable nucleo-cytoplasmic ensembles. altogether, these findings suggest that extra-mitochondrial cc interacts with nuclear and/or cytoplasmic pro-survival, anti-apoptotic proteins in both humans and plants so as to lead living cells to dye. keywords: cytochrome c, programmed cell death, signalosome. post-translational phosphorylation often modulates the function of proteins. in particular, they affect the role that cytochrome c (cc) plays in cell life and death [ ] . cc is phosphorylated in vivo in tyr and tyr residues [ , ] , but recently, two new phosphorylation sites have been described at positions and [ ] . hence, we aim at understanding the structural and functional changes induced by thr and ser phosphorylation cc. for this purpose, we designed two phosphomimetic mutants of cc by replacing either thr or ser by the canonical amino acid aspartic acid (t d and s d). as control, two other mutants at the same two positions (t a and s a) were analyzed so as to differentiate the effects due to the presence of a negatively charged residue. remarkably, the s a mutant is significantly less stable than the wild-type species. we found that phosphorylation at position thr diminishes the redox potential and oxygen consumption. in addition, t d mutation affects the ability of cc to bind the distal site pcc , thereby suggesting that phosphorylation at this position affects the electron carrier capacity of cc. mass spectrometry (ms) is widely used techniques to gain knowledge about biomolecules [ , ] . it produces a high amount of data which is often presented as a list containing thousands of proteins. that list usually contains few hits interesting for our research. the pocess to select those proteins may include integrating experimental with annotation data. it requires spending some time in both, performing calculus and searching in databases. in this poster we present msbiodata analysis tool, a web service thought to deal with this tedious work. with this tool, researchers can set rules to select the most interesting hits in his lists using both, experimental data and gene ontology [ ] annotation. the data can be upload to the web using an excel spreadsheet or a flat files in a mztab format, and rules are easily constructed by means logical sentences. those sentences are composed by one or more terms linked by logic operators (and and or). each term in the logical sentence indicates to our program the conditions that selected hits must meet. once the alysis is finished, the results are delivered by email. msbiodat analysis tool do not requires any programming knowledge to be used and is freely available at: http://msbiodata.innomol.eu keywords bioinformatics/data analysis/proteomics/data mining/ mass spectrometry. beside the rate of protein synthesis, the regulation of protein degradation plays a crucial role in the white muscle protein accumulation and overall fish growth. intracellular proteolysis in salmonid species, such as atlantic salmon, salmo salar l. and rainbow trout, oncorhynchus mykiss walb., was studied to evaluate the basic mechanisms of protein degradation that could possess a potential target to regulate the body mass accumulation in farmed fish. a number of white muscle proteases such as cathepsins b, l, and d, proteasomes, and calcium-dependent proteases (m-and m-calpains), was studied in the juvenile specimens of different size-and age-groups both wild and farmed salmonids. the correlations between the protease activity and expression levels and morphometric characteristics of fish were found. the size-and age-related differences in intracellular protease activity revealed in fish muscles indicate both general role of proteolysis regulation in salmonid growth and the specific role of the individual proteolytic enzymes as well. the data on negative correlation of cathepsin d and calpain activity in muscles and the rate of weight increase in juvenile salmonids were obtained. a revealed positive correlation of cathepsin b activity and morphometric parameters in fish young presumably indicates its primary contribution to non-myofibrillar protein turnover. ubiquitin-proteasome system seems to contribute to background protein turnover as the proteasome activity was not corresponded with growth rate. summarizing the data obtained the autophagy-lysosomal and calpain-related protein degradation pathways were recognized to be directly involved in body growth and muscle protein retention in salmonid fish. the work was carried out using technical facilities of ib karrc ras equipment centre and financially supported by the russian science foundation, grant no. - - "salmonids of the north-west russia: ecological and biochemical mechanisms of early development". solving the proteomic organization of fitness-related genes in uropathogenic escherichia coli in life threatening sepsis. nowadays, complete genomes for almost all major bacterial pathogens are available, helping researchers to identify virulence factors. however we still ignore how these genes are organized at the proteome level and how this association influences bacteria pathogenicity. we integrated available databases on upec e. coli (strain cft ) to investigate the genomic and proteomic organization of genes related to upec fitness in the host. intriguingly, we found that most fitnessrelated genes have orthologs not only in other pathogenic strains but also in non-pathogenic bacteria such as e. coli k- . these genes are organized in clusters and operons with similar structure. by integrating protein-protein interaction data we observed that genes with high impact on fitness also display a highly clustered organization when compared to other genes. overall, our results show that proteinprotein interaction clusters associated to upec fitness in the host represent a promising target for the design of new antibiotics. elucidating the molecular mechanisms by which the hnh endonuclease gp activates the terminases in bacteriophage hk ( ) . hnh endonucleases are characterized by two highly conserved his residues and an asn residue( ). gp is essential for phage head morphogenesis, likely because gp enhances the activity of the hk terminase enzymes toward the cos site ( ) . notably, enhancement of the terminase-mediated cleavage of the phage cos site requires the presence of an intact hnh motif in gp . mutation of the canonical metal binding his in the hnh motif abrogates gp mediated-terminase activity. although phages are widely studied, there is no definitive structural or mechanistic evidence as to how the hnh endonuclease within gp functionally interacts with the adjacent terminase enzymes to facilitate phage morphogenesis. previous work on hnhcontaining bacteriophage proteins does not address explicitly how the requirement for divalent metal binding at the hnh endonuclease site induces interaction with the terminase enzymes that are so crucial for phage dna packaging during morphogenesis ( , ) . in addition, gp possesses no sequence similarity to hnh proteins for which the structure has been determined ( ), making structural studies of gp necessary. toward these ends, we use nuclear magnetic resonance (nmr) spectroscopy to probe metal and terminase binding of gp in the wild type state and bearing metal binding mutations. we also report backbone resonance assignment of gp . our nmr studies have elucidated residues within gp required for metal binding and terminase activity. these data are being used to assess the role of specific gp residues in phage morphogenesis. together, this work will identify the enigmatic role describing how metal binding in hnh endonucleases is crucial in the replication and morphogenesis of phages. meat production from pigs for human consumption is a resource heavy process, indeed every part of the animal that is not used constitutes a protein food-chain loss, which is neither economically nor environmentally viable. the goal of this project is to better harness slaughterhouse waste such as the keratin rich pig bristles and nails through microbial conversion. instead of using identified single microorganisms, it is the goal to define microbial consortia where microorganisms synergistically show the ability of efficient keratin degradation/conversion. candidate consortia have been obtained by selecting for microorganisms growing on enriched media that contains milled pig bristles as sole carbon and nitrogen source. by using mass spectrometry and various biochemical analyses to investigate keratinolytic enzymes, methods will be established for identifying and characterizing suitable consortia. protein families likely to be involved are keratinases, which are specialized proteases including serine, cysteine and metallo proteases, as well as systems capable of reducing or otherwise breaking disulfide bonds which are highly abundant in hair and nails. furthermore, interactions and symbiosis of microorganisms in a consortium will be investigated at the meta-proteomics level. the project will lead to development of biotechnological degradation of keratin rich fibers, and provide new insights into functional dynamics and efficacy of microbial consortia. a comprehensive protein domain analysis to map cancer-type-specific somatic mutations interpretation of the genome-wide association studies (gwas) of cancer patients to find cancer-typespecific biomarker is challenging due to the mutational heterogeneity of cancer types. network approaches to find cancer-type-specific variants and biological pathways are increasing since genes tend to act together to display phenotypic or disease outcomes. phenotype similarity has proven to reflect the relationship of functionally related genes. we applied phenotype similarities between various diseases for expanding molecular connections of cancer-type-specific variants to discover cancer-type-specific modules. specifically, cancer-type-specific variants of cancer types from the cancer genome atlas (tcga) were analyzed to find phenotype-inferred relationships among the variants. we find that cancer variants that cause the similar disease phenotypes tend to be linked as a cluster of biological pathways or functions. moreover, cancer-type-specific modules could explain the underlying pathogenicity of specific symptoms which manifest in particular cancer types. cancer-type-specific modules and pathways found from phenotype similarity/dissimilarity based on cancer symptoms improved the discrimination performance to sort cancer-type-specific variants to accurately predict patient groups. our method will be further developed to find genetic biomarkers for the diagnosis or prognosis of specific cancer types pk- engineering a stable, symmetric membrane protein scaffold amanda duran , jens meiler computational protein engineering has the potential to contribute to various fields including drug design, protein therapeutics, and materials science. protein-ligand interface design and the construction of large, stable proteins rely on stable scaffolds. symmetry is a great tool for protein stability both in protein engineering and nature. several membrane protein structures exhibit pseudo-symmetry and are proposed to be the result of gene duplication, fusion and diversification events originating from a monomeric gene. aquaporins (aqp) are a class of membrane proteins that exhibits a two-fold inverted pseudo-symmetry. the escherichia coli aqp glycerol facilitator protein (glpf) was originally computationally engineered to be perfectly symmetric in sequence and presumably in structure. the symmetric gene was assembled, cloned, and expressed. however, after facing many challenges experimentally, the computational study has been expanded to aqps of known structure for a more extensive symmetric backbone search. mammoth structural alignment was used to align the structures to their inverted counterparts. cutpoints were calculated based on a-carbon distance. finally, the rosetta protein modeling software suite was used to refine and energetically minimize the symmetric backbones. from over generated symmetric backbones, candidates were chosen for experimental verification. these studies are ongoing.currently, the symmetric backbone models have scored to be more stable than the wild-type proteins. experimental verification of these symmetric backbones will provide valuable information for the current state of membrane protein modeling and design using computational methods. intrinsically disordered proteins drive heritable transformations of biological traits daniel jarosz , james byers , sohini chakrabortee , sandra jones , amelia chang , david garcia stanford university, whitehead institute for biomedical research, rockefeller university the transmission of information from one generation to the next generally occurs via nucleic acids. the only known protein-based molecular memories are prions, which drive heritable biological traits based upon self-templating changes in protein conformation. these protein-based genetic elements have previously been identified systematically, but at least three do not share the sequence biases or structural characteristics that have informed such studies. here we employed a comprehensive library of yeast proteins to examine the breadth of protein-based inheritance. transient overexpression of more than forty proteins created new traits that were heritable and beneficial. some shared properties of known prions, but most employed distinct genetic and biochemical mechanisms to act as elements of inheritance. traits with these characteristics were common in wild yeast strains and could also be elicited using orthologous mammalian proteins. the inducing proteins were strikingly enriched in intrinsically disordered sequences that have been widely conserved across evolution. intrinsically disordered proteins are associated with human disease and with dosage sensitivity in yeast, flies and worms. our results suggest another widespread role for such intrinsically disordered sequences: induction of heritable epigenetic switches that transform phenotypic landscapes and drive adaptation to stressful environments. prediction of binding affinity in protein complexes: contacts do matters almost all critical functions in cells rely on specific protein-protein interactions. understanding these is therefore crucial in the investigation of biological systems. despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. we assess its performance against a protein-protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. using a subset of complexes with reliable experimental binding affinities and combining our contacts-and contact types-based model with recent observations on the role of the non-interacting surface in protein-protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. free radical oxidation -a new method for obtaining stable protein coatings on magnetic nanoparticles magnetically targeted nanosystems (mtnss) are now considered to be applicable in different areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay, cell and molecular separation, a smart delivery of drugs to target cells. proteins are promising materials for creation of coatings on magnetic nanoparticles (mnps) due to their biocompatibility, an ability to protect magnetic cores from influence of biological liquids and prevent agglomeration of mtnss in dispersion, their possible functional activity as therapeutic products and biovectors. the creation of stable protein coatings with retention of native properties of molecules is still an important biomedical problem because of disadvantages of the commonly used methods such as formation of a polydisperse ensemble of particles, nonselective linking of proteins leading to cross-linking of macromolecules in solution, and desorption of coatings. a novel method in obtaining stable single-layer coatings assembled from protein molecules on the surface of magnetite nanoparticles has been developed. it is based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links. free radicals are locally generated on the surface of nanoparticles via the fenton reaction thereby proteins adsorbed on the surface are subjected to the cross-linking. o-phenylenediamine was used for detection of free radical generation initiated by nanoparticles. the proteins drastically differing in their structure and properties, namely, serum albumin, thrombin and immunoglobulin g were selected for creating the protein coatings. the properties of the obtained coatings and their stability have been studied with the help of dynamic light scattering (dls), uv/vis spectrophotometry, antibody-antigen test and the method of spectral-fluorescent probes. albumin molecules in mnps coatings have been shown to retain their capability of binding with a dye and be conformationally stable. the dye , '-di-(g-sulfopropyl) , 'diphenyl- -ethiloxacarbocyanine-betaine interacting with albumin with a growth of fluorescence and with partial cis-trans conversion of the dye has been used. it has been proven that coatings composed of protein macromolecules are ) stable, ) formed around individual nanoparticles and ) have several nanometers in thickness. the free radical linking of thrombin and immunoglobulin g on the surface of nanoparticles has been shown to almost completely keep native properties of the protein molecules. the free radical linking method reveals new possibilities for design of single-layer multiprotein polyfunctional coatings on the surfaces of all the nano-, micro-and macroobjects containing metals of variable valence (for example, fe, cu, cr). the spectral-fluorescent investigation was supported by the russian foundation for basic research, project nos. - - and - - mol_a. regulation of neuronal snares by accessory proteins shrutee jakhanwal , reinhard jahn regulation of neuronal snares by accessory proteins shrutee jakhanwal and reinhard jahn department of neurobiology, max planck institute of biophysical chemistry, fassberg, goettingen, germany- . synaptic vesicle exocytosis lies at the heart of the process of neurotransmitter release. and, the family of proteins that is central to the process of synaptic vesicle exocytosis is the family of snare proteins. there are three kind of neuronal snare proteins namely syntaxin, snap and synaptobrevin. these three snare proteins interact through their snare-motifs to form a highly stable four-helix bundle, which in turn, pulls two membranes together to mediate fusion. years of work in this field have established that the four-helix bundle is critical for the membrane fusion to occur. however, the process of regulation of snare-mediated fusion remains very poorly understood. the major regulatory proteins involved in the process are munc , munc , synaptotagmin and complexin. the major aim of my project is to obtain a closer look at the regulation process of snare-mediated fusion by focusing on the interaction between the snare proteins and the regulatory proteins. to achieve this objective, i express and purify the different proteins involved in the process of snare-mediated fusion and thereafter subject them to appropriate biochemical characterization. in order to assess the role of the purified proteins in the process of fusion, i reconstitute them into liposomes and perform in-vitro lipidmixing assays. these assays are based on f orster resonance energy transfer (fret). based on the discretion of assessing the protein-protein or protein-lipid interactions, either the proteins or the lipids can be fluorescently labeled. also, the lipid compositions can be varied in order to assess the effect of lipid on the function of the respective protein. fluorescence-based anisotropy measurements can also provide information about the degree of freedom of a protein, indirectly providing information about the kinetics of a reaction. employing these techniques, i observe that munc - leads to displacement of syntaxin from a complex of syntaxin and snap . also, a complex of syntaxin and munc is resistant to the action of the aaa-atpase, nsf and its co-factor asnap, implicating this complex as a strong candidate for acting as the starting point for the process of neurotransmitter release. munc also appears to enhance lipid-mixing by interacting with the snare-complex. further investigations on the same lines can provide very useful insights into the process and can help us unravel the secrets that underlie the beauty of the exquisitely regulated process of neurotransmitter release. binding of thymidine nucleotides to a viral thymidine monophosphate kinase aldo a. centro de investigaci on en alimentaci on y desarrollo theme: biochemistry there is great interest in the evolution and activities of fish trypsins, since they appear to have evolved into different families. the cdna for trypsin iii from the monterey sardine (sardinops sagax caerula) was obtained and its deduced amino acid sequence matched its identity with a purified protease from the fish by mass spectrometry analysis. molecular modeling of sardine trypsin iii compared to other homologs showed a typical trypsin fold with all the cognate components for catalysis, and specific amino acid distribution that are possible factors that explain the cold adaptation. from phylogenetic analysis, sardine trypsin iii belongs to the novel y family, which is proposed to have evolved for cold adaptation. the obtained recombinant trypsin iii showed a low catalytic efficiency, but it remained active at cold temperatures, similar to other cold-adapted trypsins. the cold-adaptation of sardine trypsin iii opens a wide range of biotechnological applications for this protease and is also interesting from the serine protease structure-function relationship point of view. fungicidal mechanism of scolopendin , a cationic antimicrobial peptide from centipede heejeong lee , dong gun lee drastically (from . x - to x - colonies) upon deletion of this residues domain from the full length trai. we are investigating the structure and function of this very c-terminal end of trai using nmr spectroscopy. for the backbone assignment we used slice-selectively homonuclear broadband decoupled spectra along with standard experiments. three-bond scalar coupling constants were obtained through real-time j-upscaling experiments. with the backbone assignments, we have the first hand evidence which shows that his domain is for the most part intrinsically disordered, but contains short a-helical regions. structural development, interaction studies to find the binding partner and transition of disorder to order orientation of this domain will be further investigated in this project. here we investigated a model system where mab aggregation is induced by increasing the ionic strength (nacl) at low ph. the aggregation depends both on protein and sodium chloride concentration. with nanoparticle tracking analysis (nta) and micro flow imaging (mfi) the aggregation formation was further characterized. aggregation can be partially reverted by lowering the ionic strength as determined by soluble monomer concentration measurement using se-hplc: parts of insoluble aggregates could be solubilized as soluble aggregates, dimers or even monomers. a quasi equilibrium is formed in between the subtypes. the whole aggregation process was examined by ftir and cd-spectroscopy to identify structural changes of the mab. screen of protective additives: the effect of osmolyte additives on aggregation kinetics and final aggregate concentration is investigated, revealing protective effects in both cases. in a screen with more than compounds not only the aggregation propensity was studied but also structural changes. the aggregation index (quantity for colloidal stability) and the melting point (quantity for conformational stability) measured by differential scanning fluorimetry were determined. the used mtp format screen has potential for buffer optimization and formulation development. structural biology and protein dynamics tetraspanin cd has a broad range of cellular functions, such as integrin association forming tetraspanin-enriched domains, synapse formation between b and t cells, cell adhesion, motility, invasion and signalling. furthermore, cd is one of the four receptors involved in the cell entry of hepatitis c virus (hcv) and therefore infection onset, one of the major causes for chronic liver disease resulting in cirrhosis and hepatocarcinoma. human cd large-extracellular-loop (hcd lel) is composed of a "stalk" and a "head" subdomain; with the latter interacting with hcv-e glycoprotein. we present four novel hcd lel crystal forms. analysis of the fourteen independent observed hcd lel high-resolution x-ray structures suggests that the dynamism of the hcd lel head-subdomain is an inherent molecular property, an observation supported also by molecular dynamics (md) studies. we classify the conformations in three distinct clusters (closed, intermediate and open) , which are seen both in the crystal structures and in the molecular dynamics simulations. the md simulations also show that conformational variability is modulated by ph changes, with distinct probability for each cluster at acidic and neutral ph. furthermore, in silico docking of the recent e core structure with three of the major types of hcd lel head-subdomain clusters highlights hydrophobic interactions as the major forces in the e core: hcd lel recognition mechanism. we propose that the flexibility of the hcd lel is exploited by hcv at different stages of cell entry from virus attachment to internalization and fusion with the endosomal membrane. our results provide important insights on the basic mechanism governing hcv binding to hcd , and can help structure-based drug design of entryinhibitors of hcv. allophycocyanin of gracilaria chilensis: from gene to function jorge dagnino-leone , jos e martinez-oyanedel , marta bunster-balocchi universidad de concepci on theme: structure-function relationship of proteins the phycobilisomes (pbs) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of - nm. in gracilaria chilensis, an eukaryotic red algae, pbs is composed of phycoerythrin (pe), phycocyanin (pc) and allophycocyanin (apc); these proteins possess chromophores which capture energy and then transfers it to photosytems. pbps are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab) , this trimer has discoidal shape and it is associated in hexamers (ab) , several of this hexamers forms cylinder-like structures. pbs has components: antennas and core. the antennas are composed of pe and pc, whose function is to capture energy between - and - nm respectively and transfer it to the core. the core is formed by apc, which can absorb energy in the - nm range. apc emission allows transferring energy to the photosystems with high efficiency. pbs is also composed by linker proteins which allow the correct assembly of pbs and possibly regulate the energy transfer. the main goal in our group is to build an atomic model of the gracilaria chilensis phycobilisome. we have solved the crystal structure of pe and pc and created an antenna model. at present we are working in apc and the chromophorilated linker proteins. the objective of the present work is to create a model of the core of gracilaria chilensis; to achieve these we have used molecular biology, biochemistry and bioinformatics techniques. we designed oligonucleotides primers for the four allophycocyanin subunits genes and for the globular domain of the apce linker. these primers were used in pcr experiments to obtain the genes sequences. the sequences were translated to a aminoacid sequences and used to build a d model for apc subunits and trimers using the software modeller. on the other hand we purified and analyzed the spectroscopic properties of apc from gracilaria chilensis using absorption and fluorescence spectroscopy. we also determined apc oligomerization state using gel filtration. molecular docking using the cluspro server was performed to obtain a hexamer and apc cylinder models. based on electron micrographs obtained by our lab a tri-cylindric core model was built. all the models were submitted to a molecular dynamics using gromacs software. finally we determine possible energy transfer pathways in the core model applying the extended forster equation, spectroscopic data from literature and the transition dipole moments of each of the chromophores present in the core. as conclusion of this work we built the first atomic model of gracilaria chilensis phycobilisome core and propose energy transfers pathways inside the core in the context of a phycobilisome. novel practical strategies to access artificial metalloenzymes marco filice , jose miguel palomo departamento de biocat alisis, instituto de cat alisis, csic protein chemistry and engineering since the first report, the design of artificial metalloenzymes has rapidly been converted into an important topic in biological and inorganic chemistry due to their potential applications in synthetic chemistry, nanoscience and biotechnology. the combination of a catalytically active organometallic moiety with a macromolecular host has permitted the creation of biohybrids, a new kind of heterogeneous catalytic entities combining the attractive features of both homogeneous and enzymatic systems. presenting our most recent achievements in this research area, here we describe two novel powerful and promising approaches focusing the practical synthesis and large scale production of heterogeneous artificial metalloenzymes showing chimeric activity. the first strategy is based on the in situ synthesis of noble metal nanoparticles and their supramolecular assembly with a microbial lipase from candida antarctica (fraction b) finally creating an ultra-active organometallic-enzyme heterogeneous nanobiohybrid. in the second approach, combining different protein engineering protocols (molecular biology, orienting immobilization, solid-phase bioorganic modification and bioinformatic tools), an orthogonal solid-phase strategy creating novel unnatural catalytic sites was designed and optimized. the application of such a strategy onto the structure of the lipase from geobacillus thermocatelunatus permitted the generation of a heterogeneous artificial metallolipase with chimeric activity. as proof-of-concept, the combinatorial library of generated artificial metalloenzymes obtained by both strategies was successfully assessed in a set of different synthetic reactions (selective c-c bond formation as suzuki, heck or diels-alder reactions) and also combining both activities (metallic and enzymatic) in cascade processes such as dynamic kinetic resolution of amines or production of arylamines. the obtained results were excellent in all cases. extending this strategy to other enzymes, proteins and catalytic metals, we envisage the creation of a combinatorial library of programmable artificial enzymes useful for a wide set of applications (i.e. fine organic and medicinal chemistry, bioremediation or biomedicine). proteomic examination of the yeast nuclear pore complex dynamics protein turnover and exchange nuclear pore complexes (npcs) are proteinaceous assemblies situated in nuclear envelopes of eukaryotic cells. the main function of the npc is the selective transport of macromolecules. npcs also partake in other functions, such as nuclear organization and gene regulation. the core scaffold of the npc is thought to be a stable structure, while the peripheral components exchange at various rates. however, these phenomena have not been elucidated in detail. the recent findings that yeast daughter cells get a higher proportion of the old npcs and the core scaffold hardly turns over raise the possibility that the exchange of the peripheral nucleoporins can be a repair mechanism. yeast provides a useful organism for the interrogation of nucleoporin exchange, as it performs closed mitosis; hence the only mixing of npc constituents is due to exchange. we have developed a panel of genetic tools providing for conditional induction and repression of nucleoporins. by combining these switches with stable isotope metabolic labeling and affinity capture, cross linking coupled to mass spectrometry, we are able to distinguish between pre-existing and newly synthesized proteins and quantify their relative amounts in the npc. our preliminary findings are in agreement with results obtained in other organisms: the core scaffold of the npc (inner ring, outer ring) appears to be stable, however does exchange slowly over time, while peripheral components exchange faster. by looking at the exchange rates of yeast nucleoporins we hope to gain insight into the npc biology of actively dividing eukaryotic cells. active site clustering identifies functional families of the peroxiredoxin superfamily angela harper , janelle leuthaeuser , patricia babbitt , jacquelyn fetrow department of physics, wake forest university, department of molecular genetics and genomics,-wake forest university, departments of physics and computer science, wake forest university bioinformatics understanding the relationships between proteins is vital to increasing our knowledge of the protein universe. while there are large databases of sequence information, the massive data influx over the past decade has prevented adequate classification of proteins at the molecular function level. however, it has been previously suggested that a protein's active site information may correlate with these known molecular functional differences; thus, active site profiling was developed to use residues around the active site of a protein to relate proteins. subsequently the deacon active site profiler (dasp) was developed to create these active site profiles and search them in a database, such as gen-bank, in order to find proteins with similar active site environments. by using dasp to computationally cluster proteins based on the similarity of their active site profiles, the peroxiredoxin (prx) superfamily was analyzed through active site similarity methods. the residues from the active site of each prx structure were extracted and clustered, and these profiles were iteratively searched in genbank through a multi-level iterative sequence searching technique (misst). the prx superfamily has been studied by experts, allowing the results of these searches to be compared to a well-annotated group of proteins. while previous sequence based evolutionary methods have been unable to identify functional differences between some subgroups of the prxs, notably the ahpc-prx and prx subgroups, misst discretely separates these subgroups. classifying prx proteins into functionally relevant groups using computational active site similarity methods lays the foundation for an automated process for identifying protein functional groups beyond the prx superfamily. synthesis and conformational studies of glycoprotein n homolog of bovine herpesvirus (bhv- ) by using cd, nmr and molecular modelling it serves as a chaperone for viral glycoprotein m and, in its gm-unbound form, acts as an inhibitor constraining the transporter associated with antigen processing (tap). the ul . /gm complex formation is required for the maturation and proper trafficking of both viral proteins. in the absence of gm, ul . blocks transport of antigenic peptides by tap and their mhc i-restricted presentation. the molecular mechanism of ul . activity still remains elusive. in order to investigate the structural requirements for biological function ul . study was conducted using cd, nmr and molecular dynamics methods. the data obtained with the use of high purity synthetic peptides encompassing ul . confirmed the presence of an alpha-helix structure, formed preferentially in the presence of dodecylphosphocholine (dpc) micelles as a membrane-like environment. in order to determine the three-dimensional structure of ul . protein in the present work its nmr solution structure in the presence of membrane-like environment was performed. the nmr data were used as a set of restraints for a simulated annealing protocol that generated dstructures of the colin johnson , sara codding membrane proteins resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. defects in this repair process are responsible for muscular dystrophy and cardiomyopathy. the repair pathway is triggered by the influx of calcium through lesions in the membrane, which result in membrane fusion and patching of the wound. recently dysferlin has been identified as a calcium binding protein essential for sarcolemma repair, as well as other snare mediated exocytotic events including cytokine and acid sphingomyelinase secretion. in this presentation we demonstrate a direct interaction between dysferlin and the snare proteins syntaxin and snap- . in addition, fret and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates snare heterodimer formation and snare mediated lipid mixing in a calcium sensitive manner. our results suggest a model whereby dysferlin acts as a calcium sensing snare effector for exocytosis and membrane fusion. exploring the therapeutic potential of a peptide derived from a poxviral immune evasion protein: nmr determination of the solution structure of viper and its inactive mutant toll-like receptors (tlrs) have a role in viral detection leading to cytokine and ifn induction, and as such they are targeted by viruses for immune evasion. the poxviral protein a has been identified to inhibit tlr signaling by interacting with tir domain-containing proteins of the receptor complex to collectively inhibit all tlr adaptor proteins that positively regulate transcription-factor activation ( ). one aa peptide (kysf-klilaey) termed viper (viral inhibitory peptide of tlr ) was reported to retain the inhibitory properties of full length a against tlr signaling. a r homopolymer delivery sequence at the c-terminus provided delivery of the peptide into cells. structural comparisons are presented between r-viper, which is active in preventing tlr -dependent cytokine induction in cell culture, and a mutant that exhibited loss of function ( r-viper l a,e a), through solution nmr spectroscopy. we find that despite a relatively minor sequence difference, the loss of hydrophobicity as well as negative electrostatic interactions result in subtle but potentially significant differences in the region of the peptide proposed to interface with tlr . reference: wake forest university, wake forest university, university of california san francisco protein function prediction the elucidation of protein molecular function lags far behind the rate of highthroughput sequencing technology; thus, it is essential to develop accurate and efficient computational methods to define functional relationships. protein clustering based on sequence similarity has emerged as a simple, high-throughput method for defining protein relationships, but sequence-based techniques often inaccurately define molecular function details. active site profiling (asp) was previously developed to identify and compare molecular details of protein functional sites. protein similarity networks were created using both active site similarity and sequence similarity for four manually curated superfamilies, and results demonstrate that asp-based clustering identifies detailed functional relationships more accurately than sequence-based clustering. building on this, two iterative pipelines were developed using active site profiling and profile-based searches to cluster protein superfamilies into functional groups. first, the two level iterative clustering process (tulip) utilizes active site profiling and iterative pdb searches to divisively cluster protein structures into groups that share functional site features. across eight superfamilies, tulip clusters exhibit high correlation with expert functional annotations. subsequently, the multi-level iterative sequence searching technique (misst) utilizes iterative profile-based genbank searches to identify protein sequences that belong in each tulip group. the results indicate that these asp-based methods accurately and efficiently identify functionally relevant groups through a process that can be applied systematically and on a large-scale. moreover, the approach can be applied more quickly than detailed manual curation, suggesting its value in guiding annotation efforts. dept. biochemistry and molecular biology. university of valencia, lab of peptide and protein chemistry. centro de investigaci on pr ıncipe felipe membrane proteins changes in the equilibrium between pro-survival and pro-apoptotic members of the b-cell lymphoma- (bcl- ) protein family at the mitochondrial outer membrane (mom) induce structural changes that committed cells to apoptosis. bcl- homology- (bh )-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the mom. the membrane association of bh -only proteins is a controversial issue due to the lack of a canonical carboxyl-terminal (c-terminal) transmembrane (tm) domain. we used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic c-terminal regions of the bh -members bik, bim, noxa, puma and bmf into microsomal membranes, and an escherichia coli complementation assay to validate our results in bacterial cells. furthermore, we have fused these hydrophobic regions to gfp to investigate the subcellular sorting. these results will allow further refinement in the elaboration of the bcl- protein-protein and protein-membrane interactome network. alexis peña , flaviyan jerome irudayanathan , shikha nangia syracuse university, dept. of biomedical and chemical engineering computational modeling, biostatistics, biomedical and chemical engineering tight junctions (tj) are vital intracellular barriers that are responsible for regulating paracellular transport. claudins, a family of abstract small transmembrane proteins with approximately members, are an integral part of the tj strands. tight junctions provide molecular-level protection and prevent infection and toxins from entering the body; in the same sense tjs allow nutrients and vital solutes to pass through. claudins are associated with various diseases including metastatic cancer as well as an entry point for many viruses. despite their importance and abundance in all cell membranes and their ubiquitous nature, the exact -d structure of claudins has remained elusive to traditional x-ray crystallographic and nmr studies. in this investigation, a computational approach was used to determine the claudin structure of claudin - . homology modeling, molecular dynamic simulations, and reverse mapping were employed to predict the protein structures with relative accuracy. understanding structure of claudin proteins and its interaction at the molecular level can lead to effective drug delivery technology. determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) idania emedith quintero reyes , francisco javier castillo y añez , enrique fernando vel azquez contreras , roc ıo sugich miranda , david octavio corona mart ınez , aldo alejandro arvizu flores , ivet cervantes dom ınguez protein kinetics determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) trypsin is the most studied alkaline protease and it s very common to found isoforms from this protein as the case for monterey sardine (sardinops sagax caerulea); as it shows an expression of trypsin i and trypsin iii according to the cdna characterization. trypsin i was determine to be a cold adapted enzyme as it shows a higher catalytic efficiency (kcat/km) than the mesophilic counterparts. the kinetic parameters were obtained by spectrophotometric essays, which are not fallible for all the enzymes because native, recombinant or mutant enzyme activity could be below the detection limit of the assay, opaque or turbid solutions interfere with spectrophotometric detection, etc. alternative tools as the isothermal titration calorimetry (itc) can measure enzyme kinetics using thermal power generated by the enzymatic conversion of substrate to product; were the rate of reaction is directly proportional to thermal power. the objective of this study was to stablish the optimum conditions to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine using itc. to reach the objective trypsin i was purified from viscera of monterey sardine using molecular exclusion and affinity chromatography obtaining a yield of . mg/ml. at c kcat and km of tryipsin i form monterey sardine were . s- and . mm respectively. at c were . s- and mm (kcat and km) and at c kcat was . s- and km . mm. the kinetic parameters obtained by spectrophotometric assay at c were kcat and km s- and . mm respectively. at c the kcat was . s- and km . mm and at c kcat s- and km mm. comparing the values obtained for kcat with the spectrophotometric essay were higher fold than those obtained by itc and the values in km were similar by both methods. even though the differences in kcat, we can reassert the psychrophilic behavior of trypsin i as the catalytic efficiency is higher by both methodologies. in the understanding that the kinetic behavior of enzymes is important to not only understanding biochemical pathways and catalytic mechanisms but is again a fruitful area for drug discovery and development; so the itc provides a universal approach to determining the kinetic behavior of enzymes and can yield in a single experiment a complete set of kinetic parameters for an enzyme-catalyzed reaction that can be applied for the different alkaline proteases from pyloric caeca of monterey sardine (sardinops sagax caerulea). mysterious world of stress-responding sigma factors in bacillus subtilis olga ramaniuk protein-dna interaction bacterial transcription is mediated by the rna polymerase holoenzyme containing sigma factors -essential proteins for the initial step of transcription that recognize and bind to promoter dna. the primary sigma factor is essential in exponential phase of growth while alternative sigma factors are active during transcription under stress conditions. this project has three main aims. the first aim is to explore the binding properties of b. subtilis alternative sigma factors; specifically, whether sigma factors lacking the autoinhibitory domain . can bind to promoter dna in the absence of rnap. the second aim explores whether rnap associated with alternative sigma factors is regulated by the concentration of the initiation nucleoside triphosphate. the third aim is to define the regulon of sigma i. in order to achieve our aims, out of alternative sigma factors were successfully purified using affinity chromatography and ion exchange chromatography. we set up in vitro transcription system with selected sigma factors and initiated experiments with sigma i regulon determination. results named above and our future findings will help to better understand gene expression regulation on the level of transcription initiation. this work was supported by grant no. p - -g from the czech science foundation. assessing the costs and benefits of protein aggregation protein aggregation and cell fitness protein aggregation has been associated with numerous diseases but also with important cellular functions such as epigenetic inheritance. here we present a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. this information is crucial to understand how cellular systems tolerate the formation of protein deposits and which factors modulate this event. using our experimental system, we measured different protein aggregation effects (deleterious, neutral or beneficial) within the same genomic background. single cell analyses, within the same population, showed stochastic variability in the aggregate's size and in its effect on cell fitness. our data indicates that, in certain conditions, protein aggregation can enhance population variability and survival expectancy. overall, these results suggest that the presence and formation of protein aggregates could be almost harmless whereas the associated gain and loss of function are critical for the cell. revealing the key role of negatively charged residues of heme sensor proteins involved in geobacter sulfurreducens' signal transduction pathways marta a. silva , telma c. santos , teresa catarino , carlos a. salgueiro ucibio-requimte, departamento de qu ımica, fct-unl., instituto de tecnologia qu ımica e biol ogica, unl signal transduction proteins bacterial chemotaxis systems sense and regulate the microbe mobility in response to environmental conditions. such mechanisms constitute a striking example of cell motility to gain advantages for cell survival and permit the bacteria to fill important niches in a diversity of anaerobic environments [ ] . geobacter sulfurreducens (gs) is an anaerobic bacterium with a considerable respiratory versatility whose genome encodes for an unusual family of methyl-accepting chemotaxis proteins (mcp), each containing at least one heme c-binding motif [ ] . these sensor proteins, gsu and gsu , are involved in signal transduction pathways mediated by chemotaxis-like systems [ ] . the thermodynamic and kinetic characterization of the sensors gsu and gsu by visible spectroscopy and stopped-flow techniques, at several ph and ionic strength values revealed that sensor gsu midpoint reduction potentials are lower than those of gsu at all ph and ionic strength values and the same were observed for the reduction rate constants [ ] . the origin of the different functional properties of these closely related sensor domains are rationalized in the structural terms showing that gsu has two extra negatively charged residues in the vicinity of the heme group, which have no counterpart in gsu : glu and asp . residue asp is less exposed compared to glu and it was suggested that its carboxylic group might have a role in the modulation of the heme reduction potential of gsu . to investigate this, both residues were replaced by a positively charged amino acid (lysine) and by a neutral one (asparagine or glutamine). for the mutants with enough expression, a functional characterization was carry out, using several spectroscopic techniques, including uv-visible and cd, together with kinetics and potentiometric measurements. significant changes on the reduction potential values are observed when a negative charge is replaced by a positive one at position or . therefore, the decrease of the reduction potential in asp and glu mutants reinforces the hypothesis that the higher reduction potential observed for heme sensor domain gsu is related with the less negative electrostatic surface around the heme. this work provides, for the first time, evidence for the co-existence of two similar methyl-accepting chemotaxis proteins functioning in different working potential ranges. these proteins are responsible to allow geobacter sulfurreducens triggering an adequate cellular response in different anoxic subsurface environments. national autonomous university of mexico, faculty of medicine, national autonomous university of mexico, faculty of chemistry, national autonomous university of mexico, institute of chemistry molecular evolution the glycolytic enzyme triosephosphate isomerase (tim) is an oligomeric (b/alpha) barrel that catalyses the interconversion of d-glyceraldehyde -phosphate and dihydroxyacetone phosphate in a diffusion-limited reaction. although each subunit has its own active site, naturally occurring monomeric tims have not been reported; in fact, monomer association is very tight. tim topology is well conserved among the three domains of life. nevertheless, their folding mechanism and inhibition properties vary across species. comparative studies of proteins have proved to be very useful in understanding the relationship between sequence and physicochemical properties, however, they lack the capacity to give a more integrative and evolutive correlation. in order to elucidate how the catalytic properties, the oligomerization state and the stability of extant tims arose, in this work we examined the molecular history of eukaryotic tim through ancestral protein reconstruction methods (maximum likelihood) and the subsequent physicochemical characterization of the resurrected enzymes. we first characterized in detail the protein corresponding to the last common ancestor of animals and fungi (tim ). the cd and fluorescence spectra of tim are similar to those of extant tims. secondary structure is lost in a cooperative transition with tm . c. the enzyme loses activity upon dilution suggesting that only the dimer is active. dilution experiments followed by isothermal titration calorimetry indicate that dissociation enthalpy is small; moreover the heat capacity change observed is three times higher than the one predicted for a rigid body dissociation process, suggesting partial unfolding of the monomers. when compared with extant tims, the catalytic efficiency of tim is reduced -fold, whereas binding of pgh, a transition-state analogue, shows a similar thermodynamic signature. these data indicate that although monomer association may have been less tight in ancestral tims, catalysis has been always linked to oligomerization. analysis of the crystal structure of tim , obtained at . Å resolution, suggests that the lack of four salt bridges observed in the interface of extant tims is responsible for the low dimer stability. in order to test this hypothesis we also studied the stability of four younger reconstructed ancestors that acquired the salt bridges in two different phylogenetic lineages. we found a correlation between the appearance of stabilizing interactions in the interface, dimer stability and catalysis; suggesting that these salt bridges are partially responsible for extant dimer stability and shed light on the dimeric nature of extant tims. receptor protein-tyrosine phosphatases: dimerization, receptor kinase interaction and allosteric modulation elizabeth dembicer , damien thevenin department of chemistry, lehigh university theme: receptor tyrosine kinase and receptor protein phosphatase signaling many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins, which is controlled by the counterbalanced actions of two key enzyme families: protein tyrosine kinases and protein tyrosine phosphatases. interestingly, both families include transmembrane receptor-like enzymes, namely the receptor tyrosine kinases (rtks) and the receptor-like ptps (rptps). while the regulation and actions of many rtks are well characterized, the mechanisms controlling the enzymatic activity of rptps and how they interact with their substrates remain to be fully explained. thus, understanding how these receptors function and interact will give fundamental insights into how tyrosine phosphorylation is finely tuned in cells, and how it can be modulated. increasing evidence indicates that rptps, like rtks, are regulated by homodimerization. however, it appears that homodimerization inhibits the activity of most rptps. even though the transmembrane (tm) and the juxtamembrane domains have been proposed to be involved in this process, there is no clear structure-based proposal for the role of these regions. moreover, several rptps have been identified as candidate regulators of rtks. in particular, the receptor-type tyrosine-protein phosphatase eta (ptprj; also known as dep or cd ) is capable of attenuating egfr tyrosine phosphorylation. physical interactions of egfr with ptprj at the cell surface have been documented, but the basis for these interactions is unknown. here, using a dominant-negative transcriptional activator-based assay (dn-aratm), and mutagenesis analysis, we show that: ( ) ptprj has a strong tendency to homodimerize, ( ) ptprj heterodimerizes with egfr through tm-tm interactions, ( ) these interactions are mediated by specific residues, and can be modulated by the delivery of peptide binders. this work represents the first structure-function study of rptp-rtk interaction, and may not only result in significant progress towards a better understanding of the basic biology of rptps in cancer cells, but also offer new possibilities for targeting protein tyrosine phosphatases for therapeutic modulation of egfr in oncology. inhibiting egfr dimerization and signaling through targeted delivery of juxtamembrane domain peptide mimics using phlip anastasia thevenin , kelly burns , janessa guerre-chaley , damien thevenin regulating receptor tyrosine kinase signaling the elevated phosphorylation of key regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein tyrosine kinases activity plays well-abstract established roles in promoting tumorigenesis and in the high frequency with which resistance arises to existing therapeutic treatment. for instance, this is the case for the epidermal growth factor receptor (egfr). thus, there is a clear need for novel specific targeting methods to inhibit the activity of receptor protein tyrosine kinases, such as egfr, in cancer. egfr becomes activated upon ligand binding to the extracellular domain, leading to receptor dimerization. the juxtamembrane (jm) domain of egfr is critical for intrinsic tyrosine kinase activity and receptor dimerization by stabilizing the active conformation of egrr through the formation of a antiparallel helical dimer. therefore, peptides mimicking the jm domain -if specifically delivered to cancer cells -have the potential to prevent egfr dimerization, receptor activation, downstream signaling, and thus to attenuate aberrant egfr activity in cancer cells. here, phlip (ph low insertion peptide), a peptide that can selectively target cancer cells and tumors based solely on their extracellular acidity, is used to selectively translocate the jm domain of egfr in cancer cells to prevent egfr dimerization. at ph above , phlip is soluble and unstructured, however, when exposed to lower ph such as observed in tumors, phlip inserts as a transmembrane (tm) alphahelix, allowing the direct translocation of cargo molecules into the cytoplasm. using the dominant negative arac-based transcriptional reported assay (dn-aratm), which assesses jm and tm domain interactions in cells membranes of e. coli, we show that phlip-jm is able to disrupt egfr dimer by %. current work is focused on testing the ability of such phlip-jm peptide conjugate to perturb egfr homodimerization and decrease downstream signaling through soluble kinases, such as akt and erk, in cancer cells. the thumb subdomain of yeast mitochondrial rna polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding gilberto velazquez , luis brieba , rui sousa universidad de guadalajara, langebio cinvestav, university of texas healthsscience center at san antonio dna protein interaction abstract single subunit rna polymerases have evolved two mechanisms to synthesize long transcripts without falling off a dna template: binding of nascent rna and interactions with an rna:dna hybrid. mitochondrial rna polymerases share a common ancestor with t-odd bacteriophage single subunit rna polymerases. herein we characterized the role of the thumb subdomain of the yeast mtrna polymerase gene (rpo ) in complex stability, processivity, and fidelity. we found that deletion and point mutants of the thumb subdomain of yeast mtrna polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (mtf ). the latter suggests that the thumb subdomain is part of an extended bindingsurface area involved in binding mtf . design principles of membrane protein structures vladimir yarov-yarovoy , diane nguyen membrane protein structure membrane proteins play key role in cellular signaling and ion transport. statistical analysis of expanding database of high-resolution membrane protein structures in protein data bank (pdb) provides useful information about membrane protein structure and function. we used rosettamembrane software (yarov-yarovoy v et al ( ) proteins) to analyze unique alpha helical membrane protein structures in pdb and derive knowledge based energy function for membrane protein structure prediction, membrane protein-protein docking, and membrane protein design. the rosettamembrane residue environment energy term is based on amino acid propensities in hydrophobic, interface, and water layers of the membrane and depends on the residue burial state -from being completely buried within a protein environment to being completely exposed either to the lipid or water environments. residue buried state is determined from the number of residue neighbors within and Å spheres. the rosettamembrane residue-residue interaction term is based on the propensities of amino acid pairs to be in close proximity to each other within hydrophobic, interface, and water layers. results of our statistical analysis reveal fine details of favorable and unfavorable environments for all amino acids types in all membrane layers and residue burial states. we find that large hydrophobic amino acids are favorable facing the hydrophobic core of the lipid bilayer. small amino acids are favorable facing the protein core within the hydrophobic layer of the membrane. aromatic or positively charged amino acids and favorable facing the lipid head groups. residue-residue interactions are often favored between polar and charged amino acids and also between some of small and large hydrophobic amino acids inside of the protein core within the hydrophobic layer of the membrane. these data will be useful for rational design of novel membrane protein structures and functions. coordinated gripping of substrate by subunits of a aaa proteolytic machine ohad yosefson , andrew nager , tania baker , robert sauer protein quality control' or 'protein degradation' hexameric aaa protein-remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. an open question in the aaa field is whether pore loops from different subunits of the hexameric ring grip the substrate coordinately (all six subunits involved), independently (one subunit at a time involved), or partially coordinated (two or three subunits at a time). to answer this question, we studied covalently linked hexamers of the e. coli clpx unfoldase bearing different numbers and configurations of wild-type and mutant pore loops and challenged these variants with protein substrates with a broad range of stabilities. we find that successful unfolding of increasingly resistant substrates requires the coordinated action of a greater number of wild-type pore loops. our results support a mechanism in which a power stroke initiated in one subunit of the clpx hexamer results in the simultaneous movement of all six pore loops, which coordinately grip and apply force to the substrate. structure and function of the toc m-domain, and its role in targeting the preprotein receptor to the chloroplast outer envelope membrane matthew smith , shiu-cheung lung , prem nichani , nicholas grimberg , j. kyle weston , shane szalai , simon chuong deartment of biology, wilfrid laurier university, department of biology, university of waterloo chloroplast biogenesis and function rely on the import of thousands of nucleus-encoded preproteins from the cytosol. preprotein import is supported by the toc and tic (translocon at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively to translocate preproteins across the double-membrane envelope to the chloroplast interior. toc is one of the preprotein receptors of the toc complex, is also encoded in the nucleus and post-translationally targeted to the chloroplast, and is comprised of distinct domains: ) the intrinsically disordered n-terminal acidic (a-) domain; ) the central gtpase (g-) domain; and ) the c-terminal membrane (m-) domain that anchors the protein to the chloroplast outer membrane (com) through an unknown mechanism. the m-domain has no known homologues and does not contain a predicted trans-membrane domain, but does contain intrinsic chloroplast targeting information at the extreme c-terminus. the m-domain also contains a predicted b-helix motif, which may be important for anchoring the protein to the com. we are interested in characterizing the structure of the m-domain and determining the nature of its association with the com, as part of our larger goal of understanding the role toc plays in protein import into chloroplasts. we are also interested in defining the precise nature of the targeting information contained within the extreme c-terminus of toc , elucidating the targeting pathway that is used, and whether other com proteins use this pathway. we will present our most recent data on the structure, function and targeting of the toc m-domain. structural investigation of nlpc/p protein acquired by trichomonas vaginalis through a lateral gene transfer event jully pinheiro , , augusto simoes-barbosa , david goldstone microbiology, school of biological sciences, university of auckland, structural biology, school of biological sciences, university of auckland trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes the most common non-viral sexually transmitted disease, with approximately million cases worldwide annually. nevertheless, the biochemical processes behind t. vaginalis infection and its interaction with the vaginal microbiota are still not well defined. in the draft genome sequence of trichomonas vaginalis strain g was described, identifying , protein-coding genes. of these, nine genes encode nlpc/p -like members. this superfamily is widely represented in the different kingdoms of life and has diverse enzymatic functions, such as amidases, endopeptidases and acetyltransferases. previous studies have shown that members of this superfamily hydrolyze specific peptide linkages in bacterial cell walls affecting germination, vegetative growth, sporulation and division or cell lysis/invasion. as a typical eukaryote, the protozoan parasite t. vaginalis does not have a cell wall itself. previous studies suggest that the t. vaginalis nlpc/p genes were acquired via lateral gene transfer from bacteria and must have an important function, possibly controlling the vaginal microbiota and aiding parasite invasion and infection. to investigate the function of the nlpc/p family of proteins in t. vaginalis we have expressed, purified and crystallized a member tvag_ and report its three-dimensional structure, determined at . Å resolution, by x-ray diffraction. the structure of the protein reveals a typical papain-like fold resembling peptidoglycan hydrolases from the nlpc/p family with a conserved cysteine and histidine; forming the catalytic residues. the protein contains two bacterial sh domains at the n-terminus. this domain acts as a general binding domain and is likely to aid the interaction of the nlpc/p domain with substrate components. combined with biochemical and enzymatic characterization, the structure of this nlpc/p protein will help to elucidate the molecular origin of its hydrolase activity and to decipher their putative role in the parasite infection. novel dna polymerases from red sea brine-pools: new potential polymerases for pcr application masateru takahashi , etsuko kimura , mohamed salem , ulrich stingl , samir hamdan protein biotechnology the polymerase chain reaction (pcr) is a key tool in medical and biological research. the most common pcr reaction relies on the thermal cycling method that consists of repeated cycles of heating and cooling steps for dna melting and extension by the dna polymerase, respectively. the introduction of new dna polymerases to the market is a major area of development that tremendously helped in improving the performance and quality of pcr. nonetheless, pcr still requires optimization of salt and metal ion concentrations leaving a room in the market for introducing new dna polymerases that are robuster in their salt and metal ion concentration dependence. in this study, we will present the characterization of a novel archaeal dna polymerase from the red sea brine-pool (termed br ) and demonstrate how its enzymatic activity reflects on every aspects of the environment of the brine-pool -high tolerance to concentrations and types of salts and metal ions including utilization of zn ions in its active site. these results suggest that the brine-pool microorganisms are likely to contain novel chemical pathways to deal with its exterior harsh conditions. we will further show the mechanism of br polymerase how it was adjusted to be active in harsh condition. structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid protein as an antiviral target ming-hon hou , shing-yen lin , chia-ling liu , yu-ming chang , jincun zhao , stanley perlman institute of genomics and bioinformatics, national chung hsing university., institute of biological chemistry, academia sinica., department of microbiology, the university of iowa drug discovery coronaviruses (covs) cause numerous diseases, including middle east respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. covs encode the nucleocapsid (n) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral rna through the n protein's nterminal domain (n-ntd). using human cov-oc (hcov-oc ) as a model for cov, we present the d structure of hcov-oc n-ntd complexed with ribonucleoside '-monophosphates to identify a distinct ribonucleotide-binding pocket. by targeting this pocket, we identified and developed a new coronavirus n protein inhibitor, n-( -oxo- , -dihydrophenanthridin- -yl)(n,n-dimethylamino)acetamide hydrochloride (pj ), using virtual screening; this inhibitor reduced the n protein's rna-binding affinity and hindered viral replication. we also determined the crystal structure of the n-ntd-pj complex. on the basis of these findings, we propose guidelines for developing new n protein-based antiviral agents that target covs. thermal and structural stability of ß-glucosidases gh maira artischeff frutuoso departamento de bioqu ımica do instituto de qu ımica da universidade de são paulo enzymology we compared the stability of thermophilic b-glucosidases gh to mesophilic ones in the presence of denaturants as urea and high temperature by following the transitions between the native and unfolded states by tryptophan fluorescence, enzymatic activity and differential scanning fluorimetry (dsf). the bacterial b-glucosidases (bgla) and (bglb) of the mesophile paenibacillus polimyxa and bglucosidase (bglthm) of the thermophile thermotoga maritima were expressed as recombinant proteins in novablue (de ) and purified by affinity chromatography (ni-nta resin). these recombinant enzymes have very similar folding type structure (b/a) barrel, as shown in crystal structures and exhibited a characteristic peak between and nm in the tryptophan fluorescence spectra, indicating that those proteins are folded. circular dichroism analysis in the far-uv region ( nm to nm) also showed typical spectra of folded proteins with secondary structure composition of % of a-helix and % of b-sheets for bgla, % of a-helix and . % of b-sheets for bglb and % of a-helix and % of b-sheets for bglthm. the average degree of accessibility to the exposed tryptophan residues in the native enzyme to increasing concentrations of the acrylamide suppressor (stern-volmer constant -ksv) is greater to bgla ( . ), but similar to bglb ( . ) and bglthm ( . ). the thermal stability determined by dsf was higher for bglb (tm . c) than for bgla (tm . c) . the bglthm was stable at c and remained stable for up to h at c. in addition the thermal inactivation kinetics at c evaluated by the relative remaining activity showed that bgla denaturation (kinactivation of . s- ) is faster than bglb (kinactivation of . s- ). on the other site, bglthm inactivation at c was a two-step process, which exhibited an initial fast step (kinactivation of . s ) followed by a slow step (kinactivation of . s- ). the chemical denaturation by urea followed using tryptophan fluorescence showed a transition pl- covalent structure of single-stranded fibrinogen and fibrin oligomers cross-linked by fxiiia. the influence of free radical oxidation anna bychkova , vera leonova , alexander shchegolikhin , marina biryukova , elizaveta kostanova , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences protein structure and function native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing the cross-linked fibrin clots under the effect of thrombin and fibrin-stabilizing factor (fxiiia). fxiiia-mediated isopeptide g-g bonds are known to be produced between g polypeptide chains of adjacent fibrinogen or fibrin molecules. but there are apparently conflicting ideas regarding the orientation of g-g bonds. in this study several peculiarities of self-assembly of fibrin(ogen) and induced oxidation of the proteins have been studied with the aid of elastic and dynamic light scattering, uv-, ftir-and raman spectroscopy methods. in the presence of fxiiia both the non-oxidized and oxidized fibrinogen molecules has been shown to bind to each other in the "endto-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. to identify the orientation of g-g bonds in fibrin protofibrils a novel approach based on self-assembly of soluble cross-linked fibrin protofibrils and their dissociation in the urea solution of moderate concentrations has been applied. the results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated the protofibrils to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures entirely brought about by g-g bonds. the results of this study provide an evidence to support the model of the longitudinal g-g bonds that form between the g chains end-to-end within the same strand of a protofibril. since fibrinogen is known to be sensitive to ros the mechanisms of fibrinogen and fibrin self-assembly under induced oxidation have been investigated. in both cases the polypeptide chains of the oxidized fibrin(ogen) proved to be involved in the enzymatic cross-linking more readily than those of unaffected molecules. the enhancing role of the d:d interaction under oxidation could be considered as an compensatory mechanism in the assembly of fibrin when the d:e interaction is impaired. the experimental data on fibrinogen and fibrin oxidation acquired in the present study, being combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some ros actions detrimental to the protein function. the study was supported by the rfbr, research projects - - mol_a and - - a. structural and thermodynamic analysis of co-stimulation receptor cd phosphopeptide interactions with grb , gads, and pi -kinese sh domains in addition to the signaling produced by the binding of antigen-major histocompatibility complex to tcell receptors, co-stimulatory signals from other receptor-ligand interactions are required for full activation of t-cells. the cd receptor on the t-cell surface has been well characterized, and the binding of ligand to cd is critical for producing co-stimulatory signals. cd has no enzymatic activity and its cytoplasmic region consists of amino acids that contain the sequence ymnm, in which the tyrosine residue is phosphorylated by kinase. the phosphorylated sequence, pymnm, is recognized by src homology (sh ) adaptor proteins, such as growth factor receptor binding protein (grb ), grb related adaptor downstream (gads), and the phosphatidylinositol -kinase (pi -kinase) regulatory subunit, p . the consensus sequence for the binding of grb sh and gads sh is pyxnx, and that of p n-terminus sh (nsh ) and c-terminus sh (csh ) is pyxxm. we reported the high-resolution crystal structure of grb sh in complex with the cd phosphopeptide [higo et al., plos one , e , ] , and recently determined those of gads sh , p nsh , and p csh . these data along with the results of binding thermodynamics analyzed using isothermal titration calorimetry, helped to elucidate the molecular recognition mechanisms of cd by adaptor proteins. the sh proteins were overexpressed in escherichia coli, and were purified using affinity and gel-filtration chromatography. the cd phosphopeptides, -residue (octp) and -residue (ddcp ), were synthesized using the solidphase supported technique, and were purified using reversed-phase chromatography. the crystals were obtained by the hanging-drop vapor diffusion method. x-ray diffraction data were collected at synchrotron radiation facilities, and the structures were determined by the molecular replacement method. the models of grb sh , gads sh , p nsh , and p csh in complex with octp were refined at . , . , . , and . Å resolutions, respectively. the crystal structures showed that the phosphotyrosine phosphate moiety directly interacted with the side-chain of arginine in sh , which is common in all complex structures. in the grb sh and gads sh complexes, the side-chain of asparagine at the py position forms a pair of hydrogen bonds with the main-chain amide and carbonyl groups of lysine in sh . alternatively, in the p nsh and csh complexes, the side-chain of methionine at the py position is located in hydrophobic pockets of nsh and csh , in which the hydrophobic interactions of csh would be stronger than those of nsh . this idea is supported by the observed binding thermodynamics. the binding affinity of csh to ddcp , because of a favorable enthalpy change, is about -fold higher than that of nsh . the binding affinity of grb sh to ddcp is similar to that of gads sh to ddcp , and is about -fold lower than that of nsh to ddcp . these results indicate that the contribution of hydrophobic interactions of nsh and csh at the py position are stronger than those of hydrogen bonds of grb sh and gads sh at the py position. novel kinetochore protein complex from silkworm holocentric chromosomes takahiro kusakabe , hiroaki mon , jaeman lee the kinetochore, which consists of centromere dna and a multilayered protein complex, plays important roles in chromosome organization and segregation. interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell, and to segregate the sister chromatids into daughter cells in mitosis, which is followed cytokinesis. in contrast to monocentric chromosomes, in which the centromere is normally present at a single region on each chromosome, the holocentric chromosomes have centromeric activity along the entire length of the chromosome. it has been known that the silkworm, bombyx mori, has holocentric chromosomes since s, none of silkworm kinetochore proteins, however, have been identified so far. here we report the identification of a novel set of genes for outer kinetochore proteins in silkworm by using bioinformatics and rna interference-based screening. under the hypothesis that depletion of essential kinetochore genes causes cell cycle arrest in mitosis, we performed rnai in the silkworm cell line, bmn -sid , targeting a set of candidate genes. knockdown of five genes caused significant cell cycle arrest at the g /m phase. we also found that these five proteins make a complex, and that all of them are localized along the chromosome arms, indicating that the silkworm kinetochore extends along the chromosome. inactivation of bine aldehyde dehydrogenase from spinach by its physiological substrate bine aldehyde to contend with osmotic stress caused by drought, salinity, or low temperatures some plants synthesize the osmoprotectant glycine bine (gb) from bine aldehyde (bal). the last step-the irreversible nad dependent oxidation of bal-is catalyzed by aldh enzymes that exhibit bine aldehyde dehydrogenase (badh) activity. we here report that the spinacia oleracea badh (sobadh) is reversibly inactivated by bal in the absence of nad in a time-and concentration-dependent mode to approximately % of the original activity. inactivation kinetics are consistent with a partial reversible, two-steps mechanism that involves the formation of an active non-covalent enzyme•bal complex before formation the inactive enzyme-bal complex. crystallographic evidence indicates that in the enzyme previously inactivated by bal the aldehyde forms a thiohemiacetal with the nonessential cys (sobadh numbering) located at the aldehyde-entrance tunnel, thus totally blocking the access to the catalytic cysteine. accordingly, bal does not inactivate the c s sobadh mutant. two crystal structures of the inactivating enzyme-bal complex showed that the trimethylammonium group of bal is inside the active-site aromatic box, as in the productive way of binding. this explains why the inactivation of the a i mutant-where the binding of the trimethylammonium group is hindered-requires non-physiologically high bal concentrations, while the a c mutant-where the binding is allowed-is inactivated similarly to the wildtype enzyme. cys- is conserved in most plant aldh enzymes of known sequence, and in all of them with proven or predicted badh activity. inactivation by bal appears therefore to be a common feature of plants badhs. this short-term regulation may be of great physiological importance since the irreversibility of the badh-catalyzed reaction would unbalance the nad /nadh ratio if the aldehyde concentrations are high, the nad concentrations low and the reaction is not slowed down. plants badhs are prone to this situation since they work under osmotic stress conditions, when high bal concentrations are required for the synthesis of high levels of the osmoprotectant gb. the partial nature of the decamer possesses a donut shaped structure with calcium ions on the surface available for interactions with carbohydrate molecules. binding specificity was evaluated for carbohydrates using differential scanning fluorimetry (dsf) that showed bjcul interacts with galactose and lactose but less with glucose and sacarose. surprisingly, high levels of thermostabilization of bjcul was achieved with the antibiotic aminoglycosides geneticin (g ) and gentamicin in a calcium concentration dependent manner, but not kanamycin. intriguingly, while lactose and galactose inhibited erythrocyte agglutination by bjcul, g and gentamicin did not affect hemagglutination implying a second site of binding. dsf analysis also suggested the presence of a second binding site for the antibiotics and crystallization of the complexes are in progress in order to understand fully this new binding mechanism of c-type lectin with antibiotics. ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic institute of integrative biology, university of liverpool ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic * institute of integrative biology, biosciences building, university of liverpool, crown street, liverpool l zb, united kingdom * mara.kozic@liverpool.ac.uk antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. antimicrobial peptides (amps) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. an understanding of the structure of a protein can lead us to a much improved picture of its molecular function. furthermore, an improved understanding of structure-function relationships facilitates protein design efforts to enhance their activity. currently, the d structures of many known amps are unknown. to improve our understanding of the amp structural universe we have carried out large scale ab initio d modelling of structurally uncharacterised amps. such ab initio modelling is facilitated by the typical small size of amps as well as their tendency to contain disulphide bonds, these providing valuable additional information to simulations. preliminary results reveal unexpected similarities between the predicted folds of the modelled sequences and structures of well-characterised amps. for example, lacticin q was revealed to contain a helical bundle fold that bears a striking resemblance to enterocin a. we also found a remarkable similarity between the predicted structure of silkworm peptide and b-hairpin amps such as tachyplesin i. our results improve the understanding of the structure-function relationship of amps. surface aggregation-propensity as a constraint on globular proteins evolution susanna navarro , marta diaz , pablo gallego , david reverter , salvador ventura institut de biotecnologia i biomedicina and departament de bioquimica i biologia, institut de biotecnologia i biomedicina, universitat aut onoma de barcelona in living cells, functional protein-protein interactions compete with a much larger number of nonfunctional interactions. theoretical studies suggest that the three-dimensional structures of present proteins have evolved under selective pressure to avoid the presence of aggregation-prone patches at the surface that may drive the establishment of anomalous protein contacts. however, no experimental evidence for this hypothesis exists so far. the a-spectrin sh domain (spc-sh ) has been used as a protein model to decipher the sequential aggregation determinants of proteins. here we use it to address the structural determinants of protein aggregation and their link to protein evolution. to this aim we exploit aggrescan d (a d), a novel algorithm developed by our group, which takes into account both protein structure and experimental data to project aggregation propensities on protein surfaces. we used a d to design a series of spc-sh variants with progressively stronger aggregationprone surfaces and characterized their thermodynamic, structural and functional properties. our data support evolution acting to constraint the aggregation propensities of globular protein surfaces in order to decrease their potential cytotoxicity and the protein quality control machinery acting to buffer this negative selective pressure. utilizing d structure for the annotation of structural motifs in the conserved domain database narmada thanki-cunningham , noreen gonzales , gabriele marchler , myra derbyshire , james song , roxanne yamashita , christina zheng , stephen bryant , aron marchler-bauer , farideh chitsaz conserved domain database, structure group cbb/ncbi/nlm/nih the conserved domain database (cdd) is a protein classification and annotation resource comprised of multiple sequence alignments representing ancient conserved domains. cdd protein domain models are curated by ncbi and use d protein structure explicitly to define domain extent and the location of conserved core structures, and to provide accurate alignments between diverse family members via structure superposition. cdd also imports external collections such as pfam and tigrfam. recently, a novel class of annotation labeled as "structural motifs" has been introduced to supplement current capabilities. these annotations define compositionally-biased and/or short repetitive regions in proteins, which are difficult to model as functional domains conserved in molecular evolution. structural motifs include transmembrane regions, coiled coils, and short repeats with variable copy numbers. for many types of short tandem repeats, a few position-specific score matrices (pssms) suffice to annotate more than % of the known instances of that structural motif. unfortunately, a lack of sequence similarity within coiled-coil regions prohibits the development of only a few generic models; therefore, models for coiled-coil regions in the context of specific families have been developed using the spiricoil database as a reference. increased coverage of coiled-coil regions in cdd, specific site annotations of these structural motifs as well as their representation on the webpages will be discussed. specific in vivo ultrasound imaging of e-selectin expression in tumors using a microbubble contrast agent covalently attached to the peptide ligand iellqar, known to bind to e-selectin [ ] . however, it was observed that this probe has a limitation in the imaging of cardiovascular diseases where higher shear stresses prevent microbubbles from remaining attached to the target. therefore, peptides with higher eselectin affinity are needed to design probes capable of imaging these diseases. in this context, automated docking and molecular dynamics methodologies were combined and applied to different e-selectin binding peptides. these studies predicted the energetically more favorable binding mode as well as the key interactions between the peptide ligands and the e-selectin receptor. some of these peptides were prepared by solid-phase peptide synthesis and their interactions with e-selectin analyzed by surface plasmon resonance technique. the results showed that these peptides have different affinities for e-selectin. these data were correlated with the computational studies and evaluated to obtain crucial information of the key recognition elements needed for higher e-selectin affinity. these recent results will be presented. burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive disease of animals and humans in tropical and subtropical areas. sedoheptulose- -phosphate isomerase from b. pseudomallei (bpgmha) is the antibiotics adjuvant target for melioidosis. in general, bpgmha converts dsedoheptulose- -phosphate to d-glycero-a-d-manno-heptopyranose- -phosphate (m p). this is the first step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for searching novel antibiotics increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. the crystal of this enzyme has been solved at . Å resolution. there is an active site pocket where a putative metal binding site is located. to find out inhibitors of bpgmha, in-silico virtual screening with zinc, a free database of commercially-available compounds, has been performed. tens of thousands of chemical compounds were docked into the active site of bpgmha. a number of putative bpgmha binding compounds better than m p were found using surflex-dock included in the sybyl software package. characteristics of these compounds were surveyed and classified to identify common binding properties with bpgmha. mapping the structure of laminin using cross-linking and mass spectrometry gad armony , toot moran , yishai levin , deborah fass weizmann institute of science, department of structural biology, weizmann institute of science, israel center for personalized medicine laminin, a kda heterotrimer, is a major element in the extracellular matrix (ecm). within the ecm, laminin contributes to the adhesion and migration of cells, both in health and disease. the laminin trimer was observed by rotary shadowing electron microscopy to be cross shaped: the three short arms of the cross are formed by the amino-terminal halves of the three subunits, whereas the long arm of the cross holds the three chains together in a long coiled coil. the narrow and flexible arms of the laminin cross complicate studying its structure to high resolution by crystallography or electron microscopy single particle reconstruction. to advance our understanding of this remarkable quaternary structural assembly, we have used cross-linking and mass spectrometry to analyze the organization of the laminin trimer. this technique was validated by known crystal structures of isolated laminin domains. in all cases the crystal structure distances agree with the cross-linker length. the identified cross-links were particularly helpful in assigning the register and the subunit order of the long coiled coil due to the high content of cross-linkable residues in this region. using known x-ray crystal structures, homology modeling, and distance restraints provided by two cross-linker chemistries, a clearer picture of the laminin quaternary structure is obtained. non-sequential protein structure alignment program mican and its applications shintaro minami , george chikenji , motonori ota dept. of info. sci., nagoya univ., dept. of comp. schi. & eng., nagoya univ. in some proteins, secondary structure elements are arranged spatially in the same manner, but they are connected in the alternative ways. analysis on such non-sequential structural similarity in proteins is important because it provides a deeper understanding of the structural geometry of protein. this can be also observed even in the homologous proteins, indicating the non-sequential structural similarity is significant in the protein evolution. however, the non-sequential structural similarity in proteins is less investigated. we developed a novel non-sequential structural alignment program mican, which can handle multiple chains, inverse direction of chains, c$lpha$models, alternative alignments, and non-sequential alignments. we performed comprehensive non-sequential structural comparison among homologous proteins in the same scop superfamily by using the mican program. based on the result, we found that approximately % of superfamilies include at least one protein pairs showing non-sequential structural similarity. % nonsequential structurally similar pairs are aligned in a simple way, e.g. circular permutation, $$strand flip/ swap, but % are complicated. interestingly, most of such complicated non-sequential similarities can be explicable by combination of - simple non-sequential relationships. this result indicates that accumulation of simple structural changes in the course of protein evolution produces completely different fold homologs. as early as , ritter surmised that the cell's molecules cooperate to form a "special apparatus and an organised laboratory". despite supporting evidence from srere, mcconkey and others, efforts to understand molecular organisation in vivo are still in their infancy. however, important aspects of the cell interior have already been revealed. for example, weak molecular interactions structure the cytoplasm into time-evolving, functional zones. weak interactions are difficult to capture and can preclude protein detection in cells by many biophysical techniques, including nmr spectroscopy. , we explored the effects of cell-like milieus on the cytochrome c (cyt c)-flavodoxin (fld) interaction. these oppositely charged proteins interact weakly with a number of cognate partners. neither cyt c nor fld is detectable by nmr in escherichia coli confirming their "sticky" nature ( figure a) . the cyt c-fld interaction was assessed in buffer, % polyacrylamide gels and in solutions containing g/l of macromolecular crowders ( figure b) . h, n hsqc nmr revealed that the interaction was transient in buffer, proceeding via the known binding site for both proteins. substantial line broadening was effected in crowded and confined solutions suggesting that the cyt c-fld complex is stabilised under native-like conditions. the stabilising effect of macromolecular crowders was also observed by native gel electrophoresis and crystallization. these findings coincide with spitzer and poolman's model for cytoplasmic structuring, emphasising the role of charge-charge interactions and crowding in the formation of macromolecular "clusters". the implications for cytoplasmic structuring will be discussed alongside related investigations of cationic protein interactions in e. coli extracts. , detergent:protein ratio. the transmembrane b-barrel of bama is folded in either micelles, bicelles or nanodiscs, however an n-terminally attached single potra domain is flexibly unfolded, due to the absence of stabilizing contacts with other protein domains. measurements of backbone dynamics show distinct time scales of dynamic behavior for bama b-barrel and parts of its extracellular loop l , revealing high local flexibility within the the lid loop. this work presents the first high-resolution d solution nmr spectra of the bama barrel and establishes improved biochemical preparation schemes, which will serve as a platform for structural and functional studies of bama and its role within the bam complex. protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and dna repair. mammalian protein arginine methyltransferases include a family of nine sequence-related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (mma, type iii), symmetric dimethylarginine (sdma) and mma (type ii), or asymmetric dimethylarginine (adma) and mma (type i). while prmt , , , , , and have been characterized as type i enzymes, and prmt as a type ii enzyme, the role and activity types of the two final members of this family of enzymes, prmt and prmt , had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. both prmt and prmt are distinct members of the family with two methyltransferase or methyltransferase-like domains and containing acidic residues in otherwise well-conserved substrate double e binding motif, features not seen in the other prmt enzymes. recent work in our laboratory confirmed prmt as the only type iii mma-forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an r-x-r sequence for methylation. mutations of the acidic residues in the substrate-binding motif results in a loss of the specific r-x-r activity and the appearance of a g-r-g specificity typical of many of the other prmts. the physiological substrate of prmt has yet to be confirmed, although histone h b is an effective in vitro substrate. prmt , on the other hand, had no reported activity, until immunoprecipitation from hela cells showed it pulled down two splicing factors, sf b and sf b , in a complex. amino acid analysis showed that prmt methylates sf b to produce both mma and sdma, thus making it the second type ii enzyme in mammals. prmt knockdown results in modulation of alternative splicing events. this enzyme appears to be relatively specific for the sf b protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other prmts are not recognized by prmt . we found that the position of the methylated arginine residue in sf b is important, and the acidic residues in the substrate-binding motif also play an important role in substrate recognition. thus, prmt and prmt represent unique members of the mammalian prmt family. hydrogen peroxide levels, endogenous hormones (cytokinins, salycilic acid, as well as jasmonic acid and its conjugates), polyphenolics and terpenoids in a model system of a. alba in vitro with inhibition of rootng and stimulation of callusogenesis by means of individual and combined cytokinin and cytokinin/ auxin treatments. results: it was established that inhibition of rooting and stimulation of callusogenesis caused by benzyl adenine (ba) or combinations of ba and indole- -butiric acid (iba) in vitro were related to elevation of sesquiterpenoids in the essential oils, as well as polyphenolics content, accompanied by a drop of stress hormones, bioactive cytokinins and preservation of oxidative stress and lipid peroxidation levels, as compared with non-treated control. individual treatments with either iba or ba, also increased the sesquiterpenoid content in the essential oil of the plant, in a concentration related manner, this effect being more profound after ba treatment. in addition, ba treated plants exhibited a drop of protein levels of the aerial samples, as well as profound differences of enzymatic activity in the callus tissues, as compared with callus of plants treated with different combinations of ba and iba. conclusion: the results of the present work indicate that alterations of endogenous phytohormonal levels, caused by exogenous plant growth regulators treatment, might be the mediator between primary and secondary metabolism by means of affecting protein levels and activity of key enzymes in vitro. three different additives (( . % (v/v); formic acid, acetic acid, ammonium format with formic acid) have been investigated in response to ion intensity of esi-ms for individual hnp - in saliva. kinetex v r column separation efficiency was evaluated using two different column dimensions ( x . mm and x mm.) and two different stationary phases (c and c ). kinetex v r column (homogenous porous shell) performance was also compared to new ultra ace v r (encapsulated bonded phase) column. sample optimisation revealed that the spe method removes interference from salivary glycoproteins and consequently yields larger peak area ( - %) for all hnps. hnps were extracted by spe with a recovery of - %. the meoh: h o: acetic acid ( . %) provided enhanced (p> . ) hnp - ion intensities. the kinetex v r c ( x . mm, . mm) column facilitated a better separation efficiency of the four hnps as compared to the ultra core super c ace v r ( x . mm, mm) column, the kinetex v r c ( x . mm, . mm) and the kinetex v r c ( x . mm, mm) column. the relative levels of the hnps were determined in healthy volunteers before and after a rigorous exercise regime: it is possible that prolonged strenuous exercise will affect oral innate immunity and therefore also the level of salivary defensins. hnp - are traditionally detected in an enzyme-linked immunosorbent assay (elisa) which does not discriminate between the different hnps due to their structural similarities. there has therefore been a need to develop a mass spectrometry method that will discriminate between the defensins. as part of the method validation, the hnp - level was determined by elisa and the data was compared with the lc-ms data. here we present this cross-validation; the data revealed no significance difference between the two methods (r . ) which confirms that the developed lc-ms method is and equal sensitive method for the detection of these potential antimicrobial markers. this method can easily be adopted for similar molecular weight of peptides as hnps and also for any other biological matrix. moonlighting proteins: relevance for biotechnology and biomedicine luis franco serrano , sergio hern andez , alejandra calvo , gabriela ferragut , isaac amela , juan cedano , enrique querol institut de biotecnologia i biomedicina. universitat aut onoma de barcelona, laboratorio de inmunolog ıa, universidad de la rep ublica regional norte-salto multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. the identification of moonlighting proteins could be useful for researchers in the functional annotation of new genomes. moreover, the interpretation of knockout experiments, in which the result of a gene knocking does not produce the expected results, might be enhanced. the action of a drug can also be facilitated because it might have an off-target or side effect with somewhat hidden phenotypic traits. it would be helpful that bioinformatics could predict this multifunctionality. in the present work, we analyse and describe several approaches that use protein sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. among these approaches there are: a) remote homology searches using psi-blast, b) detection of functional motifs and domains, c) analysis of data obtained of protein-protein interaction databases (ppis), d) matches of the sequence of the query protein to d databases (i.e., algorithms like pisite), e) mutation correlation analysis between amino acids using algorithms like mistic. remote homology searches using psi-blast combined with data obtained from interactomics databases (ppis) have the best performance. structural information and mutation correlation analysis can help us to map the functional sites. mutation correlation analysis can only be used in very specific situations because it requires the existence of a multialigned family of protein sequences, but it can suggest how the evolutionary process of second function acquisition took place. we have designed a database of moonlighting proteins, multitaskprotdb (http:// wallace.uab.es/multitask/). from this database we determine the frequencies of canonical and moonlighting coupled functions (being an enzyme and a transcription factor the highest), the percentage of moonlighting proteins involved in human diseases ( % of the human moonlighting proteins in the database) and the percentage of moonlighting proteins acting as a pathogen virulence factor ( % of the moonlighting proteins in the database). correlation between potential human neutrophil antimicrobial peptides (hnp - ) and stress hormones in human saliva nadia ashrafi , frank pullen , birthe nielse , cris lapthorn , fernando naclario university of greenwich (faculty of engineering and sciene), university of greenwich (centre of sports science and human performance) numerous studies have investigated the effect of exercise on mucosal immunity but the focus has mainly been on salivary immunoglobulins lysozymes and hormones (cortisol, testosterone). this is not surprising given that iga and igg are the predominant immunoglobulins in saliva and there is a relationship between mucosal immunity and upper respiratory illness. it is well known that physical and mental stress provoke the release of cortisol from hypothalamic pituitary adrenal axis, by which stress can modulate various immune responses. in general, cortisol and growth hormones helps to induce the activation of neutrophils. to date, this study represents the first study that investigated the correlation between human neutrophil alpha defensins family against cortisol (stress hormone) and testosterone (growth hormone) in human saliva before and after exercise or training. twelve resistance trained athletes volunteered to participate in the study. participants consumed supplements during exercise and the hnp - , cortisol and testosterone response was investigated pre, post and minutes of the workout. the correlation between salivary antimicrobial peptide (hnp - ) and stress hormone (cortisol and testosterone) has been investigated using elisa. cortisol showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) conditions but a strong trend (p . ) was observed for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. testosterone showed no significant (p . ; p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) and for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. hnp - showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ; pl . . ) conditions but significant difference (p . ) was observed for (pre to min post) between cho and pl (cho: . . ; pl . . ) condition. the present findings suggested that there is no correlation between salivary hnp - and cortisol for (pl: r . and cho: r . ); hnp - and testosterone (pl: r . and cho: r . ). a worth note from previous study which suggested that using murine skin model (an increase in endogenous glucorticoids (cortisol) by physiological stress reduced mrna levels of antimicrobial peptide (cathelicidin). it is not clear that the correlation between hormones and antimicrobial peptide has been affected by the time interval of the exercise. both cortisol and antimicrobial peptide demonstrated a transient increase after exercise but it is surprising that they are not correlate to each other. one of the hypothesis from the present finding could be cortisol responses slow and it will be interesting to do further research with longer interval. the second hypothesis demands a further investigation to determine the synergism between substances. school of biomolecular and biomedical science, conway institute, ucd., king saud university, sciences, biochemistry department. the crystal structure of a human glucose -phosphate dehydrogenase (g pd) shows that each subunit has two nadp sites; in addition to a catalytic site there is a "structural" site which is distant from the catalytic coenzyme site. mutations causing severe deficiency tend to cluster round and close to the dimer interface and the structural nadp , indicating that the integrity of these areas is important for enzyme stability and therefore for maintenance of activity. in order to understand the molecular basis of g pd deficiency, and to have a clearer indication about the role of some features of the threedimensional structure, a fuller study of the second, "structural" nadp binding site is needed. human g pd controls the first committed step in the pentose phosphate pathway. it catalyses the oxidation of glucose -phosphate to gluconolactone -phosphate, generating nadph which is essential, amongst other things, for protection against oxidative stress. the human enzyme can be active in dimer or tetramer forms. human g pd of "structural" nadp per subunit of enzyme. this tightly-bound nadp can be reduced by g p, probably following migration to the catalytic site. the importance of nadp for stability is explained by the structural nadp site, which is not conserved in prokaryotes. after removing the tightly bound "structural" nadp the enzyme is still active but not stable. the effects of different nadp fragments on the stability of human recombinant g pd have been investigated. nadp is crucial for the long term stability of human g pd, and only one of nadp analogues which is adenosine diphosphate ribose - '-phosphate was able to slightly promote the stability of enzyme. . molecular characterization of specific positively selected sites in mammalian visual pigment evolution miguel a. fern andez-sampedro , eva ramon , brandon m. invergo , jaume bertranpetit , pere garriga grup de biotecnologia molecular i industrial., visual rhodopsin is a member of the g-protein coupled receptors superfamily. this membrane protein consists of a -cis-retinal cromophore bound to a seven transmembrane protein, opsin, by means of a protonated schiff base linkage. it has an important role as a dim light photoreceptor in the retina of the eye. by statistical models, where episodic selection in rhodopsin is tested on one branch of the phylogeny against a background of neutral or purifying selection on the rest of the tree, we have found some significant evidence of specific positively selected sites in early mammalian divergence. we have chosen the three amino acid sites identified with the highest posterior probability of having been targets of positive selection to perform experimental studies, i.e. (positively selected from m to f), (positively selected from r to q) and (positively selected from s to a). we have constructed, expressed, immunopurified and functionally characterized the proposed candidates, f m, q r and a s rhodopsin mutants located at the n-terminus, the transmembrane domain and the c-terminus region of the protein respectively. from the analysis of the molecular features of the f m mutant, we conclude that position is very important for protein folding and also for proper protein glycosylation, since we only could observe cromophore regeneration after its rescue in the double cysteine (n c/ d c) mutant background that stabilizes the n-terminal extracellular domain of the protein. our results also show that mutants q r and a s alter the g-protein activation rate, and hydroxylamine susceptibility in the dark-adapted state. in the case of q r, disrupting critical interactions with the neighbouring y of the conserved d/ery motif, critical in gt activation, could cause the lower gt activation ability. the mutant a s would create a potential additional phosphorylation site in the protein which could affect rhodopsin phosphorylation after photoactivation and, in turn, could affect the binding affinity of arrestin, a regulator of rhodopsin deactivation. this extra phosphorylation site could provide an evolutionary explanation for the enhanced response observed in the case of gt activation. in conclusion, these results highlight the importance of molecular investigations of positive selected sites in rhodopsin evolution and the relevance of structural and functional analysis of these sites in unravelling the molecular basis of visual pigment evolution. natural evolution sheds light on modern drug resistance in protein kinases marc hoemberger , christopher wilson , roman agafonov , dorothee kern the anti-cancer drug imatinib exhibits highly specific binding to the human kinase and oncogene abl with a three thousand fold weaker affinity for the structurally and functionally very similar kinase src. it has been shown recently that the major difference in binding of imatinib to abl and src stems from an induced fit after binding of the drug. to further understand the mechanism of imatinib binding to its target we used ancestral sequence reconstruction (asr) and resurrected enzymes along the node from the common ancestor of abl and src up to the extant kinases. we show that imatinib affinity is gained towards the evolution of extant abl while it is lost towards evolving src. the combination of asr and crystallographic data of the ancestors in addition to kinetics data allowed us to identify a subset of residues involved in imatinib specificity sufficient to switch from an intermediate binder to a tight binder. preliminary data shows that a network of hydrogen bonds and packing interactions stabilize the kinked p-loop conformation for tight binders thus allowing for more interactions between the kinase and the drug. strikingly, many of these residues were identified in human cancer patients as "hot spots" for the development of resistance mutations. further investigation into the identified subset of residues in combination with these commonly found imatinib resistance mutations will allow us to understand emerging drug resistances better. an evolutionary view of the cold adapted catalysis of enzymes vy nguyen , christopher wilson , dorothee kern the diversity in protein function that we see today arose as a result of life adapting to a cooling earth. how did enzymes, the catalysts of many crucial cellular processes, achieve this cold adaptation? this is a challenging question to answer because ancient sequences of proteins that existed billions of years ago are not available. to address this question we used ancestral sequence reconstruction to create adenylate kinase (adk) enzymes from the divergence of anaerobic and aerobic firmicutes towards modern day thermophilic, mesophilic and psychrophilic organisms. adk is a phosphotransferase that catalyzes the conversion of two adp molecules into atp and amp. we make the following observations. first, all ancestral enzymes are active with optimal catalytic rates linearly corresponding to the temperature of the environments where these proteins would have been found. most strikingly, the catalytic rate of our oldest adk ancestor exhibits a higher enthalpy of activation at low temperatures as compared to the modern thermophilic adk. this suggests a large enthalpic penalty had to be paid for reactions to occur at cold temperatures in an ancestor that existed in a hot environment. second, several high resolution crystal structures of extant proteins that we solved ( . Å - . Å), show that the oldest ancestors were more rigid than the modern adks due to an intricate salt-bridge network. this work, thus shows for the first time, the molecular and thermodynamic determinants of cold adaptation in an enzyme over a time period that spans billions of years. induced oxidative modification of plasma and cellular fibrin-stabilizing factor anna bychkova , tatiana danilova , alexander shchegolikhin , vera leonova , marina biryukova , elizaveta kostanova , alexey kononikhin , anna bugrova , evgeny nikolaev , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences, institute for energy problems of chemical physics, russian academy of sciences the main function of plasma fibrin-stabilizing factor pfxiii is to catalyze the formation of the intermolecular covalent cross-links between both gand afibrin polypeptide chains. the crosslinking crucially affects mechanical strength of fibrin and its resistance against fibrinolysis. the precise role of cellular fibrin-stabilizing factor cfxiii remains poorly understood. pfxiii is a heterotetramer (fxiii-a b ) consisting of two single-stranded catalytic a subunits (fxiii-a ), and two identical single-stranded inhibitory/ carrier b subunits (fxiii-b ). the subunits are held together by weak non-covalent bonds. contrary to plasma fxiii, cfxiii is a dimer (fxiii-a ) devoid of b subunits. as well as many other proteins circulating in the bloodstream, pfxiii is known to be a target for reactive oxygen species (ros) causing processes of protein oxidative modification. since the conversion of pfxiii to the active form of the enzyme (fxiiia) is a multistage process, ozone-induced oxidation of pfxiii has been investigated at different stages of its enzyme activation. the biochemical results point to an inhibition of enzymatic fxiiia activity depending largely on the stage of the pfxiii conversion into fxiiia at which oxidation was carried out. uv-, ftir-and raman spectroscopy demonstrated that chemical transformation of cyclic, nh, sh and s-s groups mainly determines the oxidation of amino acid residues of pfxiii polypeptide chains. conversion of pfxiii to fxiiia proved to increase protein susceptibility to oxidation in the order: pfxiii < pf-xiii activated by thrombin < pfxiii in the presence of calcium ions < fxiiia. with the aid of massspectrometry it has been demonstrated that oxidation leads to decreasing fxiii-a and fxiii-b coverage both in the forms of zymogen and in the presence of calcium ions. a group of amino acid residues involved in oxidation modification of pfxiii is identified in this study. the oxidation of either cfxiii or cfxiiia has revealed an almost complete loss of enzyme activity caused by dramatic changes in the primary and secondary structure of the proteins detected by the ftir data. taking into account these new findings, it seems reasonable to assume that the inhibitory/carrier fxiii-b subunits can serve as scavengers of ros. hypothetically, this mechanism could help to protect the key amino acid residues of the fxiii-a subunits responsible for the enzymatic function of fxiiia. the study was supported by rfbr, research project no. - - - a. mass spectrometry study was supported by the russian scientific foundation grant no. - - . performance and quality. making microcalorimetry simple with microcal peaq-itc natalia markova , ronan o'brien , mark arsenault microcal, malvern instruments ltd. dynamic interactions involving biomolecules drive and regulate all biological processes. studies of biomolecular interactions are fundamentally important in all areas of life sciences. data provided by isothermal titration calorimetry (itc) enables scientists in academia and industry to directly and quantitatively characterize these interactions in solution. microcal peaq-itc, the latest generation of microcal itc instrumentation, offers a whole range of solutions for addressing current bottlenecks associated with interaction analysis. among the most recognized challenges are the needs to adequately address a broad range of binding affinities and to reliably interpret binding data complicated by the presence of inactive protein fraction or inherent uncertainty in the concentration of a ligand. consistently high performance of microcal peaq-itc enables increased confidence and data resolution when measuring low heats at low or uncertain sample concentrations and complex binding modes. the new microcal peaq-itc analysis software allows for utomated data analysis, minimizing analysis time and user subjectivity in assessing data quality. data quality is determined and advanced fitting performed in a few seconds per experiment allowing for analysis of large data sets of or more experiments in a matter of seconds. glutamine-rich activation domain of transcription factor sp -biochemical activity and structure jun kuwahara , chisana uwatoko , emi hibino , katsumi matsuzaki , masaru hoshino faculty of pharmaceutical sciences, doshisha women's university, graduate school of pharmaceutical sciences, kyoto university transcription factor sp is ubiquitously expressed in a mammalian cell, activates reasonably large subset of mammalian genes, and is involved in the early development of an organism. the protein comprises two glutamine-rich (q-rich) regions (a and b domains) located in its n-terminal half, while three tandem repeats of c h zinc finger motif at its c-terminus binds directly to a gc-rich element (gc box) of dna. in general, q-rich domain is one of the typical motifs found in trans-activation domain of transcription factors together with acidic and proline-rich domains. transcriptional signal of sp are transmitted via interaction between q-rich domains of sp and different classes of nuclear proteins, such as tata-binding protein (tbp) associated factors (tafs) in components of basic transcription factor complexes (tfii). in addition, self-association of sp via q-rich domains is also important for its regulation of transcriptional activity. it has been considered that an sp ! molecule bound to a 'distal' gc-box synergistically interacts with another sp molecule at a 'proximal' binding site. although formation of multimers via q-rich domains seems functionally important for sp , little is known about relevance between biological activity and structural nature of q-rich domains. we analyzed nature of glutaminerich domains of sp by biochemical and physicochemical methods. we found that q-rich domains do not have clear secondary structure whereas they can indicate biochemical activity. detailed analysis of nmr spectra indicated interaction between the domains. the q-rich domains of sp might be one of the intrinsically disordered proteins (idp). chipping away at the yeast proteome: redesigning an e ubiquitin ligase for targeted protein degradation michael hinrichsen , lynne regan one of the central goals of synthetic biology is to exploit biological systems in order to produce compounds of therapeutic or industrial value . often, these efforts are complicated by the many natural biochemical pathways in cells that can compete for the same small molecule precursors. currently, the most common solution is to simply delete the genes coding for the competing enzymes . while such an approach has been successful, it is only applicable to nonessential genes and can produce unintended off-target effects such as decreased cell viability . an alternative strategy is to instead target proteins directly for degradation. using this strategy, scientists would first grow cultures of engineered cells to high densities under permissive conditions (i.e. targeted proteins are stably expressed). then, once sufficient cell density has been reached, enzymes of competing pathways would be rapidly degraded, resulting in the rapid production of high concentrations of the compound of interest. we propose to create such a tool by reengineering the c-terminus of hsp interacting protein (chip), an e ubiquitin ligase. chip recognizes substrate proteins through a short c-terminal peptide tag on target proteins . we have shown that fusing this tag to non-native substrates is sufficient for ubiquitination in vitro (data not published). cellular assays have also been performed in s. cerevisiae, a model organism commonly used in metabolic engineering applications . as a number of native yeast proteins possess c-termini similar to that of chip's native substrates (data not published), it was necessary to develop an orthogonal chip-peptide pair. this was achieved by replacing chip's natural tpr ligand-binding domain with a ligand-binding domain engineered previously in the regan lab . the altered chip construct has been shown to be active both in vitro and in vivo, and produces an altered growth phenotype when targeted against an enzyme involved in uracil biosynthesis. future work will focus on further kinetic characterization of the engineered enzyme, increasing its activity, and introducing the system into a proof of concept synthetic biology application. advances in modern sequencing techniques have resulted in an explosion of genomic data. correctly classifying this new wealth of information can be daunting not only because of the sheer volume of sequence data, but also because the propagation of erroneous and less-than-ideal names and functional characterizations in the current databases gets in the way of functional classification by mere sequence similarity. we are investigating the extent to which protein domain architecture can be utilized to define groups of proteins with similarities in molecular function, and whether we can derive corresponding functional "labels", starting with some of the most common domain architectures found in bacteria. to this end, we have developed an in-house procedure called sparcle ('specific architecture labeling engine') that lets us track and examine specific or sub-family domain architectures, resulting from annotating protein sequences with domain footprints provided by the conserved domain database (cdd), which includes hierarchical classifications for many common domain families. we will discuss how the proteins are grouped into specific architectures, our successes in assigning functional labels, and the major limitations we have encountered to date. while we will be able to assign functional labels to a large fraction of protein models derived from genome sequences, this effort has the added benefit of pointing out insufficient coverage and resolution of the current protein domain model collections that constitute cdd. we will also discuss alternative procedures that utilize pre-computed domain annotation for clustering protein sequences at a level that is well suited for functional labeling. we hope that this preliminary study will help to identify approaches that facilitate rapid and accurate annotation of genomes with a minimum of manual intervention. pegylated amyloid peptide nanocontainer delivery and release system self-assembly of telechelic peg end-capped with hydrophobic dipeptides collagen stimulating effect of peptide amphiphile c -kttks on human fibroblasts self-assembly of palmitoyl lipopeptides used in skin care products bioactive films produced from selfassembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions influence of elastase on alanine-rich peptide hydrogels interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity self-assembled arginine-coated peptide nanosheets in water toll-like receptor agonist lipopeptides self-assemble into distinct nanostructures approved drugs containing thiols as inhibitors of metallo-ß-lactamases: a strategy to combat multidrug-resistant bacteria references leukotriene a hydrolase -an envolving target. inflammatory diseases -immunopathology, clinical and pharmacological bases the bifunctional enzyme leukotriene-a, hydrolase is an arginine aminopeptidase of high efficiency and specificity lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease a critical role for lta h in limiting chronic pulmonary this work was supported by the czech science foundation (project p / / ) and czech academy of sciences the tn antigen-structural simplicity and biological complexity bel b-trefoil: a novel lectin with antineoplastic properties in king bolete (boletus edulis) mushrooms acknowledgements: cynthia leyva-arg€ uelles is supported by a personal grant from conacyt, mexico. this work is supported by conacyt grant ' : and papiit grant in sequence information-based deciphering of biofunctionalities using ism-based techniques has fetched calculation of biological functionalities, designing of biomedical device called computer-aided drug resistance calculator, the understanding of the mechanism of hiv progression to aids [ ], and others. they have compared the efficacies of drugs and vaccines, which formed the basis for the innocentive award (id ) for assessing vaccine potency. conclusions: deciphering biological features without engaging reagents, equipments and animal tissues but biological data such as sequence information is one novel, feasible genotypic hiv-coreceptor tropism prediction with geno pheno [coreceptor]: differences depending on hiv- subtype a reliable phenotype predictor for human immunodeficiency virus type subtype c based on envelope v sequences available: http:// istree.bioprotection.org signal processing-based bioinformatics methods for characterization and identification of bio-functionalities of proteins an empirical framework for binary interactome mapping estimating the size of the human interactome coming to peace with protein complexes? th capri evaluation meeting pj- cabs-dock web server for protein-peptide docking with significant conformational changes and without prior knowledge of the binding site while other docking algorithms require pre-defined localization of the binding site, cabs-dock doesn't require such knowledge. given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), cabs-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone cabs-flex: server for fast simulation of protein structure fluctuations cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site mechanism of folding and binding of an intrinsically disordered protein as revealed by ab initio simulations modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-flex: server for fast simulation of protein structure fluctuations aggrescan d (a d): server for prediction of aggregation properties of protein structures cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site staphylococcal pathogenicity island dna packaging system involving cos-site packaging and phage-encoded hnh endonucleases the etiology of asdis unknown, but it is believed that it involves genetic and environmental components. the purpose of this work is to assess the possible involvement of food contaminants, such as mycotoxins, in the etiology of asd. the hypothesis is that the mycotoxins ingested with the diet could bind to proteins and expose the entire organism,including cns, to the negative effects of xenobiotics, in genetically predisposed patients. in this study some possible protein targets for the mycotoxinswere identified to evaluate if the bond between any protein target and the mycotoxin in exam could play a role in asd. twelve mycotoxins were selected (ochratoxin a, gliotoxin, aflatoxin b , aflatoxin b , aflatoxin m , aflatoxin m , aflatoxicol, a-zearalanol, b-zeralanol, zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals.for each of these molecules,possible protein targets were searched by a reverse docking approach using the idtargetserver[ ].from the results given by idtarget, human protein targets expressed in the brain or involved in brain diseaseswere selected. subsequently, a direct docking was made using auto-dock . [ ], in orderto verify the strength of the interaction between selected proteins and each mycotoxin, and to identify the mycotoxins' binding site on each of the selected protein. finally, the bond of some mycotoxins to selected protein targets has been experimentally tested. for each mycotoxin, idtarget returned thousands of possible protein targets,and only those with the best binding energy were selected and evaluated. among them, human protein targets that are expressed in the brain or that are involved in cerebral diseases,have been selected; moreover the protein targets that were not human but that idtargetselected for five or more mycotoxins, were replaced with their human counterparts. at the end of the procedure, nineteen protein targets have been identified for the following direct docking approach. from the docking results, eight proteins have been selected for experimental tests, having a predicted binding energy lower than kcal/mol. finally, the interactions between acetylcholinesterase (ache), b-secretase (bace ) and neuroligin- , x-linked (nlg x) with aflatoxin b , aflatoxin b , gliotoxin, ochratoxin a and deoxynivalenol, were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. these experiments confirmed the presence of an interaction between bace and aflatoxin b idtarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach the calculation of spatial structure and "assembling" of the whole protein from the obtained peptide structures were performed by using molecular dynamics of the protein in the fully hydrated -palmitoyl- -oleoyl-sn-glycero- -phosphatidylcholine (popc) [ ]. the obtained structural model may contribute to identification of ul . active sites and elucidation of its mode of action nmr structural studies of membrane proteins acknowledgments: polish national centre for research and development -grant number pl- functional and mechanistic studies of dysferlin, an essential protein in cell membrane repair references moreover the 'm' parameter, which represents the denaturant effect on the protein stability, is cal•mol- for bgla and cal•mol- for bglb albert einstein college of medicine protein structure modeling, protein-protein interaction computational modeling of ini /smarcb and novel insights into its interaction with hiv- integrase savita bhutoria epsteain bar virus, nuclear antigen) . ini /smarcb has no known structural homologues, and its amino-acid sequence yields little insight into its function. a detailed understanding of structure-function relationships is hampered by the lack of structural information for ini . computational methods that model protein/peptide structures with sufficient accuracy to facilitate functional studies have had notable successes. we carried out combination of sequence analysis ab initio structure modeling and dynamics studies of integrase binding domain of ini and found it to be similar to that of phospholipase a activating protein, plaa. structural similarity with this distant protein suggests divergent evolution of the two proteins. the modeled structure sheds light on various protein-protein interactions of ini . by integrating the experimental studies about the binding, we have shown through docking, how a fragment of ini binds to the hiv- in. molecular docking and experimental studies indicated that two proteins bind tightly through charged/polar residues surrounding a hydrophobic cleft. these studies provide first modeled structure of ini /smarcb or any component of the swi/snf complex, and provide structural basis for in-ini interactions. this molecular interpretation of the intermolecular interactions is expected to facilitate design of inhibitors as novel class of anti-hiv- therapeutic agents ): e . with their catalytic activity towards rna substrates, other biological properties have been reported and evolution studies suggest an ancestral host-defence function in vertebrates. indeed, genetic studies confirmed a rapid molecular evolution within the family, a distinctive trait for host defence proteins exposed to a changing pathogen environment. previous studies from our laboratory characterized the wide spectra antimicrobial activity of two highly cationic human rnases: the eosinophil rnase and the skin derived rnase ribonucleases and have antimicrobial function in the human and murine urinary tract structural determinants of the eosinophil cationic protein antimicrobial activity two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (rnase ) and rnase the regulatory mechanism that we are reporting will contribute to prevent both nad exhaustion and accumulation of the toxic bal. to the best of our knowledge, this is the first report of a novel reversible covalent modification of an aldh enzyme involving its own substrate anna lewandrowska , aldona jeli nska , agnieszka wi sniewska acknowledgement: this research was supported by the inactivation of the fxr gene reduces aqp expression and impairs urine concentrating ability, which leads to a polyuria or urine dilution phenotype. we have previously found that pon -/-mice exhibit a polyuria phenotype and produce twice as much -h urine as their wild type pon / littermates (borowczyk k et al. metabolism and neurotoxicity of homocysteine thiolactone in mice: evidence for a protective role of paraoxonase development and application of novel non-ewald methods for calculating electrostatic interactions in molecular simulations ikuo fukuda , narutoshi kamiya the most time-consuming part of molecular simulation is the calculation of long-range interactions of the particles. in particular, appropriate treatment of the electrostatic interaction is critical, since the simple truncation cannot be used due to the slow decay of the coulombic function. thus, it is highly demanded to calculate the electrostatic interactions with high accuracy and low computational cost. for this purpose we have developed the zero-multipole (zm) summation method [ ]. in this method the artificial periodic boundary conditions are not necessary and the fourier part evaluations are not needed, in contrast to the conventional ewald-based methods. instead, a pairwise function that is suitably redefined from the coulombic function is used with a cutoff scheme. the underling physical idea is simple: (a) in a biological system, a particle conformation for which the electrostatic interactions are well cancelled is more stable than other conformations [ ]; (b) since such well-cancelled conformations are essentially physical, we should clip a subset of such a conformation out of the conformation within an ad-hoc given cutoff sphere and calculate the interactions only from this subset. this idea is realized by a rigid mathematical consideration that leads to the deformation of the coulombic function. the efficiency of the zm method has been validated in applications to fundamental systems sema a) is a protein originally described as an axonal chemorepellent cue involved in many physiological processes ranging from embryonic development to bone homeostasis or immune responses sema a signal transduction requires the formation of a heteromeric complex with neuropilin- (nrp ) and plexina [ ]. in addition, sema a interaction with nrp is modulated by the furin protease cleavage at its c-terminal basic domain this c-terminal basic domain has also been suggested to mediate the binding to glycosaminoglycans (gags), an association that locates sema a to perineuronal nets and enhances its function in restricting neuronal plasticity and inhibiting axonal regeneration in the central nervous system two peptides corresponding to the highly positively charged regions on the domain were shown to bind to immobilized heparin by surface plasmon resonance (spr) and the affinity dramatically increased when the complete domain was assayed. the binding was confirmed by nuclear magnetic resonance (nmr) and circular dichroism (cd) the conserved cysteine within this motif, necessary for the dimerization of sema a [ ], is also critical for the helix formation. in addition, fluorescence spectroscopy studies showed that the n-terminal region also has a contribution in the binding to gags. we acknowledge the financial support from the european union seventh framework programme (fp / - ) under the project vision semaphorin a: a new player in bone remodeling neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex furin processing of semaphorin f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin semaphorin a displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix semaphorin a binds to the perineuronal nets via chondroitin sulfate type e motifs in rodent brains mechanistic basis for the potent anti-angiogenic activity of semaphorin f. biochemistry collapsin- covalently dimerizes, and dimerization is necessary for collapsing activity prior attempts to create functionally relevant groupings of proteins in the crotonase superfamily suggest that this superfamily is difficult to cluster functionally due in part to the functionally diverse nature of the protein superfamily. we have developed two novel procedures to combat this difficulty: tulip (two-level iterative clustering process), a process that utilizes structural information from active sites to cluster protein structures into hypothesized functional groupings, and misst (multi-level iterative sequence searching technique), a process that uses the protein groupings created in tulip as a starting point for iterative genbank searches and further clustering after each search. through these two methods, the total coverage of the crotonase superfamily has increased, and the generated groups contain proteins from subgroups and families that did not have a structural representative. novel hypothesized functional protein groupings have been created, most notably for a large number of proteins that lack annotation data at the subgroup or family level, and for proteins of the enoyl-coa hydratase family fernandes , teresa sorbo , ivan duka , lia christina appold , marianne ilbert , fabian kiessling cnrs, umr , ucibio-requimte, faculdade de ciências e tecnologia e-selectin is a cell-adhesion molecule induced on the surface of endothelial cells in response to cytokines. its upregulation has been reported in many disorders, including inflammatory and cardiovascular diseases, tumor angiogenesis and metastasis [ ]. this profile suggests e-selectin as a promising target to develop molecular imaging probes for the detection of these diseases cyt c with equivalent of fld in media mimicking the cytoplasm. these include % polyacrylamide gel, g/l bovine serum albumin or polyvinylpyrrolidone , and buffer alone for comparison. electrostatic surface representations of the proteins are shown with their in-cell spectrum pl- a search for anti-melioidosis drug candidates targeted to d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia pseudomallei bpgmhb converts dglycero-d-manno-heptose- b, -bisphosphate to d-glycero-d-manno-heptose- b-phosphate. this is the third step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for inhibitors increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. to find inhibitors of bpgmhb, we performed homology modeling of bpgmhb and in-silico virtual screening with zinc, a free database of commerciallyavailable compounds. tens of thousands of chemical compounds were docked into the active site of bpgmhb. a number of putative bpgmhb binding compounds better than d-glycero-d-manno-heptose- b, -bisphosphate were found using surflex-dock included in the sybyl software package crystal structure of dimeric d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis ewha womans university we have solved the crystal structures of d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis (btgmhb) catalyzing the removal of the phosphate at the position of d-glycero-d-manno-heptose- , -bisphosphate. it belongs to the haloacid dehalogenase (had) superfamily with an a/b rossman fold composed of six parallel b-strands sandwiched between two sets of three a-helices it reveals a conventional rossman-like a-b-a sandwich fold with a novel b-sheet topology. its c-terminus is longer than its closest relatives and forms an additional b-strand whereas the shorter c-terminus is random coils in the relatives. interestingly, its core structure is similar to that of enzyme iib(cellobiose) from e. coli (eciib(cel)) transferring a phosphate moiety. in the active site of the closest eceiib(fruc) homologues, a unique motif cxxgxaht comprising a p-loop like architecture including a histidine residue is found. the conserved cysteine on this loop may be thiolated to act as a nucleophile similar to that of eciib(cel). the conserved histidine residue is presumed to accommodate negatively charged phosphate during enzymatic catalysis leonor morgado , kornelius zeth , , , bj€ orn m. burmann , timm maier bama is a b-barrel membrane protein with five periplasmic n-terminal polypeptide transport associated (potra) domains. the bama structure has been determined recently by x-ray crystallography ( , ), however its functional mechanism is not well understood. this mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and nmr spectroscopy is thus a method of choice for its elucidation we demonstrated that knocked down autophagy by shrna (shatg , shbecn , and shatg ) and chloroquine (cq) could enhance high dose of uvb induced cell death in odc overexpressing hela and mcf- cells. here, we also observed that knocked down odc in odc overexpressing hela and mcf- cells inhibited autophagy and enhanced high dose of uvb radiation. because of atg can regulate cell apoptosis and utophagy. site directed mutagenesis was used to mutant the amino acid which can regulate cell apoptosis and autophagy on atg , respectively in these two odc overexpressing cells. according to the results fish ß-parvalbumin acquires allergenic properties by amyloid assembly using atlantic cod b-parvalbumin (rgad m ) displaying high ige crossreactivity, we have found that formation of amyloid fibers under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species at gastrointestinal relevant conditions and for the ige-recognition in allergic patient sera. incorporation of the anti-amyloid compound epigallocathequin gallate prevents rgad m fibrillation, facilitates its protease digestion and impairs its recognition by ige. conclusions: rgad m amyloid formation explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and the epitope architecture as allergen autophagy could degrade the citrullinated and unfolding protein. herein, padi could enhance autophagy in jurkat t cells and lead to a degradation of p and the accumulation of lc -ii. autophagy and apoptosis are two critical mechanisms which participate against cellular stress, cell activation, survival and homeostasis. pad -overexpressed jurkat t cells caused the activation of th cells to increase mrna expression of cytokines, such as il- , il- , il- and tnfa. cytokines provoked caspase expression and led to caspase-mediated cleavage of beclin- which was an important factor of apoptotic signaling. knockdown of bcen rescued cell survival due to the increase of bcl-xl and the decrease of caspase- . we suggested that padi participated in the activated t cell-induced autonomous death through triggering er stress pathway studies on secondary metabolites production and proteins and enzymes of in vitro cultivated artemisia alba turra and relations with some endogenous phytohormones yuliana raynova , krassimira idakieva , vaclav motyka , petre dobrev , yuliana markovska , milka todorova , antoaneta trendafilova , ljuba evstatieva switzerland aim: artemisia alba turra is an essential oil bearing shrub, characterized with great variability of the essential oil profile of wild grown plants, related to genetic, geographic and environmental factors. it was previously established that inhibition of rooting in vitro caused by cytokinin/auxin treatment affected the essential oil profile of the plant and these changes were also related to bioactive endogenous cytokinin levels in vitro ( , ) cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of artemisia alba terpenoid profile of artemisia alba is related to endogenous cytokinins in vitro salivary hnp - are conventionally measured using an enzyme-linked immunosorbent assay (elisa) which does not discriminate between individual hnps due to their structural similarities. considering the biological importance of salivary human neutrophil a-defensin (hnps), there is therefore, a need to develop an analytical method that will discriminate between the defensins. an lc-ms method has been established for the separation and detection of hnp - . the method has been optimised, validated and applied to examine the relative level of hnp - in participants undertaking a circuit resistance training workout. to date, no studies have systematically investigated the effect of acute (min to hours) and chronic (days to weeks) change in salivary adefensins family before and after exercise by lc-esi-ms systems and models calorimetry showed no difference in dissociation constants at these ph values, while the binding stoichiometry is increased . fold. furthermore, the binding stoichiometry varied fold among the two alginates corresponding to their difference in average molecular weight and in addition fold higher binding affinity was found with the high as compared to the low molecular weight alginate. in conclusion, the binding stoichiometry of b-lactoglobulin with alginate increases by a factor that correlates to the average molecular weight of the alginate and also a much higher affinity was found for the high molecular weight alginate. acknowledgements: this work is supported by the danish council for the presence of mucins and other high molecular weight glycoproteins in saliva makes the direct analysis of defensins difficult. the lc-ms method was linear for concentrations of hnp- between . and ng/ml (r . ) with a lod of . ng/ml. inter and intra assay precision was . - %, respectively. saliva sample were clean up by solid phase extraction (spe) and without-solid phase extraction (wspe) . mm internal diameter column in relation to the method transfer . during lc-ms optimisation genome-wide docking database (gwidd) provides the most extensive data repository of structures and models of ppi on a genomic scale. currently, we are expanding the gwidd dataset to , ppi in , organisms, up from , ppi in organisms in the previous release. the ppi data were imported from intact and biogrid databases and were subjected to in-house modeling pipeline. gwidd current implementation contains , experimentally determined complexes, and , sequence homology and , structure homology models of complexes. the user-friendly interface offers flexible organism-specific search with advanced functions for a refined search for one or both proteins. the new gwidd version includes also a new interactive visualization screen that allows to view search results in different residue representations with the emphasis on the ppi interface refolding and activation of recombinant trypsin i from sardine fish (sardinops sagax caerulea) amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of s. mutans, further supporting a functional role. s. mutans lacking p demonstrates residual amyloid forming properties, however, a mutant lacking sortase, the transpeptidase which covalently links p and several other proteins to the peptidoglycan cell wall, is defective in cell-associated amyloid-like properties. the objectives of this study were to identify additional amyloid forming proteins of s. mutans and to evaluate the effects of buffering conditions and ph on the ability of the identified proteins to form amyloids. a p -deficient mutant strain was grown to stationary-phase in defined minimal media, and secreted proteins from spent culture supernatants were fractionated by ion exchange chromatography. partially purified protein fractions were tested for binding of the amyloidophilic dyes congo red (cr) and thioflavin t (tht), and for characteristic birefringent properties following staining with cr and visualization under crossed polarizing filters. proteins from fractions that tested positive for amyloid-like material were separated by sds page, and identified by lc/ms. these included wapa, gbpa, gbpb, smu_ c and smu_ c. recombinant proteins were expressed in escherichia coli, and purified for confirmation and characterization of individual amyloidogenic properties in vitro. recombinant wapa and smu_ c displayed all the biophysical characteristics of amyloid, including visualization of fibrillar aggregates when viewed by transmission electron microscopy. in contrast, gbpa and smu_ c produced amorphous aggregates. wapa and smu_ c form amyloid at different ph, smu_ c under acidic conditions and wapa under neutral to basic conditions. this suggests that the prevailing environmental ph may represent different in vivo triggers for amyloid fibrillization of different s like other small gtpases, the activity of rheb is dictated by its guanine nucleotide binding states: it is active in its guanosine -triphosphate (gtp) bound form and inactive in the guanosine diphosphate (gdp)-bound form. rheb proteins play critical roles in regulating growth and cell cycle, and this effect is due to its role in regulating the insulin/tor/s k signaling pathway rheb interacts directly with fkbp and prevents its association with mtor in a gtp-dependent manner. moreover, fkbp bound to gtp-g-s, a nonhydrolyzable gtp analogon, has a much higher binding affinity for rheb than the gdp-bound form the second study contradicted both studies, since they could not detect any interaction between rheb and fkbp [ ]. to clarify whether there is an interaction and if it is nucleotide dependent, nmr monitored interaction studies were performed employing a c-terminal truncated construct of human rheb ( - rhebdct) that cannot be farnesylated and the biochemically defined binding region on fkpb (fkbp -like fkbp -bd). based on our data rhebdct -gdp does not significantly interact with fkbp -bp. n-fkbp -bd titrated , we observed a weak interaction between rhebdct bound to a gtpanalogon (gppnhp) and fkbp -bd. mapping of the observed spectral changes on the structure of rheb-gtp suggests that fkbp targets the switch region, loop - and the neighboring b-sheet region. we further analyzed the backbone dynamics of rhebdct -gdp and -gppnhp using n relaxtion data (t , t and heteronuclear noe) .based on these data the phosphorylation loop, the switch regions and the loop around residues - show increased backbone dynamics that modulated by the nucleotide binding international centre for genetic engineering and biotechnology tdp- is an rna processing protein that can form inclusions of debatable nature implicated in neurodegenerative diseases. within the putative aggregation domain, repeats of residues - can recruit endogenous tdp- into aggregates inside cells . recently, we showed that a coil to b-hairpin transition in a short peptide corresponding to tdp- residues - enables oligomerization . we have used a broad battery of biophysical experiments, including chromophore and antibody binding, electron microscopy (em), circular dichroism (cd), solution and solid-state nmr, and x-ray to shed light on the nature of these aggregates. based on these findings, structural models for tdp- ( - ) oligomers have been constructed, refined, verified, and analyzed using computational methods, ranging from docking and molecular dynamics simulations to semiempirical quantum mechanics calculations. interestingly, tdp- ( - ) b-hairpins assemble into a novel parallel b-turn configuration showing crossb spine, cooperative h-bonding and tight side chain packing cellular model of tar dna-binding protein (tdp- ) aggregation based on its c-terminal gln/asn-rich region structural characterization of the minimal segment of tdp- competent for aggregation structural evidence of amyloid fibril formation in the putative aggregation domain of tdp- kyungpook national university scolopendin , aglqfpvgrigrllrk, is a -mer peptide derived from the centipede scolopendra subspinipes mutilans. to investigate its property against fungal and bacterial pathogens, antimicrobial tests were performed. we observed that this peptide exhibited antimicrobial activity in a salt-dependent manner and showed no hemolysis. the circular dichroism (cd) analysis observed that a-helical structure properties. we determined the mechanism(s) of action using flow cytometry and investigated the release of potassium. the results showed that the microbial membrane in escherichia coli o and candida albicans was permeabilized with loss of potassium ions. additionally, the bis-( , -dibutylbarbituric acid) trimethine oxonol [dibac ( )] and , '-dipropylthiacarbocyanine iodide [disc ( )] assay showed membrane depolarization. using calcein-encapsulating giant unilamellar vesicles (guvs) and fitc-dextran containing large unilamellar vesicles (luvs), scolopendin disrupted the cell membrane and the damage size is between . to . nm against composition of microbial plasma membrane of e. coli and c. albicans. thus, we demonstrated that a cationic antimicrobial peptide, scolopendin , possesses broad-spectrum antimicrobial effects that formed pore on the cell membrane. structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy protein structural biology structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy b.krishna chaitanya, evelyne schrank and klaus zangger institute of chemistry/organic and bioorganic chemistry university of graz, austria corresponding author email id: krishna.bhattiprolu@uni-graz.at bacterial conjugation is a complex process for the horizontal transfer of single stranded dna from one cell to another. this mechanism also leads, for example, to the spread of antibiotic resistance genes and virulence factors among bacterial species. multi-protein complexes formed at the origin of transfer (orit) region of dna and at the cytoplasmic membrane of the bacterial cell, initiate this process. inside the membrane, the relaxosome identifies the single strand for transfer in a plasmid dna, relaxes and unwinds it, whereas the transferosome is involved in pilus formation (type iv secretion system) and transferring the gene through the cytoplasmic membrane. these events take place in the donor bacterial cell along with several other auxiliary proteins [ ] the bifunctional enzyme trai of plasmid r plays a crucial role in the relaxosome activity, as it contains both a relaxase and helicase domain. to exert its functions on dna, trai works in close co-ordination with other relaxosome proteins like tray, tram and the integration host factor. trai is a residual protein and contains major domains: n-terminal relaxase domain, a central helicase domain and a c terminal domain (ctd). the structure of the c-terminal domain until residue has been solved by crystallography, while the structure and function of the remaining residues remained undetermined [ ]. there are saxs models and crystallographic structures for different parts of trai and also for the full length protein. prediction of cleavage specificity in hcv ns / a serine protease and adv cysteine protease systems by biased sequence search threading gonca ozdemir isik , a.nevra ozer department of bioengineering,faculty of engineering,marmara university proteases are enzymes which recognize specific substrate sequences and catalyze the hydrolysis of designated peptide bonds to activate or degrade them. due to the biological importance of proteases, it is particularly important to identify the recognition and binding mechanisms of protease-substrate complex structures in drug development studies. the assessment of substrate specificity in protease systems is crucial, where interpreting the adaptability of substrate residue positions can be useful in understanding how inhibitors might best fit within the substrate binding sites and aid in the design of potent selective inhibitors. substrate specificity is generally determined by the amino acid profile, structural features and distinct molecular interactions. besides experimental methods, computational tools for prediction of natural substrate cleavage sites, such as threading, have emerged as useful alternative approaches to provide valuable insights into complex enzyme-substrate interactions. in this work, the substrate variability and substrate specificity of the hepatitis c virus (hcv) ns / a serine protease and the adenovirus (adv ) cysteine protease was investigated by the biased sequence search threading (bsst) methodology. using available crystal structures of the proteases, the template structures for the substrate-bound proteases were created in silico by performing various peptide building and docking procedures followed by energy minimization and molecular dynamics (md) simulations. bsst was performed starting with known binding, nonbinding and some random peptide sequences that were threaded onto the template complex structures, and low energy sequences were searched using lowresolution knowledge-based potentials. then, target sequences of yet unidentified potential substrates were predicted by statistical probability approaches applied on the low energy sequences generated. the results show that the majority of the predicted substrate positions correspond to the natural substrate sequences with conserved amino acid preferences, while some positions exhibit variability. for ns / a serine protease cleavage, the significant selection for pro at p and cys at p positions is zearalenone is a mycotoxin produced by fusarium graminearum and related fusarium species. f. graminearum is a powerful plant pathogen and infects major crop plants around the world. acute toxicity of zearalenone is low, but due to its structural similarity to b-estradiol it has binding affinity to the estrogen receptor, which results in interference with hormonal balance. typical effects seen in animals include symptoms like hyperestrogenism and reproductive disorders (reduced fertility, reduced litter size or swelling of uterus and vulva). to reduce the risk for human and animal health posed by the ingestion of contaminated food or feed different decontamination strategies have been studied, including biotransformation. today many microorganisms are known to degrade zearalenone, but for most of them the degradation pathway and formed metabolites remained unknown, hence it is unknown if this degradation also means detoxification. only for the fungal strains trichosporon mycotoxinivorans and gliocladium roseum zen degradation has been studied in detail and loss of estrogenicity of reaction products has been confirmed. we screened for, and isolated zearalenone degrading bacteria from soil samples. the most promising new bacterial isolate was taxonomically assigned to the species rhodococcus erythropolis and designated pfa d - . the zearalenone catabolism pathway of pfa d - was found to be identical as known from g. roseum. the primary reaction product, hydrolysed zearalenone, has so far only been postulated in g. roseum. we prepared hydrolysed zearalenone by preparative hplc and showed loss of estrogenicity in assays with the breast cancer cell line mcf and the estrogen reporter yeast strain yzhb . a genomic library was prepared and screened in zearalenone degradation deficient r. erythropolis pr . the gene encoding zearalenone hydrolase was found and named zena. the hydrolase was identified as member of the a/b-hydrolase family and named zena. it was cloned, recombinantly expressed in e. coli and purified by x his-tag mediated immobilised metal affinity chromatography. activity of his-tagged and untagged enzyme zena was compared in cleared lysate and zena was purified for enzyme characterisation. the influence of ph and temperature on enzyme activity and stability was evaluated and kinetic parameters were determined. a new biding site for snake venom c-type lectins?maria cristina nonato costa , ricardo augusto pereira de p adua , marco aurelio sartim , suely vilela sampaio university of são paulo, fcfrp c-type lectins are proteins that bind different glycan molecules by interactions with a calcium atom present in a carbohydrate recognition domain (crd). many organisms (plants, bacteria, virus and animals) use these proteins in various biological events like lymphocyte adhesion, erythrocyte agglutination and extracellular matrix organization. the c-type lectin fold is plastic and possible for about different sequences, what promoted its adaptation to diverse functions, similarly to the observed for the immunoglobulin fold ( - sequences). it is comprised of about - amino acid residues that folds in two fourstranded b sheets sandwiched by two alpha helices. interestingly, c-type lectins present in snake venoms are possible anti-cancer agents since they are toxic to cancer cells and inhibit the adhesion and proliferation of various cancer cell lines. therefore, we have purified a lactose binding c-type lectin from the venom of bothrops jararacussu (bjcul) to study its structure and binding properties to different sugars. bjcul crystals were obtained by vapor diffusion and the structure solved by x-ray crystallography to . Å resolution. bjcul structure is a decamer formed by a pseudo fivefold axis rotation of a dimer hold by a disulfide bond. each monomer binds a calcium atom and possibly another metal at a second and opposed binding site. key: cord- -ohdukhqt authors: patil, shital p.; goswami, ashutosh; kalia, kiran; kate, abhijeet s. title: plant-derived bioactive peptides: a treatment to cure diabetes date: - - journal: int j pept res ther doi: . /s - - -z sha: doc_id: cord_uid: ohdukhqt abstract: recent advances in analytical techniques have opened new opportunities for plant-based drug discovery in the field of peptide and proteins. enzymatic hydrolysis of plant parent proteins forms bioactive peptides which are explored in the treatment of various diseases. in this review, we will discuss the identified plant-based bioactive proteins and peptides and the in vitro, in vivo results for the treatment of diabetes. extraction, isolation, characterization and commercial utilization of plant proteins is a challenge for the pharmaceutical industry as plants contain several interfering secondary metabolites. the market of peptide drugs for the treatment of diabetes is growing at a fast rate. plant-based bioactive peptides might open up new opportunities to discover economic lead for the management of various diseases. graphic abstract: [image: see text] biomolecules play an important role in any ligand-target interaction and their recognition in any biological system starts the cascade of signalling steps, which is an important part of the normal body function. nucleic acids, carbohydrates, and lipids are the important ligands that bind and cause conformational changes in targets (receptors) but the messengers that release after binding and conformation changes are peptides. any defect in these signal transduction processes cause diseases (otvos and wade ; gautam ) . proteins, under the influence of proteolytic enzymes get fragmented at particular catalytic sites to form bioactive peptides. bioactive peptides are made up of - amino acids and lack protein-protein interaction because of their small size. they have properties like tissue affinity, specificity, and efficiency (moller et al. ) . the structure and the overall charge and hydrophobicity/hydrophilicity of the any bioactive peptide depends on the nature of amino acids, their sequences in the peptide backbone along with the n and c terminals. the relation between structure and biological activity of the bioactive peptides are yet not confirmed but the structure is an important arbitrator for their activity (li and yu ) . bioactive and nutraceutical peptides have a positive effect in the regulation of the health of an individual (kitts and weiler ) . they boost the quality of human health by preventing various ailments and by improving medical conditions related to lifestyle, metabolism, and immunity (vitetta et al. ) . peptide and protein drugs are available from the different sources for therapeutic uses, but as with many drugs, they have several advantages and disadvantages of their own. peptide drugs are effective and specific to their biological target but devoided of some of the essential qualities of a drug to withstand in the current market of therapeutics. proteins have a large chemical structure which makes their synthesis complex, tedious and expensive, the further low oral bioavailability of proteins limit their oral delivery hence they are administered through parenteral route (uhlig et al. ) . the worldwide peptide therapeutics market size valued at usd , . million in and assumes to grow at cagr of . % over the period to . ( ) . insulin is the first therapeutic peptide which has been used widely and shares good peptide market. it was first isolated by frederick banting and charles best from pancreatic islet extracts, further, j.b. collip developed the method for extraction and purification of insulin from other animal sources (quianzon and cheikh ) . eli lilly began producing insulin from the animal pancreas, later on, biosynthetic insulin was introduced in with the name humulin. novo nordisk, sanofi, and eli lilly together share . percent of the global insulin market. lantus (basal insulin) is a product of sanofi accounts for approximately . % of the total sales of top antidiabetic drug brands, in sale of lantus was usd . billion (dezzani ) . small molecules and peptide-based drug candidate belongs to the category of new chemical entities (nces) according to the food and drug administration, on the other hand, proteins used for the treatment of various diseases are classified under new biological entities (nbes). in recent years, therapeutic peptides are approved for the mitigation of various ailments mainly tumors, immunological disorders, blood disorders and metabolic disorders such as diabetes, obesity ( fig. ) (loganathan ) . the peptide drug, liraglutide (victoza) is a glucagon-like peptide- receptor (glp- r) agonist and used for the treatment of t dm has gained popularity as a top-selling drugs in recent past, which is marketed by novo nordisk (lund et al. ) . glatiramer acetate (copaxone), developed by teva is an immunomodulator and used to treat multiple sclerosis (duda et al. ) . leuprolide (lupron), developed by abbott is useful for the treatment of cancer and estrogendependent conditions that respond to hormone therapy (brito et al. ) . goserelin (zoladex) from astra zeneca is a gonadotropin-releasing hormone agonist (gnrh agonist) that suppresses the release of different sex hormones and is useful in the treatment of breast and prostate cancer (magon ) . other innovator companies include amgen, eli lilly, roche, and pfizer have peptide and protein-based drugs in different developmental stages will reach the market soon for the treatment of various complicated diseases (loganathan ) . till date, plant secondary metabolites, mainly small molecules are the established source of new drugs to treat various diseases (verpoorte ) , however, advancement in analytical technique, sophisticated purification methodology and in vitro assay system pointed out the researchers fig. uses of therapeutic peptides and proteins in various disease conditions (loganathan ) to look beyond the small molecules. identification of plant primary metabolites including proteins and peptides for the management of diseases have opened up a new horizon in the development of plant-based peptides as a drug candidate (otvos ) . this review summarizes various plant-based peptides reported for the treatment of diabetes, challenges with the development of peptide-based drugs and the future prospect of peptides as a drug. diabetes is a metabolic disorder and can hit anyone at different stages of life. though it is considered as one of the top health killers, its treatment is a huge challenge. diabetes can be characterized with symptoms of prolonged hyperglycemia, glucose intolerance, disturbance in the regulatory systems for storage and utilization of metabolic energy, including catabolism and anabolism of carbohydrate, lipids, and proteins in a diabetic individual due to lack of insulin production and impaired insulin receptor functioning or both (piero ; votey and peters ) . based on the causes and clinical survey, diabetes mellitus is categorized into four types as mentioned in table (deepthi et al. ) . ordinarily, the people below age suffer from the type diabetes, whereas type diabetes mainly occurs in adult and old age group. according to the world health organisation, approximately % of all diabetes cases are type , and the remaining % cases of diabetes worldwide are of type (baynes ) . diabetes is ruining the life of peoples worldwide. population suffering from diabetes is increasing and the imminence is expected to rise even more because of the current lifestyle issues. till the year , million people were affected by type diabetes and the number is expected to increase up to million people worldwide by (fig. ) . the data indicates that approximately % of diabetic patients will increase in the next two decades. the threat is every s a person dies with diabetes (roglic ) . from the statistics, it is clear that the population suffering from type diabetes is increasing and it requires more promising therapies. still, no promising drug is available which has the ability to completely cure type diabetes. generally, to treat type diabetes, oral hypoglycemic agents are used. the first line treatment is metformin, which is assisted with other hypoglycemic agents from different categories like thiazolidinediones, alpha-glucosidase inhibitors, sulphonylurea, dipeptidyl peptidase-iv (dpp-iv) inhibitors, sodium glucose cotransporter (sglt ) inhibitors. the risk associated with these therapies include hypoglycemia, weight gain, tiredness, diarrhea, and anemia risk, etc. over the period of time, these side effects lead to further complications for a visceral system like cardiovascular system and central nervous system (nathan et al. ). peptide drugs used for the treatment of diabetes are derived from different sources, like exendin- was initially isolated from the heloderma suspectum (gila monster) and its synthetic version had come to the market in the form of exenatide (aramadhaka et al. ) . liraglutide and semaglutide are structurally very close to glp- with improved half-life and resistant to dpp-iv mediated degradation (edmonds and price ) . nowadays, these peptides are being produced in large scale by using recombinant dna technology. the peptides which were obtained via synthesis or by using the recombinant technology are comparatively expensive than small molecules and are not affordable to most of the patients. the interest in health-promoting products from the natural origin and pharmaceutical formulations involving bioactive peptides are remarkably increasing (henninot et al. ) . numerous scientific reports have been published on bioactive peptides obtained from animal proteins. expedition by researchers in the field of bioactive peptides is of great interest as it opens the way to find economic lead from plants for the better care of diabetic patients (daliri et al. ). plentiful bioactive peptides have been reported from the animal and plant sources. most of the peptides that had bioactivity are being derived from animal products such as milk, eggs, meat, and fish. plant peptides are still under exploration and few bioactive peptides are reported from soy, wheat, etc (hartmann and meisel ) . in animals, proteins are associated with high-fat content and lead to diseases, including high blood pressure and heart diseases if consumed in the large amount. on the other hand, plant proteins neither have associated fat nor have any side effects (nehete et al. ) . previously, plant hormones were considered the only player through which cells communicate, but from the last decade as research was driven towards an understanding of plants signaling, secreted peptides, small rnas and transcription factors emerging as a new and well-defined player in the cell to the cell communication network (lindsey et al. ; van norman et al. ; murphy et al. ) . however, it is now clearly defined that signaling peptides of plant and animal origin are biosynthesized through the pathway that is evidently similar, although some aspects of the biosynthesis are still not identified. peptides secreted by plants have well recognized role in preliminary processes of development like the growth of meristem, organ shedding, cell elongation, cell multiplication, cell differentiation, geotropism and protection from the invader (ghorbani ) . the ubiquitous presences of proteins, peptides as mediator components convey to the proposals that these messenger peptides may have emerged evolutionary in microbes and were further integrated into the complicated mediator systems of complex organisms during evolution (roth et al. ) . existence of hormone-like peptides in plants including insulin-like peptide in spinacia oleracea l. (spinach) and lemna gibba g- , prolactin-like inhibitor in alfalfa, a substance with luteinizing hormone-releasing activity from leaves of avena sativa l. (oat) and somatostatin-like material in spinach support this assumption, it is speculated that the signaling peptides in plants existed from about billion years ago (collier et al. ; fukushima et al. ; leroith et al. ; morley et al. ) . plant proteins containing essential amino acids are important in the food chain to fulfill human physiological needs. the proteins from plants are derived from leaves, seeds, and fruits, out of which seeds are considered as an economical source of proteins. as compared to vegetables and fruits, seeds (legumes and cereals) contain a higher amount of protein (creighton ) . proteins in a mature seed represent - % in pisum sativum l. (pea), vicia faba l. (faba) bean and till - % in lupinus albus l. (lupin) and glycine max l. (soybean) (gueguen and cerletti ) . many peptides are reported from different plants for the treatment of diabetes as shown in tables and through various known targets such as (a) alpha-glucosidase inhibitors (b) alpha-amylase inhibitors (c) dipeptidyl peptidase-iv inhibitors (d) inhibitors of the glucose transporter system (e) insulin mimetics alpha-glucosidase inhibitors competitively inhibit small intestinal enzyme alpha-glucosidase, which converts nonabsorbable polysaccharides into absorbable monosaccharide, these effects together postpone and minimize the rise in postprandial plasma glucose level. inhibitors of this enzyme decrease the glucose toxicity (improves insulin sensitivity), decreases stress on beta cells (decreases post-meal hyperglycemia), increases glucagon like peptide- (glp- ) production hence increases insulin secretion (scheen ; mooradian and thurman ) . in general, normalization of postprandial hyperglycemia is difficult as compared to fasting hyperglycemia, inhibitors act specifically on the postprandial part of h blood glucose curve and causes a reduction in diabetes-associated hyperglycemia which is responsible for macrovascular complication in patients (mooradian and thurman ; ceriello et al. ). glycaemic control is achieved with these agents reduces glucotoxicity which increases insulin secretion from the beta cell (scheen ; salvatore and giugliano ) . all marketed alpha-glucosidase inhibitors are from natural origin, however, none of them belongs to peptide class. few research groups have reported peptides from plants having alpha-glucosidase inhibitory activity. peptides obtained from cannabis sativa l. (hemp) seeds contains hydrophobic amino acids (pro and leu), essential amino acids and branched chain amino acids in their structure and have reported to have enzyme inhibitory activity for this target (ren et al. ) . oat seed proteins hydrolysate obtained by protease (alcalase) digestion gives bioactive peptides which inhibits this enzyme. in vivo study also revealed that oat peptides, at a higher dose, showed the hypoglycaemic effect on stz-induced diabetic mice, reduces the food intake, stimulates insulin secretion, improves insulin sensitivity and elevate glycogenesis (zhang et al. ) . walnut hydrolyzed peptides (whps) from the fruit proteins of juglans mandshurica maxim. (walnut) showed anti-diabetic activity by inhibiting the enzyme. in vitro study of whps showed that peptides of molecular weight ( - kda) inhibit alpha-glucosidase with the inhibitory rate of ( . %) and they remarkably raise extracellular glucose consumption in insulin-resistant hepg cells. whereas, in vivo results showed that whps reduce . % of fasting blood glucose level by increasing insulin secretion, liver glucokinase, and glycogen level by . %, . %, and . % respectively (wang et al. ) . vigna angularis wild. (adzuki bean) proteins have reported enzyme inhibitory properties in mice model kk-ay of diabetes. the extract reduces the postprandial ( ) hook.f. in vitro and in vivo marella et al. ( ) cucurbita pepo l. in vitro (vaštag et al. ) lam. in vitro (gonzalez garza et al. ) l. in vitro arise ( ) theobroma cacao l. in vivo sarmadi et al. ( ) l. in vitro mojica et al. ( b) willd. in vitro nongonierma et al. ( ) l. in vivo and in vivo yibchok-anun et al. ( ) momordica charantia l. insulin mimetic in vivo (khanna et al. ) momordica charantia l. seed protein expressed in e.coli stimulated the phosphorylation of pdk and akt, enhanced expression of glut- , stimulated both the uptake of glucose in cells and the clearance of glucose in vitro and in vivo lo et al. ( ) hook.f. in vivo rajasekhar et al. ( ) hook.f. molina. duchesne. in vivo teugwa et al. ( ) zea mays l. enhance the secretion of glp- in vivo hira et al. ( ) glycine max l. increases the glucose uptake, enhance the expression of p-ir, p-irs , p-akt and membrane glut protein improve the insulin resistance hypoglycemic in vitro and in vivo lu et al. ( ) oryza sativa l. improve insulin resistance in vivo boonloh et al. ( ) roxb. in vivo poovitha et al. ( ) blood glucose in two sucrose challenge model i.e. normal and streptozocin-treated rats by . % and . % respectively. in vitro study of extruded adzuki bean proteins at mg/ml, concentration inhibits . % of rat intestinal alpha-glucosidase (yao et al. amylase (alpha- , -glucan- -glucanohydrolase) is an important enzyme that speeds up the hydrolysis of (alpha- , ) glycosidic linkage of carbohydrate and starch present in the diet and helps in absorption of non-absorbable polysaccharides after their conversion into absorbable monosaccharide (alagesan et al. ). inhibitors of amylase minimize the hydrolysis of (alpha- , ) glycosidic linkage and help in slow digestion of carbohydrate and thus delay the absorption of glucose and reduces the postprandial glucose level in blood (jayaraj et al. ). the pinto beans are a rich source of proteins and other nutrients but are not well explored for its therapeutic benefits. pinto bean protein fraction was digested enzymatically using protease (protamex) at ph . , and for the incubation period of -h with (e/s) ratio of : at °c, hydrolysed fraction was dialysed using molecular weight cutoffs and the fraction obtained with molecular weight < kda showed . ± . % of enzyme inhibition (ngoh and gan ) . similarly digestion with protease (bromelain) and dialysis with molecular weight cut-off < kda showed . ± . % of enzyme inhibition (oseguera-toledo et al. ) . the peptide obtained from cuminum cyminum l. (cumin) seeds, cumin seed peptide (csp ) had shown . % of alphaamylase inhibition (siow and gan, ) . in vitro study of triticum aestivum l. (wheat) albumin extract showed alphaamylase inhibition, wheat albumin restrained the peak of postprandial blood glucose levels in a dose-dependent manner. it showed the reduction in postprandial blood glucose by %, %, and % after administering . g, . g, and . g of wheat albumin respectively. in long-term administration study, . g of wheat albumin did not affect fasting blood glucose levels, while it reduces hemoglobin a c levels which reflects the metabolic control of individual suffering from diabetes (kodama et al. ) . a study revealed that different protein fractions isolated from the hordeum vulgare l. (barley) flour have been subjected to pancreatic hydrolysis and the obtained peptides have shown - % alpha-amylase inhibitory activity (alu'datt et al. ). cucurbitin protein was obtained from the cucurbita pepo l. (pumpkin) seeds, alcalase and pepsin hydrolysed fraction of it showed the enzyme inhibition (vaštag et al. ) . oligopeptides from the mulberry showed highest enzyme inhibition with ic . µg/ml. protein hydrolysates prepared from the seeds of moringa oleifera lam. by enzymes like trypsin, chymotrypsin and pepsin-trypsin for . and h. pepsin-trypsin digested fraction showed alpha-amylase inhibition and the ic was found to be . and . µg/ml for . h and h of hydrolysis respectively (gonzalez garza et al. ) . protein hydrolysate of citrullus lanatus l. (watermelon) seeds was prepared by using enzymes like pepsin, trypsin and alcalase where alcalase hydrolysate gives the highest enzyme inhibition and the ic was found to be . mg/ml (arise ) . autolysates of theobroma cacao l. fruits were prepared at ph . and . . autolysate at ph . showed highest inhibition of alpha-amylase as well as helps to release the insulin, in vivo study with autolysates showed decrease in the blood glucose level (sarmadi et al. ) . quinoa proteins were enzymatically hydrolyzed to obtain bioactive peptides, hydrolyzed fraction thus obtained showed . % inhibition of alpha-amylase at µm concentration (vilcacundo et al. ). two main gut-derived hormones glp- and glucosedependent insulinotropic polypeptide (gip), also known incretin hormones are responsible for the attainment of normoglycemia. they stimulate insulin secretion, decrease glucagon, inhibit gastric emptying, reduce food intake and appetite (drucker and nauck ) . in diabetic individuals, reduced secretion of incretin hormones have been observed. these gut hormones bind with their respective receptors and show their biological action (barnett ) . treatment of diabetes requires to restore either normal secretion or reduce the degradation of these hormones. dipeptidyl peptidase-iv (dpp-iv) is a protease enzyme that cleave the glp- hormone within minutes after its secretion and makes it inactive for biological function. thus, inhibition of dpp-iv has the potential to revert the hyperglycaemic condition. dpp-iv inhibitors block the rapid degradation of glp- and enhance the postprandial level of active glp- which further reduces the liver glucagon production and stimulate beta cells of the pancreas and increase insulin level (ahrén ) . dipeptides obtained from oryza sativa l. (rice) bran protein hydrolysates showed inhibition of dpp-iv enzyme and have ic . ± . mg/ml (hatanaka et al. ) . in another study, rice bran protein fractions have been defatted and hydrolyzed with umamizyme g and bioprase sp. the dipeptides obtained after digestion with umamizyme g showed inhibition of enzyme and the ic was found to be . ± . mg/ml (hatanaka et al. ) . the proteins extracted from amaranthus hypochondriacus l., have been subjected to simulated gastrointestinal digestion resulted into bioactive peptides. these peptides fraction have shown dose-dependent enzyme inhibition and have ic . mg/ml (velarde-salcedo et al. ) . glycine max l. (soybean) and lupinus albus l. (lupin) protein hydrolysate have been screened for the presence of bioactive peptides, and soy and lup were found to be efficient inhibitors of dpp-iv with ic values and μm respectively (lammi et al. ) . peptides obtained (cpgnk and ggglhk) from the protein digestion of common beans showed enzyme inhibitory activity and the ic (mg dw ml − ) for the pure peptides were found to be . ± . and . ± . respectively (mojica et al. b) . gastrointestinal digestion of proteins obtained from phalaris canariensis l. (canary) seed showed . % inhibition of dpp-iv (estrada-salas et al. ) . enzymatic digestion of quinoa proteins with papain showed enzyme inhibition and the ic for the hydrolysate-papain was found to be . ± . mg/ml (nongonierma et al. ) . study was performed on quinoa protein simulated duodenal digestion and the fraction (˃ kda) obtained after min of digestion showed highest dpp-iv inhibition and the ic (mg protein/ml) was found to be . ± . (vilcacundo et al. ). glucose, the metabolic fuel of any living cell, unable to cross lipid bilayer of the membrane due to its polar nature and membrane proteins (glucose transporter) associate with lipid bilayer helps glucose to travel across (abdul-ghani et al. ) . glut transporters facilitate the transport of glucose passively across the membrane along with its concentration gradient and do not require energy to operate, on the other hand sglt transporters actively transport glucose across the membrane against the concentration gradient thus these types of transporters require energy to operate. in order to receive energy for operation, sodium is transported along with its concentration or electrochemical gradient thus provide the needed amount of energy for the transportation of glucose across the membrane (musso et al. ; brown ) . in the human body, at least different glut transporters and sglt transporters exist which belongs to membrane class of proteins. excreted glucose is reabsorbed up to % in a convoluted segment of proximal tubule via high capacity, low-affinity sglt transporter while remaining % of glucose is reabsorbed in the distal segment of proximal tubule via high affinity, low capacity sglt transporter. in the case of diabetes, hyperglycemia causes the high load of glucose in order to compensate for this high load of glucose, an increase in expression of a glucose transporter gene in proximal tubule occurs (abdul-ghani et al. ) . plant peptides targeting glucose transporters are less explored. however, peptides having amino acid sequences aksplf, atnplf, feeln, and lsvsvl, isolated from the black beans have shown the ability to block the glucose transporters glut- and sglt- . the results of in vitro studies by using the caco- cell model have shown reduced glucose re-absorption by . % after h treatment of these bioactive peptides. the oral glucose tolerance test in rats has shown a . % decrease in postprandial glucose (p < . ) ( mg hydrolyzed protein isolate/kg bw) (mojica et al. a ). insulin hormone trigger several signaling events that control the metabolic fate of nutrients. insulin binds with alphasubunit of insulin receptor which causes the conformational changes in β-subunit of the insulin receptor, and these conformational changes in receptor activate the cytoplasmic tyrosine kinase domain. this activation further leads to the auto-phosphorylation which in turn trans-phosphorylate various intracellular substrate of the insulin receptor. these effects collectively control metabolic and mitogenic processes (nankar and doble, ) . tyrosine kinase domain activation by insulin mimetic, cause the auto-phosphorylation of receptor and trigger downstream signaling requires for metabolic activity of insulin. the other mechanism by which insulin mimetic (vanadate) works is by inhibiting the dephosphorylation of the insulin receptor via inhibition of tyrosine phosphatase (stapleton ) . the reports suggest that plant extracts of spinach and lemna gibba g- mimics like mammalian-insulin, which was confirmed by the radioimmunoassay. in an in vivo study on young rats indicated that the plant insulin-like material binds to insulin receptors on im- lymphocytes and stimulates glucose oxidation and lipogenesis in isolated adipocytes (collier et al. ) . vigna unguiculate l. (cowpea) containing bio-molecules have contributed in reducing the blood glucose level and enhancing the antioxidant status of patients. study on l rat skeletal muscle cells was performed where cells were treated with cowpea peptides at different doses ( . , , and ng) for h or insulin ( nm) for min. further, from the treated cells proteins were isolated for western blot analysis to check the phosphorylation of akt (a form of protein kinase b; pkb). the results of the study showed that the cowpea peptides have the ability to phosphorylated akt in the cell culture. this observation suggests that the treatment of cowpea peptides to the skeletal muscle cells can initiate the insulin signaling cascade. there is a possibility that cowpea peptides have the ability to mimic like insulin by inducing the same signaling cascade (uruakp, ) . linum usitatissimum l. (flax) seed peptides obtained from the protein hydrolysate ( kd) increases glucose uptake in l cells ). mc - - peptide fraction was obtained from momordica charantia l. showed hypoglycaemic effect in in vivo study. mc - - fraction decreases the blood glucose level by . % and . % in alloxan-induced diabetic mice with a time interval of and h respectively (yuan et al. ) . study on protein from the pulp of m. charantia l. showed increase glucose uptake in c c myocytes and t l adipocyte, the in vivo studies showed that the protein obtained from pulp helps in secretion of the insulin as well as act like insulin to lower the glucose level (yibchok-anun et al. ). polypeptide-p was isolated from the m. charantia l. acts like insulin mimetic (khanna et al. ) . another -residue insulin receptor (ir)-binding protein (mcirbp) was reported from the plant upon in vitro digestion yields mcirbp- , spanning residues - of mcirbp which increases the binding of insulin to its receptor, stimulate the phosphorylation of pdk and akt, induces the expression of glucose transporter , and stimulate both the uptake of glucose in cells and the clearance of glucose in diabetic mice (lo et al. ) . a novel protein called mcy having molecular weight kd was isolated from momordica cymbalaria l. fruit which showed hypoglycemic effect in in vivo study (rajasekhar et al. ) . the peptide fraction obtained from the soybean showed increase in glucose uptake in muscular cells, it is reported that obtained peptide fraction enhances the glucose uptake via amk activation (roblet et al. ) . protein extract of cucurbitaceae family containing seeds like telfairia occidentalis hook.f., citrullus lanatus l., lagenaria siceraria molina., and cucurbita moschata duchesne., showed a significant decrease in blood sugar level in in vivo study (teugwa et al. ) . peptide reported from the leaves of bauhinia variegata l, mimics like insulin which is evidently proved from the immunological reactivity and via in vivo study (azevedo et al. ) . zea mays l. (zein) seed protein hydrolysate increase the secretion of glp- directly in the ileum and indirectly in the duodenum in the rat and also confirmed with the help of glutag cell line study (hira et al. ). aglycine which is a bioactive peptide obtained from the soy bean increases the glucose uptake in c c cell, whereas in in vivo study on diabetic model it was observed that treatment with aglycine increases the expression of p-ir, p-irs , p-akt and membrane glut protein which results into hypoglycemia (lu et al. ) . soymorphin- also called µ-opioid obtained from soy bean decreases the blood glucose level via activation of adiponectin and pparα systems in diabetic kkay mice (yamada et al. ) . rice bran protein hydrolysate helps to improve insulin resistance and prevent metabolic diseases (boonloh et al. ) . protein extracted from the fruit pulp of momordica dioica roxb. decreases the blood glucose level in a diabetic model (poovitha et al. ) . plant scientist believe that there are more plants left unexplored which may contain insulin-like peptides or peptides which will mimic insulin (xavier-filho et al. ) . the success of any therapy and its acceptability depends on the ease of its delivery. the oral route of drug administration remains the most accepted route of drug delivery and most of the available drugs in the market are delivered through the oral route (morishita and peppas ) . an orally active drug should be stable enough to withstand the gastric microenvironment and also it should be permeable through the gastrointestinal membrane. small molecules are well tolerant to the gastric ph and permeated through the membrane whereas the large macromolecules are vulnerable to degradation in the gastrointestinal tract and are not permeable in their intact form to reach specific target successfully without being degraded in git (morishita and peppas ) . as the medical field advances, the peptide drugs are widely explored for their therapeutic potential in the treatment of various diseases. though these drugs are specific to their target, efficacious and potent, the oral bioavailability of peptide and peptide-based drugs always remains a challenge that limits their wide acceptance and market value. high cost of therapy is a major limitation associated with peptide drugs (ayoub and scheidegger ) . with the advancement of biotechnology, the recombinant synthesis of a peptide of interest opens a door for cheap and cost-effective peptides. also, the commercial interest for the production of bioactive peptides from the natural sources is elevated to reduce the cost but due to the lack of suitable technologies which helps to retain or increase the activity of bioactive peptides, the large-scale production gets hampered (craik et al. ) . another limitation associated with peptide-based drugs is their short half-life. when the peptides come in contact with intestinal mucosa, gastric acid in stomach and peptidases that are present in the blood convert peptides into amino acids and gets quickly eliminated from the body, thereby reducing its half-life. in order to achieve prolonged action and to reduce the frequent dosing, it is necessary to increase the half-life of the peptides. as peptide therapeutics advances, several non-degradable polymers and polymeric matrix are available to increase the half-life of peptides and proteins thus reducing the frequent dosing (brown ) . another strategy to increase the half-life of the peptide is to modify the amino acids which are susceptible to cleavage by enzymes (adessi and soto ) . oral delivery of the peptides is explored in past years but limits its use as peptides are degraded in the git tract (lau et al. ) . novo nordisk developed a semaglutide oral formulation which is currently in clinical trial phase . other non-invasive and suitable routes for the peptide and protein-based drugs are under investigation includes the nasal and pulmonary route which directly deliver the drugs to the blood component hence, reduces the overall drug degradation which is the main drawback associated with oral delivery of peptides (davies et al. ). different therapeutic peptides have made their way to the market in recent years, and it is assumed that the market demand will increase further as peptides are specific, selective and safe in comparison to small molecules (fosgerau and hoffmann ; loffet ) . traditional synthesis and drug design approaches are used by researchers and pharmaceutical companies for the development of the peptide-based drug candidate. the recombinant protein is another tool for the synthesis of the desired peptide in bacteria, yeast, and fungi. the market of peptides has now moved from the isolation of peptides from an animal source that can mimic the endogenous peptide to synthetic, semi-synthetic and recombinant peptides. further, development in this area is drug conjugated peptides and multifunctional peptides (fosgerau and hoffmann ) . peptides are susceptible to degradation when given orally, which is supposed to be the most convenient route of administration. the preferred route for the peptide delivery is intravenous, however, more research is directed for the delivery of peptides. recently, alternative routes are explored, which include oral, transdermal and intranasal route. as peptides are excellent biomarkers, they can also be utilized for diagnosing diseases. further, peptides are also found to be advantageous in vaccine development (brown ; sheridan ). plant peptides have been isolated and characterized in different ways as described in fig. . x-ray crystallography and nmr have contributed enormously in the field of structure elucidation of pure peptides (gomathi and subramanian fig. the general scheme of isolation, purification, and characterization of plant peptides (gomathi and subramanian ; krishnan and rupp ; sinz et al. ; wysocki et al. ) ; krishnan and rupp ) . other techniques like electron microscopy, fluorescence resonance energy transfer, chemical cross-linking emerged in recent year for the characterization of proteins and peptides (sinz et al. ) . mass spectrometry is frequently used technique in analyzing peptide mixtures and identifying their proposed structures. amino acid sequencing in mass spectrometry can be performed by two methods namely top-down sequencing and down-top sequencing. in top-down sequencing, the proteins analysis is performed without hydrolysis and the molecular weight can be detected by fragmentation pattern of the whole protein. a common approach is down-top sequencing in which proteolytic digestion is performed followed by ms analysis. software based on the algorithm of the amino acid sequence is available for protein identification (wysocki et al. ). plants remain a valuable source for developing a new drug candidate. they are still sharing their fair share of the new drug candidates and lead. as research interest is now shifted from small molecules to bio-molecules by virtue of several added benefits, plants are now under exploration for the presence of proteins and bioactive peptides for the disease management. though plant proteins and their functions have not been defined earlier, plant hormones were only considered through which cells communicate, as our understanding to these signalling pathways become clear, several secreted peptides and small rnas now known which regulates molecular recognition and thus, controls cell communication. from our diet we consume proteins which after gastrointestinal digestion get converted to bioactive peptides with more tissue affinity to specific receptor. several research publications in recent years claimed plant proteins and their bioactive peptides as a potential candidate in the treatment of various diseases specially in metabolic and life style borne diseases including cancer, diabetes and obesity. sophisticated instrument and advancement in technology leads easy isolation, purification and characterization of proteins and peptides in a short period of time, which further reduces the overall time of discovery. apparently, most of the bioactive peptides available for the treatment of diabetes have emerged from the synthetic route or derived from recombinant technology, which further adds in the overall cost of the treatment. to make peptide therapy economic there is a demand to explore plant-based biomolecules. this review discusses bioactive peptide isolated from various plant parts like leaves, fruits and seeds. the trend indicates that most of the bioactive peptides are obtained from seeds, although reports are available where bioactive peptides have been also isolated from leaves, whole fruits and pulps. it was observed in various in vitro and in vivo studies that protein hydrolysate obtained from aqueous extracts of plant are capable of inhibiting the enzymes and transporter systems responsible for the diabetes. expedition to find molecular targets and mechanism of action of plant based bioactive peptides is needed to use these bioactive peptides as a potential drug candidate. epidemiological data reveals that type diabetes requires promising therapy as the available synthetic drugs for the treatment have moderate to severe adverse effects. plant-derived bioactive peptides inhibit the enzymes like alpha-glucosidase, alpha-amylase, dipeptidyl peptidase-iv and glucose transporter systems involved in type diabetes. in vivo studies of a certain plant peptides fraction showed insulin-mimetic action in animal models. it is clear that plant-derived bioactive peptides are not explored to their full potential in comparison to other classes of natural products. the reason might be the lack of suitable instrumental techniques to identify, purify and characterize the peptides from plant source. however, the recent advancement in lcms, lc-nmr, and x-ray crystallography has bridged the gap and this would be the right time to embark on plant peptides. in future, systematic studies 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diabetic kk-ay mice slow acting protein extract from fruit pulp of momordica charantia with insulin secretagogue and insulinomimetic activities purification and characterisation of a hypoglycemic peptide from momordica charantia l. var. abbreviata ser peptides derived from oats improve insulin sensitivity the authors want to thank niper ahmedabad, under the aegis of the ministry of chemicals and fertilizers, department of pharmaceuticals for providing facilities and research fellowships. key: cord- -zp q ztr authors: moll, gert n.; kuipers, anneke; rink, rick; bosma, tjibbe; de vries, louwe; namsolleck, pawel title: biosynthesis of lanthionine-constrained agonists of g protein-coupled receptors date: - - journal: biochem soc trans doi: . /bst sha: doc_id: cord_uid: zp q ztr the conformation with which natural agonistic peptides interact with g protein-coupled receptor(s) (gpcr(s)) partly results from intramolecular interactions such as hydrogen bridges or is induced by ligand–receptor interactions. the conformational freedom of a peptide can be constrained by intramolecular cross-links. conformational constraints enhance the receptor specificity, may lead to biased activity and confer proteolytic resistance to peptidic gpcr agonists. chemical synthesis allows to introduce a variety of cross-links into a peptide and is suitable for bulk production of relatively simple lead peptides. lanthionines are thioether bridged alanines of which the two alanines can be introduced at different distances in chosen positions in a peptide. thioether bridges are much more stable than disulfide bridges. biosynthesis of lanthionine-constrained peptides exploiting engineered gram-positive or gram-negative bacteria that contain lanthionine-introducing enzymes constitutes a convenient method for discovery of lanthionine-stabilized gpcr agonists. the presence of an n-terminal leader peptide enables dehydratases to dehydrate serines and threonines in the peptide of interest after which a cyclase can couple the formed dehydroamino acids to cysteines forming (methyl)lanthionines. the leader peptide also guides the export of the formed lanthionine-containing precursor peptide out of gram-positive bacteria via a lanthipeptide transporter. an engineered cleavage site in the c-terminus of the leader peptide allows to cleave off the leader peptide yielding the modified peptide of interest. lanthipeptide gpcr agonists are an emerging class of therapeutics of which a few examples have demonstrated high efficacy in animal models of a variety of diseases. one lanthipeptide gpcr agonist has successfully passed clinical phase ia. today nearly g protein-coupled receptor (gpcr) peptide drugs have been approved, and the pipeline is filled with an increasing number [ ] . many therapeutic peptides on the market are agonists of gpcrs [ , ]. gpcrs contain seven transmembrane segments and extracellular and intracellular loops. gpcr activity can be modulated by ligands that bind to their extracellular or transmembrane parts [ , ] . gpcrs can be activated by a variety of extracellular stimuli, among which interaction with peptides. gpcrs and their selectivity are very susceptible to allosteric modulation [ , ] . extracellular gpcr stimulation activates g proteins that initiate intracellular signal transduction cascades. in addition, other gpcr-linked pathways, independent of g proteins, can be activated as well [ ] . structural stability of peptides can be imposed by hydrophobic or electrostatic intramolecular interactions and/or cross-links and may lead to increased gpcr-binding affinity by decreasing the entropic cost to adopt the gpcr-binding conformation. in the case of gpcr agonists, a conformational constraint imposed on a peptide, may induce a structure which is suitable or required for functional binding to a gpcr. importantly, lanthionine-induced cyclization of a peptide may enhance the receptor specificity [ ] or even the receptor subtype specificity [ ] . a lanthionine somatostatin showed a similar high affinity for rat somatostatin receptor (rsstr ) compared with somatostatin [ - ] and sandostatin. however, it exhibited about times weaker binding affinity for mouse somatostatin receptor b (msstr b) than sandostatin [ ] . lanthionine enkephalin analogs with d-ala in position bind to both mand δ-opioid receptors and showed picomolar potencies [ ] . the receptor specificity of a cyclized ligand has an additional impact, when the ligand up-regulates the gpcr level as reported for the angiotensin ii type receptor [ ] . furthermore, in specific cases, cyclized peptides can exert biased receptor agonism. biased agonism by lanthi-apelins via the apj receptor has been observed as witnessed by relatively stronger stimulation of the g protein pathway compared with the arrestin pathway [ ] . this can translate to improved efficacy in the clinic by selectively stimulating therapeutic actions but avoiding activating detrimental β-arrestin-dependent pathways [ ] . selected lanthi-apelins, which more strongly stimulated the g protein pathway than the β-arrestin pathway did neither cause vasodilation nor increase heart rate in rats (lanthio pharma unpublished data, figure ). in addition to the primary importance of conformational constraints of receptor agonists for their interaction with the receptor, several secondary advantages may occur such as the enhanced capacity to pass membranes and resistance to breakdown by peptidases and increased physical stability. resistance to breakdown by peptidases in organ homogenates has been demonstrated amongst others for lanthionine-stabilized angiotensin-( - ) [ , ] and for lanthionine-stabilized analogs of gonadotropin-releasing hormone i [ ] . the capacity to pass membranes and the property of proteolytic resistance may lead to increased bioavailability after oral and cartoon of hypothetical stimulation of the apj receptor by a strictly biased agonist that stimulates g protein-dependent pathways but not β-arrestin dependent pathways. apj apelin receptor; at r angiotensin ii type receptor; hne sodium/hydrogen exchanger ; ncx sodium-calcium exchanger; ac adenylyl cyclase; pka protein kinase a; mek / dual specificity mitogen-activated protein kinase kinase / ; pkcε protein kinase c epsilon type; mlck myosin light-chain kinase; mlc myosin light-chain; plc phospholipase c; ip inositol trisphosphate; sr sarcoplasmic reticulum; aqp aquaporin ; bp blood pressure. pulmonary delivery as observed for lanthionine-stabilized angiotensin-( - ) [ ] , less frequent administration, lower doses and prolonged shelf life. these features apply also to conformationally constrained antagonists/ blockers [ ] , which are outside the scope of this minireview which focusses on agonistic peptides. some naturally occurring peptide gpcr agonists are constrained by disulfide bridges, e.g. vasopressin, oxytocin, cortistatin, urotensin ii, endothelin i, somatostatin, adrenomedullin and calcitonin. gpcr . known lanthionine rings in lanthipeptide gpcr agonists range from i, i+ (two amino acids between the lanthionine) to i, i+ (five amino acids between the lanthionine) [ , , , , , ] . a thioether bridge is shorter than a disulfide bridge and a lysinoalanine. the exact structures resulting from the introduction of a lanthionine depend on the amino acid sequence and the position of the lanthionine and the resulting ring-size. bacterial production of lanthionine-constrained agonistic peptides is a convenient method in the discovery of drug candidates. obviously, the introduction of a lanthionine, either by insertion into the existing amino acid sequence or by replacing existing amino acids with (half of ) a lanthionine, may strongly modulate the agonistic activity of the peptide, ranging from completely loss of activity to enhanced activity. screening of lanthionine-containing variants will be required to select pharmacologically valuable peptides. in many therapeutic peptides, the c-terminus is amidated which precludes carboxypeptidase action and may contribute to optimal receptor interaction. peptides can be enzymatically amidated by peptidyl-glycine alpha-amidating monooxygenase or carboxypeptidase y, which is likely compatible with bacterial production of lanthionine-containing peptides. following bacterial production chemical and enzymatic amidation has been demonstrated for gonadotropin-releasing hormone i [ ] . as broadly applicable, highly convenient methods have been developed for the bacterial synthesis of lanthionine-stabilized peptides, we here provide an overview on their emerging application in the discovery of lanthionine-constrained gpcr agonists. the word lanthionine stems from the latin word lana which means wool, in which lanthionines were first observed, and thiol (sulfhydryl). lanthionines are thioether bridged amino acids such as alanine-s-alanine, aminobutyric acid-s-alanine or alanine-s-aminobutyric acid. depending on the distances between the thioether bridged residues, smaller or larger ring structures are formed in the peptide. lanthionine-containing peptides, so-called lanthipeptides [ ] are naturally produced for instance by several gram-positive bacteria [ ] . lanthipeptides are synthesized as precursor peptides that contain an n-terminal leader peptide and a modifiable core peptide. this leader peptide enables the interaction with modification enzymes that can dehydrate serines and threonines in the core peptide and couple the formed dehydroamino acids to cysteines thus forming lanthionines and methyllanthionines. the cyclization is stereospecific: either d,l (methyl)lanthionines or l,d (methyl)lanthionines are formed. the enzymatic cyclization is also regiospecific. this means that a dehydroamino acid in a certain position is coupled to only one cysteine position yielding only one ring position structure and, in case of more than one lanthionine in one peptide, only one ring pattern. in lactococcus lactis, the leader peptide allows the export of the modified precursor peptide out of the cell. these leader peptides and the corresponding modification and transporter enzymes have been subject of a detailed review [ ] . for some of these enzyme systems, a membrane-bound enzyme complex has been described which is composed of the modification enzymes and a dedicated transporter. the leader peptide is cleaved off either intracellularly by a bifunctional transporter/leaderpeptidase or extracellularly by a leader peptidase or non-dedicated peptidases. the best-known lanthipeptide is the pentacyclic lantibiotic nisin [ ] which is produced by l. lactis. the nisin precursor peptide is dehydrated by the dehydratase nisb [ , ] , after which a cyclase nisc [ ] couples dehydroamino acids to cysteines followed by export by the transporter nist and removal of the leader peptide by the extracellular leader peptidase nisp [ ] . following the elucidation of the precursor sequence of the lanthipeptide gallidermin, as early as , it was recognized that the introduction of (methyl)lanthionines into therapeutic peptides, such as agonistic peptide hormones might constitute a valuable opportunity to discover specific and stable lanthionine-containing therapeutics [ ] . however, this concept was entirely dependent on the yet unknown substrate specificity of the complex of lanthipeptide modification and transport enzymes. in , the first gpcr agonist peptide, an angiotensin-( - ) variant fused to the nisin leader, which was totally unrelated to nisin, was modified by the nisin dehydratase and exported out of l. lactis by the nisin transporter nist [ ] . subsequent studies demonstrated that the dehydratase nisb and the nisin cyclase nisc could modify a broad range of peptides unrelated to nisin, and that also the transporter could accommodate the export of a variety of fusion peptides of the nisin leader peptide and a peptide of interest [ ] [ ] [ ] [ ] . in the c-terminal part of the nisin leader peptide, general cleavage sites could be engineered to allow convenient clean removal of the leader peptide from the produced peptide of interest [ ] . by engineering a signal sequence at the n-terminus of the leader peptide constructs peptides could also be exported out of l. lactis via the sec secretion system provided they had limited bulkiness [ , ] . excellent in vitro studies by wilfred van der donk and co-workers furthermore demonstrated that a bifunctional lanthionine-introducing lanm enzyme, which performs both the dehydration as well as the cyclization step, also has a broad substrate specificity [ ] . several types of lanthionine-introducing enzymes were demonstrated to be functional in escherichia coli [ , ] allowing intracellular modification and subsequent harvesting after disruption of the cells. the use of either gram-positive or gram-negative bacteria would thus constitute a cheap and effective method for producing a variety of rationally designed lanthipeptides to subsequently pursue the discovery of gpcr agonists with interesting pharmacokinetics and -dynamics. an excellent, comprehensive and thorough review focuses on lanthionine-introducing enzymes, their structure and mechanism of action [ ] . here, we summarize some of the progress made with respect to engineering novel lanthipeptides mainly by using bacteria containing the nisin modification enzymes. while the attainment of strict rules with respect to the substrate specificities of lanthionine-introducing enzymes has been challenging, some practical guidelines for the design of modifiable peptides have been reached for some lanthipeptide biosynthesis systems [ ] . the substrate specificity of lanthionine-introducing enzymes was first investigated via an in silico study on all available lanthipeptide structures and subsequently experimentally validated with the nisin modification enzymes [ , ] . directly flanking residues of serines and threonines clearly had an impact on the extent of dehydration by nisb (figure ). hydrophobic amino acids, especially when present on both a threonine in the core peptide of interest preceded by a cleavable leader peptide (lp) is dehydrated by a lanb dehydratase forming a dehydrobutyrine (dhb) after which a lanc cyclase couples the formed dehydrobutyrine to a cysteine forming a d,l methyllanthionine (dabu-s-ala). directly flanking amino acids (light green) of the thr and of the cys (dark green) may affect the extent of dehydration and cyclization [ , , ] . the enzymes lanb and lanc can also introduce a lanthionine via lanb-catalyzed dehydration of a serine yielding dehydroalanine and subsequent lanc-catalyzed coupling of the dehydroalanine to a cysteine. however, dehydroalanines are much more reactive than dehydrobutyrines and can spontaneously react with cysteines without stereospecificity. sides of the ser/thr favored dehydration whereas hydrophilic amino acids did not. hydrophilic amino acids, especially arg, asp, glu and gly, simultaneously present at both sides of the ser/thr, prevented dehydration. the requirement of hydrophobic amino acids directly flanking dehydrateable serines/threonines might be caused by the negative charge of glutamyl trna which acts as cofactor in the dehydration process [ ] . the influence on dehydration of the combination of one hydrophobic and one hydrophilic amino acid as directly flanking residues of ser/thr differed from case to case, and often led to partial dehydration. thr was generally dehydrated to a larger extent than ser. ser/thr at the very c-terminus escaped dehydration possibly as a result of the hydrophilicity of the carboxyl group. the presence of an already present lanthionine ring might in specific sequence contexts hamper the dehydration of ser/thr by a lanm enzyme [ ] . dehydration at a position as remote from the leader as positions could still be dehydrated [ ] , but it is not known yet where the upper limit is. interestingly, an elegant high-throughput screening system allowed to obtain mutant nisb enzymes with adapted substrate specificity with respect to the amino acids that directly flank ser/thr [ ] . the substrate specificity of nisc was first investigated with model peptides. dehydrolanine appeared to be very reactive leading to spontaneous lanthionine formation in bacterially produced peptides even in the absence of nisc as evidenced by comparing cells expressing nisbtc with cells with only nisbt [ ] . in the case of spontaneous cyclization in which highly reactive dehydroalanines readily couple to cysteines, more than one isomer can be formed. to be complete it should also be mentioned that in specific sequences spontaneous cyclization between dehydroalanines and cysteines can be stereospecific as well. in these cases, the sequence imposes the stereochemistry. for nisc-catalyzed cyclization, it appears complex to generate guidelines for the design of peptides that are well cyclized or escape cyclization. bulky residues in small ring structures appear to be unfavorable [ ] . glycines appear to be favorable and the impact of prolines is hard to predict. for cyclization, negatively charged amino acids as n-terminally directly flanking residues of a cysteine seem to be favored over positively charged amino acids. the stereospecificity of nisc-cyclized gpcr agonist, , lanthionine-angiotensin-( - ) was investigated using the ni b method, which opens the ring structure while retaining the d or l conformation [ ] . using reference peptide with combinations of d-ala/l-ala in positions and , the nisc-cyclized cang-( - ) isomer turned out to be d,l [r. rink unpublished data]. using the same method it was demonstrated that also , nisc-cyclized lhrh had the d,l conformation [ ] . this is in accordance with the d,l stereospecificity of all the lanthionine and methyllanthionine rings in nisin. designed, nisc-cyclized intertwined lanthionine structures of different sizes have been synthesized [ ] . the substrate specificity of the nisin transporter is not precisely known yet. generally, positively charged peptides [ ] seem to be better exported than negatively charged peptides in correspondence with the cationicity of the natural substrate, nisin. the mechanism of export and the involvement of nist in the observed export of a large cell-wall spanning protein out of l. lactis may require detailed mechanistic analyses [ ] . generally, the efficiency of nist-mediated export of peptides that are longer than nisin decreases significantly with increasing peptide length. the membrane-bound leader peptidase nisp appears to be highly specific for lanthionine-containing precursor nisin [ ] . in contrast, an engineered water-soluble truncated nisp was able to cleave off the leader peptide from a variety of substrate peptides [ ] . apart from the nisin biosynthetic machinery, many other lanthipeptide biosynthesis systems exist. interestingly, the single bifunctional enzyme procm naturally introduces lanthionine rings in different substrate peptides [ ] . clearly, this enzyme is a valuable tool in the design and synthesis of lanthionine-containing gpcr agonists. recently, a powerful high-throughput screening method of nisb mutants with adapted substrate specificity has been demonstrated, thus opening the way to analogously obtain many tailor-made modification enzymes [ ] . lanthipeptide gpcr agonists are an emerging class of peptide therapeutics. a number of them is listed in table . most data are available on , lanthionine-constrained angiotensin-( - ) which has shown high efficacy in many animal disease models such as acute respiratory distress syndrome [ , ] , acute lung injury [ ], pulmonary arterial hypertension [ ] , myocardial infarct [ ] , diabetic nephropathy [ ] , type and diabetes [ ] and cerebral stroke [ ] . it is completely resistant to breakdown by ace [ ] and pulmonary delivery is feasible [ ] . its ex vivo vasodilatory action was inhibited by two peptides, d-pro -ang-( - ) and d-ala -ang-( - ), that are considered antagonists of the mas receptor [ ] . lanthionine naturally occurs in lanthionine-ketimine present in the human brain [ ] ; thioethers are ubiquitously present in the human body in methionines. the lanthipeptide at r agonist lp [ , ] has been tested in clinical phase i where it showed at the tested doses safety and favorable pharmacokinetics. also complex lanthipeptides, other than receptor agonists, like the lantibiotics nbv ( [ ] , biocentury|august , ) and duramycin [ ] , have been successfully tested in clinical safety trials. duramycin interacts with phosphatidylethanolamine and it can uncouple mitochondrial respiration in isolated mitochondria [ ] . duramycin activates an alternative chloride channel in cystic fibrosis nasal and airway epithelia. by this, duramycin bypasses the transmembrane conductance regulator, a chloride channel, which is dysfunctional in cystic fibrosis. under therapeutic conditions after administration via inhalation, duramycin plasma concentration stays below . nm [ ] . until now no data directly point at any intrinsic toxicity of lanthionines. next to potentially high value as therapeutics lanthipeptide, gpcr agonists may have value as stable research tools with higher gpcr specificity than the corresponding natural linear peptides. natural peptides such as those derived from angiotensin i are in vivo rapidly degraded, making it difficult to assign their precise effects. in this respect, stable analogs may allow more reliable conclusions as to which peptide acts via which receptor leading to which effects. high gpcr specificity of lanthionine-constrained agonists may also prove useful in the elucidation of gpcr heterodimerization which occurs for instance for mas and at receptors [ ] . while biosynthesis methods are very convenient in the discovery process of therapeutic candidates, upscaled chemical production methods [ ] [ ] [ ] [ ] are in development and base-assisted sulfur extrusion has already successfully been applied for bulk production of gmp material of the clinically tested lp . • the target specificity, stability and safety of lanthipeptide gpcr agonists make them an important class of candidate therapeutics. • currently, it is clear that several biosynthetic systems have relaxed substrate specificity which allows their exploitation in lanthipeptide drug discovery. in addition, the feasibility of highthroughput screening for tailor-made lanthionine-introducing enzymes with adapted substrate specificity for rational design and biosynthesis of lanthipeptide gpcr agonists has recently been demonstrated. • future developments comprise the discovery and clinical development of multiple lanthipeptide gpcr agonists as therapeutics for treating human diseases. all authors are employees of lanthiopep b.v., which organization is the owner of patents on the biosynthesis of lanthipeptides and specific lanthipeptide gpcr agonists. g.n.m. is the director of lanthiopep b.v. g.n.m. contributed to the overall outline of the manuscript; r.r. with unpublished data, a.k., r.r. and t.b. to the paragraphs on biosynthesis, r.r. to the chemical synthesis, a.k., l.d.v. and p.n. to gpcrs; p.n. made figure . gpcrs, g protein-coupled receptors; msstr b, mouse somatostatin receptor b; rsstr , rat somatostatin receptor . mechanistic understanding of lanthipeptide biosynthetic enzymes mechanistic aspects of lanthipeptide leaders biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin in vitro activity of the nisin dehydratase nisb structure and mechanism of the trna-dependent lantibiotic dehydratase nisb structure and mechanism of the lantibiotic cyclase involved in nisin biosynthesis requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings nist, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes post-translational modification of therapeutic peptides by nisb, the dehydratase of the lantibiotic nisin production of dehydroamino acid-containing peptides by lactococcus lactis nisc, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed nonlantibiotic peptides sec-mediated transport of posttranslationally dehydrated peptides in lactococcus lactis translocation of a thioether-bridged azurin peptide fragment via the sec pathway in lactococcus lactis investigation of the substrate specificity of lacticin synthetase by using nonproteinogenic amino acids production of lantipeptides in escherichia coli lanthionine introduction into nukacin isk- prepeptide by co-expression with modification enzyme nukm in escherichia coli mechanistic dissection of the enzyme complexes involved in biosynthesis of lacticin and nisin high-throughput screening for substrate specificity-adapted mutants of the nisin dehydratase nisb structural characterization of lacticin , a two-peptide lantibiotic with synergistic activity activity and export of engineered nisin-( - ) analogs bacterial display and screening of posttranslationally thioether-stabilized peptides substrate specificity of the secreted nisin leader peptidase nisp structural characterization of four prochlorosins: a novel class of lantipeptides produced by planktonic marine cyanobacteria use of lantibiotic synthetases for the preparation of bioactive constrained peptides acute respiratory distress syndrome leads to reduced ratio of ace/ace activities and is prevented by angiotensin-( - ) or an angiotensin ii receptor antagonist does activation of the protective renin-angiotensin system have therapeutic potential in covid- ? dose-dependent, therapeutic potential of angiotensin-( - ) for the treatment of pulmonary arterial hypertension the effect of the thioether-bridged, stabilized angiotensin-( - ) analogue cyclic ang-( - ) on cardiac remodeling and endothelial function in rats with myocardial infarction effect of a stable angiotensin-( - ) analogue on progenitor cell recruitment and cardiovascular function post myocardial infarction addition of cyclic angiotensin-( - ) to angiotensin-converting enzyme inhibitor therapy has a positive add-on effect in experimental diabetic nephropathy efficacy of lanthionine-stabilized angiotensin-( - ) in type and type diabetes mouse models cyclic angiotensin-( - ) contributes to rehabilitation of animal performance in a rat model of cerebral stroke angiotensin ii type receptor ligand pd attenuates hyperoxia-induced lung and heart injury at a low dose in newborn rats detection of cystathione ketimine and lanthionine ketimine in human brain current developments in lantibiotic discovery for treating clostridium difficile infection a phase i trial of intranasal moli for cystic fibrosis duramycin effects on the structure and function of heart mitochondria. ii. energy conversion reactions effects of duramycin on cardiac voltage-gated ion channels evidence for heterodimerization and functional interaction of the angiotensin type receptor and the receptor mas understanding base-assisted desulfurization using a variety of disulfide-bridged peptides chemical synthesis and biological activity of analogues of the lantibiotic epilancin x selenocysteine derivatives for chemoselective ligations lanthipeptides: chemical synthesis versus in vivo biosynthesis as tools for pharmaceutical production key: cord- - d f k authors: mall, sanjay; malcolm east, j.; lee, anthony g. title: transmembrane α helices date: - - journal: curr top membr doi: . /s - ( ) - sha: doc_id: cord_uid: d f k this chapter discusses effects of intrinsic membrane proteins on lipid bilayers and model transmembrane α helices. incorporation of a protein into a lipid bilayer has significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer tends to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface reduces the extent of these motional fluctuations. however, the chains tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein leads to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein are different; for the bulk phospholipids, the disorder is dynamic, whereas, for the boundary lipids the disorder is static. bilayer of /~, about residues will be required to span the core of the bilayer. the residues in the transmembrane region will be predominantly hydrophobic. however, for membrane proteins such as transporters and ion channels the transmembrane ot helices will also have to contain polar groups; the transmembrane helices will then be amphipathic rather than just hydrophobic. an extreme case could be a transmembrane a helix totally surrounded by other t~ helices in the center of a helical bundle: such a helix would not need to be hydrophobic at all, because it is not in direct contact with the lipid bilayer. however, although small in number, the available high-resolution structures for membrane proteins show no evidence for the presence of such purely hydrophilic transmembrane ot helices. it may be that the process of insertion of membrane proteins into the membrane during biogenesis requires all the transmembrane c~ helices to be relatively hydrophobic. analysis of the compositions of a large number of membrane proteins predicted to contain single transmembrane ot helices has shown that the amino acid composition of a transmembrane ot helix is distinctly different from that of hydrophobic a helices in water-soluble proteins. as expected, hydrophobic residues make up the bulk of the residues, the most common being leu (landolt-marticorena et al., ; wallin et al., ) . amino acids essentially excluded are the basic (arg and lys) and acidic (asp and glu) amino acids and their amide counterparts (asn and gin). transmembrane c~ helices are, however, relatively rich in bulky residues, such as ile, val, and thr, which, in water-soluble proteins, are classed as membrane destabilizers (their bulky side chains interfere sterically with the carbonyl oxygen in the preceding turn of the ot helix and thus destabilize the helical conformation). thus, factors such as residue volume and packing, which are important in determining helix stability in water-soluble proteins, are not so important for transmembrane ct helices, at least for membrane proteins containing single transmembrane c~ helices; effects of large residue volume will, in the membrane, be balanced by the favorable hydrophobic interactions of a large side chain with the fatty acyl chains. in water-soluble proteins, the conformationally flexible gly residue is also classed as a helix breaker, because it is an intrinsically flexible residue with the potential to adopt most of the dihedral angles available in a ramachandran plot. the observation that gly is quite common in transmembrane ot helices suggests that its potential flexibility is constrained in the bilayer environment. because gly possesses the smallest of all the side chains, it may play a role in mediating helix-helix interactions and packing in the membrane. the polar amino acids most commonly found within transmembrane ot helices are cys, thr, and ser. these residues can be stabilized within a hydrophobic environment by hydrogen bonding between their polar side chains and the peptide backbone at positions i - and i - (eilers et al., ) . figure shows the positional preferences of the residues in the transmembrane ot helices of type i membrane proteins with a single transmembrane c~ helix oriented figure positional preferences for amino acids in the transmembrane domains of human type i membrane proteins with single transmembrane a helices. modified from landolt-marticorena et al. ( ) . with its c-terminus on the cytoplasmic side of the membrane (landolt-marticoreno et al., ) . the amino-terminal end of the transmembrane domain contains an lie-rich region followed by a val-enriched region. the carboxyl-terminal half of the transmembrane a helix is leu-rich. ala is found randomly distributed throughout the transmembrane domain. aromatic residues are found located preferentially in the boundary regions, with trp at either end of the transmembrane domain, but with tyr and phe only at the carboxyl-terminal boundary. unlike the other aromatic amino acids, phe, is also found in the hydrophobic segment as well as in the boundary region. the polar regions flanking the transmembrane domain are enriched in arg and lys on the c-terminal side; asn, ser, and pro are enriched in the n-terminal flanking region. the presence of a positively charged c-terminus (cytoplasmic) could play a role in the process of insertion into the membrane, according to the inside positive rule of von heijne ( ) . the presence of particular residues at the n-and c-terminal ends of the helices could also be important in meeting the requirement to "cap" the ends of the ot helices; the initial four --nh and final four --c=o groups of an ot helix have no hydrogen-bonding partners provided by the peptide backbone of the a helix itself, and so suitable hydrogen-bonding partners have to be provided in some other way. one way is to extend the helix by three or four residues at each end with polar residues containing suitable hydrogen-bonding partners such as pro and ash. alternatively, if the hydrophobic, nonpolar residues in the transmembrane c~ helix extend into the headgroup region, hydrogen bonds could form with suitable groups in the glycerol backbone and headgroup regions of the lipid bilayer. either way, if about residues are required to span the hydrophobic core of the bilayer, the total helix length could be up to residues. the result is that there is a degree of indeterminacy in where the ends of transmembrane helices should be drawn; the precise ends of transmembrane ~ helices are often not known. the observed preference for trp and tyr residues for the ends of transmembrane ct helices agrees with measurements of the binding of small peptides at the lipid-water interface, which show that aromatic residues have a preference for the interface (wimley and white, ) . further, a number of small tryptophan analogues have been shown to bind in the glycerol backbone and lipid headgroup region of the bilayer, stabilized partly by location of the aromatic ring in the electrostatically complex environment provided by this region of the bilayer, and partly by exclusion of the fiat, rigid ring system from the hydrocarbon core of the bilayer for entropic reasons (yau et al., ) . thus, although it is agreed that aromatic residues at the ends of transmembrane a helices probably act as "floats" at the interface serving to fix the helix within the lipid bilayer, it is unclear whether the aromatic rings are located in the hydrocarbon or the headgroup region of the bilayer. this uncertainty is also apparent in the crystal structures of a number of membrane proteins. for example, the trp residues in the bacterial potassium channel kcsa (doyle et al., ) are found clustered at the ends of the transmembrane ot helices, forming clear bands on the two sides of the membrane, as shown in fig. . however, the tyr residues clearly form a band on the periplasmic side of the membrane above the band formed by the trp residues. similarly, in the bacterial photosynthetic reaction center (rees etal., ) the majority of the trp residues are found near the periplasmic side of the protein near the ends of the transmembrane c¢ helices, as shown in fig. . however, the band of trp residues is more diffuse than in kcsa, and some trp residues are likely to be located in the hydrocarbon core and some in the headgroup region. the average number of figure the crystal structure of the potassium channel kcsa. a cross section with just two of the four identical subunits is shown. trl o residues are shown in space-fill representation and tyr residues are shown in ball-and-stick representation. two potassium ions in space-fill representation are shown moving through the channel. the separation between the two planes representing the outer edges of the trp residues is /~. (protein data bank [pdb] file ib .) p ¢ figure the structure of the l and m subunits of the photosynthetic reaction center of rhodobacter sphaeroides. trp residues are shown in ball-and-stick representation. an approximate location for the hydrophobic core of the bilayer of thickness a is shown, as defined by the surface covered by detergent. (pdb file aij.) residues in the transmembrane c~ helices of the bacterial photoreaction center is , corresponding to a length of about /~. the stretch of hydrophobic residues in these helices is, however, only about amino acids or about . /~ long (ermler et al., ; michel and deisenhofer, ) . this matches the thickness of the nonpolar region of the complex (about /~) as defined experimentally as the part covered by detergent in the crystal (roth et al., (roth et al., , . detergent is seen to cover some of the trp residues on the periplasmic side of the membrane, but not others (roth et al., ) . the distribution of trp residues on the cytoplasmic side of the complex is much less distinct than on the periplasmic side (fig. ). if the hydrocarbon core of the bilayer around the complex does have a thickness of ]~, then again the trp residues on the cytoplasmic side of the membrane will be located in both the hydrocarbon core and the headgroup regions of the bilayer (fig. ) . in the ca +-atpase of skeletal muscle sarcoplasmic reticulum the situation is more complex, as shown in fig. (toyoshima et al., ) . many of the transmembrane o~ helices extend above the membrane surface to form a central stalk linking the transmembrane region to the cytoplasmic head of the protein. as a consequence some of the helices are very long; helix m , for example, contains residues. a ring of trp residues can be seen on the cytoplasmic side of the membrane helping to define the location of the membrane surface (fig. ) . a lys residue (lys- ) in transmembrane c~ helix m can be seen pointing up from the hydrophobic core of the bilayer like a snorkel. because the cost of burying a charged residue in the hydrophobic core of a bilayer is very high (about kj mo - for a lys residue ; engelman et al., ) , it is likely that the amino group on lys- will be located at the interface; the trp residues in the ca +-atpase will then be located in the headgroup region of the bilayer. the structure of the ca +-atpase is also unusual in that the first transmembrane ot helix contains two polar residues, asp- and arg- , pointing out into the hydrocarbon core; presumably, stacking of asp- against arg- allows formation of an ion pair. the distribution of trp residues on the lumenal face of the ca +-atpase is much more diffuse than on the cytoplasmic side. the hydrophobic thickness of the ca +-atpase could be expected to be about /~, because that is the hydrophobic thickness of a bilayer of di(c : )pc, the phospholipid that supports highest activity for the atpase . however, as shown in fig. , this definition locates the lumenal loops between transmembrane ~ helices m and m i phospholipid designations are pc, ps, and pa for phosphatidylcholine, phosphatidylserine, and phosphatidic acid, respectively. fatty acyl chains are given in the format m:n, where m is the number of carbon atoms and n is the number of double bonds. thus, for example, dioleoylphosphatidylcholine is di(c : )pc. and between m and m within the hydrocarbon core. further, it locates a lys residue (lys- ) totally within the hydrocarbon core, which seems unlikely. the hydrophobic thickness of the bilayer would have to be about a to locate the two interhelical loops and lys- at the lumenal surface (fig. ) ; this is close to the thickness of a bilayer of di(c :i)pc ( a). the crystal structure shown in fig. corresponds to the ca +-bound, e conformation of the atpase. it has been shown that the e conformation of the atpase is favored by di(c : )pc, whereas di(c : )pc favors the other major conformation of the atpase, e (starling et al., ) . thus, it is possible that conformational changes within the transmembrane region of the ca +-atpase lead to changes in the interhelical loops and thus to changes in the effective hydrophobic thickness of the atpase (lee and east, ) . incorporation of a protein into a lipid bilayer can be expected to have significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer will tend to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface would be expected to reduce the extent of these motional fluctuations. however, the chains will have to tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein will lead to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear, therefore, that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein (the boundary or annular lipids) are different; for the bulk phospholipids the disorder is dynamic, whereas for the boundary lipids the disorder is static. an example is provided by the bacteriorhodopsin trimer, whose crystal structure is unusual in showing a few well-defined lipid molecules (belrhali et al., ; luecke et al., ) . figure shows some of the lipids located at the surface of the trimer. the electron densities for the chains are well defined, but the headgroups are disordered, so that the headgroups could not be identified; the lipids were therefore modeled simply as , -di-o-phytanylsn-propane (belrhali et al., ) . the considerable static disorder of the chains is clear in fig. , the rotational disorder of the chains being necessary to obtain good van der waals contacts with the molecularly rough surface of the bacteriorhodopsin trimer. lipids on the extracellular side of the membrane are better resolved than ~ figure structures of four phospholipid molecules identified in x-ray crystallographic studies of bacteriorhodopsin (belrhali et al., ) . the lipids have been modeled as , -.di-o-phytanyl-snpropane. (pdb file lqhj.) those on the cytoplasmic side; the degree of order of the lipids parallels that of the protein, which is also greater on the extracellular side (grigorieff et al., ) . the average distance between the glycerol backbone oxygens for phospholipids on the two sides of the membrane was . /~ (mitsuoka et al., ) . as expected, this closely matches the hydrophobic length of the transmembrane helices of bacteriorhodopsin; the mean helix length is residues, corresponding to a length of about a. the crystal structure also makes clear the very different conformations adopted by the various lipid molecules located on the surface of the tfimer. for example, one lipid molecule forms a hydrogen bond from its ether oxygens to a tyrosine --oh group at the end of a transmembrane a helix (fig. ; belrhali et al., ; essen et al., ) . the result is that the strength of the interactions of individual boundary lipid molecules with the protein will be different. the disorder of the chains seen in fig. is consistent with the results of molecular dynamics simulations of the bacteriorhodopsin trimer in a bilayer of diphytanyl phosphatidylglycerophosphate (edholm et al., ) . the molecular dynamics simulation agrees with experiment in predicting higher order for both the lipids and the protein on the extracellular side of the membrane; fluctuations in the loops and the ends of helices on the cytoplasmic side of the membrane are greater than on the extracellular side. this is also seen in fluctuations of the lipids, with lipids on the cytoplasmic side of the membrane fluctuating more strongly than those on the extracellular side (edholm et al., ) . the calculated order parameters for the chains are low, mainly due to a static tilt of the chains necessary to allow them to nestle against the rough surface of the protein. the chains in the purple membrane behave more like parts of the protein than parts of a fluid lipid phase, consistent with the idea of boundary lipids (edholm et al., ) . the boundary lipids and the bulk lipids in a membrane can be distinguished experimentally in many systems, because of the static disorder of the boundary lipids and the dynamic disorder of the bulk lipids. static and dynamic disorder give rise to very different electron spin resonance (esr) spectra for spin-labeled lipids, and esr spectra for membrane protein systems usually show two components, one "immobilized," corresponding to boundary lipid, and one relatively mobile, corresponding to bulk lipid (devaux and seigneuret, ; marsh, ) . studies with oriented samples have confirmed a wide range of orientational distributions for the boundary lipid, in contrast to the bulk lipid phase, where motion of the lipid long axis is about the bilayer normal (jost et al., ; pates and marsh, ) . a particularly important feature of a membrane protein as far as the lipid bilayer is concerned is the thickness of the transmembrane region of the protein. the cost of exposing hydrophobic fatty acyl chains or protein residues to water is such that the hydrophobic thickness of the protein should match that of the bilayer. the question then is how the system responds when these do not match. most models of hydrophobic mismatch assume that the lipid chains in the vicinity of the protein adjust their length to that of the protein, with the protein acting as a rigid body. when the thickness of the bilayer is less than the hydrophobic length of the peptide the lipid chains must be stretched. conversely, when the thickness of the bilayer is greater than the hydrophobic length of the peptide the lipid chains must be compressed (fig. ) . stretching the fatty acyl chains will effectively decrease the surface area they occupy in the membrane surface, and, conversely, compressing the chains will increase the effective area occupied in the surface (fig. ) . thus, figure the result of a mismatch between the hydrophobic length of a peptide and the hydrophobic thickness of a lipid bilayer. left: positive hydrophobic mismatch (dp > dl). right: negative mismatch (dp< de). the top shows a "side" view of the chain packing around the peptide and the bottom shows a top view, illustrating the variation in chain cross-sectional area with distance from the peptide. figure based on fattal and ben-shaul ( ) . changes in fatty acyl chain order are linked to changes in average interfacial areas per lipid molecule. a number of terms have been suggested to contribute to the total free energy cost of deforming a lipid bilayer around a protein molecule (fattal and ben-shaul, ; nielsen et al., ): . loss of conformational entropy of the chains imposed by the presence of the rigid protein wall . bilayer compression/expansion energy due to changes in the membrane thickness . surface energy changes due to changes in the area of the bilayer-water interface . splay energy due to changes in the cross-sectional energy available to the chains along their length, resulting from curvature of the monolayer surface near the protein a number of models have been proposed to estimate these terms. fattal and ben-shaul ( ) calculated the total lipid-protein interaction free energy as the sum of chain and headgroup terms. for the chains the loss of conformational entropy imposed by the rigid protein wall was positive (unfavorable) even for perfect hydrophobic matching. the other contribution to the chain term arose from the requirement for hydrophobic matching and the consequent stretching or compression of the chain. the term due to the headgroup region was treated as an interracial free energy, including an attractive term associated with exposure of the hydrocarbon core to the aqueous medium and a repulsive term due to electrostatic and excluded-volume interactions between the headgroups (excluded-volume interactions signify that no two atoms can occupy the same position in space). the resulting profile of energy of interaction as a function of hydrophobic mismatch was fairly symmetrical about the point of zero mismatch. the lipid perturbation energy f (in units of kt per angstrom of protein circumference) calculated by ben-shaul ( ) fits to the equation where dp and dl are the hydrophobic thicknesses of the protein and lipid bilayer, respectively, and the unperturbed bilayer thickness is . /~. the hydrophobic thickness of a bilayer of phosphatidylcholine in the liquid crystalline phase is given by where n is the number of carbon atoms in the fatty acyl chain (lewis and engelman, ; sperotto and mouritsen, ) . a simple, but crude calculation gives an idea of the size of the effect that can be expected from hydrophobic mismatch. it is assumed that the protein is very large and so appears fiat to a lipid molecule. it is also assumed that all the lipid perturbation energy is concentrated in the first shell of lipids around the protein. equation ( ) then shows that if a lipid occupies a of the protein circumference, the lipid-protein interaction energy would change by . kj tool - for a hydrophobic mismatch of a, corresponding to an increase in fatty acyl chain length of carbons, and by . kj mol -z for a hydrophobic mismatch of . a, corresponding to an increase in acyl chain length of carbons. changes in interaction energies of . and . kj mo - correspond to decreases in the lipid-protein binding constant by factors of . and n, respectively. if the change in lipid-protein interaction energy were to propagate out from the protein surface to affect more than the first shell of lipids, effects of hydrophobic mismatch would be reduced. for example, if effects were averaged over three shells of lipids, changes in fatty acyl chain lengths by and carbons from that giving optimal interaction would decrease lipid-protein binding constants by factors of . and , respectively. energies of the magnitude calculated by ben-shaul ( ) are easily sufficient to result in conformational changes on a protein. if a protein conformational change results in a change in the hydrophobic thickness of the protein, the change will result in a deformation of the adjacent bilayer. because the equilibrium constant describing the equilibrium between two conformational states of a protein is determined by the total free energy difference between the two states, the energetic cost of the membrane deformation will contribute toward the equilibrium constant. the approach adopted by nielsen et al. ( ) came to rather similar conclusions. the most important energy terms were found to be the splay energy and the compression-expansion term, the splay energy term being most important close to the protein, with the compression-expansion term being more import'ant further from the protein. even though the bilayer deformation was calculated to extend some a from the protein, most of the deformation was found concentrated in the component immediately adjacent to the protein. an alternative model for mismatch is the mattress model of bloom ( , ) . this again expresses mismatch as two terms. the first is an excess hydrophobic free energy associated with exposing either lipid chains or the protein surface to the aqueous medium. the second is proportional to the contact area between the lipid chains and the hydrophobic surface of the protein. the calculations showed that, for a protein of hydrophobic thickness a, which matches a bilayer ofdi(c : )pc in the liquid crystalline state, the binding constant for di(c : )pc is about a factor of . greater than for a bilayer of di(c : )pc, which will give a bilayer too thick by a (sperotto and mouritsen, ) . thus, effects of mismatch calculated in this way are similar to those calculated using the approach of fattal and ben-shaul ( ) . the importance of hydrophobic matching has been confirmed in a number of experimental studies (dumas et al., ; killian, ) . for example, although bacteriorhodopsin has relatively little effect on the phase transition temperatures of di(c : )pc or di(c : )pc (alonso et al., ) , it increases the transition temperature of di(c : )pc and decreases that of di(c : )pc (piknova etal., ) . this is consistent with hydrophobic matching models; because di(c : )pc gives a too thin bilayer in the liquid crystalline phase, bacteriorhodopsin favors the gel phase, whereas di(c : )pc gives a too thick bilayer in the gel phase, so that bacteriorhodopsin favors the liquid crystalline phase. hydrophobic matching could also be the explanation for the unexpected observation that, in mixtures of di(c : )pc and di(c : )pc at temperatures where the mixture contains both gel and liquid crystalline phases, bacteriorhodopsin partitions equally between the two phases (piknova et al., ; schram and thompson, ) . this contrasts with the observed exclusion of bacteriorhodopsin from gel-phase lipid in mixtures containing single species of phospholipid (alonso et al., ; cherry et al., ) . it has been suggested that this shows that the requirements of hydrophobic matching are of prime importance; the hydrophobic thickness of bacteriorhodopsin is intermediate between the hydrophobic thickness of di(c : )pc in the gel phase and di(c : )pc in the liquid crystalline phase, so that bacteriorhodopsin shows little preference between the two. in mixtures of di(c : )pc and di(c : )pc, where, at low temperatures, two separate gel phases are formed, one enriched in di(c : )pc and one enriched in di(c : )pc, bacteriorhodopsin partitions very strongly into the di(c : )pc-enriched domains; this could be because the hydrophobic thickness of bacteriorhodopsin is better matched to gelstate di(c : )pc than to gel-state di(c : )pc (dumas et al., ) . however, in studies of the ca +-atpase of sarcoplasmic reticulum using either spin-labeled (london and feigenson, ) or brominated phospholipids , strengths of binding of liquid crystalline-phase phospholipids to the atpase were found to be independent of fatty acyl chain length. these results are not consistent with the expectations of hydrophobic matching theory and suggest that, in liquid crystalline bilayers, u-helical membrane proteins are not rigid, but, in fact, can distort to match the thickness of the bilayer. such a distortion could explain why bilayer thickness affects the activity of membrane proteins such as ca +-atpase (lee, ) . the structural distortion could take the form of a change in the tilt of the transmembrane ~ helices with respect to the bilayer normal or could be a change in the packing of the transmembrane c~ helices. an alternative approach to these questions, avoiding the complexity of real membrane proteins, is to use simple model transmembrane ~ helices, which can be synthesized chemically in large quantity. a number of studies have used peptides of the type ac-k -g-ln-k -a-amide (pn) consisting of a long sequence of hydrophobic leu residues capped at both the n-and c-terminal ends with a pair of charged lysine residues. the poly(leu) region forms a maximally stable u helix, particularly in the hydrophobic environment of the lipid bilayer. the charged lys caps were chosen both to anchor the ends of the peptides in the lipid headgroup region and to inhibit the aggregation of the peptides in the membrane. the peptide has been shown to adopt the expected u-helical structure in both liquid crystalline-and gel-phase bilayers (davis et al., ; huschilt et al., ; zhang et al., a) . rates of hydrogen/deuterium exchange for the peptide p in lipid bilayers suggest that at least % of the peptide is in an u-helical conformation in the bilayer, meaning that the whole of the poly(leu) core must be u-helical (zhang et al., b) . rates of hydrogen/deuterium exchange were greater at the n-and c-termini of the peptides than in the middle, suggesting some unraveling of the peptide at its ends (zhang et al, b) . experiments with the peptides of this type suggest that about lipid molecules are required for complete incorporation of the peptide into a bilayer of the appropriate thickness (webb et al., ) . this agrees with estimates from molecular modeling that about - lipid molecules will be required to form a complete bilayer shell around the peptide. at molar ratios of lipid less than this, non-bilayer phases can be induced, particularly when the hydrophobic length of the peptide is less than the hydrophobic thickness of the bilayer and when the peptide contains interfacial aromatic groups (de planque et al., ; killian et al., ; morein et al., ) . effects of single transmembrane u helices on lipid bilayers are likely to be less than those of a protein containing a bundle of transmembrane u helices. the cross-sectional area of a single transmembrane u helix is not much greater than that of a phospholipid molecule in the liquid crystalline phase, so that the hydrophobic surface presented to the lipid molecules is rather small. the structure of the lipid bound to bacteriorhodopsin shown in fig. shows the two chains interacting predominantly with two different transmembrane u helices. this kind of interaction will obviously not be possible with a single transmembrane u helix. less extensive interactions between lipids and single transmembrane u helices than between lipids and membrane proteins is suggested by esr experiments. whereas esr spectra of spin-labeled lipids in the presence of membrane proteins typically show two-component spectra, as described above, esr spectra for lipid bilayers containing the peptide l and for a tryptophan-containing peptide of the type aw (la)nw a are single-component (de planque et al, ; subczynski et al., ) . this means either that the lipid fatty acyl chains are not "immobilized" on the peptide surface or that the rate of exchange between bulk and boundary lipid is fast on the esr time scale (i.e., exchange is faster than s-l). effects of peptides on chain order in di(c : )pc or di(c : )pc measured using deuterium nuclear magnetic resonance (nmr) methods are small in both the liquid crystalline and the gel phases (davis et al., ; de planque et al., de planque et al., , roux et al., ) . thus, addition of a peptide aw (la)tw a to bilayers of di(c : )pc in the liquid crystalline phase resulted in only a . -/~ increase in thickness (de planque et al., ) , whereas about an -/~ increase would be necessary for the bilayer thickness to match the hydrophobic length of the peptide. similarly, nezil and bloom ( ) estimated that the peptide p increased the thickness of a bilayer of (c : ,c : )pc by just . /~, despite the hydrophobic mismatch between the peptide and the lipid bilayer being ca. a. increases in chain order caused by p in (c : ,c : )pc in the liquid crystalline phase were detected using esr, but again the effects were small (subczynski et al., ) . effects of peptides on chain order will depend on the relative hydrophobic length of the peptide compared to the hydrophobic thickness of the bilayer, with long peptides decreasing order and short peptides increasing order, and such effects have been detected using infrared (ir) spectroscopy, but again effects were small (zhang et al., a) . thus it appears that lipids will distort slightly to improve the match between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, but the extent of these modifications is very limited and much less than required to produce full matching. a number of studies have been published on the effects of these peptides on the phase transition properties of lipid bilayers. addition of the peptide pi to bilayers of di(ci : )pc both broadens the main gel-to-liquid crystalline phase transition and decreases the enthalpy of the transition (morrow et al., ) . similar effects have been seen on incorporation of membrane proteins such as bacteriorhodopsin and ca +-atpase (alonso et al., ; gomez-fernandez et al., ) . the decrease in enthalpy of the transition has often been taken to mean that the lipids adjacent to the protein (the boundary lipids) are very strongly perturbed by the peptide and so are unable to take part in the normal phase transition: they are effectively withdrawn from the transition. however, deuterium nmr spectra of mixtures of p and di(c : )pc above and below the phase transition are typical of liquid crystalline-and gel-phase lipids, respectively, with no evidence for any "special" lipid unable to take part in the phase transition . similarly, as already described, esr spectra of spin-labeled lipids show the presence of a single type of lipid in the system, not separate bulk and boundary lipids in slow exchange (subczynski et al., ) . thus the peptides (or proteins) do not remove lipid from the main transition, but, rather, perturb the whole lipid bilayer. the peptide decreases the enthalpy difference between the liquid crystalline and gel phases, whereas the lipids in the bilayer remain recognizably liquid crystalline or gel (morrow et al., ) . morrow et al. ( ) showed that mixtures of lipids and peptides can be modeled in terms of regular solution theory (lee, ) . unfortunately, the number of free parameters in fitting to regular solution theory is high, so that little useful information is obtained from the analysis, apart from showing that the data are consistent with regular solution theory. effects of peptides or proteins on phase transition properties have therefore been interpreted qualitatively in terms of a twocomponent model in which one component is more or less unperturbed bulk lipid and the other is highly perturbed boundary lipid, which undergoes a broad phase transition of low enthalpy; this approach works for peptides of the poly(leu) type, but, for some reason, does not work with peptides of the k (la)nk type (zhang et al., a (zhang et al., , . differential scanning calorimetry (dsc) thermograms for mixtures with the poly(leu) peptides have been fitted to two components, attributed to the phase transitions of peptide-free and boundary lipid, respectively. the phase transition temperature for the peptide-free component is slightly less than that for pure lipid; this is probably due to the normal colligative effects that will follow from mixing the "pure" lipid phase with the boundary lipid, the latter acting as an "impurity." the phase transition temperature for the boundary lipid is higher than the bulk transition temperature for short-chain lipids, but is lower for long-chain lipids (zhang et al, a) . the same observation has been made for membrane proteins (dumas et al., ; piknova et al., ) . this is consistent with the idea that short fatty acyl chains have to stretch to match the hydrophobic thickness of the membrane protein, whereas long fatty acyl chains have to compress. however, the experimental changes are much smaller than expected from models ofhydrophobic matching. further insights into how transmembrane ot helices might interact with lipid molecules in a bilayer have come from molecular dynamics simulations. one study was of a transmembrane ot helix of alanine residues in a bilayer of di(c : )pc in the liquid crystalline state (shen et al., ) . the peptide is not an ideal model for a transmembrane ot helix, because it lacks charged groups at each end to interact with the polar headgroups of the phospholipids. nevertheless many features of the simulation are informative. the simulation was started with the peptide as a pure ot helix. the central residues (ala- - ), which interacted just with the lipid fatty acyl chains, remained as a stable t helix. the n-and c-terminal regions of the ot helix, located in the lipid headgroup region, were less stable and fluctuated more, because of transient hydrogen bonding between the peptide bond amide hydrogen and the phosphate or fatty acyl ester oxygen atoms and the water; as a result, the ends of the helices become frayed. the length of the central helical region oscillated slightly about a -a average expected for an ot helix, varying between and a. the helix was tilted up to ° with respect to the bilayer normal. because the helix contains no resides that would make strong contacts in the headgroup region or with water, there is no reason for it not to tilt (shen et al., ) . tilting in fact allows more hydrophobic contact by allowing more of the ala residues to be located in the core of the bilayer. the presence of the peptide had little effect on the calculated properties of the bilayer. the average bilayer thickness was not significantly changed, although the average order parameter for the ch groups in the chains decreased in the presence of the peptide. many different lipid molecules contributed to the immediate surroundings of the peptide (shen et al., ) . even if the fatty acyl chain of a particular lipid was immediately adjacent to the peptide, the headgroup of the lipid could be a substantial distance away. given the diameter of the helix and the size of the phosphate group, a phosphate immediately adjacent to the helix would be between and a away from the center of the helix (shen et al., ) . on average, five lipid molecules having their chains adjacent to the helix also had their headgroups adjacent, using this definition. however, the headgroup, as given by the position of the phosphorus atom, could be up to - a away. since the average distance between lipid headgroups is a, this puts these lipids in the "second" shell around the peptide. other lipids existed between these extremes, suggesting a very diffuse environment around the protein rather than a discrete set of well-ordered shells of lipid. only rarely did an entire lipid molecule pack tightly around the helix. the shell around the helix contains contributions from a large number of lipid molecules, each contributing a small number of atoms (shen et al., ) . thus there is no evidence for a distinct shell of lipids around the peptide and any perturbation of the lipids extends out just a few angstroms. the lack of a clear shell of lipids around the poly(ala) peptide contrasts with the boundary lipids observed for membrane proteins and illustrated for bacteriorhodopsin in figs. and . in part this could be an intrinsic feature of single transmembrane a helices, which will not be able to present a large surface area on which fatty acyl chains could be immobilized. the regular structure of a poly(ala) peptide compared to the rough surface of a typical or-helical peptide might also contribute to the lack of an immobilization shell of lipids. however, a significant factor is also likely to be the lack of polar groups at the ends of the peptide able to interact with the phospholipid headgroups. the importance of polar groups at the ends of the peptides has been shown in a simulation of isolated helices from bacteriorhodopsin (woolf, ) . the simulations show that a small number of the lipids surrounding a helix interact with it much more strongly than other lipids, due to a combination of van der waals (mediated by chains) and charge interactions (mediated by beadgroups) (woolf, ) . a molecular dynamics simulation of the peptide el in a bilayer of di(c : )pc in the liquid crystalline phase showed that the peptide tilted with an average angle of . ° with respect to the bilayer normal, even though the thickness of the hydrophobic region of the bilayer ( a) was a good match to the length of the ~ helix, a (belohorcova et al., ) . a molecular dynamics simulation has been reported for pf coat protein in di(c : )pc (roux and woolf, ) . the coat protein contains an amphipathic a helix on the bilayer surface and a hydrophobic transmembrane a helix. fatty acyl chains next to the transmembrane helix were slightly more ordered than bulk lipids (roux and woolf, ) . similarly, in a simulation of the seventh transmembrane c~ helix of the serotonin ht receptor in di(c : )pc, lipids in contact with the peptide had slightly higher order parameters than bulk lipids (duong et al., ) . the theories described above show that there will be an energetic cost associated with any change in the thickness of a bilayer. this would be reflected in values for the equilibrium constant describing the binding of lipids to the protein. a lipid that can bind to a protein without a change in bilayer thickness would bind more strongly to the protein than one for which binding required a change in bilayer thickness. the strength of interaction between a peptide and a phospholipid in a bilayer can be measured using a fluorescence quenching method (webb et al, ) . peptides used are of the type kkgl wl kka (l ) and kkgl wl kka (l ) containing a central trp residue as a fluorescence reporter group. the peptide is incorporated into bilayers containing the brominated phospholipid dibromostearoylphosphatidylcholine (di(br c : )pc); di(br c : )pc behaves much like a conventional phospholipid with unsaturated fatty acyl chains, because the bulky bromine atoms have effects on lipid packing similar to those of a cis double bond . contact between the bromine atoms in the lipid and the trp residue in the peptide leads to fluorescence quenching. in mixtures of brominated and nonbrominated phospholipids, the degree of quenching of the fluorescence of the tryptophan residue is related to the fraction of the surrounding (boundary) phospholipid molecules that are brominated, and thus to the strength of binding of the nonbrominated lipid to the peptide. an example of the method is shown in fig. . the fluorescence intensity for the peptide l incorporated into bilayers of di(br c : )pc at a molar ratio of lipid to peptide of : is % of that in di(c : )pc, demonstrating highly efficient quenching of the tryptophan by the bromine-containing fatty acyl chains (fig. ) . the fluorescence intensity in mixtures of di(br c : )pc and di(c : )pc decreases with increasing content of di(br c : )pc, reflecting the increasing number of boundary lipids that will be di(br c : )pc. as shown in fig. , fluorescence quenching curves for l in mixtures of di(br c : )pc and (c : )pc show more fluorescence quenching at intermediate mole fractions of di(br c : )pc than in mixtures with di(c : )pc. this shows that di(c : i)pc binds more strongly to the peptide than does di(c : )pc. the results can be analyzed to give relative lipid-binding constants, as described in webb et al. ( ) . these lipid-binding constants for l and l are given in table i. for l strongest binding is seen with di(c : )pc, for which the relative binding constant is about double that for di(c : )pc (table i ). the hydrophobic length of the peptide l is about a, calculated for a stretch of hydrophobic residues in total, with a helix translation of . a per residue. thus, strongest binding is seen when the hydrophobic length of the peptide matches the hydrophobic thickness of the bilayer, as expected from theories of hydrophobic mismatch. however, relative binding constants do not continue to decrease with decreasing chain length from di(c : )pc to di(c : )pc as would have been predicted (table i ). an even larger deviation from theoretical predictions is observed with the short peptide l (table i ). in this case strongest binding is observed with di(c : )pc, with binding decreasing with decreasing chain length to di(c :i)pc, as expected. however, the peptide was found not to incorporate at all into bilayers of di(c :l)pc, instead forming aggregates of peptide separate from the bilayer. ren et al. ( ) obtained very similar results, except that under their conditions, unincorporated peptide bound to the surface of the lipid bilayer, with the long axis of the peptide parallel to the surface. similarly, l was found to be only partly incorporated into ~bilayer hydrophobic thickness d calculated from d = . (n - ), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, ) . bestimated hydrophobic length is a for li and a for l . bilayers of di(c : )pc (webb et al., ) . thus, a short peptide cannot incorporate into a too-thick bilayer. it is suggested that a too-thin bilayer can match to a too-long peptide both by a slight stretching of the lipid and by tilting of the long axis of the helix with respect to the bilayer normal so that its effective length across the bilayer is reduced. however, a too-thick bilayer can only match a too-thin peptide by compression of the lipid, which becomes energetically unfavorable when the difference between the bilayer thickness and the peptide length exceeds about /~ (webb et al., ) . possible effects of aromatic residues at the ends of transmembrane ~ helices have been studied using peptides k gfl wl fk a (f l ) and k gyl wl yk a (y li ), in which one leu residue at each end of the poly(leu) stretch is replaced by either a phe or a tyr (mall et al., ) . in contrast to the results with l , peptide f l incorporated fully into bilayers of di(c : )pc, and y li partitioned partially into di(c : )pc. the fluorescence quenching method was again used to obtain binding constants for phosphatidylcholines to the peptides, measured relative to the binding constant for di(c : )pc (table ii) . the effective hydrophobic length of the peptide y li might be expected to be somewhat greater than that of l ; if the peptide is modeled as an c~ helix with the two tyr residues oriented to be roughly parallel to the long axis of the c~ helix, the distance between the two tyr--oh groups is ca. a, about /~ greater than the hydrophobic length of l . the hydrophobic length of y l calculated in this way matches the hydrophobic thickness of a bilayer of di(c : )pc, whereas the relative lipid-binding constants increase from di(c : )pc to di(c : )pc (table ii) . similarly, relative binding constants for f li increase with increasing chain length from di(c :i)pc to di(c : i)pc. the results with y l and f li show that introduction of aromatic "hydrophobic thickness of the bilayer calculated from d = . (n - ), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, ). residues at the two ends of the hydrophobic sequence increases the ability of the short peptides to partition into thick lipid bilayers. the observation that the highest relative binding constant is obtained with bilayers considerably thicker than the calculated hydrophobic length of the peptides suggests that the presence of aromatic residues at the ends of the helices could lead to marked thinning of the bilayer around the peptides (mall et al., ) . the chain length dependence of lipid binding to y l is much less marked than for the shorter peptides; the relative binding constant increases from di(c : )pc to di(c :i)pc, but then hardly changes with increasing chain length between di(c :i)pc and di(c :)pc (table ii) . this contrasts with l , which shows markedly stronger interaction with di(c :l)pc than with phospholipids with shorter or longer chains. this again suggests that the introduction of the two tyr residues leads to an increase in the thickness of the bilayer with which optimal interaction of the peptide is observed. interactions between transmembrane c~ helices and the phospholipid headgroups also have to be considered. using the fluorescence quenching method, it was shown that a small number of anionic phospholipid molecules (possibly just one) bound strongly to the peptide l , the remaining molecules binding with an affinity equal to that of phosphatidylcholine. the binding constant for the strongly bound phosphatidic acid molecule relative to phosphatidylcholine in a medium of low ionic strength was . (in mole fraction units), corresponding to a difference in unitary binding energies of - . kj mo - . at ph . , phosphatidic acid bears a single negative charge (cevc, ) . the binding constant for phosphatidic acid changed little with ionic strength, suggesting that the interaction with the positively charged peptide did not follow simply from a high positive potential in the vicinity of the positively charged lys residues on the peptide, increasing the local concentration of anionic phospholipid. the energy of interaction between two ions u is given by where zl and z are the charges on the two ions eo is the permittivity of a vacuum, er is the relative permittivity (dielectric constant) of the medium, and r is the distance between the two ions. assuming a dielectric constant of . (water), we find that an energy of interaction of . kj tool - corresponds to a distance of separation between two monovalent ions of . /~. this therefore suggests that strong interaction requires the anionic headgroup of phosphatidic acid to be in close contact with one of the lys residues on the peptide. once this strong interaction with a single phosphatidic acid molecule has been made, other phosphatidic acid molecules will then interact with el relatively nonspecifically, with a binding constant relative to phosphatidylcholine close to . the relative binding constants for phosphatidylserine were less than for phosphatidic acid and are more sensitive to ionic strength . for phosphatidylserine, the presence of the positively charged ammonium group as well as the negatively charged carboxyl group in the headgroup region may reduce interaction with the positively charged peptide. in contrast to l , the binding constants for anionic phospholipids to l are very similar to those for zwitterionic phospholipids, with a relative binding constant close to . it could be that tilting of l in the bilayer, necessary to match the hydrophobic length of l to the hydrophobic thickness of a bilayer of di(c : )pc, locates the lys residues on the peptide too far from the lipid headgroup region to allow a strong interaction between the anionic phospholipid and the peptide. both phosphatidylserine and phosphatidic acid bind more strongly to the peptides yell and yel than does phosphatidylcholine, the effect of anionic phospholipid decreasing slightly with increasing ionic strength. however, in this case the experiments are consistent with a model in which the binding constants for all the anionic phospholipid molecules binding to the peptide are increased slightly (mall et al., ) . this suggests that the presence of the tyr residues prevents close association of the anionic phospholipid group with the cationic lys residues. these results suggest that the effects of charge on the interactions between phospholipids and transmembrane ot helices will often be rather small and will be strongly dependent on the detailed structure of the peptide and its orientation in the membrane. this picture is consistent with the results of the molecular dynamics simulations of individual ~ helices of bacteriorhodopsin in bilayers of di(ci : )pc, which showed that a small proportion of the lipid molecules interacted with the o~ helices much more strongly than the others, and that these strong interactions were dominated by electrostatic terms rather than van der waals terms (woolf, ) . in general, binding constants for phospholipids to membrane proteins also show relatively little selectivity for anionic phospholipids. for example, binding constants for phosphatidic acid and phosphatidylserine relative to phosphatidylcholine are close to for the caz+-atpase (dalton et al., ) , and binding constants for phosphatidic acid and phosphatidylserine for the (na+-k+)-atpase are about twice those for phosphatidylcholine (esmann and marsh, ) . however, there is evidence for the presence of a small number of "special" anionic phospholipids binding to some membrane proteins, acting as "cofactors." an example is provided by cytochrome c oxidase, whose crystal structure shows the presence of a lipid molecule bound between the transmembrane a helices (iwata et al., ) . interaction between an anionic phospholipid and a binding site on a membrane protein would be specific if strong binding requires close interaction between the anionic headgroup and a positively charged residue on the protein, as suggested by the results presented here. the phase of the phospholipid is important in determining interactions with transmembrane ot helices. as shown in fig. , fluorescence quenching is much more marked in mixtures of di(br c : )pc and di(c : )pc at temperatures where both liquid crystalline and gel phases are present than in mixtures of di(brac : )pc and di(c : )pc (mall et al., ) . thus, l is excluded from regions of lipid in the gel phase and accumulates in regions in the liquid crystalline phase. the binding constants of l| and l z for di(c : )pc in the gel phase relative to di(c :i)pc in the liquid crystalline phase are ca. . (mall et al., ) . this is consistent with the expectation that van der waals contacts between an all-trans fatty acyl chain and the molecularly rough surface of a peptide will be poor; one way that this poor packing can be overcome is by exclusion of the peptide from the gel phase. quenching plots for y l are very similar to those of l (fig. ) , showing that the presence of bulky aromatic residues does not have any significant effect on the selectivity for liquid crystalline-over gel-phase lipid (mall et al., ) . further, since y li shows a preference for longer chain phospholipids than l , y li might have been expected to show a greater preference for gel-phase lipid than l , because phospholipid in the gel phase gives a thicker bilayer than the corresponding lipid in the liquid crystalline phase. because y l and l show equal preferences for liquid crystalline-over gel-phase lipid, any effects of hydrophobic matching between the peptide and the bilayer must be small compared to effects of lipid phase on the interaction energies between the peptides and the lipid (mall et al., ) . preferential partitioning of proteins from domains in the gel phase into domains of liquid crystalline lipid has been demonstrated for a variety of membrane proteins, including bacteriorhodopsin (cherry et al., ) and ca +-atpase kleeman and mcconnell, ) . effects of sphingomyelin at °c are very similar to the effects of gel-phase di(c : )pc ( fig. ; mall et al, ) . mixtures of bovine brain sphingomyelin and di(c : )pc are in a two-phase region at °c, with gel-phase domains enriched in sphingomyelin (untracht and shipley, ) . thus, partitioning of the peptides between gel-and liquid crystalline-phase lipid shows little dependence on the structure of the phospholipid. it has been suggested that plasma membranes of mammalian cells contain domains or "rafts" enriched in sphingomyelin and that particular enzymes, particularly those associated with cell signaling, are concentrated within the rafts (simons and ikonen, ) . the results presented here suggest that membrane proteins containing transmembrane ot helices will tend to be excluded from these rafts, and it may therefore be significant that many of the signaling proteins suggested to be contained within the rafts are anchored to the membrane by glycosylphosphatidylinositol anchors (harder and simons, ) . the presence of cholesterol has a marked effect on incorporation of the peptides into phospholipid bilayers (webb et al., ) . incorporation of cholesterol at a : molar ratio to phospholipid leads to a general reduction in incorporation of the peptides l and l , but superimposed on this effect is a chain length effect. in the presence of cholesterol, the binding constant of p for di(c : )pc relative to di(c : )pc increased from . to about , as expected if the presence of cholesterol increases the effective chain length of the c chain so that it more nearly matches the hydrophobic length of the peptide (fig. ) . consistent with this interpretation, the presence of high molar ratios of cholesterol prevented the incorporation of p into bilayers of di(c :i)pc. nezil and bloom ( ) showed that incorporation of cholesterol at mol% increases bilayer thickness by about ,~. studies with brominated analogues of cholesterol showed that cholesterol binds to the peptides with a binding constant only a factor of about two less strongly than di(c : )pc . this is rather surprising, given the relatively rigid structure of the steroid ring of cholesterol and the molecularly rough surface of the peptide. in other studies, it has been shown that cholesterol binds relatively weakly at the lipid-protein interface of the atpase (simmonds et al., (simmonds et al., , ; comparison with the peptide studies reported here suggests that weak binding of cholesterol to the atpase involves interactions in the lipid headgroup region rather than interactions between the sterol ring and the hydrophobic transmembrane helices. the requirement to match the hydrophobic thickness of a membrane protein to that of the surrounding lipid bilayer could be important in a number of ways. targeting of proteins to their correct final destinations in a cell is essential in maintaining cell integrity. in the bulk flow model, the vast majority of proteins synthesized in the endoplasmic reticulum (er) are believed to leave the er by default and flow along the exocytic pathway until they reach the plasma membrane (nilsson and warren, ) . some proteins, however, have to be retained at particular points along the exocytic pathway. compartmental localization could be achieved in one of two ways. the first involves a retention signal in the protein, which, at the appropriate point in the exocytic pathway, prevents forward movement of the protein by denying it access to budding transport vesicles of the onward pathway. the second involves a retrieval signal, leading to recapture of the protein after it has left the compartment in which it resides. the classical retrieval signal is the kdel sequence found in many er-resident proteins; the situation appears to be different for golgi-resident proteins, where membrane-spanning domains act as retention signals (nilsson and warren, ) . despite the extensive flux of proteins through the golgi, the golgi maintains its own distinctive population of resident proteins. furthermore, the distribution of enzymes within the golgi is organized according to function, so that, for example, the distributions of glycosyltransferases and glycosidases, although overlapping, are distinct (colley, ; roth, ) . many of the proteins in the golgi membrane are predicted to contain a single transmembrane ot helix, oriented with the n-and c-termini on the inner and outer faces of the membrane, respectively. the golgi retention signal in such proteins has been shown to involve the membrane spanning domain (munro, (munro, , nilsson et al., ; swift and machamer, ) . however, the membrane-spanning domains show no sequence homology, and it has not been possible to identify any particular motif leading to retention (bretscher and munro, ; colley et al., ; munro, ) . thus, sialyltransferase remains localized in the golgi even when its -amino-acid transmembrane domain is replaced by leu residues (munro, ) . however, a longer stretch of leu residues did not provide an efficient retention signal (munro, ) . similarly, a -residue insertion into the transmembrane domain of galactosyltransferase reduced its retention in the golgi (masibay et al., ) . the reverse effect has been shown with the influenza virus neuraminidase, which shifted from the plasma membrane to the golgi and er when the number of residues in the transmembrane domain was reduced (sivasubramanian and nayak, ) . the lack of a clear retention motif, together with the inability to saturate the mechanism for golgi retention by overexpression, suggests that retention is not a receptor-mediated event (nilsson and warren, ) . one possible model is then retention by preferential interaction with membranes of optimal thickness (nilsson and warren, ) . both bretscher and munro ( ) and masibay et al. ( ) showed that transmembrane domains of golgi proteins are shorter (average residues) than transmembrane domains of plasma membrane proteins (average residues). it has therefore been suggested that if the golgi membrane is thinner than the plasma membrane, membrane proteins with short transmembrane domains will interact "more strongly" with the lipid bilayer of the golgi than with that of the plasma membrane, leading to retention in the golgi (bretscher and munro, ; masibay et al., ) . the studies with model peptides described above show that a protein containing a transmembrane ot helix with a hydrophobic length greater than the hydrophobic thickness of the golgi membrane will be able to move out of the golgi into the plasma membrane. however, a protein whose transmembrane ot helix has a hydrophobic length less than the hydrophobic thickness of a particular membrane will not be able to enter that membrane, and such a protein would then be retained in the golgi (webb et al., ) . studies of targeting of proteins in yeast are also consistent with a lipid-based model (rayner and pelham, ) . the length of the transmembrane domain is important in targeting with long helices ( residues), ensuring transport to the plasma membrane. however, for proteins with shorter transmembrane domains, the relative hydrophobicity of the transmembrane domain has been suggested to be important as well as its length, this determining targeting to the golgi and the vacuole (rayner and pelham, ) . retention of some membrane proteins in the er could also depend on the length of the transmembrane domain of the protein. an important class of er membrane proteins are those with an n-terminal catalytic domain exposed to the cytoplasm and a c-terminal membrane anchor. such proteins are inserted into the er membrane post-translationally by a signal-recognition-particle-independent pathway. no er retrieval signals have been identified in these proteins. instead, it has been observed that the hydrophobic domain is rather short. for example, cytochrome bs, a protein of this type, has a transmembrane domain containing just hydrophobic amino acid residues (pedrazzini et al., ) . if the length of the hydrophobic stretch is increased to residues, the protein is transported out of the er along the secretory pathway (pedrazzini et al., ) . it could therefore be that matching of the thickness of the lipid bilayer and the transmembrane length of the protein is important in retention in er, as was suggested for the golgi complex. although the length of the transmembrane domain appears to be the most important factor, the structure of the c-terminal, lumenal, region has also been shown to contribute to retention (honsho et al., ) . experiments with another c-terminalanchored protein, the ubiquitin-conjugating enzyme ubc from yeast, suggest that the thickness requirements of the er and golgi membranes may be different, explaining targeting between these two organelles (yang et al., ) . whereas ubc containing the wild-type -residue transmembrane domain targets to the er, increasing the length of the transmembrane domain to residues results in movement to the golgi, and increasing the length further to residues allows movement to the plasma membrane. these experiments show that the length of the transmembrane ot helix is often an important factor in targeting, although it is likely to be only one of a number of important factors. the lengths of the transmembrane ot helices are also likely to be important in the proper function of membrane proteins containing multiple transmembrane ot helices. an example already described is that of the ca +-atpase, which shows highest atpase activity in di(c : )pc and lower activities in bilayers of phospholipids with longer or shorter fatty acyl chains (lee, ) . changes in the atpase underlying these changes in atpase activity are complex (lee, ) , but all must be mediated by the transmembrane ot helices, because these are the parts of the atpase that can "sense" the change in bilayer thickness. in the case of the ca +-atpase it seems that, as described above, the two major conformational states of the atpase (el and e ) have different preferences for bilayer thickness, the e conformation favoring thin bilayers and the e conformation favoring thick bilayers (lee, ) . changing the bilayer thickness could change the tilt of the transmembrane u helices in the ca +-atpase, it could change the packing of the helices, and, possibly, it could lead to changes in the structures of the loops connecting the helices, changing the effective lengths of the helices. all these changes could be linked to changes in the phosphorylation domain of the ca +-atpase, located well above the surface of the membrane. if, as seems likely, the various membranes in a cell have different thicknesses because of their different lipid compositions, the structure of each membrane protein will have evolved to match the thickness of the membrane in which it resides. protein-lipid interactions and differential scanning calorimetric studies of bacteriorhodopsin reconstituted lipid-water systems structure and dynamics of an amphiphilic peptide in a lipid bilayer: a molecular dynamics study protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at . a resolution molecular theory of chain packing, elasticity and lipid-protein interaction in lipid bilayers cholesterol and the golgi apparatus membrane electrostatics temperature-dependent aggregation of bacteriorhodopsin in dipalmitoyl-and dimyristoyl-phosphatidylcholine vesicles golgi localization of glycosyltransferases: more questions than answers the signal anchor and stem regions of the fl-galactoside ot , -sialyltransferase may each act to localize the enzyme to the golgi apparatus interaction of phosphatidic acid and phosphatidylserine with the caz+-atpase of sarcoplasmic reticulum and the mechanism of inhibition interaction of a synthetic amphiphilic polypeptide and lipids in a bilayer structure influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers: a h nmr and esr study using designed transmembrane alpha-helical peptides and gramicidin a different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane alpha-helical peptides specificity of lipid-protein interactions as determined by spectroscopic techniques the structure of the potassium channel: molecular basis of k + conduction and selectivity molecular sorting of lipids by bacteriorhodopsin in dilauroylphophatidylcholine/distearoylphosphatidylcholine lipid bilayers is the protein/lipid hydrophobic matching principle relevant to membrane organization and functions? molecular dynamics simulation of membranes and a transmembrane helix lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid structure and fluctuations of bacteriorhodopsin in the purple membrane: a molecular dynamics study internal packing of helical membrane proteins identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins structure and function of the photosynthetic reaction center from rhodobacter sphaeroides spin label studies on the origin of the specificity of lipid-protein interactions in na+,k+-atpase membranes from squalus acanthias lipid patches in membrane protein oligomers: crystal structure of the bacteriorhodopsin-lipid complex a molecular model for lipid-protein interaction in membranes: the role of hydrophobic mismatch proteinlipid interaction. biophysical studies of (ca + + mg +)-atpase reconstituted systems electroncrystallographic refinement of the structure of bacteriorhodopsin caveolae, digs, and the dynamics of sphingolipid-cholesterol microdomains retention of cytochrome b in the endoplasmic reticulum is transmembrane and luminal domain-dependent phase equilibria in an amphiphilic peptidephospholipid model membrane by deuterium nuclear magnetic resonance difference spectroscopy orientation of u-helical peptides in a lipid bilayer structure at . /~ resolution of cytochrome c oxidase from paracoccus denitrificans identification and extent of fluid bilayer regions in membranous cytochrome oxidase hydrophobic mismatch between proteins and lipids in membranes induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane alpha-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans interactions of proteins and cholesterol with lipids in bilayer membranes non-random distribution of amino acids in the transmembrane segments of type single span membrane proteins calculation of phase diagrams for non-ideal mixtures of lipids, and a possible nonrandom distribution of lipids in lipid mixtures in the liquid crystalline phase how lipids interact with an intrinsic membrane protein: the case of the calcium pump what the structure of a calcium pump tells us about its mechanism lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles fluorescence quenching in model membranes. . determination of local lipid environment of the calcium adenosinetripfiosphatase from sarcoplasmic reticulum structure of bacteriorhodopsin at , ~ resolution lipid-protein interactions in the membrane: studies with model peptides effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers specificity of lipid-protein interactions mutational analysis of the golgi retention signal of bovine t- , -galactosyl transferase the photosynthetic reaction center from the purple bacterium rhodopseudomonas viridis: aspects of membrane protein structure the structure of bacteriorhodopsin at . a resolution based on electron crystallography: implication of the charge distribution influence of membrane-spanning alpha-helical peptides on the phase behavior of the dioleoylphosphatidylcholine/water system simultaneous modeling of phase and calorimetric behavior in an amphiphilic peptide/phospholipid model membrane mattress model of lipid-protein interactions in membranes models of lipid-protein interactions in membranes sequences within and adjacent to the transmembrane segment of alpha- , -sialyltransferase specify golgi retention an investigation of the role of transmembrane domains in golgi protein retention combined influence of cholesterol and synthetic amphiphilic peptides upon bilayer thickness in model membranes energetics of inclusion-induced bilayer deformations retention and retrieval in the endoplasmic reticulum and the golgi apparatus the membrane spanning domain of ~-l, -galactosyl transferase specifies trans golgi localization lipid mobility and order in bovine rod outer segment disk membranes:. a spin-label study of lipid-protein interactions a mutant cytochrome b with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane hydrophobic mismatch and long-range protein/lipid interactions in bacteriorhodopsin/phosphatidylcholine vesicles fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin transmembrane domain-dependent sorting of proteins to the er and plasma membrane in yeast membrane protein structure and stability: implications of the first crystallographic analyses control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length hydrophobicity of the peptide c=o. • -h--n hydrogen bonded group subcellular organization of glycosylation in mammalian cells detergent structure in crystals of a bacterial photosynthetic reaction centre structure of the detergent phase and proteindetergent interactions in crystals of the wild-type (strain y) rhodobacter sphaeroides photochemical reaction center molecular dynamics of pfl coat protein in a phospholipid bilayer conformational changes of phospholipid headgroups induced by a cationic integral membrane peptide as seen by deuterium magnetic resonance influence of the intrinsic membrane protein bacteriorhodopsin on gel-phase domain topology in two-component phase-separated bilayers transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations annular and non-annular binding sites on the (ca + + mg +)-atpase interactions of cholesterol hemisuccinate with phospholipids and (ca +-mg +)-atpase functional rafts in cell membranes mutational analysis of the signal-anchor domain of influenza virus neuraminidase dependence of lipid membrane phase u'ansition temperature on the mismatch of protein and lipid hydrophobic thickness lipid enrichment and selectivity of integral membrane proteins in two-component lipid bilayers characterization of the single ca + binding site on the ca +-atpase reconstituted with short and long chain phosphatidylcholines molecular organization and dynamics of -palmitoyl- -oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide a golgi retention signal in a membrane-spanning domain of coronavirus e protein crystal structure of the calcium pump of sarcoplasmic reticulum at . /~ resolution molecular interactions between lecithin and sphingomyelin principles of membrane protein assembly and structure architecture of helix bundle membrane proteins: an analysis of cytochrome c oxidase from bovine mitochondria hydrophobic mismatch and the incorporation of peptides into lipid bilayers: a possible mechanism for retention in the golgi experimentally determined bydrophobicity scale for proteins at membrane interfaces molecular dynamics simulations of individual alpha-helices of bacteriorhodopsin in dimyristoylphosphatidylcholine. ii. interaction energy analysis the transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum the preference of tryptophan for membrane interfaces interaction of a peptide model of a hydrophobic transmembrane a-helical segment of a membrane protein with phosphatidylcholine bilayers: differential scanning calorimetric and ftir spectroscopic studies ftir spectroscopic studies of the conformation and amide hydrogen exchange of a peptide model of the hydrophobic transmembrane a-helices of membrane proteins peptide models of helical hydrophobic transmembrane segments of membrane proteins. . differential scanning calorimetric and ftir spectrooscopic studies of the interaction of ac-k -(la) -k -amide with phosphatidylcholine bilayers we thank the biotechnology and biological sciences research council (bbsrc) for financial support of the original studies reported here. key: cord- -himray q authors: nan title: abstracts of oral presentations date: - - journal: biopolymers doi: . /bip. sha: doc_id: cord_uid: himray q nan s. tchertchian, f. oplinger, m. paolini, s. manganiello, s. raimondi, b. depresle, n. dafflon, h. gaertner, and p. botti geneprot inc., geneva branch, , pré de-la-fontaine, meyrin, switzerland the last decade has provided extensive demonstration of the key role played by native chemical ligation (ncl) for the preparation of small and medium size proteins [ ] . yet the requirement for cysteine at the site of ligation in standard ncl has limited its flexibility. recently, different types of auxiliary groups [ , ] have been developed to extend the application of ncl to other ligation sites. however, the generally slower ligation rates especially with large fragments and the additional step required to cleave the auxiliary post-ligation have reduced their utility. here we present a novel strategy to synthesize proteins through a chemical ligation using unprotected peptide segments. our scheme does not make use of auxiliary groups [ , ] , instead originally exploits the features of some side chain removable functionalities. ligation rates are high, comparable to ncl and the residues available for ligation are more frequent than cysteine. furthermore the whole process is "one pot" and at the end a native polypeptide is obtained directly in the ligation mixture. the total chemical syntheses of c a ( - ) using both ncl and our method will be presented and compared. [ during the biosynthesis of glycopeptide antibiotics of the vancomycin family, several oxidative phenol coupling reactions take place. the enzymes catalyzing these reactions are of interest from structural and mechanistic viewpoints. in this work [ , ] , it is shown that the oxygenase oxyb from the vancomycin producer only catalyzes a phenol coupling reaction when the putative peptide substrate is linked as a thioester to a peptide carrier domain (pcd) derived from the nonribosomal peptide synthetase. an efficient access is described to representative free linear peptide substrates, which makes use of alloc-solid phase peptide chemistry, but largely avoids the use of amino acid side chain protecting groups. in this way, the target linear peptides can be released from the resin under very mild conditions, and then be activated as thioesters, prior to loading onto the pcd. [ we have recently discovered a new nonenzymatically-formed product from n-( -oxododecanoyl)-l-homoserine lactone. interestingly, both the n-acylhomoserine and its novel tetramic acid degradation product are potent antibacterial agents. bactericidal activity was observed against all tested gram-positive bacterial strains, while no toxicity was seen against gram-negative bacteria. we propose that p. aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization. using short portions ( - amino acid) rich in positively charged residues from either human lactoferricin or the marcks protein as templates, a panel of peptides each possessing a specific chemical structure was synthesized. these included amino acid omissions, substitutions, and insertions in the aim to modify the peptides overall charge, hydrophobic core, and/or amphipathicity. the peptides antimicrobial activities against a large panel of bacteria were assessed using both conventional tests (mic, mbc) and non-conventional assays (mic quantified by an automated turbidimetry-based system and mbc measured on resting cells suspended in low-ionic strength medium-"survival assay"). furthermore, the membrane permeabilizing activity of the peptides on strains of several gram negative bacterial species was quantitated by measuring their ability both to decrease the mic of novobiocine and to promote the uptake of the hydrophobic fluorescent probe npn. while the mic determined by turbidimetry or by the conventional method did not significantly differ, bactericidal activity of the peptides measured by the survival assay was to orders of magnitude higher than that measured by the conventional mbc test. on the other hand, the two assays used to measure the permeabilizing activity of the peptides rendered similar results. interestingly, the most potent permeabilizers did not correspond with the peptides exhibiting the highest bactericidal activity thus indicating that these two activities have different structural bases. protein farnesyl transferase (pftase) catalyzes the attachment of farnesyl diphosphate (fpp) to proteins that contain a caax-box sequence at their c-termini [ ] . several analogues of fpp that incorporate azide functional groups have been synthesized and shown to be incorporated into peptides using pftase as a catalyst. in particular, it has been shown that the prenyl azide moiety from or related analogues can be transferred to the peptide substrate, n-dansyl-gcvia to yield the corresponding thioether-linked products. the resulting azide-containing peptides have been derivatized with a triphenylphosphine-based reagent to generate o -alkyl imidate-linked products rather than the amide-linked material expected via a staudinger reaction [ ] . since caax-box sequences can be appended to the c-termini of many different proteins, these analogues provide a simple and general method for incorporating orthogonal azides into proteins at unique sites. subsequent functionalization of such azide groups via staudinger or "click" chemistry should provide a convenient method for linking proteins with a diverse array of probes, biomolecules, surfaces and other materials under mild conditions. chemoselective glycosylation, acylation, and alkylation of completely unprotected peptides can be accomplished by incorporating n-alkylaminooxy amino acids into the peptide sequence. the n-alkylaminooxy side chains react selectively with reducing sugars, activated alkyl halides, and various acylating agents in mildly-acidic aqueous buffers (ph ) to furnish neoglyco-and neolipopeptides. a key feature of the approach is that a single parent peptide can be quickly reacted with a variety of agents to provide a large number of "post-translationally"modified peptides. the ability to easily synthesize arrays of modified peptides allows comprehensive studies of the effects that glycosylation and lipidation have on peptide structure and function. here we present an overview of the methodology and initial results on its application to studying problems of biological interest. this presentation describes cyclic peptides that fold into well-defined ␤-sheet structures in aqueous solution and can dimerize through ␤-sheet interactions. the cyclic peptides contain the unnatural amino acid hao, which mimics the hydrogen-bonding pattern of one edge of a peptide ␤-strand, and ␦-linked ornithine, which mimics a ␤-turn and provides enhanced water solubility or a linkage point for creating multivalent structures. institute for molecular bioscience, the university of queensland, queensland , australia the human genome project and other major sequencing projects have rapidly provided a vast array of new protein/peptide sequences. in contrast, many other new proteins/peptides are also being uncovered from plant and animal sources whose genomes are yet to be tapped. in the post-genomic era, the physical form of many of these gene-encoded sequences will be vital for biomedical research and drug development. moreover, the advantages of peptide and protein chemical synthesis over recombinant-dna methods are increasingly being used to provide rapid structure-activity information of complex bioactive peptides, small proteins and functional receptor domains. in a program designed to exploit the potential of australian conus species we have isolated, characterised and chemically synthesised a wide range of novel conotoxins. these cysteine rich microproteins have well-defined tertiary structures with considerable rigidity and stability and contain many elements of protein secondary structure. of particular interest are the two disulfide bond containing conotoxins (examples below) which target transporters, ion channels and receptors at nanomolar potencies. in this presentation i will describe some of our research on controlling the shape and potency/selectivity of these microproteins through intramolecular native ligation chemical approaches. it appears that there is considerable scope to control the properties of these native sequences which in some cases may prove useful in the development of these molecules as therapeutic candidates. despite identical amino acid composition, differences in the properties of class a amphipathic helical peptides due to differences on the hydrophobic face results in substantial differences in anti-inflammatory properties. one of these peptides is an apolipoprotein a-i mimetic, d- f. when given orally to mice and monkeys, d- f caused the formation of pre-␤ hdl, improved hdl-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity and converted hdl from pro-inflammatory to anti-inflammatory. in apoe null mice d- f increased reverse cholesterol transport from macrophages. oral d- f reduced atherosclerosis in apoe null and ldl receptor null mice. in vitro d- f caused the formation of pre-␤ hdl, reduced lipoprotein lipid hydroperoxides and converted hdl from pro-inflammatory to anti-inflammatory. physical properties and the ability of various class a amphipathic helical peptides to activate enzyme lcat in vitro did not predict biologic activity in vivo. in contrast, the use of cultured human artery wall cells in evaluating these peptides was more predictive of their efficacy in vivo. thus, anti-inflammatory properties of different class a amphipathic helical peptides depends on subtle differences in the configuration of the hydrophobic face of the peptides. physical-chemical properties provide an explanation for the mechanism of action of the active peptides. peptides to ameliorate atherosclerosis and other inflammatory diseases can be designed using this strategy. inflammatory diseases. this chemokine belongs to the family of cxc chemokines, its response is mediated through binding to seven transmembrane helical g-protein coupled receptors cxcr and cxcr . in order to investigate the relevance of selected protein segments for biological activity we synthesized chemically modified and biologically active analogues of the -mer of hil- by expressed protein ligation (epl). for ligation naturally occurring cysteine at position was chosen. c-terminal peptides carrying an n-terminal cysteine were synthesized by solid phase peptide synthesis (spps) applying the fmocstrategy and used to introduce modifications. ligation of the recombinantly produced thioester with synthetic peptides yielded in full length hil- that finally was correctly folded and stabilised by two disulfide bridges as in the native protein. in addition to fluorescent and photoactivatable analogues, we produced variants that contain a ␤-peptide helix instead of the naturally occurring ␣-helix. thus, for the first time, we received a protein containing a whole ␤-peptide segment and still showing high biological activity. depending on the linker between ␤-sheet and ␤-peptide helix of interleukin we could discriminate between active and inactive proteins suggesting that the overall orientation of the c-terminal segment is highly relevant for the folding of the protein and subsequently for the signalling of interleukin . a peptide based on residues - of the syrian hamster prion protein (h ) forms ␤-sheet aggregates in solution, which grow to form large fibers. isotope-edited infrared spectroscopy has shown that the initial antiparallel ␤-sheet formed by this peptide is disordered. a slow rearrangement occurs to form a structure in which the hydrophobic core of the strands (residues - ) pack together, resulting in the alignment of residue across the sheet. the kinetics of the realignment have been monitored for h and for peptides with mutations at residue (a i, a l and a b where b is aminobutyric acid). h and a i align with non-exponential kinetics. at low concentrations h aligns via the repeated detachment and annealing of strands, whereas at higher concentrations a reptation mechanism is observed. a b aligns instantaneously within the dead-time of our experiments. a l does not align at residue but some undefined reordering can still be observed as a shift of the c band. these data are the first experimental probes of the types of intersheet rearrangements which are required for the nucleation of fibrous peptide aggregates, and the evidence for strand reptation within the ␤-sheet confirms observations in molecular dynamics simulations. [ , ] . we have since established the microfluidic peptide chip as a miniaturizing platform. the challenging issues in making peptide chips a practical tool for understanding biology, drug discovery, and diagnostics are quality of synthesis, specificity in reported activities, and ability for quantitative measurements. we will present the results of the work which involves our intensive effort in: • development of the method for monitoring and analyzing quality of peptide chip synthesis • improvement in peptide chip synthesis • development of the methods for quantitative analysis of (a) the specific binding of antibodies/proteins to peptides on chip and (b) kinase enzymatic activities against substrate peptides on chip. our presentation should demonstrate novel applications of peptide chips that can be implemented as routine laboratorial processes. [ ] gao, x., pellois, j. p., kim, k., na, y. , gulari, e., and zhou, x. to prototype this approach we developed a protein array consisting of the ras-binding domain of craf- (rbd) that was c-terminally modified with a mer oligonucleotide and immobilized via hybridization with the complementary oligonucleotide on a wafer [ ] . the rbd-dna conjugate was generated by native chemical ligation using a recombinantly produced rbd-thioester and an oligonucleotide carrying a '-cystein residue [ ] . incubation of the immobilized rbd with ras(gtp), the activated rbd-binding form, retains sufficient amounts of ras on the protein array to allow detection by mass spectrometry with high sensitivity. controls carried out with inactive ras(gdp) did not produce any signal, demonstrating a sufficient selectivity for biotechnological applications. protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. such protein microarrays ('protein chips') can be used very efficiently to analyze all kind of protein interactions en masse. the present work describes a general method for the selective attachment of proteins to solid surfaces through its c-termini that can be used for the creation of protein chips. our method is based in the chemoselective reaction between a protein c-terminal ␣-thioester and a modified surface containing n-terminal cys residues. ␣-thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing modified inteins. we also present an efficient solid-phase approach for the rapid synthesis of cys-containing linkers that can be used for the modification of au-and si-based surfaces. this new method was used to immobilize two fluorescent proteins and a functional sh domain. a series of glycopeptides based on the leu-enkephalin analogue yt-gfs*-conh led to greatly enhanced stability in vivo and effective penetration of the bbb. transport through the bbb hinges on the biousian nature of the glycopeptides-the glycopeptides have two conflicting conformational manifolds, a h o soluble state, and an amphipathic state at h o-membrane phase boundaries. multiple lines of evidence suggest that the bbb transport mechanism is absorptive endocytosis. mixed /␦-agonists showed antinociceptive potencies greater than morphine, and lacked many of the side effects generally associated with classical -selective opiate analgesics. the biousian design was extended to larger glycopeptides ( residues) related to ␤-endorphin, which also penetrated the bbb and produced antinociception in mice. plasmon waveguide resonance (pwr) studies showed that the amphipathic helices bound to membrane bilayers with m to low nm k d 's. the presence of diverse endogenous neuropeptide transmitters and neuromodulators in the human brain is potentially applicable to the treatment of a wide range of behavioral disorders. clemencia pinilla, mireia sospedra, yindong zhao, we have recently demonstrated the feasibility of utilizing the ligase activity of inteins for the in vivo backbone cyclization of peptidic chains. this procedure -called siclopps for split intein circular ligation of peptides and proteins-provides a biosynthetic pathway for peptides that are metabolically stable, and can be produced with spatial and temporal control [ ] . to screen for bacteriotoxic peptides, a sic-lopps library was introduced into an escherichia coli population, such that each bacterium encodes a different peptide sequence. siclopps library over-expression afforded six distinct bacteriostatic peptides that reduce cell growth. one of these peptides (ln ) also caused cell aggregation. an e. coli genomic library was introduced into cells encoding ln . co-expression of the genomic library and ln peptide rescues growth only in cells expressing genomic fragments able to counteract peptide toxicity. genomic library and ln co-expression resulted in enrichment of a single genomic construct, a fragment of the narz gene. narz is part of a nitrate reductase complex and has a role in tuberculosis persistence [ ] . ln production in mycobacterium smegmatis resulted in a slow-growth phenotype. [ ] abel-santos, e., scott, c. p., and benkovic, s. j., methods in the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different (meta) stable conformations. thus, amyloid-forming proteins that mainly contain ␣-helical structures in their native conformation must undergo an ␣-helix ␤strand conversion before or during fibril formation. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of the environmental conditions such as ph, ionic strength, metal ions, protein concentration, oxidative stress, free radicals, action of physiological, or pathological chaperones. alternatively, the introduction of a small quantity of protein polymer may act as a structural template and initiate the disease. therefore, the development of model systems which allow the investigation of the complex folding mechanisms that lead to ␤-sheet aggregation appears to be one of the main challenges in the detailed understanding of the pathways from incubation to mortality. in order to create an ␣-␤ switch system we designed ideal ␣-helical parallel coiled coil peptides, introduced trigger functions by mutations in the primary structure, and studied the consequences that these mutations have on the secondary structure properties of the resulting peptides under certain environmental conditions. based on these results we continued to change the primary structure of the coiled coil system subsequently by mutations in the heptad repeat untill we ended up with soluble ␤-sheet peptides. the most important feature of these new ␤-sheet peptides is that they still follow the characteristic hydrophobic heptad repeat of an ␣-helical coiled coil and that all of the positions which are part of the dimerization domains of the coiled coil remained untouched. thus, these new peptides bear all of the requirements which are necessary for formation of cooperatively interacting helical structures and, furthermore, contain domains for cooperative sheet aggregation as well. the folded structure will now strongly depend on the environment. this peptidic model system allows a systematic study of the subtle influences that environmental conditions may have on protein folding stepwise, which means changing these conditions one after one or in all of the possible combinations that nature applies in vivo. plasmon waveguide resonance (pwr) spectroscopy is a powerful new biophysical method which allows us to examine structural changes, kinetics and thermodynamics of anisotropic biological systems and processes such as proteolipid membranes. this method has probed the mechanisms of g-protein coupled receptor (gpcr) signal transduction, and has obtained new insights into specific signaling pathways of agonists and antagonists with gpcrs. we now have extended these studies to examine the effects of lipid microdomains (rafts) on the binding, signaling and transduction pathways. using a : mixture of palmitoyloleoylphosphatidyl-choline (popc) and sphingomyelin (sm), we have directly observed the formation of two lipid bilayer microdomains, and the preferred segregation of the delta opioid receptor (hdor) into sm lipid rafts when the agonist ligand was bound, but not for the unoccupied receptor which preferentially incorporated into the popc-rich domain. furthermore, we can demonstrate directly that, g-proteins bind much more strongly to the hdor receptor in the lipid raft (sm-rich) environment than in the fluid non-raft (popc-rich) domain of the lipid bilayer. the implications of these findings for novel design of drugs, and drug screening will be emphasized. † supported by grants from the u.s.p.h.s., national institute of drug abuse and national science foundation. their measurement in high resolution nmr requires partial molecular alignment. for proteins in aqueous solution a number of standard methods exist to achieve such small alignment. we found that swelling of cross-linked polymers inside the nmr tube results in anisotropic gels. peptides in such a gel phase exhibit well resolved spectra of the partially aligned molecules. this allows to scale the orientation depending on the cross-linking of the polymer, the thickness of the unswollen stick or the temperature. rdcs can now easily be measured in natural abundance in peptides ] all common nmr solvents can be used. with the chiral gel gelatin it is possible to discriminate d-and l-alanine to determine enantiomeric purity. the procedure is demonstrated to refine the solution structures of peptides such as cyclosporin a, somatostatin analogues and others galia blum, georges von degenfeld, kinneret keren, misregulation of cysteine protease activity is associated with numerous pathologies ranging from cancer to autoimmune disease. protease activity is controlled by a delicate balance of many factors such as levels of natural inhibitors and posttranslational modifications. thus developing a detection method for monitoring protease activity rather than abundance is desirable. here we describe the design and synthesis of a novel class of chemical tools, activity based probes (abps), that detect protease activity. these probes are composed of a fluorescent tag and its cognate quenching group, a peptide recognition scaffold, and a reactive "warhead". these fluorescently quenched "smart probes" covalently modify protease active sites in a fashion that is dependent on activity of the protease. this results in loss of the quenching group, producing a fluorescent signal. we report the production of selective, cell permeable activity based probes for the study of papain family cysteine proteases in cells and whole animals. these probes are used to monitor real time protease activity in living cells using fluorescence microscopy techniques as well as standard biochemical methods. key: cord- -josb pi authors: kumaraswamy, priyadharshini; sethuraman, swaminathan; yakhmi, jatinder vir; krishnan, uma maheswari title: hierarchical self-assembled peptide nano-ensembles date: - - journal: handbook of nanomaterials properties doi: . / - - - - _ sha: doc_id: cord_uid: josb pi a variety of peptides can be self-assembled, i.e. self-organized spontaneously, into large and complex hierarchical structures, reproducibly by regulating a range of parameters that can be environment driven, process driven, or peptide driven. these supramolecular peptide aggregates yield different shapes and structures like nanofibers, nanotubes, nanobelts, nanowires, nanotapes, and micelles. these peptide nanostructures represent a category of materials that bridge biotechnology and nanotechnology and are found suitable not only for biomedical applications such as tissue engineering and drug delivery but also in nanoelectronics. self-assembly is defined as a process where individual components form organized structures via specific and local interactions without any external intervention [ ] . molecular self-assembly is a spontaneous process where the molecular components organize into ordered structures through non-covalent interactions such as van der waals, hydrophobic, capillary forces, electrostatic forces, or hydrogen bonds [ ] . although these interactions are relatively weak when compared to covalent bonds, they form reasonably stable higher-order structures through self-assembly due to the additive effect of these secondary forces. in other words, these self-assembled structures are thermodynamically more stable due to lower values of gibbs free energy when compared to that of the individual components (building blocks). since the underlying interactions are rather weak, any external stimulus can alter the self-assembled structures. however, once the stimulus is removed, they can revert back to their original structure. molecular self-assembly is ubiquitous in nature, and it has evolved in many areas including chemical synthesis, nanotechnology, polymer science and materials science, and engineering [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the molecular level self-assembly is a typical example of the 'bottom-up' approach where molecules in the sub-nm range come together to form assemblies that are in nm or bit larger in dimensions [ , ] . numerous self-assembling systems ranging from diand tri-block copolymers, complex dna structures, simple and complex proteins, and peptides have been developed [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . complex and intricate monodisperse structures can be obtained through self-assembly with high precision and reproducibility. biomolecules possess an inherent ability to form hierarchical self-assemblies in aqueous medium. biomolecules such as proteins, deoxyribonucleic acid, and lipids have been widely investigated for their self-assembling properties [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in fact, these three biomolecules form the 'molecular trinity' of biomolecular self-assembly. peptide systems have been especially popular self-assembling systems due to the large number of structures that can be generated by slight modification of the number and nature of amino acid residues in the sequence [ , , ] . the stability, ease of synthesis, and controlled self-assembly regulated by various physicochemical parameters have resulted in the popularity of selfassembled peptide systems [ ] . since peptide self-assembly is a bottom-up process where amino acids form the building blocks, it is easy to introduce functionalities on the carboxyl or amine terminal groups, opening up the possibilities of a wide range of chemical interactions leading to specific functions. though peptides containing naturally occurring l-amino acids have been widely investigated for their self-assembling characteristics, d-amino acid containing peptide systems have also been explored due to their stability against proteases [ ] . self-assembling peptides may vary in the number of amino acids starting from to as high as . the simplest building block reported thus far is the dipeptide (diphenylalanine -ff) from the core recognition motif of alzheimer's amyloid beta peptide [ ] . this dipeptide is reported to form different structures based on the ph that is employed (fig. . ). for instance, at a ph lower than the isoelectric point of the peptide, it forms nanofibrils, whereas at a ph higher than its isoelectric point, the peptide forms nanotubes [ ] . despite the numerous advantages of self-assembling peptides, there are several challenges associated with their use in biomedical applications, which include problems related to processability, control of size, functionalization, and stability in aqueous media [ ] . for example, biosensing platforms employing self-assembling peptides require electric contacts between the selfassembled nanostructures and transducers, which become tedious due to the small dimensions involved. however, with the advancement in micro-and nanofabrication techniques, such problems are being overcome, paving the way for use of peptide nanostructures in molecular electronics. another impediment relates to the low conductivity of the self-assembled peptide nanostructures, which limits their use in sensing and diagnosis. however, by the introduction of conductive polymers, enzymes, and metallic particles, the electrical current conductivity can be enhanced [ , ] . the self-assembly process is influenced by many factors that can be grouped into any of the three categories, namely, environment-driven factors, substrate-driven factors, and peptide-driven factors. the ph, temperature, solvent, nature of ions, and ionic strength are factors that influence the self-assembly process. the ph of the medium alters the charge status on the peptide and hence the electrostatic forces between the peptide molecules. for instance, the peptide stviie forms beta-sheets when its net charge is + , whereas in its zwitterionic state, it forms random coils and it exists as a mixture of random coils and beta-sheets when the net charge is À . this is because when the net charge is zero as in the zwitterionic form, the packing of the peptides could happen in many ways leading to an amorphous structure. the presence of a net charge gives directionality to the associations as well as determines the distance between the peptide chains [ ] . the charge distribution in the peptide also influences the self-assembled structures formed. for example, when the peptides eak -i (aeakaeakaeakaeak), eak -ii (aeaeakakaeaeakak), and eak -iv (aeaeaeaeakakakak) were self-assembled, it was found that eak -i and eak -ii formed fibrillar assemblies, while eak -iv formed globular structures between ph . and . and fibrillar structures at other ph due to the neutralization of charges in the beta-sheet structures formed, which promotes aggregation at ph away from the neutral ph [ ] . the nature of anion also was found to influence the structures formed by the eak -ii peptide in the presence of cu + ions. while so caused formation of nanofibers, the monovalent cland no caused formation of short fibrils with a mixture of alpha helix and random coils. this is due to the ability of the divalent sulfate anions to act as an electrostatic bridge between two lysine residues unlike the monovalent ions. higher ionic strength of the medium contributes the shielding of the electrostatic charges on the ionizable groups present in the peptide sequence, thereby altering the critical aggregation concentration as well as the ph required for association or dissociation of the self-assembled structure [ ] . factors like the solvent polarity, surface tension, hydrogen bond-forming ability, and dielectric constant influence the peptide self-assembly and strength of the self-assembled structures. introduction of methanol as cosolvent contributed to the formation of nanofibers of diphenylalanine (ff) on a glass substrate. this was attributed to the high hydrogen bond donor and acceptor property of methanol that promoted formation of highly crystalline nanofibers [ ] . on increasing the methanol content, solvation of the peptide molecules occurred, which prevented aggregation of the solvated peptide. it was also observed that organic cosolvents with higher surface tension contributed to reduction in fiber dimensions to the nm range. the dielectric constant of the solvent has been found to influence the peptide substrate binding affinities [ ] . the surface tension, hydrophobicity, and surface texture of the substrate influence the self-assembly process. hydrophobic substrates promote better spreading of peptide sequences that have greater number of hydrophobic residues. surface topography, on the other hand, directs the orientation as well as fiber dimensions. the dipeptide ff was found to self-assemble into nanofibers with a well-spread morphology on poly(vinyl chloride), whereas in silicon, which had a periodic rough texture, finer fibers were observed along with vertically aligned hollow nanotubes of larger dimensions suggesting that the rough morphology retards the stacking interactions between the peptide molecules [ ] . peptide-driven factors that direct self-assembly are the number and nature of amino acid residues in the sequence, the isoelectric point, and the peptide concentration [ ] . aromatic residues led to the formation of rigid structures that possessed nanotape or nanoribbon morphology [ ] . reduction in the surface tension of the peptide molecule can lead to the formation of globular assemblies instead of fibrillar structures as observed with the peptide eak -iv at neutral ph. peptide aggregates are formed above a particular concentration known as critical aggregation concentration (cac), which in turn is dependent on the peptide sequence. below the cac, the seeding and nucleation occur, while above cac, the aggregated ensembles are discernible [ ] . in the case of surfactant-like peptide amphiphiles, if the surfactant number is between ⅓ and ½, then cylindrical micelles and nanofibers are observed ( fig. . ). however, if the surfactant number is between ½ and , then bilayer formation occurs. in the case of micelle-forming peptides, increase in the intermolecular cross-links has been found to reduce the curvature leading to the formation of cylindrical micelles as observed in the hexadecyl-modified peptide sequence ccccggg phosphoserine-rgd [ ] . peptides that self-assemble are amphiphilic and are classified based on their nature of self-assembly. peptide lego systems consist of both hydrophilic and hydrophobic residues that form beta-sheet structures and well-defined nanofiber matrices with an average pore size of - nm in aqueous solution. these peptides are termed as molecular lego peptides as they possess alternating charged and hydrophobic amino acids like the pegs and holes of lego blocks. for example, in the peptide rad -i, the sequence is radaradaradarada, where r (arginine) is a cationic amino acid and d (aspartate) is an anionic amino acid. these oppositely charged amino acid residues are separated by a hydrophobic amino acid residue, alanine. the charge status of the peptide sequence will therefore be represented as (+ À + À + À + À + À + À + À + À), and such sequences are referred to as modulus i peptides. similarly, modulus ii (++À À++À À) and modulus iii (+++À À À+++À À À) have also been reported based on their charge pattern. these peptides spontaneously form nanofibers of nm length in the presence of cations of alkaline earth metals due to electrostatic forces. since ionic interactions are involved in the self-assembly process, the molecular lego peptides readily form hydrogels [ ] . the hexadecapeptide dar -iv with the peptide sequence dadadadarararara has similar amino acid residues as rad, with the only difference that the sequence of the charged amino acid residues varies. this difference is markedly reflected in the self-assembled structures formed by the two peptides. the self-assembled structures formed by dar -iv can transform from an alpha helix to a beta-sheet depending on the ph, ionic strength, and temperature i does not form alpha helical structures under any condition. this is because in the case of rad -i, if the peptide assumes an alpha helical structure, the positively charged side groups in arginine (r) will be repelled by the positively charged n-terminus, and the anionic aspartate (d) will experience electrostatic repulsion from the like-charged carboxylate in the c-terminus. hence, it always remains in the beta-sheet form. in the case of dar -iv, the negatively charged aspartate will stabilize the positive n-terminus, and the positively charged arginine will exhibit electrostatic attraction with the negative c-terminus when it adopts an alpha helical form. thus the nature, number, and sequence of amino acids are critical parameters that determine the type of self-assembled structures that can be formed by peptides [ ] . the molecular lego peptides are also known as ionic self-complementary peptides due to their pattern of electrostatic association that contribute to their stability. zhang and his coworkers identified the first molecular lego peptide eak- from a z-dna binding protein zuotin from yeast [ ] . the ionic self-complementary peptides initially form beta-sheets, which later form a fibrous network, progressively by undergoing sol-gel transition. the substitution of a basic amino acid with another basic amino acid (for instance r with k) or an acidic amino acid with another acidic amino acid (e.g. d with e) does not bring about significant changes in the self-assembly pattern. however, substitution of an acidic amino acid with a basic amino acid and vice versa was found to alter the self-assembly pattern. such structures were found to form beta-sheets but did not form higher-order structures. substitution of the alanine residues with more hydrophobic residues such as leucine, valine, and isoleucine accelerates the self-assembly process [ ] . surfactant-like peptides self-assemble either into nanotubes or nanovesicles [ ] [ ] [ ] [ ] (fig. . ). they are termed as surfactant-like due to the presence of a hydrophilic head comprising charged amino acids (lysine, arginine, glutamic acid, aspartic acid, etc.) and a hydrophobic segment comprising nonpolar amino acids (alanine, leucine, valine, etc.). intermolecular hydrogen bonding plays a major role in determining the structures formed by the self-assembly of these peptides. vauthey et al. were the first to design a self-assembling peptide that can self-assemble into nanotubes and nanovesicles [ ] . some common examples of surfactant-like peptides include a d, v d, v d , l d , and g d . these peptides initially self-assemble to form a bilayer, which later undergo further associations to form nanotubes. the nature of the amino acids in the sequence has an important role in dictating the type of self-assembled structures formed. the peptide sequences a k and a d both formed nanotubes often exhibiting twisted tape or fibrillar morphology. a heptapeptide, namely, ac-gavilrr-nh , formed donut-like ring structures [ ] . peptides like v d , v dvd, and v d v have been reported to form fibers, tapes, and twisted ribbons rather than nanotubes. the differences in the self-assembled structures formed could be attributed to the variations in the packing density of the peptide aggregates [ ] . the number of hydrophobic amino acids in each surfactant-like peptide also influences the final self-assembled structure. three peptides a k, a k, and a k, with different hydrophobic chain lengths were investigated for their self-assembling properties [ ] . while a k formed stacked bilayers, a k formed nanofibers and a k formed nanorods. the absence of higher-order structures in a k peptide was attributed to the absence of measurable critical aggregation concentration (cac), probably due to the short hydrophobic segment. several surfactant-like peptides found in nature also exhibit similar self-assembling characteristics to form vesicular structures that may be relevant to prebiotic enclosures that sequester enzymes from their environment [ ] . the lipid-like peptides are a subtype of surfactant-like peptides and consist of a hydrophilic head group and a tunable hydrophobic tail. though the lipid-like peptides possess different composition, sequence, and packing, they share several similarities with phospholipids that self-assemble to form lipid bilayers. the length of the peptide is about . nm, which is comparable to natural phospholipids. both systems self-assemble in water to form nanovesicles with an average diameter of - nm. a point of distinction between the two systems is in the nature of association between the individual components. in phospholipids, the acyl chains in the hydrophobic tails compactly pack together to displace water molecules from the interior and hydrophobic forces drive this process, which impedes formation of hydrogen bonds. however, in the case of lipid-like peptides, in addition to the hydrophobic tail packing, intermolecular hydrogen bonds are formed in the backbone. the presence of charged side chains in these peptides confers ph sensitivity as well as responsiveness to change in the ionic strength of the medium. carpet peptides also known as molecular paint peptides were first developed by zhang et al. [ ] . these peptides can undergo self-assembly and form monolayers, a few nanometers thick on a surface. these peptides thus act as a carpet for the attachment of cells or they can trap other molecules, thereby providing molecular recognition ( fig. . ). these peptides consist of three segments. the first segment or head contains ligands that serve as molecular recognition motifs for cell surface receptors. the middle segment serves as a linker that allows the head to interact at a distance away from the surface and also provides certain degree of flexibility to the peptide structure. the last segment or tail enables covalent binding with the surface. these peptides are widely used to study cell-cell communication. the peptide sequence rgdaaaaac is a typical example of a molecular paint peptide [ ] . the rgd segment serves as a recognition motif for the cell surface receptors integrins and hence can promote cell adhesion. the five alanine residues (aaaaa) serve as linkers, while the lone cysteine residue can enable anchoring of the peptide to gold substrates through its sulfhydryl group. similarly, the tetradecapeptide radsradsaaaaac that also possesses a ligand (rads) for cell recognition has been developed for painting gold surfaces [ ] . the switch peptides possess a unique ability to transform its molecular structure in response to environmental stimuli. for example, the hexadecapeptide dar -iv can form beta-sheet structures at ambient temperatures but transforms to an alpha helix when the temperature or ph of the system is modified. this suggests that secondary structures of sequences flanked by the negative charges on n-terminus and positive charges on c-terminus may undergo drastic changes if the ph and temperature are changed. these peptides were converted to electronically responsive structures through incorporation of metal nanocrystals [ ] . an undecapeptide with the sequence ac-qqrfqwqfeqq-nh was found to self-assemble into structures with progressively increasing order -tapes, ribbons, fibrils, and finally fibers [ ] (fig. . ) . the side chains of the glutamine (q) residues involve in hydrogen bonding and promote formation of b-sheets. the arginine (r) and glutamate (e) residues facilitate electrostatic interactions with the complementary countercharges on the neighboring chains leading to stabilization of antiparallel b-sheets forming a tape-like structure. the phenylalanine (f) and tryptophan (w) residues contribute to hydrophobic forces that drive the formation of ribbons. in the ribbon-like morphology, two tapes associate face-to-face stabilized by the hydrophobic forces leading to a twist. at higher concentrations of the peptide, the ribbons stack together to form fibrils. the substitution of the glutamine residues with glutamate (e) in the peptide sequence induces ph responsiveness in the peptide. at acidic ph (< ), the glutamate residue is protonated and hence will exhibit associative interactions promoting the existence of a nematic phase. as the ph is increased, the glutamate residues get deprotonated, and hence greater repulsive forces are introduced leading to transformation of the nematic phase to an isotropic fluid phase. thus the peptide acts as a molecular switch in response to ph changes by transforming reversibly between nematic and isotropic fluid phases. the design of cyclic peptides, whose dimensions and assembly could be tailored as desired, was inspired from the tubular pores formed by the tobacco mosaic virus [ ] . ghadiri and his coworkers were the first to report the self-assembly of a rationally designed cyclic octapeptide cyclo(l-gln-d-ala-l-glu-d-ala-) [ ] . these cyclic peptides have alternating d-and l-amino acids, which interact through intermolecular hydrogen bonding to form an array of self-assembled nanotubes with an internal diameter of - Å (fig. . ). the diameter of the tube depends on the number of amino acid residues forming the cyclic peptide. at alkaline ph, the carboxylate groups of glutamate residues become negatively charged as a result of deprotonation and prevent stacking associations due to strong electrostatic repulsive forces. at acidic ph, the carboxylate groups become protonated and hence favor association through extensive hydrogen bonding between the amide carbonyl and -nh-groups in the backbone. each cyclic peptide forms a flat ring that stacked over one another and is stabilized by hydrogen bonding resulting in a hollow nanotube. the side chains of the amino acids face the exterior of the tube to minimize steric repulsions. these side chains can also be functionalized to incorporate desired properties to the nanotubes. the cyclic octapeptide lanreotide nh -(d)naphthylalanine-cys-tyr-(d) trp-lys-val-cys-thr-conh , an analogue of somatostatin , also self-assembled into tubular structures with a diameter of nm and length running to several microns [ ] . this category of peptides represents a hybrid molecule formed from oligonucleotides and amino acids. the sequence of both components influences the nature of self-assembly. gour et al. have reported the self-assembly of a nucleopeptide formed by grafting the dipeptide ff to the -mer oligonucleotide with sequence ctctctctcttt [ ] . the diphenylalanine (ff) is part of the core recognition motif of the amyloid peptide and self-assembles to fibrillar structures in its pristine state. however, the nucleopeptide formed using this peptide motif self-assembled to spherical structures, which may be attributed to the hydrogen bonding interactions and amphiphilicity of this hybrid molecule. li et al. had developed nucleopeptides that self-assemble to form supramolecular hydrogels using the dipeptide ff conjugated to a nucleobase (a, g, t, or c). these nucleopeptides served as hydrogelators forming entangled nanofibers in water and could lead to many interesting biomedical applications [ , ] . peptide amphiphiles comprise of a hydrophilic head and hydrophobic tail that self-assemble in aqueous solution to form well-defined nanostructures like peptide bilayers, micelles, nanotubes, nanorods, and nanovesicles [ ] [ ] [ ] . these molecules can be chemically modified easily to tailor their properties for specific applications such as cell adhesion and internalization. the mechanistic insights into the self-assembly of peptide amphiphiles using v d as a model have suggested that the peptide amphiphile initially self-assembles into a bilayer and then into a cyclic vesicular form that undergoes stacking to form nanotubes. it has also been suggested that higher-order structures could be obtained by interconnection of these tubes through three-way junctions [ ] . most of the peptide amphiphiles form betasheet containing nanofibrils that can be induced either by addition of divalent salts or by altering the ph of the solution. the divalent cations form an ion bridge leading to stronger intra-and interfibrillar associations. experiments have revealed a high degree of solvation in the interior of the self-assembled structures formed by peptide amphiphiles [ ] . incorporation of a cysteine residue in a peptide sequence promotes reversible cross-linking of the peptide leading to modification of the stiffness of the peptide chain. another strategy to impart amphiphilic character to the peptide sequence is to incorporate a fatty acyl chain to the n-terminus of a peptide sequence. the acyl chain contributes to the hydrophobic character to the peptide amphiphile. using an elegant set of experiments, lowik et al. demonstrated the influence of alkyl chain length on the self-assembly of the peptide ganpnaag [ ] . the peptide molecules modified with c , c , and c acyl chains self-assembled into random coils independent of temperature, while those containing c and c acyl chains underwent a transition from b-sheets to random coils on increasing the temperature. increasing the acyl chain length contributes to enhanced hydrophobicity leading to differences in the thermal stability. in a seminal work, hartgerink et al. developed a peptide amphiphile with four distinct domains that self-assembled into cylindrical micelles in aqueous solution [ ] . the n-terminus of the peptide sequence cys-cys-cys-cys-gly-gly-gly-phosphoser-arg-gly-asp was modified with a -carbon alkyl chain that forms the hydrophobic component. the cysteine residues contribute to the formation of b-sheets and stabilize the self-assembled structure by covalent capture where superstructures are transformed into a supramolecule through covalent bonding. the disulphide bridges formed due to the oxidation of the cysteine residues confer rigidity to the supramolecular structure. further, stabilization of the structure is provided through extensive hydrogen bonding. the glycine-rich segment forms the flexible spacer domain. the phosphoserine residue confers charge and hence ph responsiveness to the sequence, while the rgd serves as a recognition motif for cell adhesion. this peptide amphiphile associated at acidic ph and dissociated at alkaline ph ( fig. . ). similar analogues have now been developed for many biological applications. reverse peptide amphiphiles like c o-veve with a free n-terminus were prepared using unnatural amino acid (ornithine, o) modified with a fatty acid chain and were mixed with conventional peptide amphiphiles containing free c-terminus. such de novo designed peptide amphiphiles formed nanobelts [ ] . a twisted nanoribbon morphology was observed when the cell adhesion motif rgd was incorporated in the c-terminus (c o-vevegrgd). apart from normal method of peptide synthesis, recombinant dna techniques have also been employed to produce two amphiphilic peptides, namely, ac-a v l wg -cooh and ac-a v l wg -cooh, which self-assembled into nanovesicles [ ] . a bolaamphiphilic peptide consists of two hydrophilic terminals linked through a hydrophobic segment. this class of peptides derives its name from the south american hunting weapon that consists of two balls linked by a string. stupp and his coworkers reported the self-assembly of a bolaamphiphilic peptide consisting of glycylglycine (gg) residues at either end linked through a -carbon acyl chain [ ] ( fig. . ) . the bolaamphiphilic peptides were ph responsive and formed helical ribbon-like structures at alkaline ph. the hydrophobicity of the acyl chain in the middle influences the twist in the structure so as to minimize contact with the polar environment. at acidic ph, the peptide self-assembles to form nanotubes presumably due to the additional hydrogen bonds formed through the protonated carboxylic groups of the amino acid residues. crick, in , first reported coiled-coil structures, commonly seen in many proteins. the major driving force is the hydrophobic interaction among the helices and a typical coiled structure consists of - left-handed alpha helices containing seven amino acid residues (heptad), each wrapped around each other to form a supercoil. according to the peptide velcro (pv) hypothesis, there are three major requirements for formation of such structures [ ] . the first and fourth residues must be hydrophobic to facilitate dimerization of the peptide chains along one face of the helix. the length of the hydrophobic side chain dictates the formation of dimers, trimers, or tetramers. increasing hydrophobicity stabilizes the self-assembled structure through van der waals' and hydrophobic interactions. the fifth and seventh amino acid residues should have charge to promote electrostatic interactions between the peptide chains. in order to facilitate attractive associations, it is important to have an acidic and a basic amino acid residue at these positions. leucine zipper proteins and cartilage oligomeric matrix proteins exhibit such type of coiled-coil structures. a right-handed alpha helical coiled-coil structure has also been identified in tetrabrachion, a protein from the bacterial staphylococcus marinus. this structure contains undecapeptide helices that possess a core filled with water, thereby exhibiting a different packing pattern with the hydrophobic and hydrophilic residues in the first, fourth, and eighth positions falling on the same face [ ] . apart from peptide velcro where one strand of acidic amino acid residues mingles with other strand of basic residues to form a parallel heterodimer, ryadnov et al. have designed belts and braces where two peptides are bound together by the third peptide of opposite charge. these belt and braces were used as a template to form colloidal gold particles and were commonly referred as peptide-mediated nanoparticle assembly [ ] . other types of self-assembled peptide systems include amphiphilic peptides in beta strand conformation which self-assemble into twisted tapes, helical dipolar peptides that undergo conformational change between a helix and beta-sheet similar to molecular switch, and surface binding peptides that form monolayers that are covalently bound to a surface. table . gives a list of some of the major peptide systems investigated for their self-assembling properties. formation of nanotubes and vesicles [ ] v k, v k , v k adsorption at air/water interface used for dna immobilization [ ] v k , l k , a k, v h, v k, h v , kv formation of nanotubes and vesicles [ ] ac-a d-cooh and ac-a k-cooh determination of critical aggregation concentration (cac) of particles formed during self-assembly [ ] mixtures of ac-a d-oh and ac-a k-nh formation of twisted fibrils [ ] ac-ga vilrr-nh formation of 'nanodonut' structures [ ] correlation of secondary structure with the morphology of nanostructures formed [ ] influence of sequence and purity on self-assembly [ ] a k, a k, a k determination of cmc, self-assembled structures, correlation with its antibacterial activity [ ] a k elucidation of nanotube structure and its mechanism of formation [ , ] ac-a v l wg -cooh and ac-a v l wg -cooh formation of vesicles [ ] chol-h r , chol-h r (chol denotes cholesterol) vehicles for delivering genes [ ] a h k , a h k , and h k (non-amphiphilic control) vehicles for delivering genes [ ] ac-(af) vehicles for delivering genes [ ] chol-g r tat, tat ¼ ygrkkrrqrrr antimicrobial activity [ ] a d and a k stabilization of g-protein-coupled receptor bovine rhodopsin against denaturation [ , ] ac-v r -nh , ac-v k -nh , ac-a k-nh , ac-i k -nh , ac-a k-oh, da -nh , ac-v d -nh , ac-a d-oh, ka -nh stabilization of protein complex photosystem-i and enhancement of activity [ ] where sl denotes the spin label acetamidoproxyl micelle aggregation and formation of particles and beads [ ] the formation of nanofibers can be promoted by peptides that have an ability to form beta-sheets. presence of branched amino acid residues confers an ability to transform from a a-helical structure to a b-sheet depending on the nature of the medium [ ] . the propensity of beta-sheet forming ability of peptide sequences can be retarded by introduction of proline residues at the n-and c-terminals. as proline lacks hydrogen bond-forming ability owing to its planar ring structure, it restricts the expansion of the beta-sheet network. this results in formation of straight nanofibers of about - nm in diameter and several mm long. the fine structure will show striations arising due to packing of tightly coiled alpha helical structures, especially if phenylalanine was one of the amino acid residues in the sequence. this is due to additional aromatic interactions contributed by phenylalanine. if phenylalanine was substituted by aliphatic hydrophobic residue such as isoleucine, the straight fibers formed revealed tape-like inner structures. attempts to functionalize these nanofibers to impart biorecognition have been made using biotin conjugation at their n-terminus [ ] . these biotin terminals can be used to tether molecules linked to anti-biotin molecules. however, such functionalization strategies resulted in a loss of the supramolecular assembly formed by the peptide. three main forces, namely, hydrophobic, hydrogen bonding, and coulombic forces, are mostly involved in controlling the self-assembly process [ ] . a combination of hydrogen bonding, p-p interactions, and van der waals' interactions promotes formation of columnar, disc-like aggregates, while hydrophobic effects and p-p stacking favor formation of d sheets and rectangular aggregates. cross-linking between peptide chains leads to rigid rods, while cross-linking in micellar assemblies leads to reduction curvature, thereby forming cylindrical micelles. rod-shaped structures can further stack to form columnar assemblies as their elongated, anisotropic geometry permits their preferential alignment along one spatial direction [ ] . hartgerink had employed three main design principles for customizing the peptide nanostructures that could be formed from cyclic peptide sequences [ , ] . the cyclic peptide should contain only eight amino acid residues as shorter sequences lead to strained amide backbone, while longer sequences will be flexible. steric interactions between the side chain and the backbone of the heterochiral alignment are prevented by designing the register of the stack in such a way that the rings will align with homochiral residues as their neighbors. side chain-side chain interactions are manipulated using glutamine residues that play a major role in intra-and intermolecular hydrogen bonding interactions. incorporation of non-peptide moieties into a self-assembled ensemble can lead to emergence of novel properties. for instance, the poor conductivity of self-assembled peptide nanostructures, which limits their use in sensing and diagnosis, can be overcome by the introduction of conductive polymers, enzymes, and metallic particles. alignment and positioning of the peptide nanostructures on a solid surface can be achieved through appropriate chemical modification of the substrate surface or peptide nanostructures, or both. atomic force microscopy (afm), dielectrophoresis, or optical tweezers have been employed for making appropriate connection between the self-assembled peptide nanostructures and transducers [ , ] . sedman et al. have used afm as a thermomechanical lithographic tool to create indents and trenches in the self-assembled nanotubes formed by diphenylalanine and dinaphthylalanine, thus utilizing them as nanobarcodes [ ] . reches and gazit have also tried manipulation of the peptide nanostructures using magnetic forces. apart from modification, functionalization of the peptide nanostructures has been carried out by rica and coworkers using dielectrophoresis (dep) for the incorporation of antibody-functionalized peptide nanotube on top of gold electrodes for the development of label-free pathogen detection chip [ ] . schnarr et al. have created coiled-coil heterotrimeric assembly by employing electrostatic forces, and this molecular self-assembly is driven by the hydrophobic forces, while the building blocks were matched via electrostatic interactions [ ] . many attempts have been made to improve stability of the self-assembled ensembles through cross-linking or enhancing associative forces. recently, selfassembled polymeric vesicles with enhanced stability, specificity, and tunability were formed from amphiphilic block copolymers with alternating hydrophilic and hydrophobic segments [ ] . introduction of a polypeptide chain in this amphiphilic block copolymer results in the formation of peptosomes [ , ] . the peptide segments are mostly associated with the hydrophobic segments resulting in the self-assembly. the peptide wnvfdflivigsiidvilse derived from the calcium channel forming protein caivs exhibits adhesive properties and thereby enhanced stability [ ] due to the cohesive forces between the chains that are responsible for driving their association even in the absence of water. gudlur et al. had designed two amphiphilic peptides with an oligolysine main chain (k ). the aand e-amino groups were both modified with either the hydrophobic nonapeptide flivigsii (h ) or the hydrophobic pentapeptide flivi (h ). these amphipathic lipid-like peptides when mixed in equimolar quantities (h h ) spontaneously self-assembled to form vesicular structures in an aqueous medium. differential scanning calorimetry studies on the h h peptide vesicles indicated good thermal stability over a wide range of temperature, and no alteration in the structure was observed [ ] . a peptide derived from human elastin consisting of hydrophobic repeats pgvgva along with the cross-linking regions composed of polyalanine interspersed with lysine [ ] formed highly insoluble nanofibers upon incubation at c. once fibers were formed, the side chain of lysine is converted to an aldehyde by lysyl oxidase enzyme, which then reacts with neighboring primary amines to form dehydrolysinonorleucine that then forms desmosine cross-links. these cross-linked fibers formed from a small peptide fragment of elastin exhibit good mechanical properties like resilience and strain at breaking point. covalent capture is a recently developed strategy that integrates the design and synthesis features offered by non-covalent self-assembly along with structural integrity. this approach involves covalent bond formation for stabilizing the self-assembled supramolecular ensembles without significantly affecting their structure. the formation of covalent bonds can occur before or after self-assembly. the order of self-assembly and the covalent bond formation play an important role in determining the physical structure of the aggregate formed. if the covalent bond is formed before self-assembly, either there will be low yield or the desired molecular aggregate will not be formed. however, if the covalent bond is formed after self-assembly, the pre-organization of reacting species happens, and the covalent bond formation is also enhanced. bilgicer et al. have employed the covalent capture method to dimerize two coiled peptides -one containing a leucine residue and the other an unnatural amino acid hexafluoro leucine at the same specific site [ ] . the dimerization of the two peptide sequences containing the natural leucine and unnatural fluorinated leucine hydrophobic residues was achieved by incubating them in a glutathione buffer. characterization of the self-assembled peptide structures for their electrical, physical, and chemical properties is essential to determine their potential in applications. a wide range of characterization tools have been employed to elicit such information on the self-assembled structures (table . ). this effort is intensifying as newer customized protocols become available with time to evaluate the performance and properties of novel peptide ensembles. different types of microscopic tools have been used to visualize the self-assemblies formed by peptides. apart from forming nanostructures, self-assembled peptides also form micrometer scale structures that can be analyzed using polarized light, epifluorescence, and confocal microscopy [ ] . while polarized light microscopy deals with birefringence and is employed to investigate the behavior of light-crystalline phases, epifluorescence and confocal microscopy are specific for the peptidic structures involving the fluorophores. polarized light microscopy is used to identify various mesophases like nematic, cholesteric, and cubic involved in the lyotropic behavior of the nanostructures formed from the amino acid units. epifluorescence microscopy is a technique that is widely applied to biological systems. the sample is irradiated with electromagnetic radiation of a particular wavelength known as excitation wavelength and the longer wavelength that is emitted from the sample is then detected. in epifluorescence microscopy, both excitation and observation of the emission occur from above ('epi') the sample. this technique has been applied to detect both intrinsic fluorescence of the peptide structures and the emission from fluorophores linked to the peptide chains. the topography of the self-assembled structures formed by two amphiphilic peptidolipids derived from the - residues of the amyloid beta peptide (c -iiglm-oh and c -iiglm-nh ) was observed using this technique [ ] . studies on the association between the ff nanotubes and pyrenyl derivatives have revealed that the final structure and its photophysical response are found to be dependent on the ph and the fluorophore concentration. when the pyrenyl concentration is low and when the ph is less or equal to , the structures formed are shorter and thinner, while at higher peptide concentrations and at alkaline ph, the fibrils formed are thicker [ ] . these findings confirm that the final structure is due to the balance between electrostatic and hydrophobic forces. at lower ph, protonation of carboxyl groups of either pyrenyl chromophore or ff molecules occurs, and hence electrostatic interactions become weakened, while the aromatic p stacking between the aromatic rings of the pyrenyl structure dominates. at neutral ph, though the amino groups are protonated, the carboxyl groups of ff and pyrenyl determination of presence of beta-sheet structures. employed in studying the mechanism of amyloid fibril formation surface plasmon resonance spectroscopy atomic force microscopy investigation of the kinetics of self-assembly, conduct structure manipulation, determine the size and morphology of the nanostructures formed during the self-assembly diffraction studies nanofiber alignment and determination of cross b-sheet structures electron microscopy morphology of self-assembled structures gel electrophoresis determination of monomers and its subsequent polymerization into dimers, tetramers, and oligomers circular dichroism determination of secondary structure and transition between secondary structures during self-assembly process fourier transform infrared spectroscopy determination of beta-sheet structures are not protonated, and hence only weak induced dipole interactions are favored. at alkaline ph, both carboxyl and amino groups are deprotonated, and hence electrostatic forces cannot compete with the hydrophobic forces resulting in side-chain contacts to form thicker fibrils [ ] . thioflavin t (tht) is a fluorescent molecule that binds to beta-sheet structures of peptide assemblies. the binding results in a red shift in the emission of thioflavin t from to nm. the presence of aromatic residues that contribute to p-p stacking interactions of the aromatic rings in tht with the peptide causes a change in the charge distribution of tht in its excited state causing the red shift [ ] . the rigidity of the peptide structure and its flatness are additional factors that contribute to tht binding. one of the limitations of tht is its poor solubility in aqueous solvents. to overcome this issue, a sulfonated analogue thioflavin s (ths) has been introduced with sulfonated groups [ ] . congo red is yet another fluorescent probe that has exhibited selective binding to the beta-sheets of amyloid and amyloid-like fibrils [ ] . confocal laser scanning microscopes (clsms) provide high in-plane resolutions by restricting entry of out-of-focus light by employing a pinhole for illumination of a sample, thus making it an attractive tool for investigating peptide self-assemblies. they are mainly used in d reconstruction of images recorded at different slices along the z direction. the imaging of the peptide nanostructures formed has been accomplished by using confocal laser scanning microscopy (clsm). ff microtubes labeled with rhodamine b were imaged using clsm [ ] , and this technique has been used to identify the hydrophobic-rich and hydrophilic-rich regions by labeling ff structures with two fluorescence dyes, namely, rhodamine and phthalocyanine. since rhodamine is relatively hydrophilic, it was localized in the inner core of the peptide assembly, which consists of hydrophilic clusters. phthalocyanine was found in the hydrophobic external wall of ff nanostructures. the clsm enabled visualization of both the hydrophilic and hydrophobic clusters by reconstructing the images in d. similarly, the distribution of the peptide amphiphiles c -vvvaaaggklakklakklakklak and c -vvvaaakkk in a hyaluronic acid membrane was imaged using clsm [ ] . the peptide amphiphiles were modified with a fluorophore at the n-terminus to enable imaging. the z-sectioning served to understand the localization of the peptide self-assemblies within the membrane. the effect of the nanofibers formed by the self-assembly of glucagon-like peptide (glp- ) mimetic peptide amphiphiles on the cell viability and proliferation of rat insulinoma cells were investigated using clsm. the clsm technique has been extensively used as a powerful tool to investigate cell morphology, migration, and proliferation. the influence of a self-assembled scaffold formed from an ionic complementary peptide modified with biorecognition motifs on the cell morphology, spreading, and migration was investigated using clsm. the results indicated that the designer peptides modified with cell adhesion motif from osteopontin and signaling motif from osteogenic signaling peptide promoted excellent growth and proliferation of osteoblasts. scanning probe microscopies, especially the atomic force microscopy (afm), have been widely used to investigate the geometry of the self-assembled nanostructures, to measure their conductivity, and to determine the young's modulus and thermal stability of the structures under dry conditions. the afm contains a probe attached to a flexible cantilever, and as the probe moves over the sample at a preset rate, the force of interactions between the probe tip and the sample surface is measured, and the topography of the sample is constructed. the deflections in the cantilever are recorded by monitoring the reflection of a laser beam focused on the cantilever and recorded through a photosensitive photodiode. this technique offers atomic level resolution and can be a very valuable tool in research on self-assembled structures. the probe tip, generally in the range of nm, can be conducting or nonconducting and can be functionalized to investigate specific interactions. the material of the probe and its geometry are vital in determining its performance. the data acquisition in afm can be made in the contact mode or noncontact mode or tapping mode. one of the challenges involved in using afm technique is to study peptide self-assembly in solution as it requires sufficient adhesion with the substrate. generally, afm had been extensively employed to study the morphology of the aggregates formed by the self-assembly of peptides. chaudhary et al. had investigated the propensity of two sequences derived from the amyloid tau protein ac-vqivyk-amide and ac-qivyk-amide to form beta-sheets in the presence of different solvents using afm [ ] . the results revealed that the ac-vqivyk-amide existed both as alpha helices and beta-sheets and the nature of the solvent was a key determinant of the form in which the peptide aggregates existed. the formation of the supramolecular assemblies by the peptide can also be monitored in a time-dependent manner using the afm probe. time-lapse liquid imaging of amyloid fibrils has been employed for kinetic studies on the rate of formation as well as morphological changes introduced in the amyloid peptide during the aggregation process under various self-assembling conditions [ ] . for the determination of young's modulus, the afm tip is positioned on the top of the structure and pressed. from the force-distance curves, the young's modulus can be directly derived using the theoretical model proposed by niu et al. [ ] . however, this model has a limitation since it depends on the type of structure that can be assumed. for example, if it is a hollow structure rather than a solid, then the young's modulus calculated using this model will exhibit significant deviations from the actual value. knowles et al. have used another approach to understand the rigidity and mechanical strength of the self-assembled amyloid fibrils on mica substrate [ ] . the topography of more than fibrils was imaged using afm and the shape fluctuations were used to compute the bending rigidity (c b ) of the fibrils. the cross-sectional moments of inertia (i) were computed for each fibril based on their height measured using afm. the young's modulus (y) was then computed as y ¼ c b /i. these values range between and gpa for most protein fibrils. the young's modulus for the completely self-assembled amyloid fibrils falls in the range and gpa. the elastic modulus of the peptide assemblies can be further dissected into contributions from the backbone as well as the side chains, i.e. y ¼ y bb + y sc where y bb and y sc are contributions from the peptide backbone and side chains, respectively. the computed results suggest that the contribution of the backbone interactions towards the elastic modulus is more than twice that of the side chains. in the case of amyloid fibrils, the contribution of y bb is about %. it is also postulated that peptide structures with moduli greater than gpa have significant contributions from the involvement of side chains in hydrogen bonding. in the absence of significant intermolecular hydrogen bonding in the peptide structures, the surface tension arising due to the hydrophobic and hydrophilic residues can also be employed to determine the young's modulus using the relation y ¼ g/h where g is the surface tension and h is the inter-sheet spacing with values in the range of and Å . for the observation of thermal stability of the nanostructures under dry conditions, the afm tip is positioned above the structures and the position of the tip is monitored with increase in temperature. at a particular temperature, as the structure degenerates, the tip will move downward which gives an indication of the maximum temperature beyond which the nanostructure will lose its stability. transthyretin fibrils have been examined for their stability after prolonged exposure to high temperatures using afm, and it was found that the thermal stability of the pre-fibrillar aggregates was poor when compared to the fibrillar assemblies [ ] . electron microscopic techniques, which include scanning and transmission electron microscopy, are commonly employed tools for imaging self-assembled structures. focused ion beam milling techniques were also recently employed to characterize the nanostructures. scanning electron microscopy (sem) is usually used to determine the geometry of the nanostructure. however, the presence of any defects or cavities in the structure can be better visualized using transmission electron microscopy (tem) where high-energy electrons pass through the nanostructures and the final image is reconstructed by mapping the intensities of electrons from each point in the sample. the more conducting regions in the sample will therefore appear dark when compared with regions with some resistance to the passage of electrons. the imaging is usually done in ultrahigh vacuum of the order of - pa. the main advantages of electron microscopy techniques when employed for visualizing the morphology of the peptide structures are ease of implementation, high magnification, resolution, and its ability to image a range of dimensions ranging from sub-nanometric to micrometric scales. these prospects are possible due to the smaller wavelength of electrons employed when compared to the visible light. the scanning electron microscopic technique when applied to nonconducting samples such as peptide nanostructures requires a thin coating of an inert metal such as platinum or gold to enable generation of the image. otherwise, the electrons will remain on the surface of the sample making the visualization of the finer structures on the sample impossible. in the case of transmission electron microscopy, there is a restriction in the thickness of the sample that can be imaged. samples less than . mm alone can be imaged using transmission electron microscopy, as thicker samples will not permit transmission of the electrons. it is not advisable to use accelerating voltages beyond kv in scanning electron microscopy for analyzing peptide nanostructures. similarly, in transmission electron microscopy, very high voltages will lead to damage of the peptide structures. apart from giving information on the morphology of the aggregates, the dimensions of the individual self-assembled structures can also be obtained from the electron microscopy techniques. the thermal stability of the structure can also be investigated using sem where after exposing the nanostructure to particular temperature, the structures can be imaged to observe any deformations postexposure to high temperature. the strength of the structure can be determined using sem combined with fib source (fib -focused ion beam). the nanostructure is placed inside fib sem and the time taken to mill the sample is analyzed [ ] . by comparing the time with the standard materials, the stability of the structure can be determined. the incorporation of metallic compounds in the peptide assemblies can be imaged using tem. for instance, tem was employed to determine the presence of cuo within the nanotubes due to the enhanced contrast provided by the metallic compounds [ ] . recently modified ff peptide nanotubes were used for the determination of the neurotransmitter dopamine. the ff nanotubes were modified with cyclic-tetrameric copper (ii) species containing the ligand ( -imidazolyl)-ethylene- -amino- -ethylpyridine [cu (apyhist) ] + (apyhist refers to the ligand -( h-imidazol- -yl)-n-( -(pyridin- -yl)ethylidene)ethanamine) in nafion membrane on a glass carbon electrode. the morphology of the modified tubes imaged using scanning electron microscopy revealed tubular structures of thickness around - nm. the deposition of the nafion membrane on the nanotubes was also clearly distinguished [ ] . circular dichroism (cd) is a technique that is applied to optically active chiral molecules such as proteins and peptides. it is based on the principle of differential absorption of right and left circularly polarized light by the chiral molecule. cd spectroscopy is an invaluable tool to identify the secondary structures adopted by a peptide during the self-assembly process. the exact location of the alpha helices or beta-sheets or random coils can be identified by this technique, and it provides information on the structural transformations that can occur during the self-assembly process under different conditions. hauser et al. have used this technique to show that the self-assembly process proceeds through structural transition that may occur in three possible steps based on the peptide concentration [ ] . the peptide monomers interact via antiparallel pairing which is followed by a structural transition to a-helical conformation. the peptide pairs assemble to form fibers and condense to form fibrils. the assembly of the peptide monomers serves as the nucleation step, which then proceeds to form fibrils via nucleation-dependent polymerization mechanism. the assembly of peptide monomers, however, requires a transition from random coil to a-helical conformation. though it is reported that short peptides of - amino acids cannot form a-helical conformation, it is indeed possible above a threshold concentration. the cd spectra also demonstrated that at low concentrations, the peptides were stable up to c and changed its random coiled structure as the temperature is increased from c to c. this change was reversed on cooling. however, once the fibril is formed, the peptide ensembles adopt b-turn structures, and no reversal in conformation is observed upon changing the temperature. circular dichroism has also been widely used to study the kinetics involved in the formation of a peptide network. it was used to show that the beta-sheet structures progressively increase with concomitant reduction in helical coils in peptides with a propensity to form fibers [ ] . zhang et al. had reported that the ionic complementary peptide eak formed a macroscopic membranous structure due to extensive beta-sheet formation [ ] . the membrane formation propensity was retarded in the dodecapeptide eak , while the octapeptide eak did not form membranous structure. investigations with cd spectroscopy revealed that the eak formed beta-sheets extensively, while eak had both alpha helix and beta-sheets and eak had only random coils. similarly cd spectroscopic studies on the self-assembly of kfe (fkfefkfe) revealed the presence of left-handed double helical beta-sheets that could represent a new category of molecular materials. the properties of the peptide bond can be studied using x-rays. linus pauling and robert corey were the first to report the length of c-n bond in the peptide link. they also found that the peptide bond is planar, i.e. all the four atoms in the peptide bond are located in the same plane and the a-carbon atoms attached to the c and n are in trans conformation. the structural and functional relationships in the fibrous protein in wool were established using x-ray diffraction patterns by william astbury [ ] . x-ray diffraction has been now employed to determine the crystal structure of any newly synthesized peptide. it has also been used to calculate the bond distances and bond angles between the moieties in the peptide. based on the torsion angle measurements, it is also possible to predict the secondary structure conformation adopted by the peptide. moreover, the forces that stabilize the crystal structure such as van der waals and hydrogen bonding interactions can also be predicted. x-ray diffraction technique is also employed to determine the ultrastructural organization found in self-assembled peptide structures. nanofibrillar arrangement results in characteristic diffraction patterns that reveal the presence of cross b-sheet structures. the meridional and equatorial reflections have been used to identify the amyloid structure. the spacing between the hydrogen-bonded b-strands results in meridional reflection at . - . Å , and the distance between the b-sheets contributes to an equatorial reflection at and Å [ ] . the main challenge in employing x-ray diffraction techniques is that it is difficult to obtain pure crystals of many self-assembled structures. the x-ray data is usually obtained using a beam wavelength of . Å and an extremely small beam size of about mm. generally, the crystals need to be cooled to about k for data recording. localized radiation damage to the crystals could be avoided by illuminating the peptide crystals at different locations. due to the high degree of complexity involved in understanding the mechanism of self-assembly, various theoretical and computational methods have also been employed. these techniques not only help in understanding the mechanism but also to design the new sequences with the selected properties for nanobiotechnological applications. in particular, molecular dynamic simulations have been used to monitor the dynamics and investigate the influence of mutations and solvent effects in the conformational transition from alpha helices into beta-sheet structures [ ] . various algorithms and theories have been developed to investigate the properties of complex biomolecular systems at different levels. due to the high degree of complexity involved along with the small time scales, coarsegrained models were used to follow the actual process [ ] . however, in this model, fine atomic details are neglected and only relevant degrees of freedom of peptide molecule are retained. activation relaxation technique is a subcategory of the coarse-grained model, where the interactions among several peptide chains were simulated ab initio with no bias in original orientation and conformation. molecular dynamics studies have now been employed to address peptide self-assembly [ ] . these simulations have certain advantages such as ability to mimic the actual self-assembly of the peptide residues, reveal the structural information at an atomic level of the peptide, and provide a dynamic molecular model at atomic level of ordered assembly. however, there are certain limitations associated with this technique, too, such as a limited resolution, strong dependency of the quality of predictions on the size of the peptide, and the limited number of peptide residues for which such predictions could be made. several computational approaches have been used to predict the aggregation propensity of the proteins. for example, a computer program named tango considers the secondary structure and the desolvation penalty of the residues for calculating the aggregation propensity [ ] . all the techniques discussed thus far are used to characterize the nanostructures in dry conditions. however, stability of the structures differs when they are analyzed in wet conditions. to determine the stability of the nanostructures in wet conditions, they are submerged in aqueous solution, and the concentration of monomers is monitored with time using high-performance liquid chromatography (hplc). the increase in the concentration of monomers with time is an indication that the nanostructures are dissolving in the medium and are, therefore, not stable. high-performance liquid chromatography has also been employed to identify impurities associated with the peptide after synthesis [ ] . microrheometry technique is widely used to perform measurement on weak hydrogels without affecting their structural components. the measurements are very fast, and the experiments are not affected by external factors as they carried out in a closed chamber. recently, multiple particle-tracking microrheology experiments were used to follow the hydrogel formation by the peptide kfe [ ] . fourier transform infrared spectroscopy (ftir) analysis reveals the presence of parallel or antiparallel beta-sheets and nature of hydrogen bonds in the self-assembled structures. differential scanning calorimetry (dsc) and thermogravimetric analysis (tga) are used to determine the effect of temperature on the self-assembled structures. while dsc gives information on the phase transition temperatures of the peptide assembly, tga provides information on the degradation profile of the assemblies. crystallographic studies have also been carried out to understand the mechanism of self-organization. biocompatibility and immunogenicity of the selfassembled peptide structures have to be evaluated before their use in biological systems. both in vitro and in vivo studies need to be carried out to evaluate the biocompatibility of a peptide assembly. the self-assembled structure fabricated using the fmoc-diphenylalanine peptide was used as a scaffold to grow chinese hamster ovary (cho) cells. the cell viability analyzed using mtt assay showed that % of the cells were viable, suggesting that the peptide scaffold is biocompatible. more in-depth biocompatibility and immunogenicity assessment is however necessary to eliminate any potential risk of using these structures for biomedical applications [ ] . the unique reproducible structures obtained through self-assembly of peptide sequences have many interesting applications in biological and nonbiological fields. figure . depicts multiple applications that can arise from a cyclic peptide. a few of them are highlighted in the following sections. peptide systems that form nanofibrils similar to those formed by the amyloid beta peptide have been investigated extensively to understand the mechanism of formation of amyloid fibrils. amyloid fibril formation has been implicated in a wide range of degenerative diseases like alzheimer's, parkinson's, type ii diabetes, and other prion-related diseases [ ] [ ] [ ] [ ] . the switch peptides have also been employed to find information about the interactions between the various proteins involved in the pathology of protein conformation diseases like scrapie, kuru, huntington's, parkinson's, and alzheimer's disease [ , ] . amyloid fibrils are formed by various peptides such as the full-length human islet amyloid polypeptide (hiapp), nfgsvq peptide fragment from medin, and ff peptide from alzheimer's amyloid beta peptide. nfgail a hexapeptide fragment from islet amyloid polypeptide is reported to form well-ordered amyloid fibrils which exactly mimics those formed by the parent peptide [ ] . two active amyloidogenic peptides, namely, nflvh fragment of hiapp and nfgsvq fragment derived from aortic medial amyloid, also formed fibrils. another peptide from the human calcitonin, namely, nh -dfnkf-cooh, is also reported to form amyloid fibrils similar to those formed by the parent protein [ ] . a short truncated tetrapeptide, namely, nh -dfnk-cooh, also formed fibrils, which clearly shows there is no correlation between hydrophobicity and amyloidogenic potential since most of the short peptides are relatively hydrophilic. these results were further supported by johansson et al., who studied charged tetrapeptides and proved hydrophobicity is not sufficient for fibril formation [ ] . peptide legos (rada, eak), peptide amphiphiles, bolaamphiphiles, peptide conjugates, ionic self-complementary peptides, and long-acting gonadotropin-releasing hormone have all been reported to self-assemble into amyloid fibril structure. many peptides derived from the alzheimer's amyloid beta peptide also been shown to form fibrils. these include aaklvff, klvffae, ßaßaklvff, yyklvffc, ffklvff-peg, ffklvff, ac-klvffae-nh , ff, and yyklvff-peg [ ] [ ] [ ] [ ] . it has been concluded that alternating binary patterns and the peptides with high beta-sheet propensity are prone to form amyloid structures. gazit and coworkers have identified that aromatic amino acids play an important role in amyloid fibril formation through p-p stacking interactions [ ] . custom-made peptides used as model systems should possess strong electrostatic binding with the negatively charged lipid membranes and should exhibit structural transition from random coil to beta-sheet on binding to lipid membranes which initiates the association and the formation of oligomers and larger aggregates. apart from utilizing the self-assembling peptides to understand the pathological mechanism, it is also used to understand the functions and mechanisms of assembly of proteins like collagen. tobacco mosaic virus (tmv) is also a form of supramolecular structure assembled from a single strand of mrna along with many copies of identical coat proteins, which organize to form rod-like shape. this tmv has been used by schlick et al. for the construction of nanoscale materials without disrupting its self-assembly [ ] . peptide molecules also have the ability to self-organize at the air-water interface, which are widely used for mineralization studies because of its resemblance to insoluble proteins found in nature. self-assembling peptides have gained considerable interest in the field of tissue engineering because functional tissue recovery has been observed in brain and heart lesions [ , ] . one common example is the incorporation of cell adhesion motif rgd in the peptide sequence, which improves the cell adhesion onto the self-assembled structures formed by peptide amphiphiles. collagen-mimetic peptide amphiphiles, which self-assemble into nanofibers that exactly mimic the structural and biological properties of the native collagen, have been widely used in tissue regeneration as natural collagen is immunogenic and difficult to process [ ] . molecular lego peptides, such as rad -i and eak , have been used as scaffolds for neurite outgrowth, hepatocyte regeneration, etc. [ ] . chondrocytes encapsulated in a peptide scaffold using a self-assembled peptide with the sequence (ac-kldlkldlkldl-nh ) exhibited excellent phenotype and secreted its own growth factors within weeks of culture [ ] . peptide nanovesicles have been employed as drug carriers and it is believed that these nanovesicles enter the cells via endocytosis mechanism and thus can deliver drugs, genes, etc. [ ] . hydrogelating self-assembling fibers (hsafs) designed by woolfson and group using coiled-coil assemblies had limited use as a drug delivery system due to the limitation of rapid drug release. however, this was overcome by incorporating an anionic polyelectrolyte in the cationic peptide [ ] . ghosh et al. designed a smart self-assembling peptide amphiphile that transforms from a linear or spherical form to fibrous form upon altering the ph for drug delivery and in vivo imaging applications [ ] . the nanotubes formed by cyclic peptides comprising d-and l-amino acids serve as ion channels and form pores in the membranes leading to disruption of the cell architecture. as d-amino acids are taken up preferentially by microbes, these systems can function as effective antimicrobial agents which kill the microorganisms through formation of pores in the cell membrane, thereby causing osmotic collapse [ ] . mackay et al. have created a chimeric polypeptide consisting of an elastin-like polypeptide (elp) fragment and a short cysteine-rich fragment for the treatment of cancer [ ] . the drug conjugated with the cysteine residue drives the self-assembly that finally forms a drug-rich core and a hydrophilic peptide corona. instead of a drug, the self-assembly can also be triggered by linking the hydrophobic cholesterol moieties with hydrophilic cell penetrating peptides consisting of six arginine residues. these peptide nanoparticles have antimicrobial properties and were shown to effectively terminate the bacterial growth in the infected brains of the rabbits. beta-sheet-rich peptide nanofibers functionalized with b-cell and t-cell epitopes have been developed for immunization [ ] . these functionalized nanofibers developed antibodies in mice when injected with saline, and the levels were comparable with those injected with complete freund's adjuvant. protection against the beta-sheet-rich peptide nanofibers lasted for a year and the antibody response is t-cell dependent. recently, self-assembling peptide/protein nanoparticles have been used as antigen display systems for the development of vaccines. for example, a fragment of the surface protein of severe acute respiratory syndrome coronavirus (sars-cov) was incorporated in a self-assembling peptide nanofiber in the native trimeric coiled conformation [ ] . antibody formation was elicited when these peptide structures were injected into the mice, and the antibodies were conformation specific as determined by qualitative enzyme-linked immunosorbent assay. peptide nanofibers have been extensively investigated as the next-generation biosensors where they are used both as a fabrication material and also as a component in the final system (biofet) [ ] . nanotubes coated with proteins, nanocrystals, and metalloporphyrins by hydrogen bonding have been employed as chemical sensors. these nanotubes also served to improve the catalytic activity of the enzymes like lipase. apart from using ff nanotubes as a template for the fabrication of nanowires, they have also been used for biosensing where ff nanotubes are deposited on the surface of screen-printed graphite and gold electrodes for improving the sensitivity. it is also reported that these electrodes exhibit greater sensitivity compared to those electrodes modified with carbon nanotubes. the possible reason may be that the ff nanotubes increase the functional surface area of the electrode. electrical and magnetic fields have been used to align ff nanotubes. apart from this, patterning of the ff tubes has been carried out using inkjet technology, machined by thermomechanical lithography via atomic force microscopy, manipulated and immobilized using dielectrophoresis, and arranged on the surfaces by low electron irradiation [ ] . nanotubes are also used in the detection of pathogens, neurotoxins, glucose, ethanol, and hydrogen peroxide and as an immunosensor [ ] . peptide nanofibers have been reported for the detection of copper, dopamine, yersinia pestis, glucose, etc. [ , [ ] [ ] (fig. . ). amyloid-forming peptides are used in the field of nanoelectronics as nanowires. for example, ff nanotubes are used as a template for silver nanowires of diameter of approximately nm. switch peptides find application in molecular electronics by serving as a nanoswitch [ ] . ryu et al. developed photoluminescent peptide nanotubes by incorporating luminescent complexes composed of photosensitizers like salicylic acid. kasotakis et al. employed self-assembled peptides as a scaffold for the introduction of metal-binding residues at specific locations within the structure [ ] . an octapeptide from the fiber protein of adenovirus was used to design cysteine-containing octapeptides that can bind to silver, gold, and platinum nanoparticles. these metal-decorated fibers were employed in photodynamic therapy and also used in the development of surface-enhanced raman-scattering biosensors for detecting dna. lipid-like peptides find application in solubilizing, stabilizing, and crystallizing membrane proteins, used for drug formulations and also as model systems for studying protein conformational diseases [ ] . velcro peptides have been mainly used to study cell-cell communication and cell behavior [ ] . ff nanotubes are used as an etching mask for the fabrication of silicon nanowires. it reduces fabrication time, cost, and use of aggressive chemicals. it is also possible to scale up these arrays of ff nanotubes by vapor deposition methods. self-assembling peptide nanostructures have also been used as photosynthetic devices, and the peptide ac-klvffae-nh is reported to form nanotubes. precise ordering of strong chromophores along the inner and outer walls of the nanotubes enables utilization of this peptide structure as nanoscale antennas and photosynthetic device [ ] . the peptide nanofibers can also serve as a template for the growth of inorganic materials such as silver, gold, platinum, cobalt, nickel, and various semiconducting materials (fig. . ). an in-depth understanding of the molecular self-assembling mechanism of peptides and various stabilizing forces associated with the overall stability of the nanostructures formed by them is essential to design novel applications using these peptides. various classes of self-assembling peptides have been developed that have led to emergence of novel applications in the fields of tissue engineering, drug delivery, electronics, etc. different characterization tools have been designed to decipher the structural and functional aspects of the self-assembled peptide structures. the field of self-assembly continues to expand and has opened up new vistas for further research. synthesis of a tubular polymer from threaded cyclodextrins self-assembly of peptide-based colloids containing lipophilic nanocrystals transition of cationic dipeptide nanotubes into vesicles and oligonucleotide delivery novel electrochemical biosensing platform using self-assembled peptide nanotubes self-assembly and application of diphenylalanine based nanostructures peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems emerging biological materials through molecular self-assembly fabrication of novel materials through molecular self-assembly design of molecular biological materials. using peptide motifs building from bottom up: fabrication of molecular materials using peptide construction motifs construction of biologically active protein molecular architecture using self-assembling peptide-amphiphiles induction of protein-like molecular architecture by self-assembly directed self-assembly: expectations and achievements designer self-assembling peptide nanomaterials self-assembly of chiral dna nanotubes design and characterization of programmable dna nanotubes lipid tubules: a paradigm for molecularly engineering structures chiral self-assembly of nanotubules and ribbons from phospholipid mixtures hydrogen-bonded sugar-alcohol trimmers as hexadentate silicon chelators in aqueous solution carbohydrate nanotubes biological and biomimetic materials biomimetism and bioinspiration as tools for the design of innovative materials and systems biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel challenges and breakthroughs in recent research on self-assembly patterning surfaces with functional polymers organogels as scaffolds for excitation energy transfer and light harvesting microcapsules containing a biomolecular motor for atp biosynthesis self-assembled peptide nanostructures: the design of molecular building blocks and their technological utilization designing peptide based nanomaterials tuning the mechanical and bioresponsive properties of peptide-amphiphile nanofiber networks self-assembling organic nanotubes based on a novel cyclic peptide architecture role of p-p stacking in the self-assembly of amyloid fibrils self-assembly of peptides: influence of substrate, ph and medium on the formation of supramolecular assemblies self-assembled peptide nanostructures for biomedical applications: advantages and challenges, biomaterials science and engineering, prof. rosario pignatello (ed) manipulation of self-assembly amyloid peptide nanotubes by dielectrophoresis zuotin, a putative z-dna binding protein in saccharomyces cerevisiae de novo designed peptide-based amyloid fibrils rational molecular design of stimulus-responsive supramolecular hydrogels based on dipeptides morphology control of one-dimensional peptide nanostructures sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures structures of helical beta-tapes and twisted ribbons: the role of side-chain interactions on twist and bend behavior concentration effect on the aggregation of a self-assembling oligopeptide self-assembly of nanodonut structure from a cone-shaped designer lipid-like peptide surfactant molecular designer self-assembling peptides dipoles of the alpha-helix and beta-sheet: their role in protein folding self-assembly at all scales molecular self-assembly of surfactant-like peptides to form nanotubes and nanovesicles self-assembly of surfactant-like peptides with variable glycine tails to form nanotubes and nanovesicles positively charged surfactant-like peptides self-assemble into nanostructures self-assembling behavior of designer lipid-like peptides self-assembly of peptide surfactants hydrophobic region induced transitions in self-assembled peptide nanostructures ) ß structures of alternating polypeptides and their possible prebiotic significance biological surface engineering: a simple system for cell pattern formation context-dependent secondary structure formation of a designed protein sequence conformational behavior of ionic self-complementary peptides peptide-based stimuli-responsive biomaterials high-resolution conformation of gramicidin a in a lipid bilayer by solid-state nmr self-assembling peptide nanotubes biomimetic organization: octapeptide self-assembly into nanotubes of viral capsid like dimension self-assembling dna-peptide hybrids:morphological consequences of oligopeptide grafting to a pathogenic amyloid fibrils forming dipeptide ) ß-peptide helix bundles long-range interactions stabilize the fold of a non-natural oligomer integration of photosynthetic protein molecular complexes in solid-state electronic devices self-assembling peptide detergents stabilize isolated photosystem i dynamic behaviors of lipid-like self-assembling peptide a d and a k nanotubes understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity tuning secondary structure and self-assembly of amphiphilic peptides self-assembly and mineralization of peptide-amphiphile nanofibers self-assembly of giant peptide nanobelts self-assembly of recombinant amphiphilic oligopeptides into vesicles label-free pathogen detection with sensor chips assembled from peptide nanotubes tetrabrachion: a filamentous archaebacterial surface protein assembly of unusual structure and extreme stability belt and braces: a peptide-based linker system of de novo design interfacial dynamic adsorption and structure of molecular layers of peptide surfactants synergistic effect and hierarchical nanostructure formation in mixing two designer lipid-like peptide surfactants peptide nanotube nematic phase structure of single-wall peptide nanotubes: in situ flow aligning x-ray diffraction cationic micelles self-assembled from cholesterol-conjugated oligopeptides as an efficient gene delivery vector self-assembled cationic peptide nanoparticles capable of inducing efficient gene expression in vitro self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect a class of cationic triblock amphiphilic oligopeptides as efficient gene delivery vectors self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent designer peptide surfactants stabilize functional photosystem-i membrane complex in aqueous solution for extended time reversible peptide particle formation using a mini amino acid sequence micro and nano techniques for the handling of biological samples biological and chemical decoration of peptide nanostructures via biotin-avidin interaction novel polymers: molecular to nanoscale order in three-dimensions supramolecular honeycomb and columnar assemblies formed by self-assembly of coil-rod-coil molecules with a conjugated rod segment electrostatic force microscopy of self-assembled peptide structures qualitative mapping of structurally different dipeptide nanotubes thermomechanical manipulation of aromatic peptide nanotubes the colloidal domain: where physics, chemistry, biology and technology meet biomimetic surfactants: biosurfactants peptide nanovesicles formed by the self-assembly of branched amphiphilic peptides sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin engineering protein specificity: gene manipulation with semisynthetic nucleases secondary structure nucleation in peptides. transition metal ion stabilized alpha-helices programmed self-sorting of coiled coils with leucine and hexafluoroleucine cores microscopy tools for investigating nano-to-mesoscale peptide assemblies peptidolipid as binding site of acetylcholinesterase: molecular recognition of paraoxon in langmuir films influence of ph and pyrenyl on the structural and morphological control of peptide nanotubes bioorganic chemistry neurotoxic effects of thioflavin s-positive amyloid deposits in transgenic mice and alzheimer's disease is congo red an amyloid-specific dye? rhodamine b and rhodamine as reference substances for fluorescence quantum yield measurements a bioactive self-assembled membrane to promote angiogenesis morphology of self-assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved time-lapse atomic force microscopy in the characterization of amyloid-like fibril assembly and oligomeric intermediates using the bending beam model to estimate the elasticity of diphenylalanine nanotubes nanostructured films from hierarchical self-assembly of amyloidogenic proteins transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils self-assembled peptide nanotubes as an etching material for the rapid fabrication of silicon wires confined conversion of cus nanowires to cuo nanotubes by annealinginduced diffusion in nanochannels electrochemical determination of dopamine based on selfassembled peptide nanostructure natural tri-to hexapeptides self-assemble in water to amyloid beta-type fiber aggregates by unexpected alpha-helical intermediate structures william thomas astbury - hierarchical self-assembly of tjernberg peptide at nanoscale conformational transition of amyloid beta-peptide connecting macroscopic observables and microscopic assembly events in amyloid formation using coarse grained simulations peptide self-assembly at the nanoscale: a challenging target for computational and experimental biotechnology prediction of aggregation rate and aggregation-prone segments in polypeptide sequences stability of diphenylalanine peptide nanotubes in solution molecular dynamics simulation of the α-helix to β-sheet transition in coiled protein filaments: evidence for a critical filament length scale rigid self-assembled hydrogel composed of a modified aromatic dipeptide from the globular to the fibrous state: protein structure and structural conversion in amyloid formation amyloid fibrillogenesis: themes and variations protein misfolding and disease; protein refolding and therapy the "correctly-folded" state of proteins: is it a metastable state? direct conversion of an oligopeptide from a ß-sheet to an a-helix: a model for amyloid formation identification of a penta-and hexapeptide of islet amyloid polypeptide (iapp) with amyloidogenic and cytotoxic properties conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state c nmr charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides self-assembly of a modified amyloid peptide fragment: ph responsiveness and nematic phase formation controlling amyloid growth in multiple dimensions templating molecular arrays in amyloid's crossbeta grooves effect of peg crystallization on the self-assembly of peg/peptide copolymers containing amyloid peptide fragments molecular self-assembly of peptide nanostructures: mechanism of association and potential uses puramatrix: self-assembling peptide nanofiber scaffolds rational design and application of responsive alpha-helical peptide hydrogels lipid-like self-assembling peptides production of self-assembling biomaterials for tissue engineering light harvesting antenna on an amyloid scaffold dual-surface modification of the tobacco mosaic virus neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering fine-tuning the ph trigger of self-assembly artificial transmembrane ion channels from self-assembling peptide nanotubes self-assembling chimeric polypeptidedoxorubicin conjugate nanoparticles that abolish tumours after a single injection a novel vaccine using nanoparticle platform to present immunogenic m e against avian influenza infection peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine development of an electrochemical metal-ion biosensor using self-assembled peptide nanofibrils self-assembled diphenylalanine nanowires for cellular studies and sensor applications an auto-biotinylated bifunctional protein nanowire for ultra-sensitive molecular biosensing remote electronic control of dna hybridization through inductive coupling to an attached metal nanocrystal antenna design of metal-binding sites onto self-assembled peptide fibrils self-assembling peptide-based nanostructures for regenerative medicine self-assembly of collagen-mimetic peptide amphiphiles into biofunctional nanofiber self-assembling functionalized nanopeptides for immediate hemostasis and accelerative liver tissue regeneration key: cord- -vy qgtll authors: nan title: proteases date: - - journal: febs j doi: . /j. - . . _ .x sha: doc_id: cord_uid: vy qgtll nan the incretin hormones glp- and gip are released from the gut during meals, and serve as enhancers of glucose stimulated insu-lin release from the beta cells. furthermore, glp- also stimulates beta cell growth and insulin biosynthesis, inhibits glucagon secretion, reduces free fatty acids and delays gastric emptying. glp- has therefore been suggested as a potentially new treatment for type diabetes. however, glp- is very rapidly degraded in the bloodstream by the enzyme dipeptidyl peptidase iv (dpp-iv; ec . . . ). a very promising approach to harvest the beneficial effect of glp- in the treatment of diabetes is to inhibit the dpp-iv enzyme, thereby enhancing the levels of endogenously intact circulating glp- . the three dimensional structure of human dpp-iv in complex with various inhibitors creates a better understanding of the specificity and selectivity of this enzyme and allows for further exploration and design of new therapeutic inhibitors. the majority of the currently known dpp-iv inhibitors consist of an alpha amino acid pyrrolidine core, to which substituents have been added to optimize affinity, potency, enzyme selectivity, oral bioavailability, and duration of action. various compound series and their sar relative to alpha amino acids will be presented. memapsin (b-secretase, bace ) is the membrane-anchored aspartic protease that initiates the cleavage of b-amyloid precursor protein (app) leading to the production of amyloid-b (ab), a major factor in the pathogenesis of alzheimer's disease (ad). since memapsin is a major target for the development of inhibitor drugs for the treatment of ad, its structure and physiological functions are topics of intense research interest currently. here we discuss the structural features of memapsin and how do they contribute to the activity and inhibition of the protease. structural and kinetic evidence support the presence of subsites for substrate or inhibitor binding in the activesite cleft of memapsin . subsites p to p ' are most useful in the design of transition-state analogue inhibitors. recent data indicated that subsites p , p and p have strong influence of hydrolytic rate or inhibition potency. these subsites are, however, too far from the transition-state isostere for the design of drug-like transition-state inhibitors but can be utilized for the design of non-transition-state inhibitors that compete for substrate binding. besides carrying out proteolytic activity, the ectodomain of memapsin also interacts with app leading to the endocytosis of both proteins into the endosomes where app is hydrolyzed by memapsin to produce ab. a phosphorylated motif in the cytosolic domain of memapsin is responsible for the recognition of gga proteins as part of the recycling mechanism that transports memapsin from endosomes to trans-golgi then back to cell surface. these interactions may also be considered for the design of small-molecular compounds that interfere with memapsin trafficking and thus reduce the production of ab. identification of human carnosinase -a brainspecific metalloprotease m. teufel biochemistry, exploratory research, sanofi aventis, strasbourg, france. e-mail: michael.teufel@sanofi-synthelabo.com metalloproteases form a large and diverse family of proteases and are molecular targets that represent an opportunity for therapeutic intervention. in particular, the development of potent inhibitors has made progress for the family of matrix metalloproteases (mmp). the sequencing of the human genome revealed that a significant percentage of the drugable genome is represented by proteases, many of them still with unknown function. in this presentation, data will be presented on the deorphanization of two previously unknown genes by means of bioinformatics and classical biochemistry. this work led to the identification of human carnosinase, a dipeptidase specifically expressed in the human brain and an ubiquitously expressed close homologue, characterized to be a non-specific dipeptidase. stimulating serpins with synthetic tailor-made oligosaccharides: a new generation of antithrombotics m. petitou thrombosis & angiogenesis, sanofi-aventis, toulouse, france. e-mail: maurice.petitou@sanofi-aventis.com we will discuss our research on synthetic oligosaccharides able to selectively activate the inhibitory activity of antithrombin towards various serine proteinases. we first synthesized pentasaccharides closely related to the antithrombin binding domain of heparin [ ] (the active site), as well as analogues displaying different pharmacokinetic profiles. selective inhibitors of coagulation factor xa were thus obtained that represent a new class of antithrombotic [ ] drugs currently being evaluated worldwide. we then designed larger oligosaccharides [ ] that inhibit both factor xa and thrombin in the presence of antithrombin. they are devoid of undesired nonspecific interactions with blood proteins, particularly with platelet factor . clinical trials are ongoing to prove the therapeutic benefits of this new type of coagulation inhibitors. slow tight binding inhibitors in drug discovery: in the case of dppiv and elastase inhibitors z. kapui, e. boronkay, i. bata, m. varga, e. mikus, k. urban-szabo, s. ba´tori and p. ara´nyi discovery research, chinoin member of sanofi-aventis group, budapest, hungary. e-mail: zoltan.kapui@sanofi-aventis.com enzyme are extremely potent causing significant inhibition at very low concentrations that may be comparable to the concentration of the target enzyme. when this inhibition is studied in vitro, complexities arise because the concentration of the inhibitor is so low that it is altered significantly as a result of combination with the enzyme. this situation is referred to as tight-binding inhibition. partly as a result of their low concentrations, tight-binding inhibitors often show slow-binding characteristics. unlike conventional inhibitors that act almost instantaneously (or at least within the ms time scale), slow-binding inhibitors may take several seconds, minutes or even hours for their effect to be fully exhibited. this association between slow-binding and tight-binding is relatively common and slow tight-binding inhibitors are extremely potent and specific. proteolytic enzymes are involved in a multitude of important physiological processes. their intrinsic properties and activities are in the focus of wide-ranging research and they have a valuable role in experimental and therapeutic purposes. serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. within the class of serine proteases, human leukocyte elastase (hle) is one of the most destructive enzymes in the body. the enzyme dipeptidyl peptidase iv (dppiv) is a serine exopeptidase that cleaves xaa-pro dipeptides from the n-terminus of oligo-and polypeptides. inhibitors of dpp iv are of increasing interest to pharmaceutical industry alike, as they may become established as the next member of the oral antidiabetic class of therapeutic agents. objective of our work was to develop reversible, slow, tight-binding inhibitors against these serine proteases. ssr is a potent inhibitor of hle, the inhibition constant (k i ) and the constant for inactivation process (k on ) being . ± . nm. this inhibitor is reversible, slow, tight-binding inhibitor with k on = . ± . /ms, and k off = . ± . ) /s. ssr inhibits the solubilization of elastin by hle with nm of ic value. this inhibitor is one of the most effective inhibitor of a serine proteinase yet described. ssr is a potent, competitive and slow tight binding type inhibitor of the human dipeptidyl peptidase-iv enzyme (k i = nm, t½ = h). on the basis of kinetic properties, ssr forms stable enzyme-inhibitor complex. these slow tight-binding inhibitors have unique inhibitory properties, they are extremely active, and selective, form stable enzyme-inhibitor complex, therefore they have long-lasting effect. their oral activity and long lasting in vivo biological potency agreed very well with stable enzyme-inhibitor complex. the advantages in drug discovery of slow tight-binding inhibitors are discussed in this presentation. enzyme inhibition trend analysis -a new method for drug design m. shokhen, n. khazanov and a. albeck the julius spokojny bioorganic chemistry laboratory, chemistry, bar lan, ramat gan, israel. e-mail: albecka@mail.biu.ac.il many of the drugs that are currently in use or at different stages of development are enzyme inhibitors. therefore, enzyme mechanism-based inhibitors could be developed into highly selective drugs. our novel enzyme inhibition trend analysis method com-bines experimental enzyme kinetics data and high level quantum mechanical modeling of enzyme-inhibitor chemical interactions. the method utilizes the principal catalytic reaction scheme of the target enzyme and does not require its d structure (a ligand based approach). the method is valid for the prediction of the trend in binding affinity of inhibitors not only for the specific enzyme for which the qsar model was optimized, but also for the whole enzyme family. the methodology would contribute significantly to overcoming the problem of fast mutational resistance developed by pathogens in response to pharmaceutical treatment. it can be used as a computational tool for expert analysis of various hypotheses about structure-activity relationships formulated for the design of new inhibitors. angiotensin-converting enzyme (ace, ec . . . ) is a key enzyme for blood pressure control and water-electrolyte homeostasis. a large number of highly potent and specific ace inhibitors are used as oral drugs in the treatment of hypertension and congestive heart failure. somatic ace consists of two homologous domains (n-and c-) within single polypeptide chain, each one containing a catalytic site. the two catalytic sites within somatic ace molecule were long considered to function independently. however, recent investigations indicate the existence of negative cooperativity between ace active sites. we studied the properties of bovine ace active centers by use of separate ace n-domain (n-ace) obtained by limited proteolysis of parent somatic enzyme and testicular ace, which represents c-domain. these results were compared with the data obtained for full-length somatic ace from bovine lungs. the results obtained demonstrate strongly dependent mechanism of action of ace active centers in the reaction of the hydrolysis of tripeptide substrates. however, the hydrolysis of decapeptide angiotensin i proceeds independently on n-and c-domains. the mechanism of inhibition of ace activity is also dependent on the length of the inhibitor: (i) random binding of the ''short'' inhibitor molecule (such as captopril, lisinopril) to one of the active sites dramatically decreases binding of another inhibitor molecule to the second site; (ii) ''long'' nonapeptide teprotid binds to both active sites without any difficulties. since the main physiological ace substrates in the organism are ''long'' peptides angiotensin i and bradykinin, the development of new class of inhibitors with prolonged structure would be beneficial for abolishing of ace activity. synthetic peptide studies on severe acute respiratory syndrome coronavirus (sars-cov) extensive proteolytic processing of the replicase polyproteins, pp a ( kda) and pp ab ( kda), by the sars-cov clike protease ( cl pro ). besides, the structural spike protein of sars-cov contains two heptad repeat regions (hr and hr ) that form coiled-coil structures, which play an important role in mediating the membrane fusion process. in this study, we focused on both cl pro and the hr regions of sars. previous studies demonstrated that the coronavirus cl pro cleaves the replicase polyproteins at no < conserved cleavage sites, preferentially at the lq sequence. the reported crystal structure of sars-cov cl pro provides insights into the rational design of anti-sars drugs. in order to understand the molecular basis of the enzyme-substrate binding mechanism, we employ the synthetic peptide and mass spectrometry-based approaches to investigate the significance of selected amino acid residues that are flanking both sides of the sars-cov cl pro cleavage site. in addition, previous studies indicated that the relatively deep hydrophobic coiled coil grooves on the surface of sars-cov spike protein heptad repeat regions (hr and hr ) may be a good target site for the design of viral fusion inhibitors. we have designed and synthesized five truncated peptide analogs derived from hr and hr peptides based on both bioinformatics and structural analysis. the biological activities of these truncated analogs will be studied using circular dichroism spectroscopy, multidimensional chromatography, protein cross-linking and mass spectrometry-based approach. the above investigation will definitely broaden our knowledge on the sars research and will reveal the feasibility of rational design of synthetic peptide-based drug in combating with sars disease. ras-transfection-associated invasion: involvement of matrix metalloproteinase(s) confirmed using a chicken embryo model and real time pcr during metastasis tumorogenic cells leave the primary tumour and intravasate into the blood/lymphatic system, exiting at a secondary site to establish a secondary tumour. ras-transfection of a parental, non-invasive mcf- a cell line, established from a patient suffering with benign fibrocystic disease, gave rise to an invasive derivative cell line (mcf- a-neot) exhibiting the phenotype of a pre-malignant, invasive tumour. invasion and metastasis are protease-assisted processes, proteases either being secreted by the tumour, or by the stromal cells under the influence of the tumour. here we demonstrate the involvement of matrix metalloproteinase(s) in the invasion of the ras-transfected mcf- a cell line. tumour cells were inoculated onto the damaged surface of the upper chorioamniotic membrane (cam) of a vasculated -day old chick embryo. the tumour cells were allowed to invade, and the number of invading cells quantified using real time pcr. inhibitors specific for various proteases were applied to the upper cam, to block invasion, and hence identify the proteinases involved. the number of tumour cells invading into the vascular system was established by sampling the lower cam and quantifying the numbers of alu sequences (present only in human cells) in the dna, isolated from the embryonic tissue, using real-time pcr. using this method, the key role of an mmp was demonstrated. spectrum ptk inhibitor, genistein ( lm) abolished the release of neutrophil mmp- , in the presence and absence of extracellular calcium, and reduced the release of timp- . both pp ( lm), a src family ptk inhibitor, and piceatannol ( lg/ml), a syk family ptk inhibitor, reduced mmp- release substantially, indicating that multiple ptk families might be involved in mmp- release. inhibition of either syk or src ptks by piceatannol or pp did not appear to influence timp- release. low levels of wortmannin ( nm, inhibition of pi k) abolished the release of mmp- in the absence of calcium, and reduced mmp- release in the presence of calcium. investigations into the signaling pathways involved in timp- release are continuing. we conclude that mmp- release induced by extracellular calcium may be mediated through pi k and multiple tyrosine kinases, including src and syk family ptks. timp- granule release may also be mediated by tyrosine kinases, although src and syk family ptks do not appear to be involved. thermodynamical and structural analysis of cruzain/cruzipain complexed with e- by molecular modeling and dynamics simulations peptidases represent one of the most relevant enzyme classes targeted by therapeutic intervention. to contribute to the assignment of a physiological role to genomic-derived peptidases and to make them more accessible for the drug discovery process, we have undertaken a program consisting of mrna expression profiling, full-length recombinant expression in insect cells, purification and determination of the catalytic activity for the human proteolytic enzymes. a milestone in the process was the construction of a non-redundant comprehensive database for all human peptidases comprising unique annotated entries, by assembling and filtering public domain information and in-house generated data. in order to get an informative picture on their expression profiling, a transcriptome database for human peptidases was created using the microarray (affymetrix tm ) and taqman Ò (applied biosystems) technologies. in parallel, we have set up the procedure for pcr amplification and cloning of the peptidase genes in mtp format and we have already created a repository of full-length human cdnas encoding for peptidases. besides, the conditions for miniaturized insect cell cultures have been established. experimental trials have defined a validated, reliable and fully-automated robotic procedure for the purification of recombinantly expressed peptidases in mtp format. in a pilot study using the high-throughput approach, % of the chosen reference hydrolases ( ) were secreted into the insect cell medium. of them, % have been proven to be catalytically active using fluorescent homogeneous assays in well format compatible with the high-throughput screening criteria. the application of this procedure to genomic-predicted peptidases is discussed. comparison of putative glutamate racemases from bacillus species glutamate racemase catalyzes the interconversion between l-and d-glutamic acid and is the cell's source of d-glutamate, a key component in the synthesis of both the bacterial cell wall and the glutamyl capsule. bacillus subtilis has two glutamate racemases in its genome, race and yrpc, while b. cerus and b. anthracis have two race genes, race and race . interestingly, race in b. subtilis is the isoform that is essential and has the greater catalytic efficiency, but both race and race have higher sequence homology to race, and % respectively and share less homology with the yrpc isoform, both at %. we have cloned, overexpressed, purified, and are characterizing the kinetic and biophysical properties of the two putative glutamate racemases, race and race from b. cereus and b. anthracis, and will utilize kinetic and biophysical information to design inhibitors that may result in a novel antibiotic. although these two isoforms share a high sequence similarity, their properties are unique. kinetic data indicates a fivefold difference in catalytic efficiency of race compared to that of race in the l-to d-glutamate reaction. also, the absence or presence of substrate has an effect on the oligomerization state, details of which will be reported. finally, our collaborators have demonstrated through genetic knock out experiments that only one of the race isoforms is essential for the growth of b. anthracis. we have crystallized the race isozyme and x-ray data have been collected to . Å . we are currently solving the structure via heavy-atom derivatives. acknowledgment: this research was funded by nih grant u ai . anti-inflammatory effects of methionine aminopeptidase inhibition on human b lymphocytes e. janas , r. priest , s. ratcliffe and r. malhotra rheumatoid arthritis biology, glaxosmithkline, stevenage, uk, high throughput chemistry, glaxosmithkline, stevenage, uk. e-mail: eva.x.janas@gsk.com processing of n-terminal methionine is an essential post-translational modification in both prokaryotes and eukaryotes regulating the subcellular localization, stability and degradation of proteins. the cleavage of the initiator methionine is catalysed by a highly conserved family of metalloproteases, methionine-aminopeptidase and (met-ap ). human met-ap is the molecular target of fumagillin, a natural product with antiangiogenic properties, which covalently binds to his in the catalytic site of met-ap . although fumagillin has been observed to inhibit proliferation and to cause cell cycle arrest in endothelial cells, the mechanism of inhibition is still poorly understood. recent studies describe high expression of met-ap in germinal centre b lymphocytes. here, we investigate the effect of the met-ap inhibitor fumagillin on b lymphocyte proliferation and cell cycle progression and compare these results to those observed in hu-vec. in addition our work sheds light on the mechanistic aspects of met-ap inhibition by fumagillin and its derivatives. effect of distal mutations on the molecular dynamics of the hiv- protease l i and l m are the most common distal mutations found in the protease gene of the drug resistant hiv- strains. these mutations do not confer resistance by themselves, however induce a large synergy effect when added to active site mutations. understanding the impact of the l m and l i mutations on the hiv- protease resistance profile is still a challenge. assuming that their contribution to the resistance profile could be mediated by conformational dynamics we have modeled l i, l m and l i/l m mutants of hiv- protease. these unbound mutated and wild type proteases were subjected to ns molecular dynamics simulations and compared using an essential dynamics (ed) analysis protocol. the first eigenvector of the native protease describes the flap openning motion. following eigenvectors describe ''the catalytic assisting motions'' (cam) of the protease that becomes dominant upon complex formation with a substrate (piana s et al. j mol biol ; ( ): - ). mutation of luecine to methionine residue at position perturbs the protein packing at the dimerization domain. such perturbations affect the dimerization domain motions which correlate with flap opening and the cam. as result the first eigenvector corresponds to the rotational of the one subunit relative to another along axis connecting residues and '. in other words l m mutation mistunes essential motions of the enzyme while retaining its flexibility. this could be the cause of the reduced structural stability of the l m mutant. in contrast, l i mutation causes only redistribution of the correlated motions amplitude. the catalytic assisting motion becomes the most influential that results in stabilization of the closed conformation. in turn, the flap opening motions are reduced in l i mutant. essential dynamics of the double mutant l i/l m could be described in the following terms. a strong propagation of the cam induced by l i mutation is coupled with the altered conformational space caused by l m mutation. as result the double mutant prefers cam motions that are close to the native protease but also account for the perturbed packing within the dimerization domain. results presented may help understanding hiv- protease resistance pathways and in developing more efficient inhibitors of known drug resistant mutants. glutamate carboxypeptidase ii as a cancer marker and therapeutical target: two faces of an enzyme glutamate carboxypeptidase ii is a membrane-bound metallopeptidase expressed in a number of tissues such as jejunum, kidney, prostate and brain. the brain form of gcpii (also known as naaladase) is expressed in astrocytes and cleaves n-acetylaspartyl glutamate, an abundant neurotransmitter, to yield free glutamate. gcpii thus represents an important target for the treatment of neuronal damage caused by excess glutamate. animal model experiments suggest that specific inhibitors of gcpii could be useful for the treatment of several neuropathic conditions, such as brain stroke, chronic neuropathic pain or amyotrophic lateral sclerosis. in the same time, the enzyme is known as prostate-specific membrane antigen since it is upregulated in prostate cancer. it is used for the diagnosis and experimental therapy of prostate cancer using monoclonal antibodies and specific inhibitors. in order to analyze this important pharmaceutical target, we established an expression system based on drosophila schneider's cells. we have also cloned, expressed and characterized its human homolog gcpiii and homologous carboxypeptidases from pig and rat. using specific monoclonal antibodies, we have been able to study the expression of gcpii in various healthy and malignant tissues. we analyzed the substrate specificity of the enzyme using peptide libraries and identified two novel peptide substrates. availability of a recombinant protein enabled to introduce a simple fluorescent activity assay and test specific inhibitors. furthermore, we have biochemically characterized the recombinant protein in terms of pharmacologic properties, oligomeric status, ph dependence and activity modulation by metal ions. we have shown that the glycosylation is indispensable for gcpii carboxypeptidase activity and analyzed the role of each specific n-glycosylation site for the gcpii activity and folding. using site-directed mutagenesis, we are able to identify the domains sufficient and necessary for gcpii activity and also suggest structural explanation for the substrate specificity of the enzyme. dozens of chemicals feature inhibition of proteolytically important tyrosine residue of s proteasome by forming covalent bond to hydroxyl group that abolished its catalytic function. in contrary, the approach we utilize here is based on hydrogen and hydrophobic interactions reversibly inactivating all three sites of s complexes. we performed flexible docking studies of analogues of a natural product tmc- a using jd crystal structure to describe the active site of protein and the position of the ligand. the search yielded several amide-like derivatives that have been screened for superimposition with tmc- a. few of them revealed similar orientation of propylene groups to the active site of s. second screen was performed to reveal the chemicals with the strongest hydrogen-bonding of the ligand to the protein backbone of the receptor. this screen resulted in two chemicals that had strong h-contacts with tyr , ser and, importantly, with proteolytically active tyr residue. to access the validity of the predicted chemicals we undertook in vitro studies measuring the hydrolyses of fluorogenic substrate by the sds activated s proteasome isolated from hela cells. we obtained more than % inhibition of s proteasome activity upon incubation the above chemicals ( . lg/ml) with proteasomes. we then demonstrated the effectiveness of the obtained chemicals to stabilize the level of oncosupressors, including p in benign (mcf a) and highly metastatic (mda ) cell lines. treatment with these compounds greatly restored the level of p in cancer cells. finally, we performed proliferation assay and proved that adding of this artificially synthesized chemicals to mda cell line significantly reduced the level of proliferation, whereas mcf a cells treated at similar conditions have not revealed any abnormal reduction of proliferation below control level. thus, we report of a strategy to predict highly suitable proteasome inhibitors that act via inhibition of protease activity and may lead to creation of a new class of drugs for cancer therapy. localization and trafficking of prostate specific membrane antigen (psma) and its variant form psḾ glutamate carboxypeptidase ii, also known as prostate specific membrane antigen (psma), is a transmembrane glycoprotein highly expressed in maligant prostate tissues. it was shown to represent very useful diagnostic marker and also potential therapeutic target for prostate cancer. two forms of the enzyme were identified in the prostate: full-length transmembrane form consisting of amino acids and a truncated form (called psḾ ), believed to represent spliced variant of psma. the cdnas of both forms are identical except for -nucleotide region near ´end of psma that is absent in psḾ . this deleted region codes for signal peptide as well as for intracellular and transmembrane domains. we are able to detect two protein forms in prostate cancer model cells (lncap cells) and we also show that both forms are glycosylated suggesting that this truncated form might originate from the processing of full length transmembrane psma. number of methods including differential centrifugation, pulse-chase experiments, immunochemistry and gfp-fusion protein analysis were used to analyze the origin, cell localization and trafficking of psma and psḾ in the mammalian cells. we have investigated the substrate specificity of the ns b(h)-ns pro protease by using internally quenched synthetic peptides representing both natural cleavage sequences and their recombinant chimeras. synthetic peptides incorporating the o-aminobenzoic acid/ -nitro-l-tyrosine fluorescence donor-quencher pair were used to analyze the minimum substrate length requirement, residue preferences and the contribution of prime side residues for enzymatic cleavage by the ns protease. a series of peptides derived from the ns /ns a cleavage site was designed for the substrate length mapping study. amino acid truncations in the non-prime and prime side region differently affected rates of substrate hydrolysis and binding as shown by their km and kcat values. the optimal substrate identified was a heptapeptide spanning p -p '. chimeric substrates with all possible combinations of non-prime and prime side sequences derived from polyprotein cleavage sites (c, a/ b, b/ , / a and b/ ) were assayed for reactivity with the ns protease. kinetic parameters revealed a strong impact of the non-prime side residues on km, whereas variations in the prime side region had greater effect on kcat. the fluorogenic derivative of tetrabasic peptide rrrr/gtgn (c/ns ) demonstrated the highest affinity, whereas the peptide kkqr/sagm ( b/c) had the highest turnover number. the one with the greatest catalytic efficiency was identified as rrrr/sltl (c/ a). in addition, we have shown that a ser at p ' is the most preferred residue. the discovery of ns substrates with maximized reactivity will be useful for inhibitor development in sensitive high-throughput assays. inhibiting the mtor pathway with cci- results in decreased production of vascular endothelial growth factor in a head and neck squamous cell cancer cell line c.-a. o. nathan , , n. amirghahari , , x. rong , and y. sun , nathan, otolaryngology, otolaryngology/head and neck surgery, louisiana state university health sciences center, shreveport, la, usa, nathan, cancer center, medicine, feist-weiller cancer center, shreveport, la, usa. e-mail: cnatha@lsuhsc.edu introduction: overexpression of the proto-oncogene eif e in surgical margins of head and neck squamous cell cancer (hnscc) patients is an independent predictor of recurrence and is associated with increase in vascular endothelial growth factor (vegf) expression. activation of eif e in margins through the mtor pathway has led us to determine that cci- an mtor inhibitor has both in vitro and in vivo growth inhibitory effects in hnscc cell lines. we wanted to determine if these effects were associated with decrease in vegf production. material and methods: a hnscc cell line fadu was treated with and ng/ml of cci- (previously established ic = ng/ml). elisa was used to determine vegf protein levels in conditioned medium at ', , , , , and h after treatment with the drug and compared to control cells treated with the diluent for each of the time points. results: a significant decrease in vegf production of % was noted at h and maintained at h in treated cells when compared to control cells at the same time points. the decrease in vegf levels ( - %) was noted within h of treatment with the drug. the percent decrease in vegf protein levels was the same for both doses of cci- . conclusions: overexpression of eif e in hnscc increases translation of mrnas with long 'utrs, one of which is an important angiogenic factor vegf. inhibiting the mtor path-way with cci- can potentially decrease vegf production. this has future clinical implications for arresting tumor progression in hnscc patients with molecular positive margins identified by cells overexpressing eif e, also known as minimal residual disease. proteases from cell culture of jacaratia mexicana m. c. oliver-salvador , g. barrera plant proteases are important in food industry and food technology. the latex of jacaratia mexicana, caricaceae, fruits contains a high level of cysteine proteases. in this work was established a cell suspension culture of j. mexicana. callus culture was initiated from stem explants of j. mexicana on medium consisted of ¼-strength and full-strength ms mineral salts (murashige and skoog, ), full-strength ms organics and g/l agar supplemented with cytokinins: -benzylaminopurine (bap) at . mg/l and -furfurylaminopurine (kinetin) at . mg/l and various concentrations ( . , . and . mg/l) of auxins: , -dichlorophenoxyacetic acid ( , -d) -amino- , , -trichloropiridin- -carboxilic acid (picloram) indoleacetic acid (iaa) a-naphthaleneacetic acid (naa). all of the treatments induced callus except for the iaa, ana and without added phytohormones. the best auxin concentration for callus development was determined to be . mg/l. and the best condition medium for callus development and proteolytic activity of callus was determined to be . mg/l , -d + . mg/l bap. cysteine proteases were produced on callus culture of j. mexicana and liberated in the medium. also in the cell suspension culture these enzymes were secreted. our results support that is possible the synthesis of proteases in vitro culture of j. mexicana. since protease is a primary metabolite, further improvement in enzyme production is possible by increasing the growth rate and yield of cell culture of j. mexicana. and arginine, from peptides and proteins at neutral ph. it is known to play an important role in the control of peptide hormones, growth factor activity at the cell surface, and in the membrane-localized degradation of extracellular proteins. therefore, the present work was carried out to clone and express carboxypeptidase m in pichia pastoris, aiming at developing specific inhibitors and to evaluate the importance of the enzyme in different physiological and pathological processes. for this purpose, the enzyme's cdna was amplified from total placental rna by rt-pcr and cloned in the vector ppic , which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in pichia pastoris. the results show that the cpm gene, after cloning and transfection, integrated in the yeast genome, which started to produce the active glycosylated protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured with the fluorescent substrate dansyl-ala-arg. the enzyme was purified by a two-step protocol including gel filtration and ionexchange chromatography, resulting in a -fold purified active protein in a concentration of mg/l of fermentation medium. sds-page showed that recombinant cpm migrated as a single band with molecular weight similar to native placental enzyme ( kda). these results demonstrate for the first time the establishment of a method using pichia pastoris to express human carboxypeptidase m. mutational analysis of active site of glutamate carboxypeptidase ii human glutamate carboxypeptidase ii (gcp ii) is a membrane metallopeptidase expressed predominantly in the nervous system, prostate and small intestine. in the brain, gcp ii catalyzes cleavage of the abundant neuropeptide n-acetyl-l-aspartyl-l-glutamate (naag) to n-acetylaspartate and glutamate. gcp ii is a type ii transmembrane glycoprotein with a short cytoplasmic nterminal region (amino acids - ), a transmembrane domain (amino acids - ) and a large extracellular domain (amino acids - ) where the active site of the enzyme is situated. gcp ii, as a cocatalytic zinc metallopeptidase, has two zn + ions in the active site which are necessary for its enzymatic activity. recently, the crystal structure of gcp ii was determined in our laboratory and amino acids arg , asn , lys and tyr were proposed to bind c-terminal glutamate of naag (mesters et al., manuscript in preparation). in the presented study, we carried out site-directed mutagenesis to assess the influence of these amino acid residues on the activity of gcp ii. in addition, glutamic acid in the position which is proposed to be involved in proton shift during the catalytical hydrolysis of peptide bond, was mutated to alanine. all the mutant proteins were expressed in insect cells, purified to near homogeneity and enzymatically characterized. it was shown that a mutation in any of these positions lead to significantly reduced naag-hydrolyzing activity. the substitution of glu almost completely abolished the enzymatic activity, thus suggesting glu is crucial for enzymatic activity of gcp ii. kinetic characterizations of mutant proteins and their substrate specificities will be presented in comparison with wild type gcp ii. comparative study of mammalian homologues of human glutamate carboxypeptidase ii glutamate carboxypeptidase ii (gcpii) is a membrane-bound metallopeptidase. in homo sapiens, gcpii was shown to be expressed in various tissues, mostly in the central nervous system, small intestine and prostate. in brain it hydrolyses n-acetylaspartylglutamate (naag), which is the most prevalent peptide neurotransmitter in the mammalian nervous system, to form glutamate and n-acetylaspartate. in small intestine gcpii plays an important role in folate absorption. in prostate its function is still unknown. it was shown that inhibition of gcpii is neuroprotective in many neurodegenerative states. according to current knowledge of this enzyme, its role may also be important in prostate (and possibly other) cancers, where its expression is dramatically changed in comparison with healthy tissue. gcpii is thus becoming an important therapeutic target and diagnostic molecule. in order to analyze structure-activity relationships in related glutamate carboxypeptidases, we set to study the mammalian homologues of human gcpii: gcpii of rattus norvegicus, sus scrofa and mus musculus, which have approximately % dna sequence similarity to human gcpii. information on the biochemical properties, expression pattern and structural similarity is crucial e.g. for testing of gcpii inhibitors in animal models. we have cloned and expressed recombinant gcpii of r. norvegicus and s. scrofa in insect cells with the aim to obtain pure recombinant protein sufficient for structural analysis. data on biochemical comparison of rat, pig and human gcpii forms will be presented and interpreted in the light of the gcpii structure. structural analysis of pla protein from y. pestis: docking and molecular dynamics of interactions with mammalian plasminogen systemz e. ruback and p. g. pascutti laborato´rio de modelagem e dinaˆmica molecular, departamento de biofisica, universidade federal do rio de janeiro, rio de janeiro, r.j. brazil. e-mail: eruback@biof.ufrj.br the plasminogen (plg) system is an important mechanism for the cell migration through the tissues in the mammalian organisms. some bacterial agents can activate this system by proteases and lead an uncontrolled degradation of extracellular matrix components (mec), and make an invasive character of these infections. the y. pestis protein pla is a plasmid coded outer membrane protein, with aspartic-protease activity and is closely related with the proteolytic activation of plg in the serine-protease form called plasmin. exactly how the pla activate plg in plasmin remains unclear. we performed in this work the predicted interaction between the plg and pla protein by rigid-body docking with hex and evaluate the complex stability by molecular dynamics (md) using the gromacs. to evaluate the docking accuracy we use the crystal structure of complex plg-streptokinase. the md results show more stability in the docked plg-streptokinase complex than in crystal complex observed by the rmsd and rmsf calculations after ns in simulation box. the pla model was constructed with spdb-viewer using the pdb structure of ompt as template and quality of model was evaluated with prochek. the docked complex of plg-pla show same interaction site predicted in mutagenesis studies. after ns md ( atoms in box), we observed the relax of beta barrel structure of pla and the progressive approximation and stabilization between the cleavage site of plg into the extracellular loops of pla, followed of the increase of hydrogen bonds number. in this study we report the possible aminoacids that can be participant in the active site and the sub sites of interaction. the total understanding of these interactions can be a important tool for drug design against bacterial proteases. glutamate carboxypeptidase ii (gcpii), also known as naa-ladase i, folylpolyglutamate hydrolase (folh) or prostate specific membrane antigen (psma) is localized in number of tissues. in brain astrocytes, it regulates neurotransmission by cleaving neurotransmitter n-acetylaspatylglutamate (naag) into n-acetylaspartate and most common excitatory neurotransmitter glutamate. inhibition of gcpii activity protects against cell death after brain stroke. in animal models it has been also shown that specific inhibitors of gcpii could be useful for the treatment of chronic neuropathic pain, amyotrophic lateral sclerosis and other pathologic situations when excess glutamate is neurotoxic. gcpii is identical to prostate-specific membrane antigen (psma), a tumor marker in prostate cancer. gcpii is also found in the membrane brush border of the small intestine where it acts as a folate hydrolase. this reaction expedites intestinal uptake of folate through hydrolysis of folylpoly-gamma-glutamates to monoglutamyl folates. gcpii inhibitors might thus be useful in the imaging and treatment of tumors where folate is required for their growth. therefore it was of interest to investigate whether gcpii might be upregulated in brain tumors as well. in order to analyze this possibility, we took samples from patients with brain tumors treated in faculty hospital motol during - and determined expression and activity of gcpii by western blots and immunohistochemistry using monoclonal and polyclonal antibodies developed against extracellular epitopes of gcpii. moreover, we characterized the enzymatic activity of the enzyme in human samples and correlated the expression of gcpii with the type and grade of the tumor. search for optimal isosteres in beta-secretase peptidic inhibitors alzheimer's disease is a widespread, neurodegenerative, dementia-inducing disorder. it is ascribed to the presence of a lesion in several brain regions, the neuritic plaques, which are extraneuronal accumulations of b-amyloid protein (ab), a -aa insoluble peptide that mixed with axons and dendrites of neurons, interrupt the synaptic process and cause neuronal death. the peptide ab is a product derived by proteolitic cleavage from a larger transmembrane cell protein termed amyloid precursor protein, app. two enzymes are involved in this cleavage: b-secretase and a-secretase. the first one cuts app between met and asp of app to generate the n-terminus of ab in the rate limiting step of the process, while the second one cleaves at various places within a sequence between amino acids and to generate the respective c-terminus. using a combination of molecular modeling techniques, we have designed a set of novel b-secretase peptidic inhibitors with a variety of isosteres starting from the available crystallographic structure of this enzyme bound to the inhibitor om - . some of the resulting ligands are predicted to have higher affinity for this enzyme than the starting compound. these inhibitors have been synthesized, their b-secretase affinity tested and cell essays have been performed to determine their ability to preclude the formation of ab peptides in cell cultures. schizophrenia and bipolar affective disorder (bd) are two neuropsychiatric diseases with high social and economic costs. in spite of the prevalence of these diseases, no effective long-term treatments are currently available. the enzyme prolyl oligopeptidase (pop) shows increased activity in both illnesses. this serine protease hydrolyzes peptide hormones and neuropeptides at the carboxyl end of proline residues. because of the relevance of pop as a therapeutic target, many specific inhibitors of this protein have been developed in recent years. the inhibitors ono- , jtp- and s- - are currently in clinical trial phase. s- - has been administered safely to humans and has been proposed as a potential treatment for cognitive disorders associated with cerebral aging. our aim is to develop new peptide human pop inhibitors. to obtain the human brain pop required for our studies, the cdna corresponding to the enzyme was cloned and subsequently expressed in e. coli. pop activity was monitored by f-nmr using a new synthesized pop substrate labeled with f. this substrate allowed us to perform the inhibition assay avoiding the interference problems of colorimetric and fluorimetric assays and was suitable for high throughput screening of new pop inhibitors. different strategies were used to find putative human pop inhibitors: in silico screening and solid phase synthesis of candidates and screening with chinese medicinal plants extracts. furthermore, nmr studies were performed with the purified human enzyme by labeling the protein isotopically with n and d o and by selective labeling of the residues methionine and tryptophan with c. nmr spectra of the labeled protein were obtained at mhz by applying trosy techniques. nmr will provide structural information to perform structure-based drug design of new pop inhibitors in the future as well as to study the interaction of the candidates with the active site of the enzyme. the crucial regulatory function of the membrane type -matrix metalloproteinase (mt -mmp or mmp- ) in connective tissue metabolism, pericellular proteolysis of extracellular matrix (ecm) components, zymogen activation and angiogenesis was demonstrated with the severe phenotype of the mt -mmp-deficient mice. this membrane-anchored enzyme is not only essential for normal development of hard tissues, but highly expressed in different human cancers where its level frequently correlates with malignant parameters. in most cases the high level of mrna or elevated level of protein can be predictive for disease development but these parameters only partly reflect the expression and forms of mt -mmp in pathological conditions. biosynthesis, trafficking, intracellular activation, internalization, protein-protein interactions, and the level of physiological inhibitors (timps) strictly influence the activity of mt -mmp in cells and tissues. in our experimental system, we followed mt -mmp processing and shedding and characterized the cell-associated and released forms of the enzyme (jbc ; : - ; jbc ; : - and biochem j ; : - ). we found active and inactive truncated forms of mt -mmp as a result of treatments or experimentally generated imbalance with timps. we have also developed approaches to identify mt -mmp forms in tumor tissues. here we present and discuss different strategies to identify mmp- in diverse biological samples. because mt -mmp endows tumor cells with the ability to invade and metastasize, these strategies can provide valuable information on the role and function of this key protease. contribution of calpain to cellular damage in human retinal pigment epithelium cultured with zinc chelator y. tamada , t. nakajima , t. r. shearer and m. azuma , research laboratories, senju pharmaceutical co., ltd., kobe, hyogo japan, departments of integrative biosciences, oregon health & science university, portland, or, usa. e-mail: yoshiyuki-tamada@senju.co.jp purpose: we previously showed involvement of calcium-dependent cysteine proteases (calpains, ec . . . ) in neural retina degeneration induced by hypoxia and ischemia-reperfusion. aged macular degeneration (amd) is one of the leading causes for loss of vision. amd showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (rpe). rpe performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. the purpose of the present study was to determine the contribution of calpain-induced proteolysis to damage in human rpe. zinc chelator tpen was used to induce cellular damage since zinc deficiency is a suspected risk factor for amd. methods: third-to fifth-passage cells from human rpe were cultured with tpen. leakage of ldh into the medium was measured as a marker of rpe cell damage. activity of calpains was assessed by casein zymography, and proteolysis of calpain substrates was detected by immunoblotting. to confirm calpain-induced proteolysis, calpain in homogenized rpe was also activated by addition of calcium. results: tpen caused ldh to leak into the medium from rpe cells, and calpain inhibitor sja inhibited the leakage. casein zymography and immunoblotting for calpain and a-spectrin showed activation of calpain in rpe cultured with tpen. proteolysis by activated calpain was confirmed by addition of calcium to homogenized rpe. conclusion: these results suggested that activation of calpain contributed to rpe damage induced by tpen in vitro. acknowledgments: dr shearer has substantial financial interest (research contract and consulting fee) in senju pharmaceutical co., ltd., and dr azuma is an employee of senju pharmaceutical co., ltd., a company that may have commercial interest in the results of this research and technology. this potential conflict of interest has been reviewed and managed by the ohsu conflict of interest in research committee. in vivo and molecular risk factors of chloroquine or pyrimethamine-sulfadoxine treatment failure in children with acute uncomplicated falciparum malaria the risk factors associated with chloroquine (cq) or pyrimethamine-sulfadoxine (ps) treatment failure were evaluated in children enrolled prospectively in six antimalarial drug trials between july and july in a hyperendemic area of southwestern nigeria. following treatment, ( %) of children given cq and ( %) of children given ps failed treatment by day or . in a multiple regression model, four factors were found to be independent risk factors for cq treatment failure at enrolment: age < years [adjusted odds ratio (aor) = . , % confidence interval (ci) . - . , p = . ], asexual parasitaemia > /ll (aor = . , % ci . - . , p = . ), presence of gametocytaemia (aor = . , % ci . - . , p = . ) and enrolment after years of commencement of the study, that is, after (aor = . , % ci . - . , p = . ). following treatment with cq, two factors were independent risk factors for failure of treatment: delay in parasite clearance > days (aor = . , % ci . - . , p = . ) and presence of gametocytaemia on day or (aor = . , % ci . - . , p = . ). in those treated with ps, two factors were found to be independent risk factors for ps treatment failure at enrolment: age < . years (aor = . , % ci . - . , p = . ) and presence of fever (aor = . , % ci . - . , p = . ). following treatment with ps, delay in parasite clearance > days (aor = . , % ci . - . , p = . ) was an independent risk factor for failure of treatment. the quintuple mutants made up of triple dhfr (asn- , arg- and ile- ) mutant alleles and double dhps (gly- and glu- ) mutant alleles were found in isolates obtained from % of patients, was significantly associated with ps treatment failure (p = . ), while pfcrt and pfmdr- mutant genes did not significantly predict cq treatment failure in these patients. these findings may have implications for malaria control efforts in sub-saharan africa where control of the disease depends almost entirely on antimalarial monotherapy. development of high-throughput assay of lethal factor using native substrate m.-y. yoon department of chemistry, hanyang university, seoul, south korea. e-mail: myyoon@hanyang.ac.kr designing of inhibitors for anthrax lethal factor (lf) is currently of interest as an approach for the treatment of anthrax because lf plays major roles in cytotoxicity of target cells. lf is a zincdependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (mapkk) family. current assay system for the screening of lf inhibitor use the optimized synthetic peptide coupled with various kinds of fluorophores, which enables fast, sensitive, and robust assays suited to high-throughput screening. however, lines of evidence suggest that the regions beside the cleavage site are also involved in specificity and proteolytic activity of lf. in the present study, we tried to develop high-throughput assay for lf activity based on native substrate, mek . the assay system relies on the ecl signal resulting from a specific antibody against the c-terminal region of native substrate. a glutathione-coated multiwell plate was used as a solid support to immobilize the native substrate by its n-terminal gst-moiety. immobilized substrate increases the specificity and sensitivity lf-catalyzed substrate hydrolysis compared to the solution phase assay. this assay system would be expected to discover a wide spectrum of anthrax inhibitor. while significant progress has been made over the past decade in elucidating the structure and enzymatic mechanism of the s proteasome, our understanding of its assembly pathway and the role of the propeptides in the maturation process is still substantially incomplete. similarly, the mechanisms involved in the translocation of substrates into the central nanocompartment are only dimly understood at present. we have used the rhodococcus proteasome to dissect the assembly pathway, combining mutagenesis and crystallographic studies. for the thermoplasma proteasome we have established a ''host-guest'' interaction system which allows us to follow the translocation of specific substrates into the interior of the proteasome by electron microscopy, mass spectroscopy and x-ray crystallography. transferring substrates to the s proteasome in the fission yeast schizosaccharomyces pombe c. gordon mrc human genetics unit, western general hospital, edinburgh, uk. e-mail: colin.gordon@hgu.mrc.ac.uk the ubiquitin pathway is found in all eukaryotes. in this pathway, target proteins are covalently modified by the addition of ubiquitin, a amino acid protein, to specific lysine residues. the ability of multi-ubiquitin chains to function as a signal to target proteins for degradation by the s proteasome is well documented. a key question is how is the multi-ubiquitin chain is recognized as a signal? fission yeast rhp /rad and pus / rpn represent two families of multi-ubiquitin chain binding proteins that can associate with the proteasome as well as some e ubiquitin ligases. they seem to provide a link to shuttle ubiquitinated substrates from the e ubiquitin ligases to the s proteasome. a detailed characterization of their proteasome binding will be presented along with their potential role in ubiquitin conjugate dynamics. finally data will be presented indicating that an additional substrate presentation pathway exists in fission yeast which is also conserved in higher eukaryotes. non-proteasomal rpn raises the threshold for association of a ubiquitin-binding protein with the proteasome the ubiquitin proteasome pathway is responsible for the removal of the vast majority of short-lived proteins in the cell. in order to be degraded, a protein substrate is tagged with polyubiquitin and delivered to the proteasome where it is proteolysed. a slew of shuttle proteins is thought to mediate the delivery of polyubiquitinated substrates, although the mechanism remains elusive. one such family of proteins is comprised of rad , dsk and ddi , which all bind polyubiquitinated substrates through a ubiquitinassociated domain (uba) as well as the proteasome through their ubiquitin-like domain (ubl). another potential shuttle structurally unrelated to the ubl-uba family is rpn . rpn is found as an integral subunit of the proteasome as well as an in an unincorporated pool. we characterized the interactions of these proteins with individual proteasomal subunits, as well as between themselves. we find unique relationships between the putative shuttle proteins and the proteasome, pointing to functional dissimilarity among them. strikingly, unincorporated rpn interferes with binding of dsk to the proteasome. thus, we propose that rpn might play a negative role in proteolysis through its action on dsk . proteins modified by multi-ubiquitin chains are usually targeted for degradation by the proteasome. in other cases, ubiquitylation mediates protein sorting or regulates other functions. a striking example for a non-proteolytic role of ubiquitin is the rad dna damage bypass at stalled replication forks. key elements of this pathway are two ubiquitin-conjugating enzymes, rad and the mms /ubc heterodimer, which are recruited to chromatin by the ring-finger ubiquitin ligases, rad and rad , respectively. moreover, also the sumo-conjugating enzyme ubc is affiliated with the pathway and we discovered that proliferating cell nuclear antigen (pcna), a dna-polymerase sliding clamp involved in dna synthesis and repair, is a substrate. pcna is (i) mono-ubiquitylated by rad /rad , (ii) modified by lysine (k) -linked multi-ubiquitylation, which additionally requires mms /ubc / rad , and (iii) sumoylated by ubc . all three modifications affect the same lysine residue of pcna, indicating that they label pcna for alternative functions. indeed, we discovered that monoubiquitylation of pcna promotes an error-prone replication bypass, whereas k -linked multi ubiquitylation mediates errorfree replication across the lesions. in contrast, sumoylation, which occurs even in the absence of dna damage, prevents recombination between homologs at the replication fork. these findings indicate that mono-ubiquitin, k -linked multi-ubiquitin chains, and sumo are crucial for decision making at the replication fork. ubiquitin-mediated proteolysis is the primary mechanism in eukaryotes for degrading unwanted and misfolded proteins. through the cascade of e , e and e enzymes, ubiquitin monomers are attached sequentially to the target proteins, which are then recognized and degraded by the s proteasome. the selection and specific timing of polyubiquitination of the target proteins are conferred by different e ubiquitin ligases. the anaphase-promoting complex (apc) is one of the most extensively studied e ubiquitin ligases that plays essential role in the cell cycle and specific developmental processes. the core apc is composed of - subunits. except for apc and apc , relatively little is known about the role of the other apc subunits or the assembly of the complex. two wd -repeat activator proteins, cdc and cdh determine stage-specific activation of the core apc as well as selection and binding of the apc substrates. in plants, the apc activators are present in multiple copies. arabidopsis contains cdc genes, cdh -type activators known as ccs a , ccs a and ccs b. our work has been focused on the function of apc activators in the cell cycle and plant development, identification of novel apc substrates and on the assembly of the apc complexes. apc activities, based on the expression profiles of the cdc and ccs genes, will be presented at organism level. by detailed protein interaction studies in yeast two hybrid system and arabidopsis protoplasts or transgenic plants, we shall demonstrate how the core apc interacts with the activators and substrates, and propose a model for apc assembly. characterization of substrate delivery to the saccharomyces cerevisiae proteasome by quantitative shotgun proteomics the proteasome is the central protein degradation machinery in the eucaryotic cell. in conjunction with the ubiquitin system, it is responsible for constitutive bulk protein turnover as well as the controlled degradation of regulatory proteins. the system is very well characterized, but the mechanism by which poly-ubiquitinated substrates are delivered to the proteasome remains unclear. recently our lab has proposed a number of proteins to be proteasome-based receptors for poly-ubiquitinated substrates in s. cerevisiae (rpn p, rad p, dsk p; verma et al., ). others (e.g. richly et al. ) have put forward a complex model for the delivery of substrates from the ubiquitinating machinery to the proteasome involving the aaa atpase cdc p. by analyzing the composition of affinity purified proteasome complexes from s. cerevisiae cells lacking these factors and/or exposed to specific proteasome inhibition, we hope to further elucidate the substrate delivery pathway. ubiquitinated proteins recruited to the proteasome are identified utilizing capillary chromatography in-line to electrospray ion trap mass spectrometry (mudpit; link et al. ). using a reference strain grown in minimal medium solely providing heavy nitrogen ( n) as an internal standard, we are able to record even gradual fluctuations in sample composition. differences in the recruitment of substrates to the proteasome in varying mutant backgrounds will shed light on the specificity of proteasome substrate receptors and the topology of the substrate delivery mechanism. oxidative protein damage by reactive oxygen species (ros) produces cross-linking, fragmentation and biochemical modification of the amino acids resulting in biological dysfunctions. quercetin, a widely distributed bioactive plant flavonoid, possesses anti-cancer, antioxidants and free radical scavenging activities, as well as it binds with dna causing dna fragmentation. a little is known about protein oxidative damage and its modifications by antioxidants. therefore, the aim of the present work was to investigate the molecular mechanisms of antioxidant and prooxidant activities of quercetin toward proteins. the antioxidant activities of quercetin, such as superoxide dismutase (sod)-and catalase (cat)-mimetic as well as hydroxyl radical (aeoh) scavenging activities were possessed. bovine serum albumin (bsa) was incubated with different concentrations of quercetin. quercetin has highly sod-and cat-like and hydroxyl radical (aeoh) scavenging activities. its activities are concentration dependent. quercetin fragmentized bsa into specific fragments which they detected by sds/polyacrylamide gel electrophoresis. oxidative protein damage was assessed as tryptophan oxidation, carbonyl, quenone and advanced oxidation protein products (aopp) generation. the increase of protein oxidation products was in concentration dependent manner. the carbonyl and quenone contents and aopp were highly significantly elevated in querce-tin-treated proteins when compared with the control sample. the tryptophan fluorescence was highly decreased in treated protein than in the control sample. the mechanisms of antioxidant and pro-oxidant activities of quercetin have been discussed. these results demonstrate that antioxidant quercetin may potentiate protein damage via oxygen free radical generation, particularly .oh radicals by quercetin. protein stability mediated by a hyaluronanbinding deubiquitinating enzyme is involved in cell viability protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. we have identified three novel genes encoding a deubiquitinating enzyme, vdub , vdub , and vdub (villi deubiquitinating enzyme , , and ) from human chorionic villi by rt-pcr. their cdnas are , bp in length and encode an open-reading frame of amino acids with a molecular weight of approximately kda. expression analysis showed that vdub transcripts are highly expressed in the heart, liver, and pancreas. in addition, they are expressed in various human cancerous cell lines. amino acid sequence analysis revealed that they contain the highly conserved cys, his, and asp domains, which are required for the formation of active site for the deubiquitinating enzymes. in vivo and in vitro deubiquitinating enzyme assays indicated that vdub , vdub , and vdub have deubiquitinating enzyme activity. here, we show that the overexpression of vdub proteins leads to irregular nuclear morphology and apoptosis, suggesting that these vdubs play an important role in regulating signal transduction involved in cell death. interestingly, the sequence analysis showed that vdub proteins contain the putative hyaluronan/mrna-binding motifs, and cetylpyridinium chloride-precipitation analysis confirmed the association between vdubs and intracellular hyaluronan and rna. chemical cleavage of peptide (amide) bonds usually requires harsh conditions. as a result of side reactions and the lack of specificity, chemical amide bond hydrolysis is not a preferred means of protein digestion. we have discovered selective cleavage of peptide bonds in proteins under milder circumstances than any previously reported chemical method. hydrolysis takes place in aqueous buffers in a ph range of , and occurs c-terminal to the proteogenic non-natural amino acid azido-homoalanine (azhal), effected by a staudinger reaction after addition of the mild and biocompatible reagent tris(carboxyethyl)phosphine (tcep). key feature in the suggested reaction mechanism is the unprecedented nucleophilic substitution of the resulting gammaiminophosphorane by the flanking c-terminal backbone amide oxygen atom. after hydrolysis, the new c-terminal peptide is present as a homoserine lactone residue and the n-terminal peptide as its free amine. this new reaction may find application as a very mild and selective bio-orthogonal degradation pathway in biochemistry and biomaterials science. overexpression of proteasome b subunit increases amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates the proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. proteasomes play a fundamental role in retaining cellular homeostasis. alterations of proteasome function have been recorded in various biological phenomena including aging. we have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of btype subunits. in this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the b subunit. stable overexpression of the b subunit in wi /t and hl cells resulted in elevated levels of other b-type subunits and increased levels of all three proteasome activities. immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of ''free''/not integrated b-type subunits identified to accumulate in vector-transfected cells. in support we have also found low pomp levels in b transfectants thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. functional studies have shown that b overexpressing cell lines confer enhanced survival following treatment with various oxidants. moreover we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. finally, as oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the b subunit into primary imr human fibroblasts and we have observed a delay of senescence by population doublings. in summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights towards a better understanding of proteasome regulation. expression levels of the components of the ubiquitin/proteasome pathway in pisum sativum seedlings under anoxia stress change in gene expression: proteins produced under aerobic conditions are no longer synthesized and are replaced by the socalled anaerobic peptides. among those proteins synthesized under o deficiency some enzymes of the glycolytic and fermentative pathways were identified in plants. upon reintroduction of air, the anaerobic mrnas disappear rapidly and the increased levels of those enzymes must return to the basal levels. the ubiquitin/proteasome system is a major pathway of proteolysis in eukaryotic cells and may contribute to controlling the intracellular levels of a variety of short-lived regulatory proteins. in this proteolytic pathway, proteins are covalently conjugated to ubiquitin, which flags them for rapid hydrolysis by the s proteasome. long polyubiquitin chains must be formed to target a protein for destruction by the proteasome. in plants, the ubiquitin-mediated proteolytic pathway is implicated in a variety of cellular processes, including stress responses. in this study, -dayold pisum sativum seedlings were subjected to: (i) h of anoxia stress; (ii) h of aerobic conditions after h of anoxia stress and (iii) h of aerobic conditions after h of anoxia stress. the levels of free and conjugated ubiquitin were detected by immunoblotting using anti-ubiquitin polyclonal antibodies. the changes in the mrna levels of some components of the ubiquitin/proteasome pathway in the seedlings were determined by relative semiquantitative rt-pcr. the results suggest an involvement of the ubiquitin-mediated proteolytic pathway in the anoxia stress response. b - p involvement of the anaphase promoting complex in plant development controlled degradation of short-live proteins via ubiquitindependent proteolysis by the s proteasome is a key mechanism in eukaryotes that regulates nearly all fundamental cellular processes including cell cycle. polyubiquitination of the protein substrate is sufficient to target it for degradation by a large atp-dependent multicatalytic protease, the s proteasome. the selection and specific timing of ubiquitination of the target proteins are conferred by different e ubiquitin ligase. the anaphase promoting complex (apc) is one of the e ubiquitin ligases, which by ordered destruction of various cell cycle proteins has fundamental roles in the regulation of mitotic and endoreproduplication cycles. the apc functions also outside the cell cycle. in post-mitotic cells, the cdh adaptor protein ensures stage specific activation and substrate selection of the apc. in plants, two classes of the cdh -type activators have been identified, ccs a and ccs b that display differential regulation during the cell cycle and plant development as well as differences in their substrate-specificities. in arabidopsis, transient and complimentary expression profiles of the atccs a , atccs a and atccs b genes indicate apc functions during flower development. to identify apc targets, yeast two hybrid screens were performed in the laboratory. out of about interacting proteins, several proteins were transcription factors including a key a regulator of flowers development. data on the interactions of the ccs proteins and transcription factors in arabidopsis protoplasts will be presented as well as a model for the apc regulated pathways. novel effects of ubiquitin system and chaperone proteins on the prion ''life cycle'' in yeast t. a. chernova , k. d. allen , e. p. tennant , k. d. wilkinson and y. o. chernoff department of biochemistry, emory university, atlanta, ga, usa, school of biology and institute for bioengineering and bioscience, georgia institute of technology, atlanta, ga, usa. e-mail: tcherno@emory.edu yeast prion [psi + ], the self-propagated aggregated isoform of the translation termination factor sup , is used as a model system to study neural inclusion disorders. prion aggregates and other neural inclusions in mammals were previously reported to sequester ubiquitin (ub). proteasome inhibitors affected the turnover of mammalian prion proteins. however, a role of ub-dependent proteolysis in the prion ''life cycle'' has not been clearly defined. chaperone proteins, which are also implicated in ub-dependent proteolysis, have been shown to influence the formation and propagation of the prion aggregates. our results uncover the connection between alterations of ub system and chaperone proteins in their effects on the maintenance of yeast prion. we have demonstrated that deletions of genes encoding deubiquitinating enzymes, that are critical for ub regeneration at the proteasome (ubp ) or the vacuole (doa ), cause pleiotropic phenotypic effects that are primarily due to decreased levels of free ub in the yeast cells. these alterations, as well as deletion of the gene encoding ub-conjugating enzyme, ubc , decreases [psi + ] curing by the overproduced disaggregase hsp , suggesting that ub system influences hsp -dependent clearance of prion aggregates. spontaneous [psi + ] formation was also increased in the ubc depleted cells. we previously demonstrated that excess of cytosolic chaperone ssa of hsp family increases de novo formation of [psi + ]. both in vivo and in vitro experiments uncover direct interactions between sup and hsp proteins. the amount of sup -bound to hsp -ssa was increased in ubc deletion strain. we propose a model to explain roles of hsp , hsp and ub system in the prion life cycle. effects of parkinson''s disease mimetics on proteasome activity and protein turnover in human sh-sy y neuroblastoma cells it has recently been suggested that impairment of the ubiquitin/ proteasomal system contributes to the degeneration of dopaminergic neurons (dn) and lewy body (lb) formation in parkinson's disease (pd). mitochondrial dysfunction is also a key factor in pd and agents such as mpp + and dopamine, which inhibit mitochondrial electron transport, produce selective degeneration of dn in animal models. in this study the effects of treating sh-sy y cells with mpp + or dopamine over h on proteasomal chymotrypsin-like activity (cla) was monitored. mpp + ( . mm) caused a sustained depletion of glutathione levels followed by a reduction in proteasomal activity. a reduction in atp levels, caused by higher levels of mpp + ( mm), exacerbated this effect. exposure to low dopamine concentrations ( . mm) led to large reductions in atp without affecting cla or glutathione levels; whilst higher concentrations ( mm) caused marked reductions in cla, glutathione and atp levels. these results suggest that, under oxidative stress, glutathione levels are important regulators of proteasomal activity in this cell line. our group has shown that mpp + can destabilize the neurofilament network in shsy- y cells, partly due to changes in phosphorylation of neurofilament (nf) chains. as nfs are important components of lbs, and their mode of turnover is uncertain, we tested the effects of proteasome inhibitors on nf levels. treatment with these inhibitors led to nf accumulation, which was enhanced when glutathione levels were artificially depleted, suggesting that nfs can be degraded via the proteasomal pathway. the effects of proteasome impairment on protein accumulation will be discussed. mitochondria and the hypoxia-inducible factor (hif- ): regulation of hif- is independent of a functional mitochondrial respiratory chain k. doege, w. jelkmann and e. metzen insitute of physiology, university of luebeck, luebeck, germany. e-mail: doege@physio.uni-luebeck.de the hypoxia-inducible factor hif- is the ''master-regulator'' in adaptation to low oxygen concentration and induces the hypoxic expression of several target genes, e.g. erythropoietin and vascular endothelial growth factor (vegf). in normoxia hif- a is constantly produced but also degraded by oxygen-dependent prolyl-hydroxylation. mitochondria consume most of the oxygen delivered to cells and have been implicated in oxygen sensing. firstly, mitochondria have been proposed to stabilize hif- a by production of reactive oxygen species (ros) in hypoxia. secondly, inhibition of the respiratory chain, e.g. by nitric oxide, has been proposed to cause redistribution of intracellular oxygen followed by reactivation of the prolyl hydroxylases and inhibition of hif signalling. we have used cells depleted of mitochondrial dna (q ) and gas permeable cell culture dishes to eliminate all oxygen diffusion gradients affecting the cells. we show that these dishes neutralize all effects of mitochondrial inhibition. additionally, cellular hypoxia as assessed by pimonidazole staining has been evaluated in human osteosarcoma cells treated with inhibitors of the respiratory chain under hypoxia. these results demonstrate an elevated po under hypoxic conditions after treatment with mitochondrial inhibitors correlating with an intracellular oxygen concentration which reduces hif- activation. thus, neither the absence of ros nor the redistribution of intracellular oxygen supply leads to the destabilization of hif- a in hypoxia. our experiments provide evidence that an increased intracellular po evoked by the absence of mitochondrial oxygen consumption reactivates the prolylhydroxylases and is therefore responsible for the degradation of hif- a under hypoxic conditions. enzyme activity is generally higher in rhizosphere than in bulk soil, as a result of a greater microbial activity sustained by toot exudates or due to the release of enzymes from roots. negative effects of heavy metals on soil microorganisms and enzyme activities have been long recognized. the aim of this study was to assess the stimulatory effects of different low molecular weight organic compounds commonly present in root exudates (mres) on microbial activity and protease activities and , and how high cd concentrations affect such stimulatory effects. soils (arenic udifluvent) were sampled from the agir long-term field trials, contaminated with cd nitrate at rates of (control soil), and mg cd per kg of soil. the mre solutions contained glucose, citric acid, oxalic acid, glutamic acid or a mixture of the four compounds, added to give a rate of mg of mre-c per kg of soil. the effects were measured at mm (bulk soil) distance from the mrs. protease activity was determined by hydrolysis of n-benzoylargininamide (baa). the results showed that different mres had different stimulatory effects on microbial growth and on the protease activities, mostly localized in the rhizosphere soil layer. in the control soil, the dsdna content was significantly increased by the addition of all mre in both rhizosphere and bulk soil layers. the and mg cd per kg of soil negatively affected on protease activity. the glucose, citric acid, oxalic acid, glutamic acid, mres mix in both rhizosphere and bulk soil layers, did not stimulate protease. the, microbial growth and protease activities were drastically reduced by high cd concentrations. participation of different digestive proteinases of the yellow mealworm, tenebrio molitor, in initial stages of hydrolysis of the main dietary protein insects generally have a wide spectrum of digestive proteinases. the knowledge about the impact of different proteinases to initial stages of hydrolysis of dietary proteins is essential for insect control by means of proteinase inhibitors and bacillus thuringiensis toxins. the larvae of a stored grain pest yellow mealworm, tenebrio molitor, were reared on milled oat flakes. the main dietary protein for these larvae was s globulin, the main storage protein of oat seeds. to study the initial stages of s globulin hydrolysis in vitro the reaction was performed in the physiological conditions of anterior midgut (am) (ph . ) by purified enzyme preparations from am: two fractions of cysteine proteinases cys ii and cys iii, chymotrypsin-and trypsin-like proteinases. total hydrolysis of s globulin was observed with cys ii. slightly less effective was hydrolysis by chymotrypsin-like enzyme. cys iii cysteine and trypsin-like proteinases produced only partial hydrolysis of seed globulin. in all cases high molecular mass (mm) intermediate products were formed testifying that hydrolysis of s globulin was sequential. incubation with both cysteine proteinase fractions led to formation of kda product, while serine proteinases pro- in contrast to ''classical'' bioregulator peptides, peptides could be generated in the course of catabolic degradation of functional proteins. for years, we have been interested in such particular group of peptides derived from blood hemoglobin, hemorphins. hemorphins consist in a family of opioid receptor-binding peptides from to amino acids that are released by proteolytic processing from the ( - ) segment of human hemoglobin betachain. they are prevalent throughout the peripheral and central nervous system and have been isolated in vivo from tissues or fluids. many in vivo physiological effects have been related (coronaro-constrictory, anti-tumorous, immunoregulatory activities) and several of the hemorphins interact at various levels of the reninangiotensin system (ras) by inhibiting angiotensin-convertingenzyme (ace), aminopeptidase n (apn) and dipeptidyl peptidase iv (dppiv) activities. in addition, some hemorphins and in particular lvv-hemorphin- (lvvypwtqrf), binds with high affinity to the brain (ic = . nm) and renal at angiotensin receptor subtype and is possible the main endogenous ligand from this receptor. in an attempt to characterize in vivo precise mechanisms for their release, our attention is focused towards tumoral and central nervous system environments. the last one is particularly interesting as all cellular components implicated in the release of hemorphins are present simultaneously: the haemoglobin precursor and localized brain proteases which might come in contact with blood haemoglobin. in this purpose, the examination of potentiality for this tissue to generate ''neuro''-hemorphins would be of interest since sources of hemorphins in the brain have not yet been definitively established. later, we showed that sgt interacts with calcyclin (s a ) and other calcium-binding proteins of the s family (nowotny et al. j biol chem ). moreover, in collaboration with dr chazin's group, we found that in vitro sgt binds to hsp (lee y-t et al. j biol chem ). in this work we studied the expression and subcellular localization of sgt in mammalian cells by means of western and northern blots. among different cell lines examined human embryonic kidney hek and human glioma t g cells exhibit highest expression of sgt protein. moreover, we found that in mouse and rat cells there is one isoform of sgt , while in human cells two isoforms of this protein were found. to study the subcellular localization of sgt we chose the cells containing moderate level of sgt such as human epidermal hep- cells. by applying immunocytochemistry we found that this protein is present not only in the cytoplasm but also in the nucleus. at present we check the effect of intracellular ca + concentration on subcellular localization of sgt and on its co-localization with target proteins. acknowledgements: this work was supported by grants: kbn p a and firca/nih r tw . combining reverse genetics, reverse chemogenomics and proteomics to assess the impact of protein n-terminal methionine excision in the cytosol of higher eukaryotes in living organisms whatever the cell compartment, proteins are always synthesized with methionine (met) as the first residue. however, this first met is specifically removed from most mature proteins. in the course of protein n terminal met excision (nme), the free n terminal met is removed by met aminopeptidase (map) cleavage. three enzymes (map a, map a and map b) have been identified in the cytoplasm of arabidopsis thaliana. by combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic nme in a. thaliana and of the respective contributions of the two types of cytoplasmic map throughout development. in the map a ko context (map a- ), modulating map activity by treatment with various concentrations of the specific drug fumagillin impaired plant development. hence, (i) cytoplasmic nme is essential in plants, (ii) plant map a and map s are functionally interchangeable as a complete block of either map type activity does not cause any visible molecular or phenotypic effect, (iii) a minimal level of cytoplasmic map is required for normal development and (iv) the plant a. thaliana appears an excellent system to study nme and the associated-role of anti-cancer agents like fumagillin. proteomics was used to assess the impact of nme blocking induced by fumagillin. we used a wild-type plant and the map a- variant grown in the presence of nm fumagillin. the map a- variant showed a dwarf phenotype. we compared by d gel electrophoresis the patterns of each protein extracts. protein spots were identified by tandem mass spectrometry. the data show that fumagillin induces many dedicated pathways, with a prevalence of those related to oxidative stress. prolyl endopeptidases from the midgut of the yellow mealworm tenebrio molitor side of proline residues. these enzymes were found in mammals, several higher plants, fungi and bacteria. it is suggested that the enzymes participate in the in vivo regulation of the action of biologically active peptides. we for the first time report about two prolyl endopeptidases in the larval midgut of a stored product pest yellow mealworm tenebrio molitor where they can participate in the proteolysis of one of the main dietary proteins of t. molitor larvae -rich in proline prolamines. characteristics of two prolyl endopeptidases are significantly different. optimum for hydrolysis of the substrate z-ala-ala-pro-pna (n-carbobenzoxy-l-alanyl-l-alanyl-l-prolyl-p-nitroanilide) by prolyl endopeptidase was at ph . , and prolyl endopeptidase -at ph . . prolyl endopeptidase displayed high phstability in the ph range . - . and the rate of hydrolysis increased in the presence of kcl and cacl . prolyl endopeptidase demonstrated low stability in the whole ph range, the rate of hydrolysis strongly decreased in the presence of above mentioned salts, but increased in the presence of high concentrations of edta. the influence of cell growth media on the stability and antitumour activity of methionine enkephalin studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. the objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. the degradation kinetics of the model peptide methionine enkephalin (met-e, tyr-gly-gly-phe-met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of foetal bovine serum (fbs). the influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the met-e degradation was also investigated. the results obtained in the dulbecco's modified eagle medium containing % fbs indicated a rapid degradation of met-e (t / = . h). pre-incubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of met-e, as shown by increased half-life to h. the in vitro activity of met-e against poorly differentiated cells from lymph node metastasis of colon carcinoma (sw ) and human larynx carcinoma (hep- ) cells was determined. tumour cells were grown weeks prior to the experiment in a medium supplemented with , or % fbs. statistically significant to mild or no suppression of cell proliferation was observed in all cultures. in both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and met-e, compared with cells exposed to the peptide alone and cells grown in the absence of met-e, was observed. this study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays. protein metabolism in whole body and skeletal muscle of laboratory rats treated by proteasome inhibitors proteasome inhibitors are new agents which may be used in treatment of cancer and other severe disorders. one of the possible side effects of their administration is disturbance in protein metabolism which may affect outcome of the illness. two separate studies were performed using wistar rats. in the first study, m. soleus (sol) or m. extensor digitorum longus (edl) were incubated in medium containing mmol/l mg or mmol/l adaahx l vs or without inhibitor (control). protein synthesis was evaluated using l-[ - c]leucine. proteolysis was determined according to the rate of the tyrosine release into the medium during incubation. in the second study, proteasome inhibitor mg diluted in dimethyl sulfoxide (dms) was administered intraperitoneally in dose mg/kg b.w. controls consisted of dms treated animals. changes in protein and amino acid metabolism were estimated in steady-state conditions using continuous infusion of l-[ - c]leucine h later. mann-whitney (in vivo study) and paired t-test (in vitro study) were used for statistical analysis. in in vitro study, both mg and adaahx l vs significantly decreased protein synthesis and proteolysis. however, in in vivo study, a significant increase in whole-body protein synthesis and proteolysis were observed in mg treated animals. acknowledgements: the study was supported by a grant of gacr no. / / . bioinformatical evidence for a prokaryotic ubiquitin-like protein modification system h. scheel, s. tomiuk and k. hofmann bioinformatics group, memorec biotec gmbh, ko¨ln, germany. e-mail: kay.hofmann@memorec.com until recently, the ubiquitin system has been considered a purely eukaryotic invention. by now, the bacterial moad/moeb and this/thif systems are known to be prokaryotic versions of a rudimentary activation system for ubiquitin-like proteins. however, similarities to the ubiquitin system end after the activation step, as moad and this are not conjugated onto target proteins but rather have a role in the biosynthesis of molybdopterin and thiamin, respectively. the eukaryotic protein urm is the closest homolog of moad and this. unlike its bacterial cousins, urm is conjugated onto target proteins and thus can be considered the founding member of the diverse eukaryotic ubiquitin family. by using a bioinformatics approach that integrates methods of sequence analysis, phylogenetics, phylogenomics and gene-order analysis, we were able to show that many bacteria possess a third ubiquitin-like activation system that most likely is used for protein modifications. the novel system uses a moad/this relative, which is more closely related to urm than the typical moad and this proteins. these bacterial urm (burm ) proteins typically require the proteolytic removal of a c-terminal extension, which masks the gg motif important for activation. many burm operons contain a mpn+/jamm domain protein (belonging to a bona fide ubiquitin-specific protease family), which is most likely responsible for this cleavage. as a third component, an e -like enzyme is also part of typical burm operons. the burm -associated e enzymes look more like uba (the eukaryotic urm -e ) than like the bacterial moeb/thif e enzymes. interestingly, the mpn+/jamm protease is also conserved in those bacteria whose burm end with gg, suggesting that burm removal is important not only for the activation step b - p non-hypoxic induction of hypoxia-inducible factors by insulin and -deoxy-d-glucose hypoxia-inducible factors (hifs) are key mediators of the cellular adaptation to hypoxia, but also respond to non-hypoxic stimuli like insulin. to clarify involvement of all known hif subtypes in conditions resembling diabetes, we determined distribution of mrnas and proteins in rats subjected to in vivo hypoglycemia and glucoprivation. wistar rats were infused with either saline, insulin, or -deoxy-d-glucose ( -dg) to provoke hypoglycemia or impaired glucose assimilation. using real-time qpcr, mrna levels of hif subunits a, a, a, b, and of the target gene glut- were determined in various organs. cellular distributions of hif-a proteins were examined by immunohistochemistry. treatments with insulin or -dg resulted in a widespread increase in hif- a mrna after h, whereas mrna expression of other hif subunits remained unaffected, except for hif- a which increased in lung and heart after -dg. in cerebral cortex and kidney, enhanced staining of all hif-a proteins was observed after insulin or -dg treatments. lung, heart and kidney showed enhanced levels of glut- mrna. both hypoglycemia and glucoprivation provoke functional activation of the hif system, with transcriptional up-regulation of hif- a representing a typical response. our data indicate an involvement of the hif system, and hif- a in particular, in the pathophysiology of diabetes. fragments of human salivary statherin and pb peptide underlying a furin-like pro-protein convertase action in the pre-secretory salivary fragmentation pathway the recent analysis of some derivatives of human salivary peptides and proteins [ ], such as acidic and basic proline-rich proteins (prp) and histatins, allowed recognizing in the presecretory salivary fragmentation pathway the action of a furinlike pro-protein convertase of the kexin-subtilisin family, often followed by a carboxy-peptidase action. on the same line, the present study was carried out to search in human saliva the fragments generated from statherin and pb peptide by the action of furin-like proteinases, utilizing a selected-ion monitoring strategy based on hplc-it ms. the fragments and post-translational derivatives detected with high frequency in multiple samples were the following: (i) statherin ( amu), des-phe- ( amu), des-thr- -phe- ( amu), des-asp- ( amu), mono-phosphor. ( amu), statherin sv (missing - residues; amu), fragm. - ( amu), fragm. - ( amu), fragm. - ( amu). moreover, the fragm. - ( amu) of pb peptide ( amu) was identified. the quantity of these fragments in salivary samples was usually < % of the parent peptide. the identified fragments confirmed the action of a proprotein convertase on furin-like consensus sequences, being the cleavage at arg- (ekflr), arg- (lrr) and arg- (rrigr) for statherin, and at arg- (rgpr) for pb peptide. detection of statherin missing n-and c-terminus residues indicated also a pre-secretory exopeptidase action, already observed in other salivary peptides. the function of these statherin and pb derivatives in the oral cavity must be elucidated. cloning and expression of a pepstatin insensitive acid protease from thermoplasma volcanium in e. coli acid proteases, commonly known as aspartic proteases, are recognized by their specific inhibition by pepstatin. acid proteases are found in microorganisms both as intracellular and extracellular enzymes. there is very limited number of thermostable, pepstatin insensitive acid proteases isolated from bacterial sources. the only example of purified and cloned acid protease from archaebacteria is thermopsin, produced by sulfolobus acidocaldarius. this thermophilic enzyme represents a new class of acid proteases. to extend our knowledge on the microbial acid proteases with thermostable properties, in this study we have undertaken the cloning and expression of a thermostable, pepstatin insensitive acid protease from themoacidophilic archaeon thermoplasma volcanium. a primer set was designed based on nucleotid sequence of the predicted thermopsin gene and pcr amplification produced a bp fragment, which covered complete thermopsin gene with some upstream and downstream sequences. the amplified thermopsin gene was cloned in e. coli, using pdrive vector. the alignment of the amino acid sequences of thermopsins from various archaea revealed the highest homology ( %) between the tp. volcanium thermopsin and putative tp. acidophilum enzyme, thermopsin . there was a low degree of similarity ( %) between the tp. volcanium thermopsin and thermopsin from sulfolobus acidocaldarius. expression of the recombinant thermopsin was attempted using qia expression kit, where the cloned gene was ligated to pqe expression vectors to be expressed under the control of t promoter. in this system the protein was tagged with xhis residue at n-terminal end so that it could be selectively isolated using ni-nta metal-affinity chromatography. include various vital proteins with discrete functions in the propagation of apoptosis. our aim is to generate a caspase cleavage site predictor specific for each member of the caspase family in order to make subtype-specific predictions of new caspase substrates. we have used a set of experimentally verified proteins to generate sequence logos and train a neural network in order to predict caspase cleavage sites. machine learning techniques, such as artificial neural networks, are often well suited to integrate the subtleties of sequence variations. this approach also enables integration of structural information in the pattern recognition procedure which could possibly increase the predictive performance of the neural network. the identification of new caspase substrates can lead to further elucidation of several cellular processes involving caspases, including apoptosis, cell cycle regulation, cellular differentiation, and pro-inflammatory responses. in addition, the generation of caspase inhibitors could be greatly aided by a caspase cleavage site predictor. regulation of protein synthesis and autophagic-lyososomal protein degradation in isolated pancreatic acini a. l. kovacs and e. papp cell physiology laboratory, department of general zoology, eotvos lorand university, budapest, hungary. e-mail: alkova@cerberus.elte.hu a series of biologically active compounds (wortmannin, ly , -methyladenine, rapamycin, okadaic acid, theophyllin, insulin, glucagon, cholecystokinin) influencing protein synthesis and autophagic-lysosomal protein degradation by interfering with important signalization pathways were investigated. our results show that in exocrine pancreas cells phosphatidyl inositolkinases (pi k-s) are activators, while the target of rapamycin protein (tor) is an inhibitor of autophagy. camp is an inhibitor of lysosomal protein degradation that acts through members of the pi k family. okadaic acid inhibits lyososomal protein degradation without inhibiting the formation of autophagic vacuoles. the inhibition of pi k-s and tor diminishes protein synthesis, inhibitors of these kinases reduce the synthesis stimulatory effect of insulin. cholecystokinin showed a biphasic stimulatory effect while glucagon was ineffective on protein synthesis. on the base of these results a possible signalization pathway is suggested for autophagic segregation and lysosomal protein degradation in pancreatic acinar cells. purification and characterization of a bifunctional protease from vibrio vulnificus in this study, we purified and characterized an extracellular protease showing dual functions as prothrombin activator and fibrinolytic enzyme from vibrio vulnificus atcc . the purified enzyme had broad substrate specificity towards various bloodclotting associated proteins such as prothrombin, plasminogen, fibrinogen and factor xa. the cleavage of these proteins could be stimulated by addition of mm mn + . the protease could acti-vate prothrombin to active thrombin. however, the thrombin activity generated from prothrombin activation by the protease seemed to be transient, with further cleavage resulting in a loss of activity. interestingly, the enzyme could enhance the activity of thrombin during the initial rate of fibrin formation when purified fibrinogen was used as substrate. it could also actively digest fibrin polymer as well as cross-linked fibrin. these results suggest that the secreted protease functions as a prothrombin activator and a fibrinolytic enzyme to interfere with blood clotting as part of the mechanism associated with its pathogenicity in human. tumor invasion and metastasis are the major causes of treatment failure and death in cancer patients. one requisite for neoplastic cell invasion during tumorigenic processes is the remodeling events that occur within the stroma or extracellular matrix (ecm). cysteine cathepsins, most likely along with matrix metalloproteases and serine proteases, degradate the ecm, thereby facilitating growth and invasion into surrounding tissue and vasculature. clinically, the activity levels and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. the aim of our study was therefore both the determination of prognostic and diagnostic impact of cathepsins b, l and h from human tissues extracts (normal and tumor tissue) and extracellular fluids (such as plasma and urine) and a -proteinase inhibitor (pi) in pathogenesis of different types of human brain tumors, and extraction and purification of cysteine cathepsin endogenous inhibitors from normal and tumor brains and studying of their physicochemical properties. it was found that the increasing of cysteine cathepsins b, l, h activity levels in brain tumors tissues depend on histostructure, histogenesis and tumor malignancy grade. increasing of cathepsins l and h activity levels was found in plasma and urine in depending on histogenesis. at the same time decrease in pi activity level was registered. besides, kinetic characteristics of extracted normal brain endogenous inhibitors of cysteine cathepsins were determined. in extracted tumor brain endogenous inhibitors, there were differences in physicochemical properties in comparison with normal. the data obtained contribute to understanding the participation of cysteine cathepsins and their inhibitors in mechanisms of cancer genesis and both become useful for solving the problem of improving of tumor therapy and provide the possibility of using their activity as diagnostic and prognostic markers. protein hydrolysates of sea origin as components for microbiological culture media dry hydrolysate was prepared from protein-containing waste of icelandic scallop chlamys islandicus processing (spw) by means of a proteinase complex from king red crabs hepatopancreas. the enzyme consist of the proteolytic enzyme complex from crab hepatopancreas, in which serine proteases dominate (collagenase, elastase and trypsin-and chymotripsin-like proteinases). as proteinases from king red crab hepatopancreas have high enzymesubstrate affinity to icelandic scallop proteins, a high degree of proteolysis can be achieved. the composition and properties of the material were investigated on enzymatic protein hydrolysate from spw obtained under the most technologically suitable conditions: - o c, ph . , h, the ratio between the protein material and the enzyme preparation being : . for comparison we examined the composition of commercial pancreatic hydrolysate from poor-quality fish species, mainly boreogadus and micromestistus. it was found that hydrolysate from spw significantly overpowered the commercial analog in the mass percentage of the target product (free amino acid and oligopeptides). the resulting product contains not < % free amino acids and oligopeptides. predominant are aspartic acid, leucine, isoleucine, arginine and lysine, which account for > % of the free amino acids. the potential usage of the protein hydrolysate as a nutrient for microorganism cultivation is estimated. microbiological studies have demonstrated that the hydrolysate from spw can be used as a protein component in nutrient media. the tested microbial strains satisfactorily grew on the media. the z variant alpha- proteinase inhibitor (a piz) misfolds in the endoplasmic reticulum (er) and is a substrate for er-associated protein degradation (erad). we report here that a piz degradation is also dependent on vps /atg , a gene that encodes a component of two pi -kinase complexes that regulate membrane traffic; complex i is required for autophagy, complex ii is required for the cpy-to-vacuole pathway. to elucidate why vps p participates in a piz degradation, we tested the hypothesis that erad was saturated at elevated levels of a piz expression and that excess a piz was targeted to one of these alternative quality control pathways. overexpression of a piz led to vacuole-dependent degradation and both complexes were required for delivery of the excess a piz to the vacuole. when the cpy-to-vacuole pathway was compromised a piz was secreted and the distribution of soluble vs. aggregated forms of a piz was comparable with that of wild type yeast. however, disruption of autophagy led to an increase in levels of aggregated a piz; suggesting that when erad is saturated the excess a piz is selectively targeted to the vacuole via the cpy-to-vacuole sorting pathway, while excess a piz that forms aggregates in the er is targeted to the vacuole via autophagy. together, these results reveal multiple pathways for recognition and removal of aberrant proteins and provide direct evidence that aggregated a piz is removed by autophagy. our findings may have application in the understanding of, and treatment for, individuals with liver disease caused by the accumulation of er aggregates of a piz. acknowledgements: the study was supported by national science foundation grants mcb- and mcb- . yeast and lactobacillus association generates peptides from acid goat whey proteins fermentation s. didelot, s. bordenave-juchereau, e. rosenfeld, l. murillo, j. m. piot and f. sannier laboratory of biotechnology and bioorganic chemistry, university of la rochelle, la rochelle, france. e-mail: lmurillo@univ-lr.fr our goal was to produce peptides from fermentation of unsupplemented acid goat whey by dairy micro-organisms. we used a lactobacillus, lactobacillus paracasei, and a yeast, candida parapsilosis, both previously isolated from a cheese microflora. when co-cultivated aerobically, both micro-organisms grew on unsupplemented goat whey and led to a medium acidification from to ph . . reversed phase (rp)-hplc analysis revealed a total alpha-lactalbumin hydrolysis after h of fermentation, a modification of the beta-lactoglobulin elution peak, and . -fold increase in peptide level compared with the non-fermented whey. in the absence of c. parapsilosis, l. paracasei grew poorly on whey and only a weak medium acidification from to . was observed after h of fermentation. rp-hplc analysis revealed a weak modification of beta-lactoglobulin elution peak, a truncated form of alpha-lactalbumin and no peptide generation. c. parapsilosis was able to grow on unsupplemented goat whey without modifying ph of the medium, but only % of proteins were hydrolysed (alpha-lactalbumin) or denaturated (beta-lactoglobulin) and, again, no peptides were detected. these results suggest that (i) c. parapsilosis is required for l. paracasei growth and (ii) the co-culture of both micro-organisms is needed to generate peptides from alpha-lactabumin hydrolysis. during co-culture on whey, the use of penicillin g and cycloheximide as bacterial and yeast growth inhibitors respectively, revealed that l. paracasei growth was required for medium acidification to ph . and alpha-lactalbumin hydrolysis. however, we demonstrated that the protease(s) responsible of alpha-lactalbumin hydrolysis was (were) synthesized by c. parapsilosis during the first stage of fermentation and that medium acidification (obtained either by l. paracasei growth or chemically) was required for yeast protease(s) activity. dengue virus causes widespread human diseases such as dengue fever, dengue hemorrhagic fever and dengue shock syndrome. the viral genome is a positive rna strand that encodes for a single polypeptide precursor. processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by ns serine protease. the three dimensional structure of ns protease domain ns pro has been elucidated [ ] . recently a new construct of the recombinant form of the ns pro, was engineered [ ] . we have expressed in e. coli the his-tag-cf .gly.ns pro protein a new construct of the recombinant form of the ns pro linked to a -residue co-factor, corresponding to a part of ns b, via a non-cleavable, flexible non-apeptide (gly sergly ), and have currently optimized the purification procedure. chemically optimized substrates, peptides and depsipeptides, were designed and tested to afford an efficient in vitro activity assay, using hplc and fret spectroscopy. the data suggest that the amino-terminal region of the -amino acid co-factor domain is involved in additional charged interactions with ns that are essential for activity as previously described. this form showed catalytic activity and spectroscopic studies were performed to identify the folding of the protein. moreover, experiments of limited proteolysis have been performed to identify the essential enzymatic domain of the protein and to stabilize the role of the cofactor in the activity and in folding stabilization of the enzyme. after h of the limited proteolysis with endoproteinase asp-n the product was analyzed by sds-page and activity assay, showing a high reduction of the molecular mass and only a loss of the activity of the %. cd and n- h-hsqc spectra of this protein fragment were performed and other functional and structural characterizations are in progress in our laboratory. it is intended to obtain the structure in solution of the essential active domain of the uniformly c, n-labeled cf .gly.n-s pro by high-field d nmr spectroscopy. the solution structure of the enzyme will be used to answer yet unresolved questions about the mechanism of action, the role of its cofactor ns b, and the observed substrate specificity. introduction: fish consumption is associated to nutritional benefits due to the presence of proteins of high biological value, minerals, vitamins and polyunsaturated fatty acids. most studies concerning the benefits of fish consumption on cancer prevention have focused on fish fatty acids but little is known about the potential bioactivity of fish peptides. the present study was then designed to assess the antiproliferative activity of various fish protein hydrolysates, in order to further purify and characterize anticancer peptides. methods: twenty-one fish hydrolysates (from seven species) produced within the framework of the european valbiomar programm. fish hydrolysates composition (protein, fat and salt content) was determined by standard methods (kjehldhal, soxhlet extraction and volhard respectively). cytotoxic and antiproliferative activity were assayed in vitro on mcf- / and mda-mb- human breast adenocarcinoma cell lines, following a cell viability colorimetric assay (promega, france). antiproliferative activity of fish hydrolysates was compared with that of reference anticancer molecules with various cellular targets, namely actino-mycine d, cytosine-beta-d-arabinofuranoside, cyclophosphamide, etoposide, kenpaullone and roscovitine. results: composition analysis revealed that most hydrolysates contained more than % protein. three blue whiting hydrolysates containing % protein, . % lipid and . % salt induced a strong breast cancer cells growth inhibition when tested at g/l for h in cell culture medium. blue whiting hydrolysates , and , respectively, induced a growth inhibition of . , . and . % on mcf- / , and . , . and . % on mda-mb- . these in vitro antiproliferative activities are in the range of that observed when the two breast cancer cell lines are treated for h with kenpaullone, roscovitine or cytosine-beta-d-arabinofuranoside ) m. further studies are engaged to fractionate and characterize the antiproliferative peptides contained in blue whiting hydrolysates. during recent years, it has been established that intracellular proteolysis in eukaryotic cells is largely accomplished by a highly selective non-lysosomal pathway that requires atp and a large ( . mda) multisubunit complex known as the s proteasome. the proteasome-mediated pathway plays vital regulatory functions. it degrades many important proteins involved in cell cycle control, in signaling pathway, and in general metabolism, including transcription factors and key metabolic enzymes. another function of the proteasomal system is the removal of abnormal, misfolded and oxidized proteins generated under normal and, in particular, stress conditions. to date, proteasomes from other than animal or plant cells were studied only in yeast. recently, in our laboratory, the proteasome-mediated pathway was shown to be involved in the regulation of ligninolytic activities in the white rot fungi trametes versicolor and phlebia radiata upon nutrient starvation (staszczak, enzyme microb technol ; : - ). it was the first report on proteasomes in fungi representing basidiomycota. white rot fungi are able to degrade lignin by the action of secreted enzymes, the best characterized of which are laccases, lignin peroxidases, and manganese peroxidases. the subject of lignin biodegradation has commanded attention for a considerable period of time mainly because of its ecological significance and wide industrial applications of bioligninolytic systems. heavy metal ions are important environmental pollutants which affect biodegradation processes performed by white rot fungi. in the present study, we investigated whether the proteasomal degradation pathway might be involved in the regulation of laccase production by t. versicolor in response to cadmium exposure. studies of cacybp/sip function using small interfering rna cacybp/sip was discovered as a protein that bound calcyclin (s a ) in a calcium-dependent manner (filipek and wojda ; filipek and kuznicki, ) and its distribution and some biochemical properties have been studied. for instance, it has been shown that cacybp/sip binds calcyclin via its c-terminal fragment (nowotny et al. ) and that, beside calcyclin, it interacts with other calcium binding proteins of the s family (filipek et al. ) . originally, we identified cacybp/sip in ehrlich ascites tumour (eat) cells but it is also present in other mammalian tissues and cells. in particular, high expression of cacybp/sip was found in neuronal cells of mouse and rat brain (jastrzebska et al. ) . at present the distribution and structural properties of cacybp/sip are quite well described but its function remains obscure. there is only one paper published concerning the possible involvement of cacybp/sip in b-catenin ubiquitination and degradation (matsuzawa and reed ). to elucidate the biological role of cacybp/sip we have designed and synthesized sirna (small interfering rna) against this protein. this sirna was then used to transfect neuroblastoma nb- a and embryonic kidney hek cells, expressing high and low amount of endogenous cacybp/sip respectively. the level of cacybp/sip was monitored in cell extracts by western blot technique. we found that sirna against cacybp/sip, which we designed, inhibited the expression of this protein, as its level in transfected cells was lower in comparison with control cells. at present, we checked the effect of diminished expression of cacybp/sip on b-catenin degradation and other cellular processes. acknowledgements: this work was supported by grants: kbn heavy metals are powerful poisons for living cells. it has been shown that exposure to arsenicals, either in vitro or in vivo, in a variety of model systems, causes the induction of a number of the major stress protein families, such as the heat shock proteins (hsp) (toxicol appl pharmacol ; : ). the reasons for heavy metal toxicity in vivo are not fully understood, but they are known to contribute to the accumulation of aberrant proteins (bba, , , ). in animal cells, arsenite has been reported to cause sulfhydryl depletion, to generate reactive oxygen species and increase the level of high molecular mass ubiquitin-protein conjugates (toxicol appl pharmacol ; : ). in cells submitted to stress conditions, several components of the ubiquitin/proteasome pathway are activated. in this major, eukaryotic proteolytic pathway, multiple ubiquitin molecules are enzymatically ligated to proteins destined for catabolism by an enzyme system composed of three types of enzymes, commonly referred to as e , e , and e . the large ubiquitinprotein conjugates thus formed are subsequently degraded by a very large protease complex, the s proteasome, in an atpdependent process. the changes in free ubiquitin (ub) and ubiquitin-protein conjugates (ub-p) levels were followed by immunoblotting during the incubation of the higher plant lemna minor l.(duckweed) in the presence of arsenite (as), at concentrations known to confer thermotolerance to the plants. the observed increase in the amount of large molecular mass ubiquitin-protein conjugates is indicative of a role for the ubiquitin/ proteasome pathway in the response of lemna to as stress. this outcome is primarily attributed to an increased availability in protein substrates during as treatment for three main reasons: an increase in protein carbonyl (a major marker for protein oxidation) content detected by immunoblotting; moderate increments (as determined by semi-quantitative rt-pcr) in the mrna levels of the codifying sequences for the ubiquitin pathway components: ubiquitin, e , e and the b subunit and the atpase subunit of the s proteasome; an identical pattern of variation for the large ubiquitin-protein conjugates is observed in the simultaneous presence of as and cycloheximide, indicating that the observed increase in ubiquitin conjugates does not depend on de novo protein synthesis. ageing and autophagy y. stroikin and a. terman experimental pathology, linko¨ping university, linko¨ping, sweden. e-mail: yurst@inr.liu.se life of aerobic cells is associated with continuous oxidative damage resulting in the formation of altered, non-functional macromolecules and organelles. intracellular accumulation of oxidized proteins defective organelles and lipofuscin inclusions are typical manifestations of ageing that preferentially affects long-lived post-mitotic or growth-arrested cultured cells. autophagy, an important biological mechanism for renewal of damaged intracellular structures, has been found decreased in ageing. to learn more about the role of autophagy in ageing, we studied the effect of the inhibitor of autophagic sequestration -methyladenine ( -ma) on human diploid fibroblasts and astrocytes. inhibition of autophagy in growth-arrested (confluent) fibroblasts for weeks resulted in the accumulation of altered lysosomes displaying lipofuscin-like autofluorescence, especially when -ma exposure was combined with hyperoxia. the findings suggest that autophagy is indispensable for normal turnover of lysosomes, and lysosomal components may be direct sources of lipofuscin. the accumulation of oxidatively damaged intracellular structures (so-called biological ''garbage'') was associated with decreased cell viability. two-week-inhibition of autophagy with -ma resulted in a significantly increased proportion of dying cells when compared with both untreated confluent cultures and dividing (subconfluent) cells exposed to -ma. similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. the results support the idea that biological ''garbage'' accumulation is essential for ageing and age-related death of post-mitotic cells, which can be prevented by cell division. recently two family members of the tumour suppressor gene p have been described, p and p , which seem to be necessary for specific p -induced stress-response pathways. furthermore, p and p appears to be crucial to determine the cellular sensitivity to anticancer drugs, particularly in tumours lacking functional p . here, we show that p and p isoforms are also regulated by proteasomal degradation. we have identified several e -ubiquitin ligases responsible for the regulation of the stability of p and p . we found that the regulation of p and p is isoform-specific. furthermore, we demonstrate that ubiquitination of p influences the cellular localization of p and of the respective e -ubiquitin ligases. finally, we show that the expression of the various e -ubiquitin ligases can be differentially induced by p -isoforms. in addition, the e -ubiquitin ligases can influence the apoptotic function of p . our findings demonstrate that p and p are sent to degradation or stabilized by e -ubiquitin ligases in an isoform-specific manner and we suggest a negative feedbackloop between p , p and their regulators, as they also influence the function of p and p . increased level of metalloproteases was shown to accompany tumor angiogenesis and active invasion in adjacent tissue [ ] . development of different types of tumors is often accompanied by increased protease activity in blood [ , ] . in the present study we compared protease activity of plasma and eluate from surface of blood cells in healthy donors and patients with breast tumor. we have demonstrated recently that in blood of healthy donors almost all circulating nucleic acids (cirna) are bound at the surface of blood cells. in patients with fibroadenoma cirna were found at cell surface whereas in breast cancer, no cell-surfacebound cirna were detected in blood [ ] . conjugates of hydrophobic and hydrophilic peptides of cd receptor with biotin were incubated with avidin-coated -well eia microplates. avidinpeptide complex was incubated with samples under investigation and serial dilutions of proteinase k solution, which was used for calibration of protease activity. undegraded peptides were visualized by incubation with goat anti-peptide antibodies followed by conjugate of anti-goat immunoglobulins with peroxidase. blood plasma and eluate from surface of blood cells of cancer patients demonstrated increased level of anti-hydrophilic protease activity compared with healthy donors. increase of protease activity against hydrophilic peptide in blood correlate with decrease of cell-surface-bound cirna, indicating that blood proteases can affect concentration and distribution of circulated na. identification of cleavage site and natural substrate specificity of prta, a serralysin-type metalloprotease from the entomopathogenic microorganism photorhabdus prta, a secreted basic metalloprotease of photorhabdus, belongs to the m b (serralysin) family of proteases. the biological function of these enzymes is not known, but in some cases they are supposed to have a role in virulence. serralysins are generally assumed to have broad substrate side-chain specificity. attempts toward the generation of a sensitive and specific substrate of these enzymes had limited success, and no such substrate is available for prt-a. through mass spectrometric analysis of prta cleavage products of oxidized insulin a and b chain, we found that prta has a welldefined cleavage site preference. based on this, we developed a sensitive and highly specific oligopeptide substrate through optimization of the amino acid composition and length. the kinetic parameters of prta isolated from photorhabdus luminescens ssp. laumondii strain brecon were measured on the best substrate, dabcyl-glu-val-tyr-ala-val-glu-ser-edans, giving a km of . · ) , a kcat of . · ) /s and a kcat/km of . · . its poor hydrolysis by various proteases proved its specificity, while it was very sensitivity in measuring prta activity in hemolymph samples from photorhabdus infected galleria mellonella larvae. the substrate preference of prt-a was determined by in vivo digestion of hemolymph proteins from manduca sexta. six minor protein components were selectively cleaved, which were provisionally dis- the epithelial sodium channel (enac) is an integral component of the pathway for na + absorption in epithelial cells. enac activity is mainly regulated by mechanisms that control its expression at the cell surface, such as ubiquitination. the ubiquitin ligases nedd and nedd - have both been shown to bind to enac and decrease its activity. conversely, the serum-and glucocorticoid regulated kinase (sgk), a downstream mediator of aldosterone, is able to increase enac activity. this effect is at least partly mediated by direct interaction between sgk and nedd - . sgk binds both nedd and nedd - but it is only able to phosphorylate nedd - . phosphorylation of nedd - reduces its ability to bind to enac, and hence increases enac activity. the impact of the interaction between nedd and sgk remains unclear. nedd -like proteins interact with enac via their ww-domains. these domains bind py-motifs (ppxy) present in enac subunits. nedd and nedd - both have four highly homologous ww-domains. previous studies have shown that interaction between nedd and enac is mainly mediated by ww-domain . sgk also has a py-motif, therefore we tested whether the ww domains of nedd and nedd - mediate binding to sgk. we show that single or tandem ww domains of nedd and nedd - mediate binding to sgk and that, despite their high homology, different ww domains of nedd and nedd - are involved. our data also suggest that ww domains and of nedd - mediate the interaction with sgk in a concerted manner, and that in vitro the phosphorylation of sgk at serine residue increases its affinity for the ww domains of nedd - . the stimulatory effect of sgk on enac activity is partly mediated via nedd - and will decrease if competition between nedd and nedd - for binding to sgk occurs. we show that nedd and nedd - are located in the same subcellular compartment and that they compete for binding to sgk in vitro. the concerted or successive action of proteolytic enzymes has been described in a number of important biological processes in which proteins are degraded or matured, such as digestion, turnover (lysosomal, proteosomal...), blood coagulation, developmental remodeling or apoptosis, among others. the complementary action of proteases belonging to different families to achieve a more efficient o a better modulated hydrolytic mechanism is well documented. specific molecular associations or shared scaffolds between the involved proteases and/or protein inhibitors and defined three-dimensional structures have also been reported. however, only in a few cases such structures involved metallo.carboxy-peptidases or their inhibitors [ ] . we shall review this subject and describe, in such a context, a new model found in a marine invertebrate organism in which such a fact takes place. in particular, the characteristics of a novel bifunctional molecule displaying the functionalities and structures of serine-and metallo.carboxy-peptidases will be presented. its structure is fully different than the ones previously reported by us and collaborative groups for metallocarboxypeptidase inhibitors [ ] [ ] [ ] . regulating the activity of herpes virus proteases c. s. craik departments of pharmaceutical chemistry, pharmacology, and biochemistry and biophysics, ucsf, san francisco, ca, usa. e-mail: craik@cgl.ucsf.edu herpesviral proteases exist in a monomer-dimer equilibrium in solution. dimerization is required for activity and a comformational change communicates the oligomerization state of the enzyme to the active site of each intact monomer. each monomer has an active site, which is spatially separate from the dimer interface. kaposi's sarcoma-associated herpesvirus (kshv), encodes a protease (kshv pr), which is necessary for the viral lytic cycle. like those of other herpesvirues proteases, the dimer interface of kshv pr is composed primarily of a helix near the c terminus, of the protein. the helix of one monomer interacts with residues in the symmetrically related helix of the other monomer across the dimer interface as well as with neighboring helices. small molecule inhibitors, site directed mutagenesis and d nmr spectroscopy were used to compare the monomeric and dimeric forms of kshv pr and to investigate the relationship of the active site and the dimer interface of the enzyme. active site inhibition was shown to strongly regulate the binding affinity of the monomer-dimer equilibrium of the protease, shifting the equilibrium completely to the dimeric form of the enzyme. a previously undetermined conformational change provided insight in to the regulation of protease activity by dimerization as well as an explanation for the weak dimerization of a family of enzymes with a disparately large dimer interface compared to their measured binding affinities. using this information as a guide, protein grafting of the interfacial helix onto a small stable protein, avian pancreatic polypeptide, generated a small macromolecular inhibitor that successfully disrupted the dimer interface and inhibited enzymatic activity. these results provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, validate the protease as a therapeutic target and suggest the dimer interface may be an alternative site for antiviral design. abteilung strukturforschung, max-planck-institut fu¨r biochemie, martinsried, germany. e-mail: huber@biochem.mpg.de proteolytic enzymes catalyze a very simple chemical reaction, the hydrolytic cleavage of a peptide bond. nevertheless they constitute a most diverse and numerous lineages of proteins. the reason lies in their role as components of many regulatory physiological cascades in all organisms. to serve this purpose and to avoid unwanted destructive action proteolytic activity must be strictly controlled. control is based on different mechanisms which i will discuss and illustrate with examples of systems and structures determined in my laboratory. the family of serine protease inhibitors known as the serpins is represented in all branches of life and predominate in the higher organisms, including man. they have evolved an extraordinary mechanism to inhibit proteases which distinguishes them from the other families of serine protease inhibitors, and renders them uniquely qualified to control of the proteolytic pathways essential to life. the mechanism is best described as a spring-loaded mousetrap, where nibbling of the peptide loop bait springs the trap and crushes the unsuspecting protease. as with a mousetrap, the active state of a serpin is metastable, and the energy released upon conversion to its more stable form is used to trap the protease. the complexity of the serpin mechanism provides many advantages over the simpler lock-and-key type mechanism, utilized by all other serine protease families. serpins provide stoichiometric, irreversible inhibition, and the dependence on serpin and protease conformational change is exploited for signaling and clearance. the potential for regulation is also an inherent part of such a complex mechanism, as illustrated by the heparin activation of serpins antithrombin and heparin cofactor ii. however, with complexity of mechanism also comes susceptibility to disease causing mutations: both through loss-of-function, as with thrombosis caused by antithrombin deficiency; and gain-of-function, as with dementia caused by neuroserpin polymerization. many crystallographic structures of serpins have been solved over the past years, and we now have a frame-by-frame cinematic view of the intricate conformational rearrangements involved in protease inhibition, modulation of specificity, and molecular pathology of the remarkable shape-shifting serpins. structural lessons of serine proteases: function and mechanism of the serine protease-like hgf as a growth factor in met signaling hepatocyte growth factor (hgf), a plasminogen-related growth factor, is the ligand for met, a receptor tyrosine kinase implicated in development, tissue regeneration and invasive tumor growth. hgf acquires signaling activity only upon proteolytic cleavage of single-chain hgf into its a/b-heterodimer, similar to zymogen activation of structurally related serine proteases. although both chains are required for activation, only the achain binds met with high affinity. recently, we reported that the protease-like hgf b-chain binds to met with low affinity this suggests that additional allosterically linked regions may be involved in the signaling process. furthermore, antibodies directed toward the b-chain or the hgf a-chain result in inhibition of met phosphorylation in a cells. these antibodies also inhibit proliferation in bxpc cells and baf cells. implications for dimerization mechanisms of hgf-dependent met receptor activation and signaling are presented. in addition, mutagenesis of the hgf b active site region has been investigated with respect to imparting enzymatic activity. thus while hgf has the function of a growth factor, the structural and receptor binding aspects of hgf are more akin to those of serine proteases. trypsinogen with a amino acid leader peptide on its n-terminus is the predominant form of the enzyme in human brain gene prss on chromosome of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen . mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mrna for trypsinogen has recently been identified in brain and other human tissues. analysis of the gene encoding trypsinogen predicted two isoforms of the zymogen: isoform a may have a amino acid, while isoform b a amino acid n-terminal leader sequence. the translation initiation site for isoform a is an atg codon, while the initiation site predicted for isoform b is a ctg codon. we measured the amount of trypsinogen mrna and the quantity of the protein as well in selected areas of the human brain. trypsinogen could be localized in glial and neuronal cells using immunohistochemical methods. we purified human trypsinogen by affinity chromatography. our results show that splice isoform b is the predominant if not the exclusive form of the zymogen in human brain. the n-terminal residue of the isolated protein was identified by amino acid sequencing as a leucine. at the same time the longest mrna we were able to isolate was barely longer than the one corresponding to splice isoform b. although the most trivial explanation of our results is that isoform a is proteolytically processed to result in isoform b, it cannot be excluded that leucine rather then methionine is used as translation initiator amino acid. search for endogenous substrates for prolyl oligopeptidase in porcine brain prolyl oligopeptidase (po) is a serine protease present in most tissues, which preferentially cleaves the peptide bond at the carboxyl site of proline residues. the function of po is unknown, but it has been associated with several disorders of the central nervous system, such as depression and alzheimer disease. the purpose was to look for endogenous substrates for the recombinant porcine po in porcine brain. we adapted a method to extract the proteins from the brain with special attention to the smaller polypeptides since po is not known to cleave peptides larger than amino acids. subsequently we looked for a method to separate the protein mixture in less complex fractions. d-gelelectrophoresis, commonly used in proteomics, is only suitable for proteins with a molecular weight between and kda and an iso-electric point between and . two-dimensional chromatography offers a suitable alternative for small peptides. we chose ion exchange chromatography as a first and reversed phase high pressure liquid chromatography as a second step. the resulting fractions were divided into two parts. one part was incubated with the purified po, the other served as a control. by looking for shifts in the mass spectrum between the control sample and the incubated sample, we identified peptides cleaved by po. different methods, such as esi-qtof-ms and maldi-toftof-ms, were used to sequence cleaved peptides by msms. these experiments allowed us to deduce the sequence requirements for po cleavage. serine protease subtilisin immobilized on novel mesoporous materials serine proteinases are widely used in protein mapping and peptide or ester bond formation. fixation of enzyme on solid support has many advantages, such as high stability, possibility of recovering and low product contamination by enzyme. subtilisin carlsberg, a protease from bacillus licheniformis, was immobilized on mesoporous silica (sba- ) and several organosilica supports via physical adsorption. the bifunctional mesoporous organosilicas containing ch -ch or ch=ch bridges in combination with organic tethers bearing amino or hydroxyl functionalities were synthesized using supramolecular templating in the presence of non-ionic triblock copolymers and exhibited high surface area and large pore diameters in the range of - Å suitable for the incorporation of subtilisin. the kinetics of immobilization was examined for six different carriers. it was shown that enzyme retained hydrolytic activity after the immobilization. the dependence of subtilisin loading on the starting concentration of the enzyme during adsorption shows the maximum loading ( mg protein/g support) at [e] = mg/ml. the ph dependences of loading and activity of immobilized biocatalysts were bell-shaped. for the organosilica support containing amino and hydroxyl groups the ph-dependence was shifted to the alkaline ph by in comparison with the support containing ch -ch bridges. the adsorbed subtilisin desorbs easily in aqueous media, while no leaching of the enzyme was observed in acetonitrile and dmf/acetonitrile mixture ( / ). the immobilized biocatalyst shows high hydrolytic activity after incubation in non-aqueous acetonitrile for week and after h incubation in % dmf/acetonitrile mixture. these data indicate a possible application of the obtained biocatalysts in low water media. purification, structural and biological characterization of protease inhibitors from acacia plumose seeds protease inhibitors have been used in many current medicines. therefore, there is a considerable interest inside the pharmaceutical industry in discovering new composites and mechanisms of protease inhibition, since these investments have led, for example, to new anti-hiv therapeutical tests, coagulation diseases treatment and tests with anti-carcinogenic drugs. serine protease inhibitors are found in all plant tissues, mostly in the seeds of the leguminosae subfamilies: mimosoideae, caesalpinoideae and papilionoideae. acacia genus is one of most important member of mimosoideae, and the presence of protease inhibitors in this genus was described in only three species and none of them were structurally characterized. in this sense, we are studying three new protease inhibitors from a. plumose seeds. from saline extract of triturated mature seeds the inhibitors were purified and presented anti-coagulant activity, serine protease inhibitory activity and action on growth of fitopathogenic microorganisms, in vitro. the purification steps included size exclusion chromatography on the superdex- column, equilibrated and eluted with pbs, a ionic exchange chromatography on mono-s (hr / ) column, equilibrated with the buffer sodium acetate mm (ph . ), and eluted with the same buffer in a gradient of - . m of nacl. three fractions (eluted around . , . and . m of nacl) that presented anticoagulant activity and serine protease inhibition were separated and denoted apia, apib and apic. their apparent mws were around kda, by sds-page in the absence of reducing agents. in the presence of reducing agents they shown two bands: between - , and - kda. the n-terminal analyze of higher mw chains were tyafl (apia); kellvdne (apib) and telhdd (apic). the circular dichroism spectra of these inhibitors were very similar, presenting a maximum around nm and a minimum in nm, compatible with presence of unordered and beta elements of secondary structure. their nterminal, cd spectra and two-polypeptide chains linked by covalent bound, are compatible with kunitz type inhibitors. probably these inhibitors are three different isoforms that present different inhibition specificity degree on the serine proteases family. the ki to different serinoproteases (trypsin, plasmatic kalikrein, elastase, quimotrypsin) and specificity to the phytopatogenic fungus are being investigated. although the proteases were initially described as enzymes involved in the non-specific degradation of dietary proteins, today it is known that they can also act as highly specific enzymes that perform selective cleavage of specific substrates. thus, alterations in the structure, regulation or function of this type of enzymes underlie serious human disorders including cancer. to date, more than protease and protease homologs are annotated in man, mouse, and rat genomes (www.uniovi.es/degradome). the increasing complexity of the proteolytic systems has led to the introduction of global concepts as the term degradome to define the complete set of proteases that are produced in a specific moment by a cell, tissue or organism. as part of our studies focused on the characterization of the mammalian degradomes, we have identified and cloned unusual mosaic proteases containing in tandem serine protease domains. the first, called polyserase- is synthesized as a transmembrane protein that undergoes post-translational events to generate three independent serine protease domains. the second polyprotease is the polyserase- , a secreted protein that remains as integral part of the initial protein product. to date, it is difficult to understand the putative functional advantages derived from the complex polyproteases and, albeit extremely unusual, it is not an unprecedented situation. thus, the amphibians ovochymase and oviductin are polyserine proteases that contain three in tandem serine proteases. in humans, angiotensin-coverting enzyme and carboxypeptidase d are polymetalloproteases that exhibit some similarities to the polyserases. all these polyproteases constitute examples that illustrate an additional strategy for increasing the complexity of the degradomes. evolution of a genetic locus, expressing several protease inhibitors with homology to whey acidic protein (wap) a. clauss and Å . lundwall department of laboratory medicine, lund university, malmo¨, sweden. e-mail: adam.clauss@klkemi.mas.lu.se we have previously described a locus on human chromosome that gives rise to proteins containing wap four disulphide core (wfdc) domains. among them are the elastase inhibitors elafin and secretory leukocyte proteinase inhibitor (slpi). both slpi and elafin are also known to be important components of the innate immune defence by displaying anti-microbial properties. in order to gain a deeper understanding of the biological role of the locus, we have now extended our investigations of its organization and evolution into non-human mammals. homologous loci were identified on mouse chromosome , rat chromosome and dog chromosome . transcript sequences were generated by race technology or retrieved from the est databases. as in humans, the murine and canine loci are divided into two sub-loci separated by approximately kb. the majority of genes are conserved in all species, but the comparison also showed gain and loss of genes, e.g. two human pseudogenes were identified due to the discovery of functional rodent genes, and in the rat several duplications has yielded four slpi genes. a most interesting finding was that there is no murine elafin gene. the different wfdc domains showed a highly variable species conservation. this was particularly striking in proteins containing multiple domains, where the aminoterminal wfdc generally displayed low conservation, whereas the opposite was true for the carboxyterminal wfdc. the difference could be due to the potential targets of the inhibitors, which might be either highly variable exogenous microbial proteases or conserved endogenous proteases. signaling mechanism of thrombin-induced human gingival fibroblast contraction thrombin is activated during gingival tissue injury and inflammation. thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of proteaseactivated receptors (pars). we noted that thrombin and par- agonist peptide ( lm) induced the gingival fibroblasts (gf)-populated collagen gel contraction within -h of exposure. however, par- and par- agonist peptide (< lm) show little effect on collagen gel contraction. u (phospholipase c inhibitor) and -apb (ip antagonist) were effective in inhibition of gf contraction. thrombin-induced gf contraction was inhibited by mm egta (an extracellular calcium chelator) and verapamil (a l-type calcium channel blocker). in addition, w ( and lm, a calcium/calmodulin inhibitor), ml- ( lm, myosin light chain kinase, mlck inhibitor), and ha ( lm, rho kinase inhibitor) completely inhibited the thrombin-induced collagen gel contraction. thrombin also induced the phosphorylation of erk /erk in gf. however, u only partially inhibited the thrombin-induced gf contraction. similarly, wortmannin ( lm), ly ( lm) (two pi k inhibitors) and genistein, also showed partial inhibition. moreover, nac was not able to suppress the gf-contraction, as supported by slightly decrease in reactive oxygen species production in gf by thrombin. these results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting gf contraction. this event is mainly mediated via par- activation, plc activation, extracellular calcium influx via l-type calcium channel, and the calcium/calmodulin-mlck and rho kinase activation pathway. survival of the anticarcinogenic bowman-birk inhibitor from soybean at the terminal ileum of cannulated pigs plant protease inhibitors (pi) of the bowman-birk class, a major pi class in legume seeds, have emerged as highly promising cancer chemopreventive agents, being capable of preventing or suppressing carcinogenic processes in a wide variety of in vitro and in vivo animal model systems. in order to exert their chemopreventive properties in vivo, plant pi have to resist and survive, at least to some extent, degradation by acidic conditions and digestive enzymes during gut passage. in this study, we have evaluated the survival rate of the bowman-birk inhibitor (bbi) in the terminal ileum of cannulated pigs fed defatted soybean. two different quantitative approaches have been carried out. firstly, a competitive indirect elisa assay using an antisera capable to detect bbi free and/or in complex with digestive proteases; secondly, we have carried out spectrophotometric measurements of trypsin and chymotrypsin inhibitory activities in ileal samples, where the presence of bbi metabolites and/or single active loops can be detected. according to the elisa method, ileal apparent digestibility of bbi was %, which resulted in a recovery of . mg out of . mg/kg feed ingested. significantly higher ileal digestibility values ( %) were found when trypsin and chymotrypsin inhibitor activities were evaluated. the results suggest that the immunoassay may be overestimating the presence of functional pi by detection of inactive bbi, but also that the presence of complexed bbi with digestive proteases, even if protein extraction was carried out under acidic conditions, could make bbi undetectable in activity assays. studies are in progress to overcome these drawbacks. the resistance of bbi to the acidic conditions and digestive enzymes of the upper gastrointestinal tract make these proteins very interesting candidates for evaluation as chemopreventive agents, in modulating cell viability and tumor progression within the gastrointestinal tract. a single amino acid change in a chymotrypsin prevents plant proteinase inhibitor binding plants have evolved economical strategies to combat insects, which on one hand involves the production of multi-domain pis that can target multiple enzymes with different specificities and on the other, pis that belong to structurally distinct families. solanaceous plants, produce both type i and type ii families of pis, which specifically target serine peptidases. this study showed that type i pis are better inhibitors of a particular class of chymotrypsins within the gut of helicoverpa species that is otherwise unaffected by the type ii class of inhibitors. homology models were used to identify a single amino acid substitution in the helicoverpa chymotrypsin that was likely to confer resistance to the type ii inhibitor. our hypothesis was further supported by recombinant expression and mutagenesis of this single amino acid in the type ii inhibitor-resistant chymotrypsin. we therefore propose that both type i and ii inhibitors are required to protect plants against lepidopteran insects. mobility of the sulphate protamin/ low molecular weight heparin complexes in an electrical field glycosaminoglycans low molecular weight heparin (lmwh) activated plasma serine proteases inhibitors. serine proteases play an important role in thrombogenesis, the process that leads to blood clotting and such as heart attack, stroke and other cardiovascular disorders. lmwh has been used to temporarily render the blood incoagulable during prophylaxis or treatment of thrombosis and sometimes result in serious bleedings and for the heparin anticoagulant activity neutralization used sulphate protamin. it was investigated relationship between new lmwh-sk derivatives (were generated through the controlled cleavage of porcine intestinal mucosa heparin with a mixture of chitinolytic complex from streptomyces kurssanovii) anticoagulant activities and lmwh-sk complexes with sulphate protamin mobility in an electrical field. with this purpose used biospecific electrophoresis in % agarose with protamin sulphate. precipitation zones (zones of the equivalent) in the ''rocket'' form were generated. scanning image was saved as jpg format. the ''rocket'' squares estimated with the help of bandscan program. results: lmwh-sk with molecular mass (mm) . ; . ; . ; . ; . ; . kd demonstrated antithrombin activities (aiia) - iu/mg, activities against factor xa (axa) has made - iu/mg, axa/aiia ratio -( . - . ). correlation coefficients between mm and precipitation zone heights or squares consist . - . (p < . ), between axa activities and precipitation zone heights or squares consist . - . (p < . ). conclusion: lmwh-sk was obtained with the chitinolytic comlex hydrolisis help has ratio axa/aiia- , , it is necessary for antithrombotic preparations. with the mm decrease axa activity increase and precipitation zone heights or squares of the lmwh-sk complexes with sulphate protamin decrease. the role of extracellular proteases in supplying filamentous fungi with nutrient compounds is well understood and experi-mentally documented. however there is no definite answer on the question on the need and role of these proteases in pathogenesis. the study of differences in the spectra of extracellular enzymes of saprotrophic and pathogenic fungi performed on fusarium species revealed that activity of secreted serine proteinases of pathogenic f. culmorum strain was much higher (up to -fold) than that of saprotrophic strain. the use of f. culmorum strains differing in pathogenicity (strongly and weakly pathogenic) demonstrated that activity of secreted serine proteases of strongly pathogenic strain was significantly higher ( . - -fold) than that of weakly pathogenic strain. this tendency was preserved in calculations of activity towards protein content and dry weight of mycelium indicating on purposeful synthesis and secretion of extracellular proteases by strains with high pathogenicity. at that these differences were much higher when the substrate for trypsin-like proteinases bz-arg-pna was used than in the case of substrate for subtilisin-like proteinases glp-ala-ala-leu-pna. according to the data obtained it is proposed that the value activity of trypsin-like proteinases secreted by the fungi correlated with the degree of their pathogenicity and plays, apparently, an important role in pathogenesis. acknowledgment: this work was supported by grants from the russian foundation for basic research. conformational adaptation of a canonical protease inhibitor upon its binding to the target protease increases specificity atomic resolution crystal structure of sgti in complex with crayfish trypsin provided further data on the molecular basis of the inhibition mechanism of pacifastin type inhibitors. in complex with crayfish trypsin, sgti exhibits more or less continuous contacts in an extended region (through sites p -p ' ) of the molecule. the comparison of this complex with a simulated bovine trypsin-sgti one shows that more than half of the interaction energy surplus is originated from the extended region of binding. some of these contacts result from a conformational change of sgti that was induced by its binding to the enzyme which is strongly supported by the critical comparison of the crystal structure of crayfish trypsin-sgti complex with the free form of sgti. alignment of the nmr structure ensemble with the x-ray structure of complexed sgti and a careful comparison of the backbone j, w angles were carried out. additionally, noe-derived restraints and corresponding distances in the complex are also compared. local conformation of both p -p and p '-p ' regions of the inhibitor shows significant changes upon binding suggesting that either or both of these regions may act as molecular recognition sites. this comprehensive analysis of the local backbone properties of sgti in the free and in the complex form made possible to identify conformational similarities and differences responsible for its efficient binding to the enzyme, and provides a good basis for further studying the structural aspects of protease inhibitor specificity. as most of serine proteases enteropeptidase light chain contains four disulfide bonds and one nonpaired cysteine at (chymotrypsinogen-derived residue numbering) position which forms disulfide bond linking the pro-and catalytic domains. a mutant of human enteropeptidase light chain cys ser was constructed by site-directed mutagenesis. the recombinant wild type and mutant proteins were produced in escherichia coli bl (de ) with expression vector pet- a. the active proteins were obtained after solubilization and renaturation of the fusion protein thioredoxin/human enteropeptidase light chain from inclusion bodies. after autocatalytic cleavage of thioredoxin the active enzyme was purified on agarose linked soybean trypsin inhibitor. the yield of refolded active enzyme increased from . to . % in case of cys ser mutant. the wild type and c s mutant showed similar kinetic parameters for cleavage of small synthetic substrate gly-asp-asp-asp-asp-lys-naphthylamide, small ester thiobenzyl benzyloxi-carbonil-l-lysinate (z-lys-sbzl) and fusion protein cleavage. both enzymes were inhibited by trypsin-like serine proteases inhibitors but not inhibitors of chymotrypsin-like, cysteineor metallo-proteinases. recombinant human enteropeptidase light chain and its mutant c s were active between ph and with a broad optimum at about ph . and demonstrated quite high stability to different denaturating agents. both enzymes demonstrated secondary specificity to chromogenic substrate z-ala-phe-arg-na with km = . mm, kcat = s- . proteinaceous low molecular serine protease inhibitors from wood rotting fungi k. j. grzywnowicz and j. zuchowski biochemistry department, maria curie-sklodowska university, lublin, poland. e-mail: grzyw@hermes.umcs.lublin.pl proteolytic enzymes have been firmly established as main regulatory components in a number of cellular and physiological processes. the most important factors influencing the proteolytic enzymes are natural, proteinaceous protease inhibitors, which form complexes with target proteases. they have been extensively investigated from the points of view on physiological functions, as tools for protease enzymology, models for protein-protein interactions and on potential medical applications. there is growing interest in new inhibitors of proteases from various sources. among known protease inhibitors from fungi are, yeasts inhibitors of proteinases a (asparagine protease) and b (serine protease), and low molecular inhibitors of serine proteinases from fruiting bodies of mushrooms -pleurotus ostreatus and lentinus edodes as well as some undefined proteinase inhibitory activities from water extracts of some species of basidiomycetes. searching for new, bioactive metabolites of basidiomycetous fungi we isolated and characterized recently some low molecular, proteinaceous, natural inhibitors of serine proteases, from mycelia of wood rotting fungi -trametes versicolor, abortiporus biennis and schizophyllum communae. isolation of inhibitors was achieved by ion exchange and size exclusion chromatography. preliminary characterization of their inhibitory activity (against some serine proteases), ph and temperature optima of action, and molecular mass, were classically analyzed. analysis of n-terminal amino acid sequences of these inhibitors suggests a new family of serine protease inhibitors from fungi. more detailed characterization of inhibitors (including molecular modeling) and preliminary experiments with laboratory animals and with lines of human cells are in progress. the role of serine proteases in the lectin pathway of complement activation p. ga´l , g. ambrus , v. harmat , b. ve´gh , g. na´ray-szabo´ , r. b. sim and p. za´vodszky institute of enzymology, hungarian academy of sciences, budapest, hungary, protein modeling group, hungarian academy of sciences, budapest, hungary, department of biochemistry, university of oxford, oxford, uk. e-mail: gal@enzim.hu the complement system is a cascade of serine proteases, and mediates essential functions during infection as a part of the innate immunity. activation of the complement system culminates in the destruction and clearance of invading microorganisms and damaged or altered host cells. our view about the complement system has changed considerably in the recent years, due to the discovery of a new activation pathway of complement: the lectin pathway. we have recombinantly expressed and characterized the mannose-binding lectin associated serine proteases: masp- and masp- . these are related mosaic serine proteases with similar domain organization but with different enzymatic properties. we showed that masp- is capable of autoactivation and it can cleave c and c complement subcomponents. masp- , therefore, can initiate the complement cascade without the contribution of any other protease. we demonstrated that the complement control protein (ccp) modules, which associate directly with the serine protease domain, stabilize the structure of the catalytic region masp- and contain exosites for the large protein substrates. these results are in agreement with the crystal structures of activated and zymogen forms of masp- . masp- is the most abundant mbl-associated serine protease but it cannot activate the complement system. we demonstrated that masp- has a more relaxed substrate specificity compared to masp- and the activity of both proteases can be blocked by c -inhibtor. we concluded that the two mbl-associated serine proteases participate in evolutionary and functionally different pathways. comparative kinetic study on s ' trypsin variants l. gombos, j. tó th, p. medveczky, a. ma´lna´si csizmadia and l. szila´gyi laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, . e-mail: gl@ludens.elte.hu by far the most serine proteases have a glycine in position , which is part of the s ' subsite (the second subsite on the enzyme surface c-terminal from the scissile bond of the substrate). in contrast, human trypsin , the trypsin isoform expressed in human brain, possesses an arginine in that position. the bulky side chain of this amino acid is responsible for the inhibitor resistance, the most striking feature of this isoform, as it interferes with the binding of polypeptide inhibitors to the enzyme surface. a chimpanzee typsin also has an arginine , while rat trypsin v bears a tyrosine in that position. there is also a snake venom plasminogen activator, a trypsin type serine protease, that contains an s ' phenilalanine. we created glycine, arginine, tyrosine and phenilalanine s ' variants of human and rat trypsins by site directed mutagenesis in order to investigate the effect of these amino acids on the kinetic behaviour. on small chromogenic substrates and synthetic inhibitors, which do not interact with the s ' residue, there is no signifi-cant difference between the various mutants in catalytic efficiency and inhibitory constants, respectively. however, on oligopeptide substrates the catalytic efficiency decreases - -fold in the nonglycine variants. this effect is even more dramatic with polypeptide partners: the catalytic efficiency drops - times while inhibitory constants increase by - orders of magnitude. we conclude that the catalytic mechanism is not fundamentally influenced by the substitution of residue , although this amino acid is part of the oxyanion hole. bulky residues in the s ' subsite hinder mainly the binding to interaction partners. structural studies on masp- : towards the understanding of the mechanism of autoactivation mannose-binding lectin-associated serine protease (masp- ), is the key enzyme of the lectin activation pathway of complement, a major element of innate immunity. a dimer of masp- complexed with mannose-binding lectin (mbl) is able to perform its biological functions: upon recognition of the pathogen by mbl masp- undergoes autoactivation, and then initializes the complement cascade by cleaving c and c . masp- is a mosaic protein containing a chymotrypsin like serine protease domain (sp) and further domains with binding sites of mbl or substrates. our present study focuses on the structural background of the ability of the zymogen form of masp- to undergo autoactivation. we solved the structures of catalytic fragment of masp- both in its zymogen and activated forms. comparison of the two structures reveals characteristic conformational differences in the classical activation domain and in some other loops lining the substrate binding region. loop shows a unique conformation with arg blocking the s pocket. we docked the activation loop of masp- in the active site of the active enzyme and built a model of the complex of the active and zymogen forms. the model reveals extended regions of molecular recognition. while this model represents the second step of autoactivation (active form cleaves zymogen), the first step (zymogen cleaves zymogen) requires the stabilization of the zymogen enzyme in active-like conformation. we built a model of a zymogen-zymogen complex. favorable and unfavorable contacts of the two zymogen molecules help us to identify possible molecular switches, as well as contact regions stabilizing an active-like conformation of the zymogen enzyme in the complex. the deg/htra proteases are atp-independent serine endopeptidases which are present in most organisms, including bacteria, humans and plants. previous work in our laboratory has shown that the deg protease of the model plant arabidopsis thaliana selectively degrades the photodamaged d protein in the reaction center of photosystem ii (psii) in vitro. therefore, deg is thought to catalyze the primary cleavage of photodamaged d protein, which is an important step of the repair mechanism that restores functional psii. our present studies aim to elucidate the regulation of the deg protease activity, especially with regard to its d degrading activity. we found deg associated to the stromal side of the thylakoid membranes and as a soluble protein in the chloroplast stroma. the amount and distribution of deg protein remained unchanged after exposure to different light intensities, which suggest either a substrate regulation or a posttranslational regulation of the d degrading activity of deg . recent advances on deg regulation and complex formation will be presented. novel peptide inhibitors of human kallikrein (hk ) human kallikrein (hk ) is a serine protease produced by the secretory epithelial cells in the prostate. it activates several other proteases that may participate in the proteolytic cascade mediating metastasis of cancer. thus, modulation of hk activity is a potential way of preventing tumor growth and metastasis. furthermore, specific ligands for hk may be potentially useful for targeting and imaging of prostate cancer. we used enzymatically active recombinant hk captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. six different peptides binding to hk were identified using libraries expressing or amino acids long linear peptides. three of these peptides were specific and efficient inhibitors of the enzymatic activity of hk . alanine substitution analysis revealed that motifs of - amino acid determined the inhibitory activity of the peptides. the peptides are also of potential utility for development of immunopeptidometric assays for hk , which is promising marker for diagnosis of prostate cancer. furthermore, these peptides are potentially useful for treatment and targeting of prostate cancer. the mechanism of autoactivation of the zymogen masp- residues on the surface of pathogens. we managed to recombinantly express and purify two forms of zymogen masp- . one form is the wild type zymogen enzyme, which can be activated, while the other one is a stable zymogen mutant form of masp- . we could prepare the zymogen form of wild type masp- under certain conditions which enabled us to examine the kinetics of activation. we demonstrated that activation of masp- is a true autocatalytic activation without the involvement of any other protease. we characterized the enzymatic properties of zymogen masp- using the stable zymogen form. we demonstrated that zymogen masp- cannot cleave small synthetic substrates but it can cleave large protein substrate (c ). a molecular model for the interaction between zymogen and activated masp- during activation has also been built based on the available d structures of zymogen and activated masp- . influence of streptokinase on the fibrinolytic system proteins the present study is dedicated to the investigation of the effect of protein by bacterial origin -streptokinase (sk) on the activity and interaction regulation mechanisms of fibrinolytic system proteins. the study was carried out with use of porcine haemostasis system which plasminogen isn't activated by sk. especially we were interested in study of the changing fibrinolytic system parameters such as tissue type plasminogen activator (t-pa), plasminogen activator inhibitor (pai- ), plasminogen, a -antiplasmin activities. also the main parameters of coagulation system such as fibrinogen, soluble fibrin, fibrin degradation products levels and thrombin activity and quantity were studied. it was used affinity chromatography, electrophoresis, western-blotting, elisa, determination of proteins activity. it has been determined an increased consumption of plasminogen on % in h after streptokinase injection. it was shown that activity and concentration of t-pa were significantly increased in . times in h. on the next stages of investigation this parameters tend to norm. after sk injection pai- quantity was increased in two times ( . ng/ml compared to normal . ng/ml). the interesting fact was the activation of prothrombin by sk without activation of coagulation system in vivo. the injection of sk causes the significant increase of t-pa activity and quantity possibly due to direct or/and indirect effect on endothelial cells. we can conclude that sk causes pai- secretion due to effect on platelets as % of pai- storage is in a-granules of platelets. thus analysis of the data displayed besides of well-known sk function the influence of sk on the changing of fibrinolytic system potential possibly due to its effect on endothelial cells and platelets. paracrystalline inclusions in the mitochondrial matrix or intermembrane compartment occur in several biochemically unrelated disorders such as myopathies, paragangliomas and steatohepatitis, and in various cell types under normal conditions, as well. however, little is known about the composition of the inclusions, the mechanism of their formation and their relation to disease processes. in this study we have described the helix-shaped structures in the intracristal compartments of rat liver mitochondria that have undergone ca + -induced permeability transition. the filaments are anchored in opposing parts of the mitochondrial membranes and appear to support the cristae mechanically. a protein, that apparently is a component of these helical filaments, has been identified as serine protease lactb. this protein shows close sequence similarity to the class c bacterial beta-lactamases and is the only member of this class in animals. since lactb has not been studied previously we cloned its cdna for expression in e. coli as c-terminal his-tagged fusion protein. lactb underwent proteolytic processing in both e. coli and in isolated mitochondria resulting in several protein fragments. this is likely to be due to autocleavage and may be an activation/maturation process. d blue native gel electrophoresis indicated that lactb was part of a > kda protein supercomplex. in summary, the presence of the serine protease motive in lactb and its supposed ability to form helical filaments suggest that lactb might function not only as a component of 'mitoskeleton' in maintaining and rearranging the mitochondrial ultrastructure under certain conditions, but also might take part in apoptotic processes. novel psychrophylic trypsin-type protease from serratia proteomaculans proteinase with trypsin specificity from psychrophylic microorganism serratia proteomaculans was partly purified. it was shown that the properties of this enzyme (temperature and ph-stability, efficiency of substrate hydrolysis) correspond with the psychrophylic character. inhibitor analysis and study of substrate specificity indicate that this enzyme is serine trypsin-type protease. at the same time this enzyme is zinc-dependent. proteases of such type were unknown till now. secondary specificity of the studied enzyme differs from the bovine trypsin specificity -this protease hydrolyses the short substrates more efficient. zinc, cadmium (ii) and copper (ii) ions in mmolar concentrations inhibit the enzyme activity. the unusual character of calcium ions influence on substrate hydrolysis and inhibition by the bovine pancreatic trypsin inhibitor (bpti) was registered for the studied enzyme. in vitro by a neutral to basic ph change [ , ] . the kinetics of the activation process can be followed by stopped flow fluorescence (sff) experiments while the structural features of the transition can be explored by in silico molecular dynamics (md) and targeted molecular dynamics (tmd) [ ] simulations. to challenge the activation process, mutants were constructed and studied by sff measurements. subsequently, on these mutants multiple md/tmd simulations were carried out. our results indicate the existence of parallel activation pathways. they demonstrate the absolute necessity of multiple simulations and of proper statistics. they reveal the pros and cons of the tmd method. a simple method for the purification of a novel serine endoprotease from wheat triticum aestivum (cv. giza ) has been developed. it consists of ion-exchange and gel filtration. the molecular mass of the enzyme was kda by sds/page under reducing conditions and kda by gel filtration on a sepharose b column. the enzyme had isoelectric point and ph optimum at . and . , respectively. the substrate specificity of the enzyme was studied by the use of synthesized and natural substrates, azocasein, azoalbumin, hemoglobin, casein, gelatin and egg albumin. the enzyme appears to prefer azocasein with km mg azocasein/ml. the enzyme had a temperature optimum at °c with heat stability up to °c. while co + and mg + accelerated the enzyme activity by and %, respectively, ca + and ni + had very little effect. the enzyme was strongly inhibited by phenylmethylsulphonyl fluoride (pmsf), but not by the other protease inhibitors, suggesting that the enzyme is a serine protease. from the results it can be concluded from the characterization that the t. aestivum serine protease may be suitable for food processing. in vitro effects of a potent, selective dipeptidyl peptidase ii (dppii) inhibitor in leukocytes and u -cells. the compound was able to penetrate the cell membrane and proved efficacy without evidence for acute cellular toxicity. there was a dosedependent inhibition of intracellular dppii activity without affecting the dppiv activity (maximal efficacy at nm). these properties enable to differentiate between dppii and dppiv in biological systems and allow further investigation of the physiological function of dppii. in a second step, we have been investigating the involvement of dppii in apoptosis in human leukocytes by using this compound. preliminar results based on annexin v-/pi-staining using up to lm inhibitor in u -cells and pbmc did not show signs of apoptosis while dppii activity was inhibited for %. effect of calcium ions on hydrolysis of peptide substrates of general formula a-(asp/glu) n -lys(arg)-b, catalyzed by enteropeptidase (ec . . . ), differs depending on substrate type. for specific enteropeptidase substrates (n = ) calcium ion exhibits the promotion of hydrolysis by the natural two-chain enteropeptidase. hydrolysis of atypical enteropeptidase substrates (n = - ) is as a rule less efficient; in addition calcium ion shows in this case the inhibition influence. therefore the regulation of the nondesirable side-hydrolysis during full-length enteropeptidase-catalyzed chimeric proteins processing is possible by means of calcium ions. on the contrary the hydrolysis of substrates of all type (n = - ) by enteropeptidase light chain as well as the enzyme containing the truncated heavy chain ( - or - fragments) is inhibited by calcium ions. hydrolysis of the natural enteropeptidase substrate, trypsinogen, is at least two orders of magnitude more efficient than any artificial substrate hydrolysis. we propose that this effect is caused by participation in trypsinogen coordination with enzyme of the addition secondary substrate binding site and/or calcium-binding site; both sites located on the n-terminal half ( - ) of the enteropeptidase heavy chain. one more mechanism of the regulation of the enteropeptidase activity by calcium ion is the unusual calciumdependent autolysis of the enteropeptidase heavy chain leading to the drastic loss of its activity towards trypsinogen. autolysis of enteropeptidase heavy chain and well-known autolysis of trypsin were compared; the second one serves as the natural defense mechanism against the undesirable premature proenzymes activation in pancreas leading to pancreatitis. the corresponding enteropeptidase inactivation in low ca + ion environment might be the component of the same protective mechanism. b - p human trypsin selectively cleaves myelin basic protein: is this brain protease involved in the pathomechanism of multiple sclerosis? demyelination, the breakdown of the major membrane protein of the central nervous system, myelin is involved in many neurodegenerative diseases. proteases participating in this process are potential targets of therapy in neurodegenerative diseases. in the present in vitro study the proteolytic actions of calpain, human trypsin and human trypsin (the product of gene prss ) were compared on lipid-bound and free human myelin basic protein as substrates. digestions only with calpain and human trypsin actions may be of some physiological or pathological relevance, since these two are expressed in human brain. the fragments formed were identified by using n-terminal amino acid sequencing and mass spectrometry. the analysis of the degradation products showed that human trypsin of these three proteases cleaved myelin basic protein most specifically. it selectively cleaves the arg -thr and arg -thr peptide bonds in the lipid bound form of human myelin basic protein. based on this information we synthesized region - of myelin basic protein, peptide ivtprtpppsq that contains the specific trypsin cleavage site arg -thr . in vitro studies on the hydrolysis of this synthetic peptide by trypsin confirmed our results with intact myelin basic protein. what lends some biological interest to the above finding is that the major autoantibodies found in patients with multiple sclerosis recognize sequence - of the protein. our results suggest that human trypsin may be one of the candidate proteases involved in the pathomechanism of multiple sclerosis. enteropeptidase is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of its activation peptide following the sequence asp-asp-asp-asp-lys. its light chain alone is sufficient for an effective cleavage of fusion proteins with trypsinogen activation peptide analog. human enzyme possesses -fold specificity coefficient compare to bovine one, and an explanation of this fact can contribute a lot to the attempts of improving or modulating enzymatic properties. highly pure and active recombinant human enteropeptidase light chain (l-hep) was obtained by renaturation from inclusion bodies expressed in escherichia coli cells and the active l-hep was purified on agarose-linked soybean trypsin inhibitor. enzymatic activity of purified l-hep was studied through the cleavage of the synthetic peptide substrates and several fusion proteins. l-hep associated with soybean trypsin inhibitor slowly and z-lys-sbzl cleavage was inhibited with ki* = . nm. comparison of l-hep and bovine enteropeptidase inhibition by bovine trypsin inhibitor aprotinin has shown almost an order difference in ki*. ph dependence of the enzyme activity was measured and ph optimum point was found to be . . enteropeptidase light chain amino acid sequence and crystal structure were analyzed for the presence of target regions for mono-and bivalent ions. unlike trypsin with predicted and experimentally proved calcium-binding sites and sodium-activated thrombin, l-hep was predicted to be deprived of any of such sites and an influence of these ions on the cleavage of different substrates was found to be confined primarily to a substrate binding. as a continuation of our efforts to fully elucidate the antisnake venom properties of mucuna pruriens and to further understand the molecular changes that occurred in mouse plasma proteome as a result of in vivo challenge test with venom and mucuna pruriens proteins (mpe), two dimensional polyacrylamide gel electrophoresis was done. plasma was pooled and gels were run in triplicate to eliminate both biological and experimental variations. analysis using imagemaster d platinum software and other statistical analysis tools showed significant differences in protein expression between all the treatments and the control group. some proteins were down regulated, some up-regulated, some completely disappeared while new protein spots were identified. the protein expression of plasma of mouse immunized with mpe for weeks before challenge with lethal dose of venom and that injected with venom alone was more complex. some venom proteins like ecarin are serine proteases that activate clotting factors like prothrombin, causing haemorrhage and disseminated intravascular coagulation, on the other hand, the protease inhibitors from mucuna pruriens must have acted to antagonize these effects by direct proteolysis (cleavage products/spots appearing in the protein map) or other immunological mechanisms. the results obtained represents the first proteomics approach in studying all the plasma proteins involved in this phenomenon. we have only concentrated on protein spots showing interesting variations with respect to control. it is also an important step in the identification of the affected proteins, the kind of modifications/molecular mechanisms involved which is likely the basis of the in vivo protection the plant extract showed against the venom. the use of enzymes at low temperatures has great potential in terms of lower energy costs, therapeutic applications and to lower microbial contamination in industrial processes. low temperature proteases (cryophilic -or psycrophilic -proteases) are of particular interest for detergents and as wound debriding agents. at present, we are studying cryophilic proteases from antarctic krill (euphausia superba), which normally lives in the sea at temperatures near °c. we have isolated several low temperature proteases by chromatography. enzyme activities and stability were characterized at low temperatures and as a function of ph to find optimum conditions for different applications. a particular enzyme, named kt , showed particularly high specific activity at °c, several times that of commercial preparations of proteases such as subtilisins. this protein showed a high degree of similarity with digestive trypsins isolated from various arthropoda species. using mrna molecules obtained from abdominal sections of e. superba and subsequently subjected to a reverse-transcription reaction, we identified, isolated and sequenced a dna molecule that codes for an inactive zymogen of the enzyme. cloning of this dna sequence in escherichia coli strains allowed the recombinant expression of the zymogen, followed by purification and activation of the zymogen, which lead to an active cryophilic trypsin. we performed a homology modeling procedure that conducted us to obtain a molecular model of the mature enzyme. the d model thus obtained was refined using energy minimization, hydrogen network optimization and residue-residue contact optimization techniques, leading to a reliable model of the enzyme. we used this model to identify many interesting and novel features of the enzyme molecule that could be related with its cryophilic character, and to propose site-directed mutagenesis strategies that could be used to improve the enzyme performance at low temperatures, its ph-activity profile, specificity, inactivation resistance and recombinant expression. in addition, the d model allowed us to design and experimentally obtain mutants that are resistant to auto-degradation and more readily activated. molecular cloning and expression of lactba mitochondrial serine protease mitochondria are thought to have originated from a symbiotic relationship between a bacterium able to perform aerobic metabolism ant the ancestor of eukaryotic cells. lactb is the only mammalian protein showing sequence similarity to bacterial serine proteases and belongs to c class b-lactamases. mouse lactb is amino acids long and compromises a predicted mitochondrial import sequence, a short putative transmembrane segment, a b-lactamase homology domain containing the serine protease motif, -sxxk-, and a c-terminal d-transpeptidase domain. the physiological role of mammalian lactb is unclear. therefore, the purpose of this research work was to clone the gene of lactb for expression of lactb in e. coli for further biochemical and cell biological study. the full length lactb gene was cloned into the entry plasmid pentr/sd/d-topo. expression clones were created performing a recombination reaction between the entry clone and four destination vectors. expression constructs resulting in n-or c-terminal gst fusion protein and in n-or c-terminal his -tag fusion protein were transformed into bl (de ) competent cells which are designed for use with bacteriophage t promoter based expression systems. when lactb was expressed as an n-terminal gst fusion protein, full-length lactb protein was recovered by glutathione-agarose affinity chromatography. expression of lactb as a c-terminal gst fusion protein or with either an n-or c-terminal his -tag resulted in proteolytic degradation of the protein and we were not able to detect full-length lactb. these results show that the n-terminal gst fragment protects lactb from proteolytic processing and that lactb can undergo autoproteolysis, which may be a part of a physiological maturation or activation process. design and synthesis of retro-binding peptides active site inhibitors of thrombin thrombin is an important pharmaceutical target for the treatment and prevention arterial and venous thrombosis. biological active peptides are recognized to have significant therapevtic potential but serious limitations especially for oral dosing. the peptide stereomers could differ when forming productive complexes with an enzyme. moreover, the replacement of l-amino acid residues forming the hydrolyzed p -p' bond by their enantiomers is known to result in either an uncleavable or a very slowly hydrolyzed analogue. this phenomenon is often used for the synthesis of the peptide's inhibitors stable to the degradation by the enzymes of organism. as the peptides containing d-amino acids, nor are subject to an enzymatic hydrolysis, the purpose of researches was synthesis of a retro -d-analogues of thrombin's substrates constructed from d-amino acids. the di-and tripeptides of the general formula x-d-arg-d-phe-ome [where x = z, tos, ac h, and z-d-arg-d-ala-(d), l-phe-ome (otbu)] were synthesized by conventional methods of peptide synthesis in solution. special features of their interaction with thrombin are investigated. their inhibitory action on reaction of splitting of fibrinogen by thrombin and on reaction of a hydrolysis by thrombin baee showed, that their inactivating action depends on the substituent on n-end of dipeptides and configuration of phenylalanine in a molecule of tripeptides. the relationship between structure and inhibitory action of the synthesized peptides is discussed. the successful application of d-amino acids for designing of biologically active peptide's analogues as a potential medicinal agent, steady to enzymatic degradation is shown. substrate specificity of mannose lectin binding associated serine proteinase n. s. quinsey and r. n. pike department of biochemistry and molecular biology, monash university, melbourne, victoria australia. e-mail: noelene.quinsey@med.monash.edu.au the innate complement system is involved with the neutralization of pathogenic microorganisms. it plays a comparative role to that of the classic immune complement cascade. in the innate complement system, the oligomers of mannose lectins are able to bind to microorganisms. these oligomers have been shown to have mannose lectin binding serine proteinase (masps) attached, which once activated lead to the activation of the c convertase complex, which finally leads to the formation of the membrane attack complex. there have been three active masps identified in the human innate immune system-masp- , masp- and masp- . there is high homology between these three serine proteases especially in the n-ter- serpins are protease inhibitors that present their reactive site loop (rsl) to target proteases, followed by drastic conformational changes that inactivate the protease. the sequence of the rsl of serpins determines the target specificity. the drosophila melanogaster gene spn encodes multiple serpin isoforms each containing an individual rsl, thus enabling the attack of different proteases. variant spn a contains a consensus recognition/cleavage sequence of furin within its rsl and is equipped with a signal peptide and an endoplasmic reticulum (er) retrieval signal (hdel). this suggested that the protein resides in the secretory pathway, like furin, a proprotein convertase that activates many cellular proteins and pathogens. our experiments demonstrate that spn a forms sds-stable complexes with human furin that is inhibited with a second order rate constant of . · /m/s. the rsl of spn a is cleaved c-terminally to arg-arg-lys-arg, in accord with the enzyme's cleavage site. furthermore, the serpin is retained in the er of transfected cos cells as shown by immunofluorescence staining. a hdel deletion mutant was detected mainly in the medium of trans-fected cos cells, demonstrating the necessity of the hdel signal for the observed cellular localization. further experiments show that furin and of drosophila melanogaster are physiological targets for spn a, since secreted forms of both enzymes form stable complexes with the serpin. together, the results demonstrate that spn a is a potent inhibitor of furin that may meet the target at its natural location. experiments with the other rsl variants show that the spn gene represents a multipurpose weapon that is directed against different families of proteases. formation of the covalent tetrahedral complex (tc) with substrate is the first step of the catalytic process in the active site of serine proteases. his (chymotrypsin numbering) plays a role of a general base catalyst, activating the ser nucleophile by abstraction of its proton. it was experimentally observed that the pka of his ne in tc formed by serine proteases with transition state analog inhibitors is about units higher than the corresponding pka in the free enzyme. this work demonstrates that the environmental change of the his in tc, induced by the substrate binding in the enzyme active site, is the dominant factor in the pka increase of his ne, and triggers the enzymatic processing of the substrate. these results are based on quantum mechanical modeling of the active site of free chymotrypsin and tc complex of chymotrypsin with trifluoromethyl ketone inhibitor in dft b lyp/ - +g** level of theory. the polar environment of the enzyme active site is accounted for explicitly in the microscopic model. the combined environmental effects of the bulk water solvation and the rest of the protein is implicitly accounted for by our scrf(vs) continuous solvation approach. the role of local polar effects, such as the oxyanion and the asp -his hydrogen bond, on the pka of his ne in tc is analyzed. genome-wide analysis of subtilase (subtilisinlike serine protease) genes in microbial genomes limited to regions surrounding the asp, his and ser catalytic residues. pattern-searching methods using hidden markov models, based on conserved sequences surrounding the catalytic residues, were used to search for subtilases encoded in > bacterial and archaeal genomes, representing species. more than subtilases were found to be encoded in genomes. subtilases are more commonly found in grampositive bacteria than in archaea or gram-negative bacteria, and it is more common to have multiple subtilase-encoding genes than a single gene. the majority of the subtilases have a predicted signal peptide for translocation across the cell membrane, and a sub-group of these secreted subtilases are predicted to have a carboxy-terminal cell-envelope anchor, mainly of the lpxtg type for covalent anchoring to peptidoglycan. the genomic context of the subtilase-encoding genes was analyzed to gain insight in putative functions for these proteolytic enzymes. by also taking into account the predicted intracellular or extracellular location of the encoded subtilases, it was possible to predict a function for many subtilases in either nutrition/growth, spore germination, surface protein processing/activation, bacteriocin/toxin processing, or sigma factor activation/regulation. the poisoning by botropics species makes a similar physiologic, one of systemic effects is the blood coagulation for several mechanisms, as direct action on fibrinogen; factor x activation or platelet activation, by toxins of venoms. in the last years were identifies in botropics venoms, serine proteases. this toxins are responsible by coagulant activity with direct action on fibrinogen. serine proteases are utility for hemostatic system studies and for therapeutics use. looking for new molecules models is very important to show the mechanism of action and search structural characteristics responsible for its activities. the present work has the objective of purification and characterization of a coagulant factor (cf) from b. pirajai venom. the purification was made using a gel filtration, hydrophobic chromatography and an affinity chromatography. the molecular filtration was made in sephadex g- with ammonium bicarbonate buffer (ambic) . m ph . , resulting four fractions (p -p ), the coagulant fraction was named p . the p fraction was submitted in phenyl sepharose chromatography using triz buffer mm ph . in a decreasing gradient of nacl ( ; ; ; ; . ; m), and to finish the chromatography it was used distilled water, resulting six subfractions (fp -fp ), the coagulant subfraction was named fp . the fp sub fraction was submitted in benzamidine sepharose chromatography and eluted in the solutions: distilled water, obtained the subfraction bfp , sodium phosphate buffer mm ph . , obtained the subfraction bfp and glycine buffer mm ph . , obtained the sub fraction bfp that is the cf. the cf displayed one band in sds-page ( %) showing a pure protein, it has kda, the minim coagulant dose is . lg and has action on fibrinogen beta chain. the genome of arabidopis thaliana encodes putative proteases from the deg/htra family. this group of atp-independent serine-proteases was well examined in other organisms, especially e. coli and humans, but only limited data is available for members from this protease family in plants. deg and deg have been shown to act as proteases in the chloroplast, but no deg/htra proteases from other compartments have been examined so far. the putative protease deg is predicted to be localized in the peroxisome. we cloned the gene encoding deg (at g ) in an overexpression vector for heterologous expression in e. coli. the tagged protein was purified by affinity chromatography and used to raise polyclonal antibodies. with these antibodies we investigated the intracellular localization of deg and the protein level under various stress conditions in order to evaluate the in planta function of this protein. the effect of site-directed mutagenesis on cold adaptation of vpr; a subtilisin-like serine proteinase from a psychrophilic vibrio-species psychrophilic enzymes have very similar d structures as their homologous enzymes from mesophilic and thermophilic organisms. main characteristics of enzymes from psychrophiles are their high catalytic efficiency (kcat/km values) and thermolability. a subtilisin-like serine proteinase from a psychrophilic vibrio-species (vpr) shows these characteristics when compared to homologous enzymes from mesophilic and thermophilic organisms. the vpr gene was cloned, sequenced and expressed in e. coli and recently the crystal structure was determined at . Å resolution [ ] . structural comparisons have been carried out which have led to hypotheses about some of the structural factors which may contribute to cold adaptation of vpr. some of these hypotheses have been examined using site-directed mutagenesis. the specific residue exchanges were selected with the objective to incorporate stabilizing interactions into the cold adapted enzyme which were deemed to be present in related thermostable homologues. these include incorporation of pro into loops, a new potential salt-bridge, as well as substitutions aimed at improving packing in the hydrophobic core and decreasing apolar exposed surface. we have also introduced ser to ala substitutions at three different locations in the cold-adapted enzyme, but these were the most frequent amino acid exchanges observed in sequence comparisons of the enzyme to those of more thermostable homologues. here we report on the catalytic and stability characteristics of the selected mutants. engineering of gfp for the screening of serine protease inhibitors site specific proteolysis has been an attractive target for the development of antiviral therapies based on selective viral inhibitors. it has been previously demonstrated that reporter proteins like beta-galactosidase could be very useful for the high-throughput screening of hiv- protease inhibitors through the display of an accessible protease target site on the enzyme surface. in this work, by using structural analysis, we have engineered the gfp protein from jellyfish aequorea victoria to accommodate in its surface the hcv virus ns a- b protease cleavage site edvvccsmsytwtg, in a manner that proper proteolysis results in a fluorescent activity decrease. the three resulting gfp constructions, carrying the protease cleavage site in positions - , - and - , were soluble expressed in escherchia coli. moreover, the hcv ns cofactor residues - fused in frame via a short linker to the amino terminus of the hcv ns protease domain (residues - ) were also expressed in e. coli and under mm iptg induction, at least % of soluble protein was recovered and further purificated by an histidin tag. the analysis of gfp proteolysis in front of hcv recombinant protease were performed either with bacteria crude extracts and purificated proteins. the results presented here indicated that proper solvent exposure of target sites on gfp carrier protein may be a critical factor for protease cleavage and for the observation of fluorescence activity variance, being an aspect of absolute relevance for further design and implementation of newer analytical tests. various kinds of stressors cause the group of metabolic changes defined as the general stress response, initiated by some intracellular signals, such as production of abnormal or denaturated proteins, enhanced generation of reactive oxygen species and others. proteolytic enzymes quickly modify proteins and as a consequence can regulate cellular metabolism. although the stress defense mechanisms have been very often described in the recent literature, in very few works were estimated stress response abilities of white-rot basidiomycetes, which produce two kinds of very important ligninolytic enzymes -laccase and peroxidases. our previous results showed that the addition of menadione to abortiporus biennis idiophasic cultures caused the significant increase of the extracellular laccase activity in comparison to the control. the aim of this study was to determine activities of serine proteinases and natural serine proteinase inhibitors in idiophasic cultures of basidiomycete a. biennis grown under menadione-mediated oxidative stress conditions. we investigated the changes of intracellular serine proteinases activities in the presence and absence of atp, using hemoglobin and fluorogenic substrates. the level of natural serine proteinase inhibitors in mycelia was also measured. a fungal inhibitor of trypsin was partially purified and used to in vitro experiments. an interesting correlations between serine proteinases, serine proteinase inhibitors and laccase activities in prooxidant treated cultures were also observed. it can suggest that the proteolytic modifications under oxidative stress conditions can act as a regulation way of laccase activity. serine proteinases, inhibitory and laccase activities were additionally analyzed by native page. calpain is a ca + -regulated cytosolic cysteine protease, functioning as a ''modulator protease'', i.e. regulating/modifying functions/activities of substrates by limited proteolysis to modulate cellular functions. human has calpain genes and potential substrates extend to various cytosolic proteins such as kinases, transcription factors, cytoskeletal and er proteins. in skeletal muscles, expression of p (also called calpain ) predominates, playing an indispensable role for muscle functions in cooperation with ubiquitously expressed conventional calpains. for, a defect of p proteolytic activity originated from gene mutations causes muscular dystrophy. p localizes in myofibrils binding to connectin/titin, a gigantic elastic muscle protein connecting the z-and m-lines of sarcomere, the repetitive unit of myofibril, with a single molecule. in mdm (muscular dystrophy with myositis) mice, connectin/titin with a small deletion caused by natural mutation of the connectin/titin gene is expressed, resulting in severe muscular dystrophy phenotypes such as body weight less than a half of that of wild type, severely affected limb muscles with impaired walking ability and only - months of life time. the deletion in the mdm allele of the connectin/titin gene overlaps one of the binding sites of p in the n -line, another electron-microscopically visible line between the z-and m-lines of sarcomere. the mdm phenotypes clearly indicate that connectin/ titin or p or both are essential for proper muscle functions. to elucidate physiological roles of connectin/titin and p , we analyzed mdm mice in relation to calpain system. as a result, mar-ps (muscle ankyrin repeat proteins) were shown to be up-regulated in mdm muscle. marps bind to the n -and z-line regions of connectin/titin and function as transcriptional regulators translocating into the nuclei. carp (cardiac ankyrin repeat protein), one of marps, binding site in the n -line region is proximate to the p binding site, thus suggesting interactions of both molecules. possible signal transduction systems to modulate muscle functions revealed by the analyses will be discussed based on the results. inhibition and activation of calpain by its disordered endogenous inhibitor, calpastatin p. tompa , z. mucsi , o. gyo¨rgy , c. sza´sz and p. friedrich institute of enzymology, biological research center, budapest, hungary, research group of peptide chemistry, university of eo¨tvo¨s lora´nd, budapest, hungary. e-mail: tompa@enzim.hu calpains are a family of intracellular calcium-activated cysteine proteinases, implicated in the regulation of key cellular processes, such as cell division and programmed cell death. their activity is under tight control by an intracellular protein inhibitor, calpastatin, an intrinsically unstructured protein that contains four equivalent inhibitory domains. each of these comprise three conserved subdomains, of which subdmomains a and c anchor the inhibitor in a calcium-dependent manner, whereas subdomain b binds at the active site and inhibits the enzyme. in this work it is shown that the consequence of this mode of binding is that isolated a and c peptides promote calcium binding to calpain and thus activate the enzyme. this activation is manifest in the sensitization to calcium ion: the calcium required for half-maximal activity is lowered from . to . lm for l-calpain and to lm for mcalpain. in the physiologically significant sub-micromolar and low micromolar calcium concentration range this sensitization leads to a more than tenfold activation, which is of potential physiological importance as isolated calpain requires high calcium concentrations never realized in vivo. here we suggest calpastatin is degraded in vivo in a way that generates the activator peptides. due to the structural disorder of calpastatin, this unprecedented mode of action raises intriguing questions with respect to the generality of this ambivalent behavior. to address this issue, we have collected extreme cases, when the same protein elicits opposing, inhibitory and activatory, responses within the same molecular setting: structural predictions show that these proteins are largely disordered. as a conclusion, the possible general implications of this finding are discussed. meprins are oligomeric, brush border membrane or secreted zinc proteases that have unique and complex structures. they are composed of multidomain, highly glycosylated evolutionarily-related a and b subunits that form disulfide-linked homo-or heterooligomeric dimers. the homooligomeric form of meprin a forms very high molecular mass multimers of - da, among the largest extracellular proteolytic complexes known. meprins cleave cytokines, growth factors, bioactive peptides and extracellular matrix proteins, important compounds in inflammatory intestinal disease and in cancer metastases. to investigate the role of meprins in intestinal immune responses, inflammation was induced in mice by oral administration of dextran sulfate sodium (dss). the results showed that wild-type mice (c bl/ · ) had a more severe reaction to dss than meprin b null mice on the same genetic background, as determined by body weight loss, intestinal bleeding and mortality. this implies that the presence of meprin b increases host damage caused by dss and that meprin b plays an active role in intestinal pathophysiology. meprins are also expressed in colon cancer cells (e.g. sw , sw , and caco- ). expression of meprin a appears to increase with increasing metastatic potential. in addition, meprin a is highly expressed in the human liver hepatoblastoma cell line hepg and abundantly secreted into culture media. examination of human tumor samples showed that meprin a is expressed in primary colon tumors and in tumors that have metastasized to the liver. this indicates that meprin a expression in gastrointestinal tumor cells contributes to the progression of the disease. biochemical pathways mediating necrotic cell death and neurodegeneration in caenorhabditis elegans n. tavernarakis, p. syntichaki, c. samara and k. troulinaki institute of molecular biology and biotechnology, foundation for research and technology, heraklion, crete greece. e-mail: tavernarakis@imbb.forth.gr necrotic cell death plays a central role in devastating human pathologies such as stroke and neurodegenerative diseases. elucidation of the molecular events that transpire during necrotic cell death in simple animal models should provide insights into the basic biology of inappropriate neuronal death, and facilitate the characterization of mechanisms underlying degeneration in numerous human disorders. various cellular insults, including hyperactivation of ion channels, expression of human beta-amyloid protein implicated in alzheimer's disease, constitutive activation of certain g proteins, hypoxia and possibly the ageing process, can trigger a degenerative, necrotic cell death in the nematode caenorhabditis elegans. we are genetically and molecularly deciphering the c. elegans necrotic death program. we have isolated mutations in several distinct genetic loci that bock degenerative cell death initiated by various genetic and environmental insults. by characterizing such suppressors, we have discovered that neuronal degeneration inflicted by various genetic lesions in c. elegans, requires the activity of specific calcium-regulated calpain proteases and acidic ph-dependent aspartyl proteases. although, it is believed that these proteases become activated under conditions that inflict necrotic cell death, the factors that govern the erroneous activation of such-otherwise benign-enzymes are largely unknown. we identified novel factors that modulate cellular ph homeostasis, which are required for necrosis and showed that targeting these factors effectively protects from necrotic cell death in c. elegans. our findings demonstrate that two distinct classes of proteases are involved in necrotic cell death and suggest that perturbation of intracellular calcium levels may initiate neuronal degeneration by compromising ph homeostasis and deregulating proteolysis. search for regulatory proteins which are controlled by proteolysis in escherichia coli based on microarray analysis j. m. heuveling ag hengge, institute of microbiology, fu berlin, berlin, germany. e-mail: joheuvel@zedat.fu-berlin.de the impact of controlled proteolysis on regulatory events in prokaryotes is increasingly recognized over the last decade. as in eukaryotic cells, proteolysis is more than just a garbage disposal but has been found to be implicated in the regulation of many vital functions of the bacterial cells, like cell cycle, stress responses and development (hengge r and bukau b. mol microbiol ) . conditional degradation of regulators shows a high potential of integrating a great variety of signals as is well studied for the degradation of sigma s. this sigma subunit of the rna polymerase, which triggers the general stress response in escherichia coli is digested rapidly by the clpxp protease in association with the phosphorylated response regulator rssb under non-stress conditions (stuedemann a. embo ). several other regulatory proteins have been found to be subjected to proteolysis, as lexa, a regulator of the sos response. also lon protease is involved for example in the degradation of rcsa, a regulator of the capsule biosynthesis and of sula, a cell division inhibitor (as a review: hengge-aronis r, jenal u. curr opin microbiol ). in order to find other regulatory processes in which proteolysis plays a role we pursued a global approach using the microarray technique. in mutants lacking functional clpp or lon proteases or either one of the clp recognition factors clpa and clpx, we searched for genes, which are differentially transcribed compared to the wildtype. we found some interesting groups of genes belonging to common regulons governed by known regulators -candidates for clp or lon mediated proteolysis. after confirmation of these results through lacz fusion studies of representative genes of these regulons, these regulators are presently examined in in vivo degradation studies using immunodetection methods. a distinct group of serine peptidases cannot hydrolyze proteins, but can readily cleave peptides that are up to about amino acid residues long. the representative member of the family, prolyl oligopeptidase is implicated in a variety of disorders of the central nervous system. the enzyme consists of a peptidase domain with an a/b-hydrolase fold and its catalytic triad is covered by the central tunnel of a seven-bladed b-propeller. this domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. in most propeller domains the circular structure is ''velcroed'' together in a mixed blade, where both amino and carboxy terminus are involved to form a four stranded antiparallel b-sheet. non-velcroed or ''open topology'' propellers are rare, and prolyl oligopeptidase was the first protein structure exhibiting a domain of this nature. the apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. two possibilities of substrate access were investigated: either blades and of the propeller domain move apart or the peptidase and/or propeller domains move to create an entry site at the domain interface. engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. this indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action. biochemical characterization of thermoplasma volcanium recombinant s proteasome and its regulatory subunit g. baydar and s. kocabiyik molecular genetics, biological sciences, middle east technical university, ankara, turkey. e-mail: gozde_baydar@hotmail.com proteasome associated energy dependent proteolysis is not only involved in rapid turnover of specific proteins that could be important during periods of stress, but also engaged in the turnover of the short-lived proteins that regulate a variety of cellular processes in both procaryotic and eucaryotic cell. the universal distribution of proteasome homologs in archaeal genome provide insight into the vital role of archaeal proteasomes. s catalytic core of archaeal proteasomes in combination with various aaa atpases and membrane associated lon proteases may play role in stress response or turnover of the regulatory proteins. however, little is known about the potential physiological roles of archaeal proteasomes. this study presents the data on biochemical and biophysical features of recombinant s proteasome of a thermoacidophilic archaeon thermoplasma volcanium (tpv). pcr was performed to amplify dna fragments containing tpv genes encoding the a -and b-subunits of the proteasome from tpv genomic dna. the amplified a-gene (tpva) and b-gene (tpvb) together with their upstream sequences were separately cloned and then combined in puc vector. the resulting recombinant puc-skba plasmid was used for heterologous production of in vivo assembled s proteasome in e. coli. the recombinant proteasome was purified by combination of ammonium sulfate precipitation, gel filtration chromatography (sepharyl s- ) and ion-exchange chromatography (q sepharose). molecular masses of purified protein subunits were estimated as . kda (b-subunit) and . kda (a-subunit). substantial post-glutamyl peptide hydrolyzing activity and chymotrysin-like activity were detected as associated with recombinant proteasome. maximum chymotrypsin-like activity was measured at °c and ph . . crystallographic studies of the gtp-dependent transcriptional regulator cody from bacillus subtilis. cody is a gtp dependent transcriptional regulator of early stationary phase and sporulation genes in bacillus subtilis. it is activated by gtp, during rapid cell growth it represses several genes whose products allow adaptation to nutrient depletion. when the cells pass from rapid growth to stationary phase, the intracellular concentration of gtp drops thus releasing the repressed genes. cod y is a -residue polypeptide containing a helix-turn-helix motif for binding to dna. it also has motifs common with small gtpases, but cody has a much lower affinity for gtp. crystals of the full-length cody have been grown in the presence and absence of gtp from sodium citrate buffered solutions using lithium sulphate as a precipitant and diffraction data have been collected to . Å resolution. attempts to solve the structure using anomalous data from the semet derivative crystals of cody have been hampered by the large number ( ) of methionines in the asymmetric unit and difficulties in reproducibility of angiotensin-converting enzyme (ace) is a zinc metallopeptidase critical for the generation of the vasoconstrictor peptide angiotensin ii. a homologue of ace, ace- , has recently been identified, which appears to play a counter-regulatory role to ace by inactivating angiotensin ii. like ace, ace- is a type i membrane protein with its active site contained within the extracellular domain. the expression of ace protein is normally low and restricted primarily to endothelial cells of the heart and kidney, kidney epithelium and testis. recent evidence from ourselves and others indicates that ace is significantly upregulated in a number of pathologies, such as myocardial infarction, renal disease and hepatitis c-induced cirrhosis. given that ace can be proteolytically released from the cell surface in culture, ace may likewise be shed into plasma or urine. detection of elevated levels of ace in plasma and urine may be a useful biomarker for the diagnosis of hepatic, renal and vascular disease. using a specific quenched fluorescent substrate, we have detected ace activity in human urine. in contrast, ace activity could not be detected in human plasma; interestingly, however, we noted that plasma markedly inhibited the activity of recombinant ace , thus compromising the possibility of measuring plasma enzyme activity. we are in the process of purifying this inhibitor, which preliminary results suggest is small and hydrophilic. we are also currently optimizing methods for its removal from plasma samples, thus allowing detection of low levels of soluble ace activity in normal human plasma. the identification of a potential endogenous inhibitor of ace , the first for this family of metallopeptidases, could have significant consequences for ace function in vivo and the regulation of angiotensin peptides. future studies will examine whether plasma or urinary levels of ace are elevated in cardiovascular, renal or liver disease. molecular determinants of proteolytic processing of non-structural polyprotein of semliki forest virus. institute of molecular and cell biology, university of tartu, tartu, estonia. e-mail: lulla@ut.ee semliki forest virus (sfv) is a positive-stranded rna virus. the replication of sfv is performed by the rna-dependent rna replicase complex (rc) and regulated by proteolytic processing. during the course of the infection template preference of rc changes from rna plus-strand to minus-strand. it has been known for several years that this preference switch is due to the proteolytic processing of sfv non-structural polyprotein p , mediated by viral cysteine protease located in the carboxy-terminal domain of the nsp protein. tight temporal regulation of this template specificity switch is crucial for the viral replication, but, nevertheless, its mechanism remains unsolved. therefore, the mapping of the essential molecular determinants of the site-specific cleavage consensuses may provide necessary information, concerning the cleavage regulation as well as regulation of the rna replication. the results of our studies indicate that as little as amino acid residues from the c terminus of nsp protein determine the specificity of the proteolytic cleavage of the nsp /nsp junction. at the same time sequences laying downstream of the cleavage point (in nsp region) have only minor effect on the cleavage efficiency. the exact region required for the cleavage of nsp /nsp junction is yet not known but the sequences, required from c-terminal part of nsp protein, are likely short as well. in contrast, sequence lying within - n-terminal amino acid residues of nsp is vital for cleavage of the nsp /nsp junction. this region may represent the cofactor of the nsp protease that activates processing at the nsp /nsp cleavage site. thus, as the result of current research, a principally new function -regulation of the proteolytic processing and rna replication -was mapped to the conserved n-terminal region of the nsp . this finding significantly improves our understanding about the role of nsp , which was enigmatic till now, in the virus life cycle. activation occurs within the specific dibasic motif hsiirrsl, suggesting the involvement of the proprotein convertases (pcs) in these process. this family of endoproteases are responsible for the activation of a large variety of regulatory proteins by cleavage at multi-basic recognition sites exhibiting the general motif (k/r)-(x)n-(k/r)(n = , , or ). cotransfection of the furindeficient colon carcinoma cell line lovo with provegf-c and different pc members revealed that furin, pc and pc are vegf-c convertases. the processing of provegf-c is blocked by the inhibitory prosegments of furin, pc and pace , as well as by furin-motif variants of alpha -macroglobulin and alpha antitrypsin. accordingly, mutation of the vegf-c pc-site (hsiirrsl to hsiisssl) inhibited provegf-c processing. following zebrafish caudal fin amputation, the injection of control vector or vector containing wild vegf-c did not affect fin regeneration. in contrast, injection of muted vegf-c (pro-vegf-c) inhibited fin regeneration. these data highlight the importance of vegf-c processing in zebrafish fin regeneration and suggest that zebrafish can be used as a simple and useful model for studying the role of protein maturation by the pcs in physiological processes. thimet oligopeptidase (top) hydrolyzes a variety of bioactive peptides and is implicated in the regulation of neurological and other physiological processes. top is composed of two ''clamshell'' domains, with the substrate-binding pocket and catalytic site lying between these domains. it is speculated that conformational changes in loops and coil regions connecting the domains lead to changes in substrate specificity. the loop region (residues - ) is close enough to the active site to interact with even the smallest substrate. it contains three glycine residues and is expected to be quite flexible. in an effort to trap intermediate conformations of the loop, we have replaced gly , , or with ala and have compared the activities of the three resulting protein constructs towards two quenched fluorescent substrates. all three enzymes had lower activity than wild type towards a bradykinin analog, with g a, the most active of the mutants, possessing / wild-type activity. however, utilizing a smaller substrate, g a was the most active, surpassing even wild type (fivefold increase in activity). g a had little activity towards either substrate. these results are consistent with data that revealed increases in activity towards the larger substrate, when the enzyme is partially denatured and presumably, more flexible and with increased accessibility of the binding loop to proteolytic enzymes, when partially denatured. acknowledgment: this work was supported by hhmi, nih-ns (mjg). the proteasome: paradigm of a self-compartmentalizing protease self-processing of subunits of the proteasome crystal structures of the rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly handbook of metalloproteins bovine chymotrypsinogen-a x-ray crystal-structure analysis and refinement of a new crystal form at . a resolution equilibrium and rate constants for the interconversion of two conformations of a-chymotrypsin. the existence of a catalytically inactive conformation at neutral ph refolding transition of alpha-chymotrypsin -ph and salt dependence e-mail: saleh @hotmail.com reference . arnorsdottir j, kristjansson mm, ficner r. crystal structure of a subtilisin-like serine proteinase from a psychrotrophic vibrio species reveals structural aspects of cold adaptation (which is an excellent substrate for adam- ), we observed % increase in enzymatic activity in the media of mm -ht-treated cells compared to untreated cells. however, we did not see any increase in the fluorescence when we used ''cate '', an adam substrate, which does not recognize adam- . to further support a role for adam- /tace, we designed silencing rnas against the enzyme, which were introduced into the mesangial cells using lentiviral infection. successful silencing was confirmed by western blotting days after infection. control and tace silenced human mesangial cells were stimulated with - lm of serotonin for min, and erk activation was assessed by western blotting baron-ruppert and e. heymann department of physiological chemistry (fb /gw) e-mail: rebecca.lew@med.monash.edu.au b - p functional properties of p /calpain and connectin/titin in mdm mouse skeletal muscle y bunkyo-ku the lectin pathway of complement system is an important component of the innate immunity. it provides the first line of defence against infection, since it is activated on the surface of invading pathogens. the activation of the complement system results in the destruction and clearance of foreign microorganisms. mannose-binding lectin-associated serine protease- (masp- ) is the enzyme which is responsible for the initiation of the lectin pathway of complement activation. masp- is a multidomain serine protease, which is synthesized as an inactive zymogen and become activated upon mbl binds to carbohydrate plant proteinase inhibitors are widely spread in the different plant species being a significant component of a defense system. somewhere a significant diversity of the proteins related to the same structural family of the inhibitors in the same species may be observed. the family of potato kunitz-type proteinase inhibitors (pkpis) exemplifies a group of proteins with the diverse properties and may be divided into three major homology groups: a, b and c. a lot of genes encoding different pkpiproteins of each group were found in various potato cultivars (solanum tuberosum l.). inhibition activity of plant invertase, cysteine and serine proteinase was found in proteins subgroup c. a set of gene copies were isolated by pcr from potato cv. istrinskii genome. dna sequencing analysis of these resulted in identification of different dna sequences with a high similarity to potato kunitz-type inhibitors of group c (pkpi-c). cluster analysis demonstrated that this clones represented multiple copies of six new genes denoted as pkpi-c , -c , -c , -c , -c and c . it can be supposed that at least two alleles containing pkpi-c genes are harbored in tetraploid genome of potato. one of new genes, namely pkpi-c , exhibited % identity with known invertase inhibitor cdna ( ) from cv. provita. another pkpi-c gene was similar ( % identical residues) with cdna (p ) from potato cv. bintje encoding for a putative trypsine inhibitor. four other new genes demonstrated as much as - % identity with known pkpi-c proteins from other potato cultivars. the n-terminal sequence of the protein encoded by the pkpi-c gene was identical to the n-terminal sequence of specific subtilisin inhibitor pksi isolated from cv. istrinskii.b - p regional distribution of human trypsinogen in human brain determined at mrna and protein level j. to´th , l. gombos , e. siklo´di , p. ne´meth , m. palkovits , l. szila´gyi and l. gra´f laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, hungary, institute of immunology and biotechnology, university of pe´cs, pe´cs, hungary, laboratory of neuromorphology, department of anatomy, semmelweis university, budapest, hungary. e-mail: july@ludens.elte.huproteases play an important role in many physiological and pathological processes in the central nervous system such as development, neurite outgrowth, neuronal plasticity and degeneration and cell signaling. a gene coding for such an enzyme might be prss on chromosome of the human genome. it encodes due to alternative splicing both mesotrypsinogen, which is expressed in pancreas, and trypsinogen whose mrna has been identified in different human tissues (initially in brain, recently in different epithelial cell lines from prostate, colon and airway). analysis of the gene prss predicted two isoforms of the zymogen: isoform a may have a amino acid, while isoform b a amino acid n-terminal leader sequence. in order to gain information on the possible role of human trypsinogen we have determined its amount at the mrna and the protein level as well in selected brain areas using real-time quantitative pcr and elisa.the highest transcript levels could be detected in cerebellar cortex, while low amounts were found, e.g. in cerebellar white matter samples. the distribution of the mrna in different brain areas measured by real-time pcr is consistent with the protein levels detected with elisa. the usage of different monoclonal antibodies specific for the amino acid leader sequence and the protease domain allowed the separate detection of the zymogen and the active enzyme. in e.g. the hypothalamus the zymogen is the dominant form, while a significant degree of activation was found in the cerebellar cortex. our data indicate that the extent of activation varies with different areas. as human trypsinogen is ubiquitous in the brain we conclude that it might play a role in general neurological processes. serine proteases are enzyme involved in the maintenance of the cell homeostasis. thus, this type of enzymes must be extremely regulated and it has been highly reported that serine proteases are involved in the growth and expansion of different cancers. in this regard, the type ii transmembrane serine proteases (ttsps) constitute a subfamily of membrane anchored serine proteases that are ideally positioned to carry out different interactions with other cell surface or extracellular proteins. among them, tmprss and tmprss proteins have been reported to be overexpressed in most prostate and ovarian cancers respectively, matriptase/mt-sp is expressed in a wide variety of benign and malignant tumors and hepsin is overexpressed in ovarian and renal cancers. desc- is a ttsp member found differentially expressed in squamous cell carcinoma (differentially expressed in squamous cell carcinoma gene ) and differentially from other ttsps, its expression is found to be reduced in tumor tissues respecting to the normal tissue at rna level in head and neck squamous cell carcinoma (hnscc), what suggests a possible tumor protective function for desc- . in order to shed light about the role of desc- in these processes, we have carried out the molecular cloning of the human full-length cdna and expression of the recombinant protein to delineate the implication of this protease in hnscc.diffracting crystals. therefore we used limited proteolysis and mass-spectrometry analysis to identify the domain boundaries of the protein and were able to determine the sequence of two principal proteolytic fragments corresponding to the n-and c-terminal domains of cody. these individual domains which were successfully cloned in escherichia coli, overexpressed as histagged proteins, isolated and purified. both domains have been crystallized. the crystals of the n-terminal domain grow from bis-tris buffered solutions at ph . containing polyethylene glycol and calcium acetate. the crystals of the c-terminal domain were obtained using ammonium sulphate as a precipitant. the crystals of n-terminus domain diffract to at least . Å and crystals of c-terminus domain -to . Å using an in-house diffractometer with a mar research image-plate as a detector. progress towards the determination of cody structure will be presented. the gram-positive bacterium listeria monocytogenes is a facultative intracellular parasite. interactions of l. monocytogenes with the host cell are provided by a number of secreted and cell surface proteins. one of the most important virulence factors, actinpolymerizing protein acta, is surface attached via the hydrophobic c-tailed membrane anchor. despite, the membrane anchor acta was found in comparable amounts both on the cell surface and in the culture supernatant. the aim of the work was to investigate the mechanism of acta release and the role of this process in l. monocytogenes virulence. maldi-tof ms analysis of trypsin released acta suggested releasing due to proteolytic cleavage between histidine and threonine residues in the close vicinity of the membrane anchor predicted by the htmm analysis. the substitution of histidine with proline prevented acta release into the culture supernatant, although did not disturb its surface presentation. in silico analysis of eight other l. monocytogenes membrane-anchored surface proteins suggested the role for asparagine and threonine residues in specific proteolysis. the prediction was experimentally tested by substitution of the residues with alanine. the l. monocytogenes spontaneous mutant strain, unable to release membrane-anchored proteins into the culture supernatant, was isolated. the mutation was mapped outside the acta gene and presumably affected the corresponding peptidase. the mutation impaired the invasion of l. monocytogenes into the human epithelial-like hela cells that suggested the effect of the released proteins on signaling events that result in induced phagocytosis of the pathogen by normally non-phagocytic cells. angiotensin ii (ang ii) has been proposed to act as a regulatory peptide in the epidermal layer of human skin. while the expression of receptors and peptide precursors have been demonstrated in epidermis, the formation of ang ii and its inactivation have not been studied in detail. thus we have established a model system with cultured keratinocytes to examine the metabolism of ang i, ii and related peptides by intact epidermal cells. cultures were incubated with peptides in a minimal medium, which sustained cell viability for at least h and the metabolism of peptides was monitored by chromatography (rp-hplc). with ang i as peptide substrate five major products were detected in keratinocyte culture media after h incubation. a half-life of about h was estimated for ang i and the slow degradation supports results of earlier studies revealing low activities of exopeptidases in a microsomal fraction from keratinocytes as compared to fibroblasts. the degradation of ang i was not affected by inhibitors of alanyl aminopeptidase, peptidyl dipeptidase a and neprilysin. since a peptide product formed from ang i in keratinocyte cultures resembled ang ii in hplc analysis, the activity of peptidyl dipeptidase a in these cells was assayed with hip-his-leu and the presence of the peptidase was confirmed by its sensitivity to captopril. further experiments showed that ang ii, iii and related peptides were degraded in keratinocyte cultures with rates similar to ang i and these reactions interfered severely with the formation of ang ii. immunohistochemical studies showed a strong positive staining for neprilysin and alanyl aminopeptidase in the dermal layer of human skin and at the epidermal-dermal junction confirming the results obtained with the cell cultures. soluble angiotensin converting enzyme- present in human plasma and urine p /calpain is the skeletal-muscle-specific calpain and is considered to be a modulator protease in various cellular processes. a defect in the p gene causes limb-girdle muscular dystrophy type a (lgmd a), suggesting that p functions are indispensable for proper muscle functions. in sarcomeres, p localizes at z-, n -and m line regions. although the binding partner for p at z-line has not been identified yet, n -and m-line localization of p are considered dependent on its interaction with the n a and m-line regions of connectin/titin, respectively. connectin is a gigantic sarcomeric protein playing an important role as a molecular template for sarcomeric organization, an elastic element generating passive tension, a platform for various protein ligands, etc. in this study, we focused on the molecular components associated with the n a region of connectin/titin to extend our understanding on p . intriguingly, a recessive mutation in the mouse connectin gene, mdm (muscular dystrophy with myositis), causes muscular dystrophy. there are two remarkable phenotypes consequential to mdm mutation. first, the mdm mutation abolishes p binding activity of connectin n a fragment. second, in skeletal muscle from mice homozygous for mdm mutation, upregulation of cardiac ankyrin repeat protein (carp) is observed. carp also binds to n a connectin at the n-terminal proximity of the region mutated by mdm. the effect of mdm mutation on p activity and the properties of n a connectin as well as carp were analyzed using both animal model and cell culture systems. semliki forest virus (sfv) is well known model virus, which has been studied for decades. the main topic of this research was characterization and analysis of sfv replication machinery using approach based on use conditional-lethal mutants of viruses. the direct aim of the present study was to sequence and functionally characterize a panel of independent sfv temperature sensitive mutants. from all putative ts-mutations, identified in this study, two were mapped to nsp protein, four were mapped to nsp protein and one was founded in nsp region. number of assays were used to verify phenotypic effects of revealed mutations: titration of virus stocks at different temperatures, leak yield experiments, analysis of viral rna synthesis and viral polyprotein processing at different temperatures. nsp mutants had clear viral protease defect and accumulated non-cleaved polyproteins on different stages. besides all, biotechnological branch of our research is already developing. it includes improving of existing sfv based expression vector system by use of ts-mutations for the temperature regulation of foreign gene expression in mammalian cells. dipeptidyl peptidase iv activity and/or structure homologues (dash) in brain tumors pathogenesis of many diseases, including cancer, often involves improper proteolytic post-translational modification of biologically active peptides. association of dysregulated expression pattern of novel group of ''dipeptidyl peptidase (dpp)-iv activity and/or structure homologues'' (dash) with cancer development and progression has been suggested by several authors, including us [ ] . dpp-iv enzymatic action as a common attribute of most of dash members modifies signaling potential of their substrates, biologically active peptides, not only quantitatively, but due to the changes in their receptor preferences also qualitatively. in this study, we have investigated expression (by real time rt-pcr and immunohistochemistry) and enzymatic activity (by biochemical assays and enzyme histochemistry) of plasma membrane localized dash members, in particular dpp-iv, fibroblast activation protein-alpha (fap) and attractin in human gliomas. it was revealed that varying quantities of dpp-iv, fap and attractin mrnas and proteins were coexpressed in the studied tumors. the majority of dpp-iv-like activity in the glioma tissue could be attributed to the canonical dpp-iv. this activity, assayed biochemically and expressed per mg of protein, was increased in high grade gliomas. inhibition studies suggested lack of enzymatically active attractin in the examined glioma tissues. the results of our pilot study demonstrate for the first time that both enzymatically active and inactive dash molecules are coexpressed in gliomas and suggest prevailing association of increased dpp-iv activity with high grade tumors. acknowledgment: this work was supported by iga nr/ - and msmt vegf-c is involved in the neovascularization processes, steps essential for wound healing, cancer progression and many other physiological functions. zebrafish vegf-c processing and key: cord- - caevjvh authors: falanga, annarita; galdiero, massimiliano; morelli, giancarlo; galdiero, stefania title: membranotropic peptides mediating viral entry date: - - journal: pept sci (hoboken) doi: . /pep . sha: doc_id: cord_uid: caevjvh the means used by enveloped viruses to bypass cellular membranes are well characterized; however, the mechanisms used by non‐enveloped viruses to deliver their genome inside the cell remain unresolved and poorly defined. the discovery of short, membrane interacting, amphipathic or hydrophobic sequences (known as membranotropic peptides) in both enveloped and non‐enveloped viruses suggests that these small peptides are strongly involved in breaching the host membrane and in the delivery of the viral genome into the host cell. thus, in spite of noticeable differences in entry, this short stretches of membranotropic peptides are probably associated with similar entry‐related events. this review will uncover the intrinsic features of viral membranotropic peptides involved in viral entry of both naked viruses and the ones encircled with a biological membrane with the objective to better elucidate their different functional properties and possible applications in the biomedical field. significant improvements have been achieved in recent years in the understanding of the multiple alternative ways of virus entry into susceptible cells. [ , ] the strategies employed by viruses to enter cells are different according to the presence or absence of a lipid bilayer surrounding the virus. enveloped viruses present a membrane bilayer while non-enveloped viruses lack this membrane and present on their surface only capsid proteins. the mechanism of cell invasion by the two groups of viruses is rather diverse and the foremost difference is the direct consequence of their distinctive physicochemical state at the interface that occurs at the time of the encounter between the virus and the cell membrane; in particular, two lipid membranes confronting each other in the case of enveloped viruses as opposed to a layer consisting only of proteins that face a lipid layer in the entry of naked viruses. enveloped viruses entry exploits direct fusion with a cellular membrane through the involvement of specialized viral fusion proteins, present on the viral membrane and the consequent transfer of the nucleocapsid into the cytoplasm. [ ] on the other hand, the entry of non-enveloped viruses, which lacking the outer viral membrane are unable to take advantage of the cellular mechanism of membrane fusion, involves the activation of viral lytic factors that induce cell membrane rupture. [ ] the major features in membrane interaction by enveloped and non-enveloped viruses are reported in figure . notwithstanding the different mechanisms of penetration, the key step is the entry process and the modification of the cell membrane which allows the viral genome to penetrate into the host cell and start the replication cycle in the appropriate cellular compartment. [ , ] the most critical barriers for viral penetration and replication are the plasma membrane, the cytoplasm, and any other membrane that needs to be crossed in order to have access to the sites where viral replication or assembly take place. the overall picture of the mechanism of viral entry is becoming increasingly complete; in fact, depending on their dimension and structure, they have acquired different strategies to penetrate and take control of cell functions. [ , ] the molecular details of the interactions at the interface of virus and cell surfaces are quite complex and highly variable, but there is a common idea that only a limited number of pathways allowing viruses to reach the sites of penetration exist, with enveloped and non-enveloped viruses presenting different and unrelated processes, but with general principles driving all fusion events. the main difference between fusion peptides of enveloped viruses and lytic factors of non-enveloped viruses is the fact that fusion peptides promote membrane fusion while lytic factors promote membrane disruption. in this review, we summarize current knowledge on the mechanism of membrane fusion of both enveloped and non-enveloped viruses with a focus on common principles. fusion peptide derived from enveloped viruses and lytic peptides from non-enveloped viruses are described with an effort to find undisclosed differences and similarities. we conclude reporting ground breaking applications of membranotropic peptides (both fusion and lytic peptides) which open a new exiting field of research. enveloped viruses depend on membrane fusion for cell penetration. [ ] the two main routes used by enveloped viruses to enter the cell are the endocytic and non-endocytic pathways. [ ] most enveloped viruses undergo endocytosis while only few are able to fuse directly to the plasma membrane. [ ] viruses that use the endocytic route for cellular internalization are able to escape into the cytosol avoiding lysosomal degradation. therefore, penetration invariably involves membrane fusion mediated by specific viral glycoproteins (catalysts) with the main difference being that viruses using endocytic pathways fuse their envelope with the endosomal membrane from the luminal side. [ ] thus, membrane fusion constitutes the essential and ubiquitous mechanism of entry of enveloped viruses, irrespective of their route of entry. [ , , ] viral fusion proceeds through a hemifusion stalk with merging of proximal leaflets, which culminates in the opening and expansion of a pore connecting the two sides of the membrane. [ ] the overall process is supported by a catalyst responsible of the lowering of the transition barriers and fusion proteins constitute the catalytic agents fulfilling this function. [ ] the entry involves several other critical steps which include cellular receptor or co-receptor binding, internalization, uncoating, and release of viral nucleic acids at the proper site of replication. [ , , ] various factors can mediate an efficient interaction between cells and viruses; for example, cholesterol rich domains are platforms for the entry of many enveloped viruses such as the influenza virus [ , ] and many viral membranes contain much more cholesterol than the mammalian plasma membranes from which they are derived. [ , ] viral fusion proteins undergo significant rearrangements from the pre-fusion to the post-fusion conformations which are triggered by either receptor binding, proteolytic cleavage or low endosomal ph, and eventually determine the exposure of previously sequestered hydrophobic peptides, loops, or patches, able to interact with and destabilize one or both the opposing membranes. [ , ] crystallographic data on pre-and post-fusion structures of viral fusion proteins has resulted in the identification of at least three distinct classes based on their three-dimensional organization and mechanism of fusion ( figure ). [ , ] class i fusion proteins includes many of the most studied human pathogens such as influenza virus, human immunodeficiency virus (hiv), and ebola virus; the key features is the requirement of a proteolytic cleavage to initiate fusion, [ ] activating these proteins which form an extended intermediate on the surface of the virion with the fusion peptide at the n-terminus of the protein engaged in the interaction with the target membrane and forming a link between the two fusing bilayers. the pre-hairpin structure is a unique state in which the fusion protein is simultaneously connecting two distinct membranes, the target membrane through the fusion peptide or patches and its own viral membrane through its transmembrane domain (tm). further conformational changes produce trimers of hairpins with a central a-helical coiled-coil structure. [ , ] the -helix formation is related to the opening of the fusion pore and provides the major driving force for the process. thus, peptides able to prevent -helix formation, interfering with refolding of the fusion glycoproteins act as potent viral entry inhibitors. [ , ] class ii fusion proteins, which include flaviviruses, alphaviruses, and bunyaviruses, are consistently different from those of class i and are mainly composed of b structures. [ ] these fusion proteins are often heterodimers or homodimers lying almost flat on the virion surface. [ ] they are organized in three globular domains: domain i is char- class iii fusion proteins have a mixed secondary structure with the central a-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated b-sheet similar to class ii fusion proteins; the main representatives of class iii fusion proteins are protein g of vesicular stomatitis virus (vsv), [ , ] gb protein of herpes simplex virus (hsv), [ ] and of epstein-barr virus (ebv), [ ] and gp from insect-cell baculovirus. [ ] the post fusion structure is characterized by the presence of an internal fusion peptide in domain i organized in two hydrophobic fusion loops flanked by a b-sheet domain with a pleckstrin-like fold (domain ii). domain ii is nested with the largely a-helical domain iii, which is composed of trimers that give rise to an elongated, rod-like shape molecule. the a-helical domain is inserted in domain iv which is made of b-sheets and a very long c-terminal extension (domain v). interestingly, the multicomponent herpesvirus fusion machinery requires the presence of gb, gh/gl, and gd and multiple cellular receptors are engaged in the entry pathway in a cascade of molecular interactions. [ ] gh/gl and gb are part of the core fusion machinery and need to cooperate in order to trigger the initial lipid destabilization which culminates in the fusion of the two bilayers. [ ] [ ] [ ] despite the fact that it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process, studies leave little doubt that also the gh/gl complex undergoes dynamic rearrangements during the fusion process. [ ] the crystallographic post-fusion structure of gb shows that it is a canonical class iii fusion protein; [ ] and several synthetic peptides derived from gb induce the fusion of large unilamellar vesicles and inhibit herpes virus infection. [ , , ] irrespective of their structural differences, the three classes of fusion proteins seem to induce membrane fusion by essentially the same generic mechanism and a common refolding pathway is highly suggestive for the conservation of several phases of the process. the non-enveloped viruses exploit a different mechanism for entry because they lack a membrane which surrounds the protein capsid; as a consequence they require capsid-dependent mechanisms for penetrating the cell membrane or for exiting from the endosome. [ , ] the entry process is much less known and involves a series of triggers producing conformational and structural rearrangements which end in the exposure and/or release of lytic factors. low ph, receptor interactions, protease cleavage, chaperone-assisted morphological changes, divalent cation chelation, or any combination of these factors which take place at the appropriate site of membrane penetration are essential triggers to produce the activated viral intermediate. [ ] the mechanism of membrane disruption involves proteolytic cleavage of the coat protein and programmed exposure/release of small membrane-lytic peptides. in this scenario, viral penetration is mediated by short, membrane interacting, amphipathic and/or hydrophobic sequences present in proteins undergoing a conformational modification which allows the exposure of these domains and their interactions with membranes. [ ] similarly to enveloped viruses, capsid proteins seem to be trapped in a metastable state waiting for a trigger to expose their membrane active peptides and the membrane penetrating ability of these sequences is fundamental for entry but at the same time their premature exposure has to be avoided before host cells provide the triggers. flock house virus (fhv) is one of the simplest and most studied non-enveloped viruses. the infection starts with the binding to one or more host cell receptors leading to a receptor-mediated endocytosis mechanism. [ ] the receptor binding induces a conformational change that initiates uncoating or viral disassembly once the particle is exposed to low ph within the endocytic pathway blocking this event often inhibits infection. the immature provirion fhv is initially assembled from copies of the coat protein alpha and, subsequently, it undergoes autoproteolytic cleavage to generate the mature infectious virion which contains a large n-terminal fragment, b ( amino acids), and a small c-terminal fragment, g ( amino acids). [ ] the capsid shell is constituted by the central region of b forms, while the g peptides are the amphipathic helices non-covalently associated with the capsid interior. the g peptides are responsible of the interaction with the membrane and its local disruption which ends in viral entry. these peptides are composed of an n-terminal amphipathic helix separated by a proline-glycine-proline turn from a hydrophobic c-terminal region. the amphipathic g helices are located in the interior of the capsid and when fhv enters cells through receptor mediated endocytosis, the g peptides are exposed and determine the disruption of the membrane with consequent release of the viral genome in the cytosol. [ ] this is a dynamic process with g peptides continuously, transiently, and reversibly exposed to the exterior of the capsid; g peptides may not only be exposed but also released from the virus particle which may represent a common paradigm of non-enveloped virus entry. in this dynamic process g peptides may continuously "sample" the environment until they encounter the appropriate cellular trigger; at this point the virus undergoes an irreversible conformational change in which the g amphipathic helices insert into the target membrane, allowing the viral rna to enter the cytoplasm. [ ] | m em b ra n otrop i c p ep ti d es many studies are devoted to the comprehension of the mechanism of insertion of fusion peptides, loops, or patches into monolayers inducing nipple formation and curvature in the target cellular membrane through many synergic interactions. the insertion in one leaflet of a closed bilayer will cause the increase of the surface area of the leaflet and the formation of a spontaneous curvature, which is one of the driving forces to reduce the energetic barrier needed for the achievement of fusion. [ ] fusion and/or membranotropic domains in viral fusion proteins contain aromatic residues which together with alanines and glycines [ ] contribute to the interaction with just one bilayer leaflet. many biophysical and structural techniques using synthetic analogues and model membranes have been used to determine physiologically relevant states during membrane partitioning. [ , ] the two structural motif widely found in fusion proteins and able to produce membrane curvature are the amphipathic a-helix and tilted peptides. [ , ] the segregation of hydrophobic and polar residues on the two opposite sides of the amphipathic a-helix is responsible of the superficial insertion into the upper monolayer which modifies the pack- similarly, also tilted peptides are characterized by an asymmetric distribution of hydrophobic residues, which again produces a modification of the organization of the membrane into which they insert. [ ] the membrane bound conformation of the influenza virus fusion peptide is characterized by the presence of a hairpin of two tightly packed, antiparallel a-helices; [ , ] the asymmetric insertion is determined by the fact that hydrophobic residues insert into the proximal membrane leaflet and polar residues project outward. the precise angle of the kink between the two arms depends on the sequence, the ph, and the lipid environment and determines the functional features necessary for activity. as a matter of fact, the compact hairpin structure drives favorable insertion, while its expanded structures promote subsequent membrane destabilization. [ ] canonical class ii and class iii fusion peptides correspond to loops which do not undergo conformational changes upon insertion into the target membrane; the interaction clearly involves few hydrophobic residues, and the insertion into the outer leaflet of the membrane is superficial and probably inadequate to destabilize membranes. thus, a cooperative effect attained upon fusion activation is the only explanation for activity. [ ] the idea of a single fusion peptide being exclusively responsible for the membrane perturbing activity has been overwhelmed by evidences supporting the concerted action of different membranotropic peptides, [ ] which together with the canonical fusion peptide are involved in the modification of membrane curvature. [ ] [ ] [ ] [ ] [ ] [ ] many viral fusion proteins present an additional hydrophobic membrane proximal region at the intersection between the ectodomain and the transmembrane anchor (tm), the so-called mper (or pre-tm); [ ] the unusual clustering of aromatic amino acid in these regions prompted the idea of their strong involvement in the fusion process. indeed, peptides corresponding to the pre-tm region partition into membrane interfaces and likely cooperate with fusion peptides and tm domains during apposition of membranes, enhancing the overall hydrophobicity of the environment and contributing to the distortion of the lipid membranes required for fusion. [ ] the pre-tm of hsv- gh interacts strongly with membranes; [ ] the pre-tm of gp of foamy virus induces fusion of model membranes; [ ] the aromatic domain of the glycoprotein s of severe acute respiratory syndrome virus (sars) partitions into lipid membranes and perturbs their integrity; [ ] the pre-tm region of the gp protein from ebola promotes perturbations of membranes when in a helical structure. [ ] hsv fusion involves both gb and gh and several membranotropic sequences are present in both glycoproteins, [ , ] although the precise role played by each of these regions remains to be elucidated. the two fusion loops at the tip of the domain ii of gb constitute a structural subdomain with the hydrophobic amino acids forming a crest lined on both sides by charged residues; [ ] the concerted use of the two peptides produced a significant distortion of the target membrane bilayer, while when they were used separately they presented a lower membrane penetration. [ ] the two charged residues represent a novel feature of fusion peptides with the presence of hydrophilic residues on either side favoring insertion. [ ] fusion of inner and outer monolayers is clearly involved but not formation of pores, indicating that bilayer perturbation not complemented by leakage is a typical feature of viral fusion peptides which violate the host membrane without compromising its integrity. [ ] several gh peptides are able to interact with membranes and play a role in the process. among these, gh , represents a key achievement in grasping the role of hydrophobic viral peptides. [ ] [ ] [ ] [ ] the peptide contains residues critical for interaction such as aromatic residues (tryptophan and tyrosines) which are known for their preferred location at the membrane interface and for their ability to facilitate oligomerization [ ] together with numerous hydrophobic residues (glycines, leucines, alanines) which are critical for membrane insertion; [ ] at the c-terminus there is an arginine residue which is key for establishing peptide-lipid interactions. gh penetrates into membranes from its n-terminal side, and assumes an amphipathic helical conformation. [ ] the n-terminal histidine acts as a switch for triggering viral fusion and strongly enhances the fusion activity; [ ] the role of the histidine has been reported also for paramyxoviruses [ ] and togaviruses. [ ] yao et al. [ ] in order to investigate how the fusion peptide (fp) and lipids also play a key role in this process, generating membrane curvature thanks to their physico-chemical properties. cholesterol is key as it selectively intercalates into the leaflet of the bilayer favoring its distortion without producing unfavorable hydrophobic/hydrophilic interactions. [ ] the secondary structure of the fusion peptide of hiv changes according to the cholesterol content in the membrane, being a-helical in the absence of cholesterol, but shifting to a b conformation with the increase of cholesterol. [ ] both the fully a-helical and the fully b-structured peptides are able to insert deeply inside the membrane, while the mixed secondary structures, present at intermediate cholesterol concentrations, are more superficially inserted into lipid bilayers and less effective in inducing membrane fusion. [ ] it is likely that different secondary structures and domains with different content of cholesterol might be involved in different stages of fusion. [ ] probably, lipid phase discontinuities between liquid ordered and disordered domains containing cholesterol produce membrane defects with exposed hydrophobic surfaces favoring a deeper insertion of fusion peptides, and promoting membrane fusion. [ ] in conclusion, the clear view is that membrane fusion is a very complex process involving several domains of the fusion proteins which interact directly or indirectly with biological membranes, and contribute to the merging of the viral envelope and cell membrane. some peptides (as the lytic peptides of nodaviruses, picornaviruses, and reoviruses) can be generated by an autocatalytic cleavage step of a precursor, whereas others can be generated from the proteolytic activity of cellular enzymes. [ , ] essentially we can classify lytic peptides in amphipathic a-helices and myristoyl groups; or we can classify them according to their mechanism of membrane action in those causing transient modification of the cellular membrane, pore formation, and total disruption of the limiting membrane. [ ] although being clearly different among themselves and with fusion peptides of enveloped viruses, they present notable similarities. incubation of hela cells with various peptides corresponding to the c terminus of the l protein of papillomavirus, determines the entrance of propidium iodide into cells. [ ] the incubation of the adenovirus internal protein vi with liposomes loaded with a fluorophore, caused the release of the entrapped fluorophore. [ ] similarly, incubation of vp * of rotavirus with liposomes entrapping a fluorophore caused its release, suggesting that vp * is sufficient to perforate the lipid vesicles. [ , ] the membrane disrupting g peptide of fhv also triggers the release of the fluorophore. [ , ] as for enveloped virus fusion peptides, the amphipathic a-helix seems to be a key structural motif also for non-enveloped viruses. the n-terminal amino acids of the fhv g peptide form an amphipathic a-helix which is commonly referred to as g , [ ] while the g peptide comprises also a c-terminal region which is commonly considered to play a supporting role for the correct positioning of g . the g peptide assumes a random coil conformation in solution, while it adopts a kinked helical conformation in model membranes; similarly to other membranotropic peptides also viral lytic factors seem to be able to adopt a membrane-active conformation when interacting with the lipid bilayer. [ , ] the g peptide is able to spontaneously partition into lipid bilayers and increases the membrane permeability of liposomes; [ , [ ] [ ] [ ] ] in particular, it mediates liposome lysis through the insertion only into the outer leaflet of the lipid bilayer, and locating parallel to the membrane surface, with the hydrophobic face of the helix packed against the membrane surface. [ , ] a concentration dependent membrane leakage process [ ] similar to other non-enveloped viruses such as poliovirus vp [ ] and reovirus l n [ ] is observed. similarly to the influenza [ ] and hiv gp [ ] fusion peptides from enveloped viruses, the amphipathic region of g peptide presents a kinked helical structure in solution. the rigid, boomerang like structure assumed by the influenza fusion peptide in lipid environment is required to promote membrane fusion; [ ] in fact, abolishing the kink in the structure or making it flexible eliminates membrane fusion [ ] probably for the failure of the fusion peptide to insert deep into the lipid bilayer and pack against the hydrocarbon moieties. at neutral ph the g peptide is able to cause membrane disruption; while at low ph it is only able to alter its location relative to the capsid, but does not increase its membrane interacting ability. [ ] surprisingly, thanks to a kinked structure and a tight alignment of the hydrophobic residues on one side of the peptide at low ph similar to influenza virus, fhv g peptide shows localized perturbation of lipid arrangements with no proof of pore formation. [ , ] while membrane destabilization requires the simple insertion of the fusion peptide into the outer leaflet of the lipid bilayer, leakage needs both a deeper insertion and an interaction between peptides inside the membrane. the amphipathic region of g peptide oligomerizes in bilayers [ ] with a low content of cholesterol, which being more fluid would promote association between peptides. [ ] the presence of the g c-terminus is absolutely necessary for virus entry; [ ] as a matter of fact, truncations, or point mutations in the c-terminal region of g determine a disordering of the pentameric bundle formed by the n-terminal amphipathic helices of g in fhv particles and hamper in vitro and in vivo membrane lysis. the pentameric bundles constitute the viral "membrane attack module," and an essential function of the g c-terminal region is to maintain this module in its correct conformation [ ] (figure ). the g and g peptides cause a similar localized disorder of the target bilayer and the presence of the cterminal hydrophobic helical region in full-length g make the peptide effective at concentrations achievable in the context of viral infections. the kinked helical structure seems to be a common trait of enveloped and non-enveloped viruses and differences in length and angularity of the helices as well as differences in the amino acid content may cause variations in the mechanism of interaction with the bilayer; the presence of this motif in viral proteins may signal a membrane associated role for this component during a certain step of the viral life cycle which adds to eventual other roles. adenovirus requires acidic ph for exposure of the amphipathic helix contained in its protein vi and disrupts endosomal membranes to release its nucleocapsid. [ ] mutations in the amphipathic helix reduce infection and endosome escape, supporting the view that both the hydrophobic character and a-helical structure are key to allow maximal membrane disruption. the n-terminal amphipathic helix of protein vi, as fhv g peptides, lies parallel to model membranes with the hydrophilic face interacting with the phospholipid head groups, and probably causes disruption of the bilayer by introducing positive curvature in figure schematic representation of fhv capsid (a). an expanded view of the crystallographic structure (pdb: ftb) of one subunit (a protein) showing the location of the amphipathic region of g peptide in yellow (b). schematic representation of a protein, which undergoes auto cleavage during maturation producing b and g (c) with relative sequence of g peptide membranes. [ ] mutations in the amphipathic region of protein vi, which prevent insertion into the membranes, severely affects membrane penetration and cellular entry. [ ] proteins vp and vp play a fundamental role in membrane penetration by poliovirus [ ] with the exposure of amphipathic a-helical nterminal approximately amino acid region of vp being necessary for liposome binding; [ ] while the n-terminus of poliovirus vp contains a hydrophobic myristoyl (c acyl) group for insertion into membranes. [ ] following receptor binding, the amphipathic a-helix within the n-terminal portion of vp , is exposed and probably forms pores to transfer the genome to the cytoplasm. interestingly, vp also performs a role in membrane penetration and the hypothesis is that vp primarily function is to secure the particle to the limiting membrane, while vp participates directly in pore formation. mammalian orthoreovirus protein l contains a small hydrophobic peptide (l n) with a myristoyl group at its n-terminus. [ ] disruption of cellular membranes, requires the complete dissociation of l n peptides from the particle; auto-cleavage of l during reovirus entry generates l n peptides that are linked to an n-terminal myristoyl group. after lipid association, the l n peptide changes conformation from an extended to a b-strand rich secondary structure. [ ] l n is able to generate size-selective pores in erythrocyte or liposomal membranes. it is likely that the b-hairpins in l n associate with the membranes forming a b-barrel pore. [ ] probably, membrane disruption caused by l n is similar to that of b-barrel toxins. [ ] it is highly probable that a future more detailed knowledge of the mechanism used by lytic peptides of non-enveloped viruses will lead to the discovery of more common features between enveloped and nonenveloped membranotropic peptides. membranotropic peptides thanks to their adaptness to interact with the membrane, are opening the way for numerous applications. [ , ] their ability to bind lipid membranes is correlated to their simultaneous hydrophobic and amphipathic nature, while their insertion into the bilayer is due to their capability to change conformation according to the environment; moreover, they are able to penetrate deep into the hydrophobic core but do not span the bilayer in a pore-like manner; on the contrary, they tend to self-associate at the interface between the membrane and the aqueous compartments. below are reported main applications. one of the main applications of fusion peptides of enveloped viruses is as inhibitors of viral penetration. their ability to directly interact with the hydrophobic surfaces present on cell membranes and/or fusion proteins allows them to interfere with virus entry. [ , ] the inhibition mechanism is still unclear but it is likely that inhibition of infectivity is correlated to inactive aggregates formed between the fusogenic stretches present in the viral protein and in the peptides. in particular, the formation of aggregates is related to their ability to oligomerize or to mimic the mode of binding of their original domains in their partner protein; thus, stabilizing a pre-fusion intermediate and preventing merging of the bilayers. [ , [ ] [ ] [ ] self-oligomerization of fusion peptides has been proposed to be responsible of inhibition by several groups. [ , ] hiv fusion peptides form structurally defined oligomeric complexes which have been considered responsible of inhibition; [ , ] moreover, mutants of the native sequence with a lower helical content and tendency to self-associate into b-sheets are able to inhibit membrane fusion with different magnitude and at various stages. [ ] virip is a peptide designed to target gp fusion peptide and thus block hiv- infection; it has undergone clinical studies and was demonstrated to be as active as peptides targeting the coiled-coil. [ , ] its clinical evaluation represents the proof of concept that membranotropic sequences could inhibit viral replication in infected individuals and may have potential clinical effectiveness. moreover, the knowledge that several domains are implicated in the fusion mechanism and may interfere with the intramolecular interactions between the several domains, clearly demonstrates that they all represent potential targets for the design of entry inhibitors. [ ] membranotropic peptides are emerging as delivery vectors. [ ] [ ] [ ] until now, the most widely used delivery vectors are cationic cell penetrating peptides (cpps), which enter essentially by endocytosis causing the entrapment of the cargo into endosomes with only minor quantities of the cargo able to reach the target where to exert the biological function. on the contrary, membranotropic peptides are internalized by direct penetration of the membrane and thus determine immediate bioavailability of the delivered molecule. fusion membranotropic peptides are particularly noteworthy because they can physically interfere with the membrane hydrophobic interior forming bulges that protrude from the membrane and ease contacts between fusing bilayers; in particular, they are able to translocate molecules through the plasma membrane directly into the cell, promoting lipid-membrane reorganizing processes, and causing local and temporary membrane destabilization with subsequent reorganization, circumventing the endosomal entrapment by favoring the escape from the endosome. [ , ] the internalization mechanism is also related to the toxicity of the internalized drug and the development of resistance. the gh is able to directly translocate across the membrane bilayer and to transport several cargos such as quantum dots, [ ] liposomes, [ ] dendrimers, [ , ] nanoparticles; [ ] it is also able to cross the bbb in vivo. [ , ] mpg is an amphipathic peptide composed of the fusion peptide of hiv- associated to an hydrophilic domain with positively charged residues derived from the nuclear localization sequence (nls) of simian virus (sv ) large t antigen (pkkkrkv), through a spacer (wsq). [ , ] in vitro mpg is able to deliver both sirna and dna after just h. [ ] the principal internalization mechanism was shown to be independent of the endosomal pathway and to involve the study of the internalization of g peptide derived from fhv [ ] revealed that this is mediated by relatively high cell surface adsorption leading to enhanced macropinocytic uptake and cytosolic distribution and also revealed a higher efficiency of internalization compared with tat. [ ] influenza virus fusion peptide has been used for increasing transfection efficiency, its ph-dependent fusogenic and endosomolytic activities are able to enhance lysosomal degradation before the contents of the endosomes are delivered to lysosomes. [ ] the development of industrial applications in drug delivery is probably one of the most exciting and fastest growing fields, with the possibility of these peptides to pass through the bbb and become an important player in the fight against all pathologies correlated to neurosciences. scientists envisage also a possible use of viral membranotropic peptides as an alternative to classical antibiotics in order to combat the antibiotic resistance problem. [ ] antimicrobial peptides (amps) are widely exploited and represent attractive candidates for the development of anti-infective agents; [ ] [ ] [ ] recently, attention has been devoted also to the exploitation of membranotropic peptides derived from viral fusion proteins as antibacterial drugs. in fact, some amps with helical structure seem to share high sequence (preference for alanines and glycines) and structure (amphipathic a-helix) similarity with fusion peptides and suggest a convergent evolution correlated to their ability to disturb lipid bilayers. [ ] the fusion domain of influenza virus was evaluated for its antibacterial activity; analysis showed that the amidation of the c-terminus is a key factor to render the fusion peptide an antibacterial peptide and optimization of the amphiphilic balance can improve efficacy. [ , ] the antibacterial activity of viral membranotropic peptides is not yet widely evaluated and much work is still open in this field; in particular, their mechanism of perturbation of membrane bilayers may allow the design of novel sequences with the ability to denature the membrane bilayer of bacteria which will add to their many roles. development of effective vaccines against viruses is another worldwide concern. a potent vaccine needs to be able to induce both [ ] in vivo prime-boost immunization enhanced humoral and cellular immune responses, suggesting the promising application of membranotropic peptides as vaccine candidates in future (table ) . the membrane entry of enveloped and non-enveloped viruses employs fundamentally different mechanisms, although common themes have emerged in the entry process. this similarity is essentially represented by the presence/exposure of small membranotropic peptides which cause membrane disruption and/or promote membrane fusion. entry involves membrane fusion versus perforation, but cellular triggering factors and structural intermediates appear to share some similarities. interestingly there is also some similarity with the mechanism used by bacterial toxins to cross biological membranes in order to reach the cytosol; in fact, many toxins, undergo conformational changes which allow them to initiate the translocation process. [ , ] how exactly both enveloped and non-enveloped viruses overcome host cell membrane barriers to deliver their genomes remains an intriguing problem. comprehensive structural and biochemical studies on enveloped viruses have brought to the conclusion that a unifying mechanism for host cell entry exists; where a membranotropic fusion loop, peptide, or patches catalyze fusion of the two membranes. in contrast, interaction of non-enveloped viruses with host cells during entry is less defined; while membrane active peptides have been discovered as necessary elements for entry in several well-studied non-enveloped virus capsids. in conclusion, it is now evident that the success of membranotropic peptides further stimulates challenging research on the unraveling of the many roles and applications that could be developed for both enveloped virus fusion peptides and small lytic peptides in nonenveloped viruses; membranotropic peptides are attracting increasing attention from the scientific community and their future will be dictated by the progresses in their industrial applications. http://orcid.org/ - - - entry of enveloped viruses into host cells: membrane fusion biophysical modulation of peptide-membrane interactions author biographies annarita falanga received the degree in food science and technology at the university of naples he earned his phd in virology from the university of cambridge (uk) in . he was appointed lecturer in microbiology in at the department of animal health of the faculty of veterinary medicine of the university of naples 'federico ii'. he moved to the faculty of medicine of the second university of naples where he was appointed associate professor and then full professor of microbiology giancarlo morelli is a full professor of chemistry at the university of naples 'federico ii she carried out research activities at the columbia university of new york in - . in she earned the position of assistant professor of inorganic chemistry at the university of naples and since she is associate professor. since , she is adjunct professor at loyola university chicago. research activities focus on antimicrobial peptides and drug delivery mechanisms. how to cite this article membranotropic peptides mediating viral entry key: cord- -df luh v authors: dos santos-silva, carlos andré; zupin, luisa; oliveira-lima, marx; vilela, lívia maria batista; bezerra-neto, joão pacifico; ferreira-neto, josé ribamar; ferreira, josé diogo cavalcanti; de oliveira-silva, roberta lane; pires, carolline de jesús; aburjaile, flavia figueira; de oliveira, marianne firmino; kido, ederson akio; crovella, sergio; benko-iseppon, ana maria title: plant antimicrobial peptides: state of the art, in silico prediction and perspectives in the omics era date: - - journal: bioinform biol insights doi: . / sha: doc_id: cord_uid: df luh v even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. these guardians include small molecules as antimicrobial peptides (amps), generally cysteine-rich, functioning to prevent pathogen establishment. some of these amps are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). when compared with other organisms, plants tend to present a higher amount of amp isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. therefore, plants arise as a rich resource for new amps. as these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. the possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated. proteins and peptides play different roles depending on their amino acids (aa) constitution, which may vary from tens to thousands residues. peptides are conventionally understood as having less than aa. proteins, on the contrary, would be any molecule presenting higher amino acid content and bothproteins and peptides-present a plethora of variations in plants. despite that, plant proteomes have been much more studied than peptidomes. it is well-known that the biochemical machinery necessary for the synthesis and metabolism of peptides is present in every living organism. from the variations of this machinery, a wide structural and functional diversity of peptides was generated, justifying the growing interest in their study. in eukaryotes, peptides are prevalent in intercellular communication, performing as hormones, growth factors, and neuropeptides, but they are also present in the defense system. besides plants and animals, several pathogenic microorganisms, peptides can serve as classical virulence factors, which disrupt the epithelial barrier, damage cells, and activate or modulate host immune responses. an example of this performance is represented by candidalysin, a fungal cytolytic peptide toxin found in the pathogenic fungus candida albicans that damages epithelial membranes, triggers a response signaling pathway, and activates epithelial immunity. there are also reports of defense-related fungal peptides. for example, the copsin, a peptide-based fungal antibiotic recently identified in the fungus coprinopsis cinerea kills bacteria by inhibiting their cell wall synthesis. regarding bacterial peptides, certain species from the gastrointestinal microbial community can release low-molecular-weight peptides, able to trigger immune responses. there are additionally peptides that act like bacterial "hormones" that allow bacterial communities to organize multicellular behavior such as biofilm formation. some peptides are known for their medical importance, as defensins that bioinformatics and biology insights present antibacterial, antiviral, and antifungal activities. for example, human alpha-and beta-defensins present in the saliva may potentially impede virus replication, including sars-cov- , besides other roles as protection against intestinal inflammation (colitis). considering the roles of plant peptides, they can also be multifunctional, and have been classified into main categories (supplementary figure s ) : ( ) peptides with no bioactivity, primarily resulting from the degradation of proteins by proteolytic enzymes, aiming at their recycling, and ( ) bioactive peptides, which are encrypted in the structure of the parent proteins and are released mainly by enzymatic processes. the first group is innocuous regarding signaling, regulatory functions, and bioactivity. so far, it has been reported that some of them may play a significant role in nitrogen mobilization across cellular membranes. the second group of bioactive peptides has a substantial impact on the plant cell physiology. some peptides of this group can act in the plant growth regulation (through cell-to-cell signaling), endurance against pathogens and pests by acting as toxins or elicitors, or even detoxification of heavy metals by ion-sequestration. comprising bioactive peptides, additional subcategorization has been proposed regarding their function. tavormina et al figure s ) based on the type of precursor: • • derived from functional precursors: originated from a functional precursor protein; • • derived from nonfunctional precursors: originated from a longer precursor that has no known biological function (as a preprotein, proprotein, or preproprotein); • • not derived from a precursor protein: some sorfs (small open read frames; usually < codons) are considered to represent a potential new source of functional peptides (known as "short peptides encoded by sorfs"). a more intuitive classification of bioactive peptides was further proposed by farrokhi et al receptors in leaves. another example is the pls (polaris) peptide that acts during early embryogenesis but later activates auxin synthesis, also affecting cytokine synthesis and ethylene response. regarding the second group, it includes peptides with signaling roles in plant defense, comprising at least subgroups, including syst (systemin) (supplementary figure s ). the syst peptides were identified in solanaceae members, like tomato and potato (acting on the signaling response to herbivory). the syst leads to the production of a plant protease inhibitor that suppresses insect's proteases. stratmann suggested that in plants, systs act to stimulate the jasmonic acid signaling cascade within vascular tissues to induce a systemic wound response. • • defense peptides or antimicrobial peptides (amps): to be fitted into this class, a plant peptide must fulfill some specific biochemical and genetic prerequisites. regarding biochemical features, in vitro antimicrobial activity is required. concerning the genetic condition, the gene encoding the peptide should be inducted in the presence of infectious agents. in practice, this last requirement is not ever fulfilled as some amps are tissue-specific and are considered as part of the plant innate immunity, while other isoforms of the same class appear induced after pathogen inoculation. plant amps are the central focus of the present review, comprising information on their structural features (at genomic, gene, and protein levels), resources, and bioinformatic tools available, besides the proposition of an annotation routine. their biotechnological potential is also highlighted in the generation of both transgenic plants resistant to pathogens, and new drugs or bioactive compounds. antimicrobial peptides are ubiquitous host defense weapons against microbial pathogens. the overall plant amp characterization regards the following variables ( figure ): electrical charge, hydrophilicity, secondary and -dimensional ( d) structures, and the abundance or spatial pattern of cysteine residues. these features are primarily related to their defensive role(s) as membrane-active antifungal, antibacterial, or antiviral peptides. regarding the nucleotide sequence, plant amps are hypervariable and this genetic variability is considered crucial to provide diversity and the ability to recognize different targets. for their charges, amps can be classified as cationic or anionic ( figure ). most plant amps have positive charges, which is a fundamental feature for the interaction with the membrane lipids of pathogens. concerning hydrophilicity, amps are generally amphipathic, that is, they exhibit molecular conformation with both hydrophilic and hydrophobic domains. silva et al with respect to their d structure, amps can be either linear or cyclic ( figure ). some linear amps adopt an amphipathic α-helical conformation, whereas non-α-helical linear peptides generally show or predominant amino acids. in turn, cyclic amps, including cysteine-containing peptides, can be divided into subgroups based on the presence of a single or multiple disulfide bonds. a peculiar feature of these peptides is a cationic and amphipathic character, what improves their functioning as membrane-permeabilizing agents. considering the secondary structures, amps may exhibit α-helices, β-chains, β-pleated sheets, and loops ( figure ). wang classified plant amps into families (α, β, αβ, and non-αβ), based on the protein classification of murzin et al, with some modifications. antimicrobial peptides of the "α" family present α-helical structures, whereas amps from the "β" family contains β-sheet structures usually stabilized by disulfide bonds. , some plant amps show an α-hairpinin motif formed by antiparallel α-helices that are stabilized by disulfide bridges. such amps present a higher resistance to enzymatic, chemical, or thermal degradation. antimicrobial peptides from the "αβ" family having both "α" and "β" structures are also stabilized by disulfide bridges. an example of amp presenting "αβ" structures are defensins, usually with a cysteine-stabilized αβ motif (csαβ), an α-helix, and a triplestranded antiparallel β sheet stabilized mostly by disulfide bonds. finally, amps that do not belong to the "αβ" group exhibit no clearly defined "α" or "β" structures. plant amps are also classified into families considering protein sequence similarity, cysteine motifs, and distinctive patterns of disulfide bonds, which determine the folding of the tertiary structure. therefore, plant amps are commonly grouped as thionins, defensins, heveins, knottins (linear and cyclic), lipid transfer proteins (ltp), snakins, and cyclotides. , these amp categories will be detailed in the next sections, together with other groups here considered (impatienlike, macadamia [β-barrelins], puroindoline (pin), and thaumatin-like protein [tlp]) and the recently described αhairpinin amps. the description includes comments on their structure, pattern for regular expression (regex) analysis (when available), functions, tissue-specificity, and scientific data availability. thionins are composed by to amino acid residues with a molecular weight around kda, considering the mature peptide. they are synthesized with a signal peptide together with the mature thionin and the so-called acidic domain. to date, there is no experimental information available about possible functions of the acidic domain, even though it is clearly not dispensable as shown by the high conservation of the cysteine residues. the thionin superfamily comprises distinct groups of plant peptides α/β-thionins and γ-thionins with distinguished structural features. the α/β thionins have homologous amino acid sequences and similar structures. besides, they are rich in arginine, lysine, and cysteine. in turn, γ-thionins have a greater similarity with defensins, and some authors classify them within this group. however, compared with the defensins, they present a longer conserved amino acid sequence. regarding the cysteine motif, it can be divided into subgroups, one with residues connected by disulfide bonds called c and the other with residues connected by disulfide bonds called c. the general designation of thionins has been proposed as a family of homologous peptides that includes purothionins. the first plant thionin was isolated in from wheat flour and labeled as purothionin. since then, homologues from various taxa have been also identified, like bioinformatics and biology insights viscotoxins (viscum album) and crambins (crambe abyssinica). they have also been isolated from different plant tissues like seeds, leaves, and roots. , thionins have been tested for different elicitors: grampositive , or gram-negative bacteria, , yeast, , insect larvae, nematode, and inhibitory proteinase. thionins are hydrophobic in nature, interact with hydrophobic residues, and lyse bacteria cell membrane. their toxicity is due to an electrostatic interaction with the negatively charged membrane phospholipids, followed by either pore formation or a specific interaction with a membrane. it has been reported that they are able to inhibit other enzymes possibly through covalent attachment mediated by the formation of disulfide bonds, as previously observed for other thionin/enzyme combinations. thionin representatives with known d structures determined by x-ray crystallography are crambin (pdb id: crn), α and β-purothionins (pdb id: phn and bhp), β-hordothionin (pdb id: wuw), and viscotoxin-a (pdb id: okh). the first to be determined was the mixed form of crambin. , it showed a distinct capital Γ shape with the n terminus forming the first strand in a βsheet. the architecture of this sheet is additionally strengthened by disulfide bonds. after a short stretch of extended conformation, there is a helix-turn-helix motif. in crambin, there is a single disulfide involved in stabilizing the helix-tohelix contacts. at the center of this motif, there is a crucial arg that forms hydrogen bonds to tie together the first strand, the first helix, and the c terminus. the first plant defensins were isolated from wheat and barley grains, initially called γ-hordothionins. due to some similarities in cysteine content and molecular weight, they were classified as γ-thionins. later, the term "γ-thionin" was replaced by "defensin" based on the higher number of primary and tertiary structures of these proteins and also on their antifungal activities more related to insect and mammalian defensins than to plant thionins. plant defensins belong to a diverse protein superfamily called cis-defensin and exhibit cationic charge, consisting of to aa with to disulfide bonds. , plant defensins share similar tertiary structures and typically exhibit a triple-stranded antiparallel β sheet, enveloped by an α-helix and confined by intramolecular disulfide bonds (figure a ). this motif is called cysteine-stabilized αβ (csαβ). the csαβ defensins were classified into groups based on their sequence, structure, and functional similarity. defensins are known for their antimicrobial activity at low micromolar concentrations against gram-positive and gramnegative bacteria, fungi, viruses, and protozoa. in addition, they present protein inhibition, insecticidal, and antiproliferative activity, acting as an ion-channel blocker, being also associated with the inhibition of pathogen protein synthesis. instead, plant defensins act in the regulation of signal transduction pathways and induce inflammatory processes, in addition to wound healing, proliferation control, and chemotaxis. in general, plant defensins do not present high toxicity to human cells, having in vivo efficacy records, with relevant therapeutic potential, and can be applied in treatments associated with traditional medicine. cools et al reported that a peptide derived from a plant defensin (hsafp ) acted synergistically with caspofungin (an antimycotic) (in vivo and in vitro) against the formation of candida albicans biofilm on polystyrene and catheter substrate, indicating that the hsafp variant presented a strong antifungal potential in the proposed treatment. other biotechnological applications of defensins are described, as in the case of ecgdf , which was isolated from a legume (erythrina crista-galli), heterologously expressed in escherichia coli and purified. ecgdf inhibited the growth of various plant and human pathogens (such as candida albicans and aspergillus niger and the plant pathogens clavibacter michiganensis ssp. michiganensis, penicillium expansum, botrytis cinerea and alternaria alternate). due to these features, ecgdf is a candidate for the development of antimicrobial products for both agriculture and medicine. non-specific lipid transfer proteins (ns-ltps) were first isolated from potato tubers and are actually identified in diverse terrestrial plant species. they comprise a large gene family, are abundantly expressed in most tissues, but absent in most basal plant groups as chlorophyte and charophyte green algae. they generally include an n-terminal signal peptide that directs the protein to the apoplastic space. some ltps have a c-terminal sequence that allows their post-translational modification with a glycosylphosphatidylinositol molecule, facilitating the integration of ltp on the extracellular side of the plasma membrane. the ns-ltps are small proteins which were thus named because of their function of transferring lipids between the different membranes carrying lipids (non-specifically, the list includes phospholipids, fatty acids, their acylcoas, or sterols). they consist of approximately aa and are relatively larger in size than other amps, such as defensins. depending on their sizes, ltps may be classified into subfamilies: ltp and ltp , with relative molecular weight of and kda, respectively. , the limited sequence conservation turned this classification inadequate. thus, a modified and expanded classification system was proposed, presenting main types (ltp , ltp , ltpc, ltpd, and ltpg) and additional types with a smaller number of members (ltpe, ltpf, ltph, ltpj, and ltpk). the new classification system is not based on molecular size but rather on ( ) the position of a conserved intron, ( ) the identity of the amino acid sequence, and figure s ) . although this latter classification system is the most recent, the conventional classification of ltp and ltp types has been maintained by most working groups. lipid transfer protein nomenclature has been confusing and without consistent guidelines or standards. there are several examples where specific ltps receive different names in different scientific articles. the lack of a robust terminology sometimes turns it quite difficult, extremely time-consuming, and frustrating to compare ltps with different roles/functions. therefore, an additional nomenclature was also proposed by salminen et al, naming ltps as follows: atltp . , osltp . , hvltpc , ppltpd , and taltpg , with the first letters indicating the plant species (eg, at = arabidopsis thaliana, pp = physcomitrella patens); ltp , ltp , and ltpc indicating the type; while the last digit (here - ) regards the specific number given to each gene or protein within a given ltp type. for the sake of clarity, the authors recommend the inclusion of a point between the type specification (ltp and ltp ) and the gene number. for ltpc, ltpd, ltpg, and other types of ltp defined with a letter, the punctuation mark was not recommended. this latter classification system is currently recommended as it comprises several features of ltps and is more robust than the previous classification systems. lipid transfer proteins are small cysteine-rich proteins, having to helices in their tertiary structure ( figure b ), which is stabilized by several hydrogen bonds. such a folding gives ltps a hydrophobic cavity to bind the lipids through hydrophobic interactions. this structure is stabilized by disulfide bridges formed by conserved cysteines, similar to defensins, although bound by cysteines in different positions. the disulfide bridges promote ltp folding into a very compact structure, which is extremely stable at different temperatures and denaturing agents. [ ] [ ] [ ] these foldings provide a different specificity of lipid binding at the ltp binding site, where the ltp structure is relatively more flexible and present a lower lipid specificity when compared with ltp . the first d structure of an ltp was established for taltp . based on d and d data of h-nmr, purified from wheat (triticum aestivum) seeds in aqueous solution. , currently, several d structures of ltps have been determined, either by nuclear magnetic resonance (nmr) or x-ray crystallography; in their free, unbound form or in a complex with ligands. the heveins were first identified in in the rubber-tree (hevea brasiliensis), but its sequence was determined later, whereas a similarity was detected to the chitin-binding domain of an agglutinin isolated from urtica dioica (l.) with cysteine residues forming a typical cys motif. the primary structure of the hevein consists of to aa, positively charged, with abundant glycine ( ) and cysteine ( - ) residues, and aromatic residues. , the chitin-binding domain is a determinant component in the identification of hevein-like peptides whose binding site is represented by the amino acid sequence sxfgy/sxygy, where x regards any amino acid. , most heveins have a coil-β -β -coil-β structure that occurs by variations with the secondary structural motif in the presence of turns in long coils in the β chain. antiparallel β chains form the central β sheet of the hevein motif with long coils stabilized by disulfide bonds ( figure c ). although the presence of chitin has not been identified in plants, there are chitin-like structures present in proteins that exhibit a strong affinity to this polysaccharide isolated from different plant sources. the presence of aromatic amino acids in the chitinbinding domain favors chitin binding by providing stability to the hydrophobic group c-h and the π electron system through van der waals forces, as well as the hydrogen bonds between serine and n-acetylglucosamine (glcnac) present in the chitin structure. , this domain is commonly found in chitinases of classes i to v, in addition to other plant antimicrobial proteins, such as lectins and pr- (pathogenesis-related protein ) members. , it may also occur in other proteins that bind to polysaccharide chitin, such as the antimicrobial proteins ac-amp and ac-amp of amaranthus caudatus (amaranthaceae) seeds which are homologous to hevein but lack the c-terminal glycosylated region. plant chitinases (class i) have the hevein-like domains, called hlds. due to the similar structural epitopes between chitinases and heveins, they are responsible for the cross reactive syndrome (latex-fruit syndrome). , among the several classes of proteins mentioned, the proteins with a high degree of similarity to hevein are chitinases i and iv. chitinases are known to play an essential role in plant defense against pathogens, also inhibiting in vitro fungal growth, especially when combined with β- , -glucanases. it also interferes with the growth of hyphae, resulting in abnormal ramification, delay, and swelling in their stretching. however, it has been shown that heveins have a higher inhibitory potential than chitinases and that their antifungal effect is not related only to the presence of chitinases ; pn-amp and pn-amp amps with hevein domains have potent antifungal activities against a broad spectrum of fungi, including those without chitin in their cell walls. , modes of action of chitinases usually include degradation and disruption of the fungal cell wall and plasma membrane due to its hydrolytic action, causing extravasation of plasma particles. , therefore, heveins have good antifungal activity, and only a few are active against bacteria, most of them with low activity. another role of hevein chitinases regards the antagonistic effect in triggering the aggregation of rubber particles in the latex extraction process in rubber trees. unlike heveins, other chitinases inhibit rubber particle aggregation. however, its action in conjunction with other proteins (β- , -glucanase) increases the effect of β- , -glucanase on rubber particle aggregation. a study by shi et al found that the interaction of the protein network related to the antipathogenic activity released by lutoids (lysosomal microvacuole in latex) is essential in closing laticiferous cells (cells that produce and store latex), not only providing a physical barrier, but a biochemical barrier used by laticiferous cells affected by pathogen invasion. knottins are part of the cysteine-rich peptides (crps) superfamily, sharing the cysteine-knot motif and therefore resembling other families as defensins, heveins, and cyclotides. their structure was initially identified by crystallography of carboxypeptides isolated from potato, showing the cysteine-knot motif with aa and cysteine residues. they are also called "cysteine-knot peptides," "inhibitor cysteine-knot peptides," or even "cysteine-knot miniproteins" because their mature peptide presents less than aa, forming interconnected disulfide bonds in the cysteine-knot motif, characterizing a particular scaffold. this conformation confers thermal stability at high temperatures. for example, the cysteine-stabilized β-sheet (csb) motif derived from knottins presents stability at approximately °c with only disulfide bonds. the knottins may have linear or cyclic conformation. however, both exhibit connectivity between the cysteines at positions - c, - c, and - c, forming a ring at the last bridge ( figure d ). knottins have different functions, such as signaling molecules, response against biotic and abiotic stresses, root growth, symbiotic interactions as well as antimicrobial activity against bacteria, fungi, virus, and insecticidal activity, among others. knottins antimicrobial activity has been attributed to the action of functional components of the plasma membrane, leading to alterations of lipids, ion flux, and exposed charge. the accumulation of peptides on the surface of the membrane results in the weakening of the pathogen membrane, resulting in transient and toroidal perforations. in the course of a large-scale survey to identify novel amps from australian plants, , an amp with no sequence homology was purified. its complementary dna (cdna) was cloned from macadamia integrifolia (proteaceae) seeds, containing the complete peptide coding region. the peptide was named miamp , being highly basic with an estimated isoelectric point (pi) of and a mass of kda. the miamp is aa long, including a aa signal peptide in the n-terminal region, bound to a aa mature region with cysteine residues. its d structure was determined using nmr spectroscopy, revealing a unique conformation among plant amps, with beta-strands arranged in greek key motifs, forming a greek key beta-barrel ( figure e ). due to its particularities, miamp was classified as a new structural family of plant amps, and the name β-barrelins was proposed for this class. this structural fold resembles a superfamily of proteins called γ-crystallin-like characterized by the precursors βγ-crystallin. this family includes amps from other organisms, for example, wmkt, a toxin produced by the wild yeast williopsis mraki. the miamp exhibited in vitro antimicrobial activity against various phytopathogenic fungi, oomycetes, and grampositive bacteria with a concentration range of . to μm generally required for a % growth inhibition (ic ). in addition, the transient expression of miamp in canola (brassica napus) provided resistance against blackleg disease caused by the fungus leptosphaeria maculans, turning miamp potentially useful for genetic engineering aiming at disease resistance in crop plants. there are few scientific publications with macadamia-like peptides, maybe because they prevail in primitive plant groups (eg, lycophytes, gymnosperms to early angiosperms as amborella and papaver), being apparently absent in derived angiosperms (eg, asteridae, including brassicaceae as arabidopsis thaliana). on the contrary, they have been identified in some monocots (as zantedeschia, zea, and sorghum). in fact, peptides similar to miamp appear to play a role in the defense against pathogens in gymnosperms, including species of economic importance (as pinus and picea) thus deserving attention for their biotechnological potential. four closely related amps (ib-amp , ib-amp , ib-amp , and ib-amp ) were isolated from seeds of impatiens balsamina (balsaminaceae) with antimicrobial activity against a variety of fungi and bacteria, and low toxicity to human cells in culture. these amps are the smallest isolated from plants to date, consisting of only aa in length. the ib-amps are highly basic and contain cysteine residues that form disulfide bonds. interestingly, they have no significant homology with other amps available in public databases. sequencing of cdnas isolated from i. balsamina revealed that all peptides are encoded within a single transcript. concerning the predicted precursor of ib-amp protein, it consists of a pre-peptide followed by mature peptide domains, each one of them flanked by propeptide domains ranging from to aa in length (supplementary figure s ) . this primary structure with repeated domains of alternating basic peptides and acid propeptide domains has, to date, not been reported in other plant species. patel et al conducted an experiment to purify ib-amp from seeds of impatiens balsamina. after purification, this peptide had its secondary structure tested by circular dichroism (cd). the results revealed a peptide that may include a β-turn but do not show evidences for either helical or β-sheet structure over a range of temperature and ph. structural information from d h-nmr was obtained in the form of proton-proton internuclear distances inferred from nuclear overhauser enhancements (noes) and dihedral angle restraints from spinspin coupling constants, which were used for distance geometry calculations. owing to the difficulty in obtaining the correct disulfide connectivity by chemical methods, the authors had built and performed separate calculations: ( ) a model with no disulfides; ( ) another with predicted disulfide bonds; and ( ) a model with alternative connectivity disulfide, as assigned from the nuclear overhauser effect spectroscopy (noesy) nmr spectra. as a result, hydrophilic patches were observed at opposite ends and opposite sides of the models, whereas in between them a large hydrophobic patch was identified. however, the study did not conclude which of the models would be the most likely representative of ib-amp , reporting only that cysteines are necessary for maintaining the structure. based on the experiment performed by patel et al, the present work built different models: model : without disulfide bonds, and the other models with different disulfide connections-model : nmr prediction by patel et al -cys; -cys and -cys; -cys, and model : disulfide bond partner prediction by dianna -cys; -cys and -cys; -cys. calculations have shown that although the peptide is small, the cysteines constrain part of it to adopt a well-defined main chain conformation. from residue to (except ), the main chain is well-defined, whereas residues to in the n-terminal region present few restrictions and appear to be more flexible (supplementary figure s ) . analyzing the rmsd (root mean square deviation), we observed that all the models lost the initial conformation and, among them, model was the most stable. models and showed a similar pattern (supplementary figure s ) , as in the models of patel et al, although model was the most flexible. little is known about impatiens-like amps mode of action. lee et al investigated the antifungal mechanism of ib-amp noting that when oxidized (bound by disulfide bridges), there occurs a -fold increase in antifungal activity against aspergillus flavus and candida albicans, as compared with reduced ib-amp (without disulfide bridges). confocal microscopy analyses have shown that ib-amp can either bind to the cell bioinformatics and biology insights surface or penetrate cell membranes, indicating an antifungal activity by inhibiting a distinct cellular process, rather than ion channel or membrane pore formation. fan et al reported the ib-amp antimicrobial activity dependent of β-sheet configuration to enable insertion into the lipid membrane, thus killing the bacteria through a non-lytic mechanism. current approaches aim to make changes in ib-amp to improve its antimicrobial activity. as an example, synthetic variants of ib-amp were fully active against yeasts and fungi, where the replacement of amino acid residues by arginine or tryptophan improved more than twice the antifungal activity. another study involving amp modification generated a synthetic peptide without the disulfide bridges (ie, a linear analog of ib-amp ), which showed an antimicrobial specificity . to . times higher than the wild-type ib-amp . puroindoline puroindolines are small basic proteins that contain a single domain rich in tryptophan. these proteins were isolated from wheat endosperm, have a molecular mass around kda, and a calculated isoelectric point higher than . at least main isoforms (called pin-a and pin-b) are known, which are encoded by pina-d and pinb-d genes, respectively. these genes share . % identical coding regions but exhibit only % identity in the ′ untranslated region. both pin-a and pin-b contain a structure with conserved cysteine residues and a tertiary structure similar to ltps, consisting of α-helices separated by loops of varying lengths, with the tertiary structure joined by disulfide bonds, of which identical to ns-ltps. the conformation of the pin isoforms was studied by infrared and raman spectroscopy. both pin-a and pin-b have similar secondary structures comprising approximately % helices, % β-sheets, and % non-ordered structures at ph . it has been proposed that the folding of both pins is highly dependent on the ph of the medium. the reduction of the disulfide bridges results in a decrease of pins solubility in water and to an increment of the β-sheet content by about % at the expense of the α-helix content. no high-resolution structure for any of the pin isoforms is available, bringing challenges to understanding the function of their hydrophobic regions, with some evidence coming only from partially homolog peptides. however, wilkinson et al proposed a theoretical model for several sequences of this amp. puroindolines are proposed to be functional components of wheat grain hardness loci, control core texture, besides antifungal activity. [ ] [ ] [ ] [ ] although the biological function of pins is unknown, their involvement in lipid binding has been proposed. while ltps bind to hydrophobic molecules in a large cavity, pins interact only with lipid aggregates, that is, micelles or liposomes, through a single stretch of tryptophan residues. this stretch of tryptophan residues is especially significant in the main form, pin-a (wrwwkwwk), while it is truncated in the smaller form, pin-b (wptwwk). [ ] [ ] [ ] puroindolines form protein aggregates in the presence of membrane lipids, and the organization of such aggregates is controlled by the lipid structure. in the absence of lipids, these proteins may aggregate, but there is no accurate information on the relationship between aggregation and interaction with lipids. the antimicrobial activity of pins is targeted to cell membranes. charnet et al indicated that pin is capable of forming ion channels in artificial and biological membranes that exhibit some selectivity over monovalent cations. the stress and ca + ions modulate the formation and/or opening of channels. puroindolines may also be membranotoxins, which may play a role in the plant defense mechanism against microbial pathogens. morris reported that the pin-a and pin-b act through similar but somewhat different modes, which may involve "membrane binding, membrane disruption and ion channel formation" or "intracellular nucleic acid binding and metabolic disruption." natural and synthetic mutants have allowed the identification of pins as key elements for antimicrobial activity. snakins are crps first identified in potato (solanum tuberosum). , due to their sequence similarity to gasa (gibberellic acid stimulated in arabidopsis) proteins, the snakins were classified as members of the snakin/gasa family. the genes that encode these peptides have ( ) a signal sequence of approximately aa, ( ) a variable region, and ( ) a mature peptide of approximately residues, with highly conserved cysteine residues. these cysteine residues maintain the d structure of the peptide through disulfide bonds, besides providing stability to the molecule when the plant is under stress , , , (figure f; supplementary figure s ). snakins may be expressed in different parts of the plant, like stem, leaves, flowers, seeds, and roots, - both constitutive or induced by biotic or abiotic stresses. in vitro activity was observed against a variety of fungi, bacteria, and nematodes, acting as a destabilizer of the plasma membrane. , , moreover, they were reported as essential agents in biological processes such as cell division, elongation, cell growth, flowering, embryogenesis, and signaling pathways. [ ] [ ] [ ] [ ] alpha-hairpinins as reported by nolde et al, alpha-hairpin emerged as a new amp with unusual motif configuration. these peptides prevail in plants and their structure was resolved based on nmr data obtained from the ecamp- peptide isolated from barnyard grass seeds (echinoa crus-galli). some α-hairpinins comprise trypsin inhibitors with helical hairpin structure and this group silva et al was recently proposed as a new plant amp family. similar to other amps, the amino acid sequences of α-hairpinins are variable. they share the conserved cysteine motif (cx cx - cx c) that form a helix-loop-helix fold and may have disulfide bridges c -c and c -c . its structural stability is maintained by forming hydrogen bonds, so that the side chains have a relatively stable spatial orientation. as reviewed by slavokhotova et al, members of alphahairpin family have been described in both mono and dicot groups, including species as echinochloa crus-galli and zea mays (both poaceae, monocot), fagopyrum esculentum (polygonaceae, eudicot), and stellaria media (caryophyllaceae, eudicot). several transcripts with α-hairpinin motif exhibit similarities to snakin/gasa genes and are sometimes positioned within this family. although the α-hairpinins structure has been published, its mechanism of action is still not resolved ( figure j , pdb id: l r). however, studies indicate they present a potential dna binding capacity. the term cyclotide was created at the end of the past century to designate a family of plant peptides with approximately aa in size and a structural motif called cyclic cysteine knot (cck). this motif is composed by a head-to-tail cyclization that is stabilized by a knotted arrangement of disulfide bridges, with conserved cysteines, connected as follows: c - , c - , c - . cyclotides are generally divided into subfamilies, mӧbius and bracelets, based on structural aspects. in addition to ccks, loops (between c - and c - ) have high similarity between both subfamilies, while the other loops (between c - and c - ) exhibit some conservation within the subfamilies , (supplementary figure s ) . to date, several cyclotides were identified in eudicot families such as rubiaceae, violaceae, fabaceae, and solanaceae, in addition to some monocots of poaceae family. in general, cyclotides may act in defense against a range of agents like insects, helminths, or mollusks. in addition, they can also act as ecbolic (inducer of uterus contractions), antibacterial, anti-hiv, and anticancer factors. all these characteristics added to the stability conferred by the cck motif turn these peptides into excellent candidates for drug development. , thaumatin-like protein thaumatins or tlps belong to the pr- (pathogen-related protein) family and received this name due to its first isolation from the fruit of thaumatococcus daniellii (maranthaceae) from west africa. thaumatin-like proteins are abundant in the plant kingdom, being found in angiosperms, gymnosperms, and bryophytes, being also identified in other organisms, including fungi, , insects, and nematodes. thaumatin-like proteins are known for their antifungal activity, either by permeating fungal membranes or by binding and hydrolyzing β- , -glucans. , in addition, they may act by inhibiting fungal enzymes, such as xylanases, α-amylases, or trypsin. besides, the expression of tlps is regulated in response to some stress factors, such as drought, injuries, freezing, and infection by fungi , viruses, and bacteria. as to the tlp structure, this protein presents characteristic thaumatin signature (ps ): , most of the tlps have molecular mass ranging from to kda, possessing conserved cysteine residues (supplementary figure s ) involved in the formation of disulfide bonds, which help in the stability of the molecule, allowing a correct folding even under extreme conditions of temperature and ph. thaumatin-like proteins also contain a signal peptide at the n-terminal, which is responsible for targeting the mature protein to a particular secretory pathway. the tertiary structure presents distinct domains, which are conserved and form the central cleft, responsible for the enzymatic activity of the protein, being located between domains i and ii. this central cleft may be of an acidic, neutral, or basic nature depending on the binding of the different linkers/receptors. all plant tlps with antifungal activity have an acidic cleft known as motif reddd due to highly conserved amino acid residues (arginine, glutamic acid, and aspartic acid; supplementary figure s ), being very relevant for specific receptor binding and antifungal activity. , , crystallized structures were determined for some plant tlps, such as thaumatin (figure g ), zeamatin ( figure h ), tobacco pr- d and osmotin, the cherry allergen pruav , and banana allergen ba-tlp, among other tlps. some tlps are known as small tlps (stlps) due to the deletion of peptides in one of their domains, culminating in the absence of the typical central cleft. these stlps exhibit only -conserved cysteine residues, forming disulfide bonds, resulting in a molecular weight of approximately to kda. they have been described in monocots, conifers, and fungi, so far. , , other tlps exhibit an extracellular tlp domain and an intracellular kinase domain, being known as pr k (pr -like receptor kinases) and are present in both monocots and dicots. for example, arabidopsis contains pr k genes, while rice has only . with the rapid growth in the number of available sequences, it is unfeasible to handle such amount of data manually. thus, amp sequences (as well as their biological information) have been deposited in large general databases, such as uniprot and trembl, which contain sequences of multiple origins. , in this sense, the construction of databases that deal specifically with amps was an important step to organize the data. during the past decade, several databases were built to support the deposition, consultation, and mining of amps. thus, these databases can be classified into groups: general and specific. the specific databases can be divided into subgroups: those containing only specific group (defensins or cyclotides) and those containing data from a supergroup of peptides (plant, animal, or cyclic peptides) (supplementary table ). in general, both types of databases share some characteristics such as the way that the data are available or the tools to analyze amps. the collection of antimicrobial peptides (campr ) is a database that comprises experimentally validated peptides, sequences experimentally deduced and still those with patent data, besides putative data based on similarity. [ ] [ ] [ ] the current version includes structures and signatures specific to families of prokaryotic and eukaryotic amps. the platform also includes some tools for amp prediction. the antimicrobial peptide database (apd) collects mature amps from natural sources, ranging from protozoa to bacteria, archaea, fungi, plants, and animals, including humans. amps encoded by genes that undergo post-translational modifications are also part of the scope, besides some peptides synthesized by multienzyme systems. the apd provides interactive interfaces for peptide research, prediction, and design, statistical data for a specific group, or for all peptides available in the database. the lamp (database linking antimicrobial peptides) comprises natural and synthetic amps, which can be separated into groups: experimentally validated, predicted, and patented. their data were primarily collected from the scientific literature, including uniprot and other amp-related databases. the database of antimicrobial activity and structure of peptides (dbaasp) contains information about amps from different origins (synthetic or non-synthetic) and complexity levels (monomers and dimers) that were retrieved from pubmed using the following keywords: antimicrobial, antibacterial, antifungal, antiviral, antitumor, anticancer, and antiparasitic peptides. this database is manually curated and provides information about peptides that have specific targets validated experimentally. it also includes information on chemical structure, post-translational modifications, modifications in the n/c terminal amino acids, antimicrobial activities, cell target and experimental conditions in which a given activity was observed, besides information about the hemolytic and cytotoxic activities of the peptides. due to the diversity of amps and the need to accommodate the most representative subclasses, several databases were established, focusing on specific types, sources, or features. there are several ways to classify amps, and they can range from biological sources such as bacterial amps (bacteriocins), plants, animals, and so on; biological activity: antibacterial, antiviral, antifungal, and insecticide; and based on molecular properties, pattern of covalent bonds, d structure and molecular targets. , the "defensins knowledgebase" is a database with manual curation and focused exclusively on defensins. this database contains information about sequence, structure, and activity, with a web-based interface providing access to information and enabling text-based search. in addition, the site presents information on patents, grants, laboratories, researchers, clinical studies, and commercial entities. , the cybase is a database dedicated to the study of sequences and d structures of cyclized proteins and their synthetic variants, including tools for the analysis of mass spectral fingerprints of cyclic peptides, also assisting in the discovery of new circular proteins. the phytamp is a database designed to be solely dedicated to plant amps based on information collected from the uniprot database and from the scientific literature through pubmed. plantpepdb is a database with manual curation of plantderived peptides, mostly experimentally validated at the protein level. it includes data on the physical-chemical properties and tertiary structure of amps, also useful to identify their therapeutic potential. different search options for simple and advanced compositing are provided for users to perform dynamic search and retrieve the desired data. overall, plantpepdb is the first database that comprises detailed analysis and comprehensive information on phyto-peptides from a wide functional range. biological data banks (dbs) are organized collections of data of diverse nature that can be retrieved using different inputs. the management of this information is done through various software and hardware resources, whose retrieval and organization can be performed in a quick and efficient way. considering biological data, information can be classified into ( ) primary (sequences), ( ) secondary (structure, expression, metabolic pathways, types of drugs, etc), and ( ) specialized, for example, containing information on a species or on a class of protein. within this third group, some references to amps can be mentioned, such as campr and apd that compile sequence data and structure retrieved from diverse sources, and also the defensin knowledgebase and the cybase which are dedicated to specific classes of peptides (defensins and cyclotides, respectively), in addition to phytamp, a specific database of plant amps (supplementary table ). the first step to infer the function of a given sequence (annotation) is to retrieve it in databases. for this purpose, approaches have been used mostly: ( ) local alignments, especially by using basic local alignment search tool (blast) and fasta ; by searching for specific patterns using ( ) regex or ( ) hidden markov model (hmm). the first approach has been widely used, since most of the information is available in databases as sequences, together with tools to align them, whereas the blast is the primary tool for doing so. this tool splits the sequence into small pieces (words), comparing it with the database. however, this approach has a limitation. small motifs may not be significantly aligned as they comprise small portions of the sequences that can be smaller than % of the total size. , due to the high variability of amps, only few highly conserved sequences can be identified using this type of inference. to reduce the effects of local alignment limitations, other strategies based on the search for specific patterns were introduced, such as regex (supplementary table ) and hmm. the regex is a precise way of describing a pattern in a string where each regex position must be set, although ambiguous characters (or wildcards) can also be used. for example, if we want to find a match for both amino acid sequences caiessk and waiesk, we can use the following expression: [cw]aies{ , }k, this expression would find a pattern starting with the letter "c" or "w," followed by an "a," an "i," and an "e," or "s," and ending with a "k." the hmms are well-known for their effectiveness in modeling the correlations between adjacent symbols, domains, or events, and they have been extensively used in various fields of biological analysis, including pairwise and multiple sequence alignment, base-calling, gene prediction, modeling dna sequencing errors, protein secondary structure prediction, noncoding rna (ncrna) identification, protein and rna structural alignments, acceleration of rna folding and alignment, fast noncoding rna annotation, and many others. using hmm, a statistic profile is included in the model, which is calculated from a sequence alignment, and a score is determined site-to-site, with conserved and variable positions defined a priori. , predicting antimicrobial activity the design of new amps led to the development of methods for the discovery of new peptides, thus allowing new experiments to be done by researchers. in this sense, the new challenge lies in the construction of new prediction models capable of discovering peptides with desired activities. the apd db has established a prediction interface based on some parameters defined by the entire set of peptides available in this database. these values are calculated from natural amps to consider features like length, net charge, hydrophobicity, amino acid composition, and so on. if we take as an example the net load, the amps deposited in the apd range from - to + . this is the first parameter incorporated into the prediction algorithm. however, most amps have a net load ranging from - to + , which then becomes the alternative prediction condition. therefore, the same method is applied to the remaining parameters. the prediction in apd is performed in main steps. first, the sequence parameters will be calculated and compared. if defined as an amp, the peptide can then be classified into groups: ( ) rich in given amino acids, ( ) stabilized by disulfide, and ( ) linear bridges. finally, sequence alignments will be conducted to find peptides of higher similarity. , , the advent of machine learning (ml) methods has promoted new possibilities for drug discovery. in ml inferences, both a positive and a negative dataset are usually required to train the predictive models. the positive data, in this case, regard preferably experimentally validated amps that can be collected in databases, whereas negative data are randomly selected protein sequences that do not have amp characteristics. , machine learning methods based on support vector machine (svm), random forest (rf), and neural networks (nn) have been the most widely used. svm is a specific type of supervised method of ml, aiming to classify data points by maximizing the margin between classes in a high-dimensional space. , random forest is a non-parametric tree-based approach that combines the ideas of adaptive neighbors with bagging for efficient adaptive data inference. neural networks is an information processing paradigm inspired by how a biological nerve system process information. it is composed of highly interconnected processing elements (neurons or nodes) working together to solve specific problems. [ ] [ ] [ ] evaluating proteomic data regarding the use of amps in peptide therapeutics, as an alternative to antimicrobial treatment, new efficient and specific antimicrobials are demanded. as aforementioned, amps are naturally occurring across all classes of life, presenting high active potential as therapeutic agents against various kinds of bacteria. the identification of novel amps in databases is primarily dependent on knowing about specific amps together with a sufficient sequence similarity. however, orthologs may be divergent in sequence, mainly because they are under strong positive selection for variation in many taxa, leading to remarkably lower similarity, even in closely related species. in this scenario, where alignment tools present limited use, strategy to identify amps is related to proteomic approaches. proteins and peptides are biomolecules responsible for various biochemical events in living organisms, from formation and composition to regulation and functioning. thus, understanding of the expression, function, and regulation of the proteins encoded by an organism is fundamental, leading to the so-called "proteomic era." the term "proteome" was first used by marc wilkins in and it represents the set of proteins encoded by the genome of a biological system (cell, tissue, organ, biological fluid, or organism) at a specific time under certain conditions. protein extraction, purification, and identification methods have significantly advanced our capacity to elucidate many biological questions using proteomic approaches. , due to the wide diversity of proteomic analysis, methods makes the choice of the correct approach dependent on the type of material and compounds to be analyzed. , two main tools are used to isolate proteins: ( ) the -dimensional electrophoresis ( -de) associated with mass spectrometry (ms) and ( ) liquid chromatography associated with ms, each one with its own limitations. [ ] [ ] [ ] obtaining native proteins is a challenge in proteomics or peptidomics, due to high protein complexity in samples, as the occurrence of post-translational modifications. alternative strategies applied to extraction, purification, biochemical, and functional analyses of these molecules have been proposed, favoring access to structural and functional information of hard-to-reach proteins and peptides. based on d gel, al akeel et al evaluated spots obtained from seeds of foeniculum vulgare (apiaceae) aiming at proteomic analyses and isolation of small peptides. extracted proteins were subjected to kda dialysis, and separation was carried out by deae-ion exchange chromatography while further proteins were identified by d gel electrophoresis. one of its spots showed high antibacterial activity against pseudomonas aeruginosa, pointing to promising antibacterial effects, but requiring further research to authenticate the role of the anticipated proteins. for amps, de is challenging due to the low concentration of the peptide molecules captured by this approach, their small sizes, and their ionic features (strongly cationic). in addition, the limited number of available specific databases and high variability turn their identification through proteolysis techniques and mass spectrometry, matrix-assisted laser desorption/ionization (maldi-ms) difficult. in addition, the partial hydrophobicity characteristics and surface charges facilitate peptide molecular associations, making analysis difficult by any known proteomic approaches. in addition, peptides are most often cleaved from larger precursors by various releasing or processing enzymes. furthermore, profiles generated do not represent integral proteome, as de has limitations to detect proteins with low concentration, values of extreme molecular masses, pis, and hydrophobic proteins, including those of membranes. due to these limitations, multidimensional liquid chromatography-high-performance liquid chromatography (mdlc-hplc) has been successfully employed as an alternative to d gels. techniques and equipments for the newly developed separation and detection of proteins and peptides, such as nano-hplc and multidimensional hplc, have improved proteomics evaluation. molecular mass values obtained are used in computational searches in which they are compared with in silico digestion results of proteins in databases. in silico approaches, usually by the action of trypsin as a proteolytic agent, may generate a set of unique peptides whose masses are determined by ms. , these methodologies are widely adopted for large-scale identification of peptide from ms/ms spectra. theoretical spectra are generated using fragmentation patterns known for specific series of amino acids. the first widely used search engines in database searching were sequest and mascot (matrix science, boston, ma; www.matrixscience.com). they rank peptide matches based on a cross-correlation to match the hypothetical spectra to the experimental one. mascot is widely used for peptidomics and proteomics analysis, including amp identification in many organisms, or to evaluate the antibacterial efficacy of new amps. evaluating new amp against multidrug-resistant (mdr) salmonella enterica, tsai et al used d gel electrophoresis and liquid chromatography-electrospray ionization-quadrupole-time-offlight tandem ms to determine the protein profiles. the protein identification was performed using the mascot with trypsin as cutting enzyme, whereas ncbi nr protein was set as a reference database. the methodology used in this study indicated that the novel amp might serve as a potential candidate for drug development against mdr strains, confirming the usability of mascot. in a similar way, umadevi et al described the amp profile of black pepper (piper nigrum l.) and their expression on phytophthora infection using label-free quantitative proteomics strategy. for protein/peptide identification, ms/ms data were searched against the apd database using an in-house mascot server, established full tryptic peptides with a maximum of missed cleavage sites and carbamidomethyl on cysteine, besides an oxidized methionine included as variable modifications. the apd database was used for amp signature identification, together with phytamp and campr . to enrich the characterization parameters, isoelectric point, aliphatic index, and grand average of hydropathy were also used (gravy) (using protparam tool), besides the net charge from phytamp database. based on label-free proteomics strategy, they established for the first time the black pepper peptidomics associated with the innate immunity against phytophthora, evidencing the usability of proteomics/ peptidomics data for amp characterization in any taxa, including plant amps, aiming the exploitation of these peptides as next-generation molecules against pathogens. other tools use database searching algorithms, such as x!tandem, open mass spectrometry search algorithm (omssa), probid, radars, and so on. these search engines are based on database search but use different scoring schemes to determine the top hit for a peptide match. general information on database search engines, their algorithms, and scoring schemes were reviewed by nesvizhskii et al. despite its efficient ability to identify peptides, database searching presents several drawbacks, like false positive identifications due to overly noisy spectra and lower quality peptides score (related to the short size of peptides). so, the identification is strongly influenced by the amount of protein in the sample, the degree of post-translational modification, the quality of automatic searches, and the presence of the protein in the databases. , in this scenario, the knowledge about the genome from a specific organism is important to allow the identification of the exact pattern of a given peptide. if an organism has no sequenced genome, it is not searchable using these methods. , once the sequences are obtained, bioinformatic tools can be used to predict peptides structure and estimate bioactive peptides. more recently, an interactive and free web software platform, mixprotool, was developed, aiming to process multigroup proteomics data sets. this tool is compiled in r (www.r-project. org), providing integrated data analysis workflow for quality control assessment, statistics, gene ontology enrichment, and other facilities. the mixprotool is compatible with identification and quantification outputs from other programs, such as maxquant and mascot, where results may be visualized as vector graphs and tables for further analysis, in contrast to existing softwares, such as giapronto. according to the authors, the web tool can be conveniently operated, even by users without bioinformatics expertise, and it is beneficial for mining the most relevant features among different samples. the central tenet of structural biology is that structure determines function. for proteins, it is often said the "function follows form" and "form defines function." therefore, to understand protein function in detail at the molecular level, it is mandatory to know its tertiary structure. experimental techniques for determining structures, such as x-ray crystallography, nmr, electron paramagnetic resonance, and electron microscopy, require significant effort and investments. all methods mentioned have their own limitations, and the gap between the number of known proteins and the number of known structures is still substantial. thus, there is a need for computational framework methods to predict protein structures based on the knowledge of the sequence. in addition, in recent years, there has been impressive progress in the development of algorithms for protein folding that may aid in the prediction of protein structures from amino acid sequence information. historically, the prediction of a protein structure has been classified into categories: comparative modeling, threading, and ab initio. the first approaches construct protein models by aligning the query sequences with already solved model structures. if the models are absent in the protein data bank (pdb), the models must be constructed from scratch, that is, by ab initio modeling, considered the most challenging way to predict protein structures. in the case of comparative modeling methods, when inserting a target sequence, the programs identify evolutionarily related models of solved structures based on their sequence or profile comparison, thus constructing structure models supported by these previously resolved models. this approach comprises main steps: ( ) fold assignment, which identifies similarity between the target and the structure of the solved model; ( ) alignment of the target sequence to the model; ( ) generation of a model based on alignment with the chosen template; and ( ) analysis of errors considering the generated model. there are several servers and computer models that automate the comparative modeling process, with swiss-model and modeler figuring as the most used. , although automation makes comparative modeling accessible to experts and beginners, some adjustments are still needed in most cases to maximize model accuracy, especially in the case of more complex proteins. therefore, some caution must be taken regarding the generated models, considering the resolution and quality of the model used, as well as homology between the model and the protein of interest. threading modeling methods are based on the observation that known protein structures appear to comprise a limited set of stable folds, and those similarity elements are often found in evolutionarily distant or unrelated proteins. the most used servers based on this approach are muster, sparks-x, raptorx, prosa-web, and most notably the i-tasser. in some cases, the incorporation of structural information to combine the sequence used in the search with possible models allows the detection of similarity in the fold, even in the absence of an explicit evolutionary relation. the prediction of structures from known protein models is, at first sight, a more straightforward task than the prediction of protein structures from available sequences. therefore, when no solved model is available, another approach is recommended, namely, the ab initio modeling. this method is intended to predict the structure only from the sequence information, without any direct assistance from previously known structures. the ab initio modeling aims to predict the best model, based on the minimum energy for a potential energy function by sampling the potential energy surface using various searchable information. , such approaches turn it challenging to produce high-resolution modeling, essential for determining the native protein folding and its biochemical interpretation. on the contrary, later resolved structures and comparisons with previously predicted proteins point to a higher successful modeling generated by ab initio methods than those generated by pure energy minimization methods, classical or even pure methods. among the most used servers and programs for ab initio modeling, we highlight the rosetta, quark, and touchstone ii. the accuracy of the models calculated by many of these methods is evaluated by cameo (continuous automated model evaluation) and by casp (critical assessment of protein structure prediction). probably the first reasonably accurate ab initio model was built in casp . since then, sustained progress was achieved in ab initio prediction, but mainly for small proteins ( residues or less). in casp , for the first time, a novel -residue protein with a sequence identity with known structures lower than % was constructed with high precision for sequences of this size. in casp , a significant improvement was reported in areas: contact prediction, free modeling, template-based modeling, and estimating the accuracy of models. the authors report that this improvement is due to the accuracy of modeling and alignment methods, as well as increased data availability for both sequence and structure. due to the number of amps deposited in the pdb (to date approximately structures), comparative modeling is the most used. however, when it comes to de novo peptide design, the most recommended choice would be ab initio or a hybrid approach that uses more than modeling method. after the generation of a model, the amp stability should be evaluated using molecular dynamics (md). molecular dynamics comprises the application of computational simulations that predict the changes in the positions and velocities of the constituent atoms of a system under given time and condition. this calculation is done through a classical approximation of empirical parameters, called "force field." if, on one hand, this approximation makes the dynamics of a system containing thousands of atoms numerically accessible, it obviously limits the nature of the processes that can be observed during the simulations. no quantum effect is visualized in a md simulation; just as no chemical bond is broken, no interactions occur between orbitals, resonance, polarization, or charge transfer effects. however, the molecules go beyond a static system. thus, md is a computational technique that can be used for predicting or refining structures, dynamics of molecular complexes, drug development, and action of molecular biological systems. molecular dynamics simulation is widely used for protein research, aiming to extract information about the physical properties of individual proteins. the results of such simulations are then compared with experimental results. as these experiments are generally carried out in solvents, it is necessary to simulate molecular systems of protein in water. these simulations have a variety of applications, such as determining the folding of a structure to a native structure and analyzing the dynamic stability of this structure. the use of md to simulate protein folding processes is one of the most challenging applications and should be relatively long (in the order of microseconds to milliseconds) to allow observing a single fold event. in addition, the force field used must correctly describe the relative energies of a wide variety of shapes, including unfolding and poorly folded shapes that may occur during the simulation. the considerable application potential led to the implementation of md simulation in many software packages, including gromacs, - amber, namd, charmm, lammps, and desmond. in addition to the above mentioned, there are other simulation types available, such as the monte carlo method, stochastic dynamics, and brownian dynamics. in the last decades, md simulation has become a standard tool in theoretical studies of large biomolecular systems, including dna or proteins, in environments with near realistic solvents. indeed, simulations have proven valuable in deciphering functional mechanisms of proteins and other biomolecules, in uncovering the structural basis for disease, and in the design and optimization of small molecules, peptides, and proteins. historically, the computational complexity of this type of computation has been extremely high, and much research has focused on algorithms to achieve unique simulations that are as long or as large as possible. the interplay between a given pathogen (eg, virus, bacteria, fungus) must be studied through a holistic approach. hostpathogen relationships are very complex and occur at diverse conceivable levels, including the cellular/molecular level of both, pathogen and host, under given environmental conditions. a most approximate understanding of these interactions at every level is the ultimate goal of "systems biology" (sb). it comprises a holistic approach, integrating distinct disciplines, as biology, computer science, engineering, bioinformatics, physics, and others to predict how a given system behaves under given conditions and what is the role of its parts. systems biology stands out because it is capable of correlating omics data for the understanding of plant-pathogen interaction. the construction of a plant-pathogen interaction network includes the reconstruction of metabolic pathways of these organisms, identification of the degree of pathogenicity, besides the expression of genes and proteins from both plant and pathogen. the networks can be classified into types: ( ) regulatory; ( ) metabolic; ( ) protein-protein interaction; ( ) signaling and regulatory; and ( ) signaling, regulatory, and metabolic. each of these networks can be plotted according to computational approaches. also, further studies are required to contemplate the construction of evolutionary in silico models and the characterization of these molecular targets in vitro. , studies of protein-protein interactions to understand the regulatory process are essential and new computational methods are necessary for this purpose with more optimized algorithms, also to remove potential false positives. thus, in-depth studies on the orientation of molecules and their linkages to the formation of a stable complex are of great importance for understanding plant-pathogen studies and also to develop new drugs. the understanding of the regulatory principles by which protein receptors recognize, interact, and associate with molecular substrates or inhibitors is of paramount importance to generate new therapeutic strategies. in modern drug discovery, docking plays an important role in predicting the orientation of the binder when it is attached to a protein receptor or enzyme, using forms and electrostatic interactions, van der walls, coulombic, and hydrogen bond as parameters to quantify or predict a given interaction. , molecular docking aims at exploring the predominant mode(s) of binding of a molecule (protein or ligand) when it binds to a protein with a known d structure based on a scoring function that has main functions: the first is to determine the binding mode and the binding site of a protein, the second is to predict the absolute binding affinity between protein and ligand (or other protein) in lead optimization, and the third is virtual screening, which can identify potential drug leads for a given protein target by searching a large ligand or protein in database. protein-protein interactions are essential for cellular and immune function. in many cases, due to the absence of an experimentally determined structure of the complex, these interactions must be modeled to obtain an understanding about their structure and molecular basis. few studies on plant-pathogen interactions include docking approaches and most studies focus on drug development for medical purposes. drug research based on structure is a powerful technique for the rapid identification of small molecules against the d structure of available macromolecular targets, usually by x-ray crystallography, nmr structures, or homology models. due to abundant information on protein sequences and structures, the structural information on specific proteins and their interactions have become crucial for current pharmacological research. even in the absence of knowledge about the binding site and limited backbone movements, a variety of algorithms have been developed for docking over the past decades. although the zdock, the rdock, and the hex have provided results with high coupling precision, the complexes provided are not very useful for designing inhibitors for protein interfaces due to constraints on rigid body docking. in this context, more flexible approaches have been developed which generally examine very limited conformations compared with rigid body methods. these docking methods predict that binding is more likely to occur in broad surface regions and then defines the sites in complex structures of high affinity. the best example is the haddock software, which has been successful in solving a large number of precise models for protein-protein complexes. a good example of its use is the study of the complex formed between plectasin, a member of the innate immune system, and a precursor lipid of bacterial cell wall ii. the study identified the residues involved in the binding site between the proteins, providing valuable information for planning new antibiotics. however, the absolute energies associated with intermolecular interaction are not estimated with satisfactory accuracy by the current algorithms. some significant issues as solvent effects, entropic effects, and receptor flexibility still need to be addressed. however, some methods, such as moe-dock, gold, glide, flexx, and surflex which deal with lateral chain flexibility, have proven to be effective and adequate in most cases. realistic interactions between small molecules and receptors still depend on experimental wet-lab validation. , despite the current difficulties, there is a growing interest in the mechanisms and prediction of small molecules such as peptides, as they bind to proteins in a highly selective and conserved manner, being promising as new medicinal and biological agents. while both "small molecule docking methods" and "custom protocols" can be used, short peptides are challenging targets because of their high torsional flexibility. proteinpeptide docking is generally more challenging than those related to other small molecules, and a variety of methods have been applied so far. however, few of these approaches have been published in a way that can be reproduced with ease. [ ] [ ] [ ] although it is difficult to use peptide docking, a recent focus of basic and pharmacological research has used computational tools with modified peptides to predict the selective disruption of proteinprotein interactions. these studies are based on the involvement of some critical amino acid residues that contribute most to the binding affinity of a given interaction, also called hot-spots. , despite the number of docking programs, existing algorithms still demand improvements. however, approaches are being developed to improve all issues related to punctuation, protein flexibility, interaction with plain water, among other issues. in this context, the capri (critical assessment of predicted interactions) is a community that provides a quality assessment of different docking approaches. it started in and since then has aided the development and improvement of the methodologies applied for docking. an evaluation was carried out for capri in , resulting in an improvement in the integration of different modeling tools with docking procedures, as well as the use of more sophisticated evolutionary information to classify models. however, adequate modeling of conformational flexibility in interacting proteins remains an essential demand with a crucial need for improvement. different docking programs are currently available, and new alternatives continue to appear. some of these alternatives will disappear, just as others will become the top choices among field users. molecular docking technique is not often used for amps, due to its standard mechanism of action based on the classical association with the external membrane of the pathogen. despite that, some amps have the ability to bind other proteins and/or enzymes, a feature still scarcely studied. in such cases, molecular docking can be useful. an example of success is the study performed by melo et al, where they showed the specific binding of a trypsin to a cowpea (vigna unguiculata) thionine, revealing that this interaction occurs in a canonical manner with lys , located in an extended exposed loop. therefore, further application of docking may bring new evidences about the antimicrobial mechanisms revealing other molecular targets of interest. it is clear that the combination of data bank information with bioinformatic tools (especially those allowing the identification of patterns, rather than sequence order) is able to revolutionize the identification of amps and prediction of their activity. the data may come from genomic, transcriptomic, or proteomic databases, or a combination of different information sources (eg, genomic and transcriptomics, transcriptomics and proteomics). supplementary figure s brings a schematic flowchart describing the steps for mining, annotation, and structural/ functional analysis of amps, in addition to some wet-lab analyses that can be integrated to assess/confirm candidate amps. similar bioinformatic approaches have been actually used to identify potential peptide candidates with anti-sars-cov- activity, especially those potentially able to interact with the spike protein and proteases involved in viral penetration. , as emphasized, plant amps show greater diversity and abundance, when compared with other kingdoms. it can be speculated that plants shelter many yet undescribed amp classes, given their vast abundance and isoform diversity. the genomic and peptidic structure of amps can be variable, with few key residues conserved, which turns their identification, classification, and comparison challenging even in the omics age. nevertheless, advances in the generation of new bioinformatics tools and specialized databases have led to new and more efficient approaches for both the identification of primary sequences and molecular modeling, besides the analysis of the stability of the generated models. despite the large availability of omics data and bioinformatics tools, most new plant peptides have been discovered by wet-lab approaches regarding single candidates. high throughput in silico methods have the potential to transform this scenario, revealing many new candidates, including some new or "non-canonical" peptides. it may be also speculated that a myriad of new peptides may exist considering even smaller peptides, still less considered and more difficult to identify. finally, in silico approaches shall in future studies be mandatory to define the design of wet-lab studies, turning the identification more efficient and requiring reasonably less time to track, identify, and confirm new candidate amps. considering the actual pandemic scenario of covid- , plant amps may be regarded as an important source of antiviral drug candidates, especially considering that some amp categories present not only antiviral effects but also a wide spectrum antimicrobial activity, act as anti-inflammatory, and also induce the immune response. of higher education personnel, biocomputational program), cnpq (brazilian national council for scientific and technological development), and facepe (fundação de amparo à ciência e tecnologia de pernambuco) for fellowships. the project is supported by the interreg italia-slovenia, ise-emh / and rc / from irccs burlo garofolo/ italian ministry of health cass performed the literature review and whore the manuscript. lz, mol, lmbv, jpbn, jrfn, jdcf, rlos, cjp, ffa, and mfo wrote specific chapters, eak and sc critically revised the text and included relevant suggestions. ambi conceived the review, wrote the introduction and concluding remarks, besides critically revising the manuscript. all authors have read the manuscript and agree to its content. supplemental material for this article is available online. prediction of protein function from protein sequence and structure plant peptides in defense and signaling plant bioactive peptides: an expanding class of signaling molecules candidalysin is a fungal peptide toxin critical for mucosal infection copsin, a 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Å resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins crystal structure of osmotin, a plant antifungal protein crystal structure of a sweet tasting protein thaumatin i, at · Å resolution crystallization and preliminary structure determination of the plant identification of conidialenriched transcripts in aspergillus nidulans using suppression subtractive hybridization analysis of the aspergillus nidulans thaumatin-like ceta gene and evidence for transcriptional repression of pyr expression in the ceta-disrupted strain the pr k receptor protein kinase from arabidopsis thaliana is structurally related to a family of plant defense proteins uniprot: the universal protein knowledgebase discovering new in silico tools for antimicrobial peptide prediction computational tools for exploring sequence databases as a resource for antimicrobial peptides camp: a useful resource for research on antimicrobial peptides camp: collection of sequences and structures of antimicrobial peptides campr : a database on sequences, structures and signatures of antimicrobial peptides apd : the antimicrobial peptide database as a tool for research and education dbaasp: database of antimicrobial activity and structure of peptides new trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches a large-scale structural classification of antimicrobial peptides defensins knowledgebase: a manually curated database and information source focused on the defensins family of antimicrobial peptides computational resources and tools for antimicrobial peptides cybase: a database of cyclic protein sequence and structure phytamp: a database dedicated to antimicrobial plant peptides plantpepdb: a manually curated plant peptide database bdbms-a database management system for biological data bioinformatics: a way forward to explore "plant omics basic local alignment search tool rapid and sensitive sequence comparison with 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multimers the haddock . web server: user-friendly integrative modeling of biomolecular complexes rdock: a fast, versatile and open source program for docking ligands to proteins and nucleic acids protein docking using case-based reasoning principles of flexible protein-protein docking plectasin, a fungal defensin, targets the bacterial cell wall precursor lipid ii variability in docking success rates due to dataset preparation development and validation of a genetic algorithm for flexible docking extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein−ligand complexes protein-ligand docking: current status and future challenges surflex-dock: docking benchmarks and real-world application docking small peptides remains a great challenge: an assessment using autodock vina advances in the prediction of protein-peptide binding affinities: implications for peptide-based drug discovery: protein-peptide binding affinities recent work in the development and application of protein-peptide docking peptide docking and structurebased characterization of peptide binding: from knowledge to know-how protein-ligand docking in the new millennium-a retrospective of years in the field unal eb, gursoy a, erman b. vital: viterbi algorithm for de novo peptide design modeling protein-protein and proteinpeptide complexes: capri th edition docking, scoring, and affinity prediction in capri potential chimeric peptides to block the sars-cov- spike receptor-binding domain peptide-like and small-molecule inhibitors against covid- the authors are very grateful to capes (coordination for the improvement key: cord- -khdvj t authors: zhang, hong; pelech, steven; ruijtenbeek, rob; felgenhauer, thomas; bischoff, ralf; breitling, frank; stadler, volker title: peptide arrays date: - - journal: microarrays in diagnostics and biomarker development doi: . / - - - - _ sha: doc_id: cord_uid: khdvj t despite the concern over the potential loss of structural information as a result of the use of peptides as opposed to proteins as molecular probes, peptide arrays have been implemented in a broad range of applications including antibody screening and epitope mapping, characterization of molecular interactions, and enzymatic activity profiling, and they have become a valuable tool for proteomics research. in this chapter, we first (sect. . ) recapitulate the development of these arrays and highlight a couple of key improvements in the array production and the application in proteomics research. for clinical and biomarker development applications, it is important to measure entities that are directly related to physiological function (and dysfunction). in this respect, the assessment of enzymatic activities is obviously preferable to genotyping, expression profiling, or even measurement of protein amounts. in sect. . , an original technology based on peptides arrayed onto a porous support allows detailed profiling of kinase activities in a biological sample. the applications described range from kinase characterization to inhibition profiles, detection of off-target effects, and drug response prediction in a clinical setting, allowing rational choice of the drug to be used. such directly functional approaches will have an important role in the transition to more personalized medicine. finally, in sect. . , a recently developed method for “laser printing” of peptide arrays that will make these approaches much more practical is presented. has proved to be unparalleled in its power in profiling gene expression and identifying single nucleotide polymorphisms (snps) in high-throughput manner, there is a poor correlation between mrna and protein expression and activity. this arises from regulation of mrna translation, for example, with micrornas, and from the involvement of posttranscriptional and posttranslational modifications (ptms), protein-protein interactions, and differences in subcellular localizations. these confounding factors have driven the development and the use of array technologies to directly study proteomes and have set the stage for the arrival of microarray-based proteomics. similarly to dna/oligonucleotide microarrays, arrays for proteomics studies feature a wide range of molecules including recombinant proteins, complex protein samples, antibodies, peptides, or small molecules that are assembled in an addressable fashion on planar surfaces to allow parallel interrogations for activity and interactions associated with biomolecules at the protein level. among various proteomics array types, protein microarrays and antibody microarrays have attracted most of attention in the field in the past decade, as illustrated by the rapid technological advancements and broad applications in numerous basic research and clinical studies (reviewed in chap. ). even though synthetic peptides together with oligonucleotides were among the first to be explored as molecular probes in array format, the development and application of peptide arrays has been sluggish and has lagged far behind arrays of many other types (frank et al. ; geysen et al. ) . one of the concerns with the use of peptides as opposed to proteins as molecular probes is the potential loss of information as a result of the missing structural context (mahrenholz et al. ). there is a much higher degree of entropy in the structures of peptides than with macromolecules such as proteins which are much more constrained. arrays of proteins have the distinct advantage in being able to mimic their physiological counterparts through presenting full-length proteins that are likely in their native d conformations and with proper ptms and associated proteins, to facilitate investigation of protein-protein interactions and assay of physiological activities. however, the molecular cloning and expression of tens of thousands of proteincoding genes with optimal covalent modifications and in combination with activating subunits has been proven to be technologically challenging, which significantly hampers the development and application of protein microarrays. in contrast, the chemistry of solid-phase peptide synthesis (spps) was well defined about half a century ago (merrifield a, b) . further technological advancement in peptide synthesis over the years coupled with the recent influx of genome sequencing information upon the completion of a number of genome-wide sequencing projects has made designing and synthesizing peptides with defined sequences corresponding to the segments of a protein easier than ever. furthermore, given the fact that the biological activity of a protein is often carried out through the coordinated actions of individual protein domains, it is reasonable to expect that the various functions of the protein can be recapitulated through the study of its constituent peptides representing individual functional domain sequences. since its advent about two decades ago, the peptide array has been implemented in a broad range of applications including antibody screening and epitope mapping, characterization of molecular interactions, and enzymatic activity profiling and has since become an increasingly important and versatile tool for proteomics research. more recently, with the improvements in array production techniques, peptide microarrays with higher peptide density and diversity can be produced en masse using the peptides generated from the parallel synthesis approaches such as the spot technology. the utility of these peptide microarrays has been demonstrated in some large-scale systems biology studies on the dynamics of protein interactions in cell signaling networks as well as kineome (also known as kinome) activity profiling. however, the clinical application of peptide arrays is still at its infancy despite potential and promises shown in early exploratory work. this chapter summarizes the development of peptide array technology and highlights the progress made toward its applications in proteomics research in recent years. a perspective of its future implementation in clinical practice is also presented. despite the fact that the concept of the array-format peptide synthesis was introduced about three decades ago, it was not until the introduction of the spot synthesis of peptides in the early s that the peptide array finally took its shape (geysen et al. ; frank ) . currently, there are two main strategies (in addition to very new approaches, see sect. . ) being used to produce peptide arrays: in situ parallel on-chip synthesis or immobilization of presynthesized peptides on the array surface. each has its own advantages and shortcomings. hence, the choice of the approach for peptide array production is largely determined by the downstream applications of the resulting arrays. the in situ on-chip synthesis of peptides is affordable as a result of the requirement of small amounts of reagents and of the fact that no purification of individual peptides is required. however, by the same token, the resulting peptides from in situ synthesis may suffer from low purity due to the variability in coupling efficiency between amino acid residues, especially in the case of long peptides (> residues) or those with residues such as cys or met, or containing multiple hydrophobic residues or phosphorylated amino acid residues. the two most common techniques for in situ synthesis that have been described and routinely used are spot synthesis and photolithography. the spot synthesis was introduced by ronald frank back in and is essentially a stepwise synthesis of peptides through sequentially delivering small amounts of activated amino acids on functionalized cellulose or polypropylene membranes using standard fmoc-based peptide chemistry (frank ) . the resulting membrane-based arrays are usually at low to medium density and can be used directly for downstream assays such as antibody epitope mapping. recently, improvements have been introduced allowing peptides either to be cleaved from the membrane using strong bases or to be recovered from individual spots as soluble peptides. for this purpose, they are synthesized on acid-labile cellulose membranes (e.g., trifluoroacetic acid (tfa)-soluble), allowing postsynthesis printing of the recovered peptides on selected array surfaces at a higher density (zander et al. ; hilpert et al. ) . another technique used in preparing peptide arrays in situ is the photolithographic synthesis developed by fodor et al. ( a, b) on the basis of the addressable surface activation concept. compared to spot synthesis, photolithographic synthesis of peptides on array surface is more suitable for generating high-density arrays. however, it is both laborious and expensive. it requires the use of special photolabile protected amino acid derivatives as building blocks and of photomasks through which a laser is then used to activate specific areas on the array to cleave photolabile protecting groups. even though the technique was initially developed for peptide synthesis, it was more easily adopted for the production of oligonucleotide arrays, due to the complexity of making masks for each of the amino acids for every coupling cycle, as opposed to only four bases in oligonucleotide array production (pease et al. ; mcgall and fidanza ) . in recent years, a number of modifications have been introduced, including the uses of conventional amino acids and photogenerated reagents, resulting in the improvement of efficiency and the reduction in cost for array production (singh-gasson et al. ; gao et al. ; bhushan ; shin et al. ) . thanks to technological advances in microarray printing and array substrate production in the field of genomics, it has become a common practice to spot presynthesized peptides onto reactive planar array surfaces. the approach is particularly useful when multiple copies of the same array with high density are required. furthermore, peptides can be purified after synthesis and prior to array printing to avoid any complications that may stem from peptide impurity. various chemistries have been utilized for immobilizing peptides onto the array surfaces. one of the popular approaches is to attach n-terminal biotinylated peptides onto avidin/ streptavidin-coated microarray slides (lesaicherre et al. ) . covalently immobilizing peptides through a terminal cys onto a surface functionalized with maleimide groups or disulfide is also a method that is being used quite commonly (inamori et al. ) . the suitability of immobilization chemistry varies according to the nature of the peptide as well as the downstream applications of the array. in our hands, attaching peptides through the n-terminal free amino groups to a surface functionalized with epoxide groups worked very well for assaying protein kinase activity (see below). thus, it is very important and necessary to determine which strategy should be used for preparing peptide arrays already prior to peptide synthesis, according to the intended application of the resulting arrays. since the advent of the technology more than two decades ago, peptide arrays have been applied in a broad range of investigations including antibody epitope mapping, protein domain-mediated interaction screening, and enzymatic activity profiling. in recent years, the utility of peptide arrays has been further extended to system-wide proteomics studies, fuelled by the advances in genomics and proteomics. this can be exemplified by their roles in cell signaling studies. kineome activity profiling reversible phosphorylation and dephosphorylation of proteins mediated by protein kinases and protein phosphatases, respectively, are recognized as one of the most important and widespread molecular mechanisms in regulating cell signaling pathways involved in cell proliferation, division, differentiation, adherence, angiogenesis, and apoptosis (brognard and hunter ) . according to our most recent tallies, the total number of phosphorylation sites in the human proteome is estimated to exceed , , which encompass over , phosphosites that have been experimentally characterized and those predicted based on evolutionary conservation (http://www. phosphonet.ca). the biological importance and potential clinical significance of protein phosphorylation, exemplified by the implication of deregulation of both kinase and phosphatase activity in a wide range of human diseases including cancer, autoimmune diseases, neurodegenerative diseases, and diabetes, has driven the development of strategies for the identification of physiological substrates for each of over protein kinases within the human kineome, as well as for systematic profiling of kinase activity in biological samples. for the identification of physiological substrates of kinases, a protein microarray featuring all the proteins representing the entire proteome would seemingly be an ideal platform. however, it is technically challenging to create such a comprehensive array encompassing all of the proteins encoded by about , genes in the human genome with current cloning and expression technologies. the most comprehensive protein microarray currently available commercially, trademarked as protoarray® by life technologies (carlsbad, ca, http://www.lifetechnologies. com), consists of only , human proteins that have been expressed and purified from a baculovirus-based expression system. moreover, issues with protein conformation, autophosphorylation, and stability on the array surface, as well as complications in data interpretation as a result of the presence of multiple phosphorylation sites within a protein potentially targeted by different kinases, have limited the practicality of this approach. as a result, a number of peptide-based strategies have been devised including peptide libraries and peptide arrays, based on the notion that the substrate specificity of the kinase is largely defined by the flanking linear amino acid sequences around its target phosphorylation site(s) on the substrates. based on the consensus recognition sequences for protein kinases derived from the peptide-based studies, one can deduce potential physiological substrates for each of the kinases in a proteome, coupled with information about protein-protein interactions, subcellular colocalization, and correlations in expression or activation. back in , luo and colleagues originally used the peptide array approach to identify and optimize substrate sequences for protein kinase a (pka) and transforming growth factor (tgf) b receptors (luo et al. ) . since then, the substrate specificities for a number of protein kinases have been elucidated and refined sequentially using peptide arrays (schutkowski et al. ) . there are two main strategies to be deployed for determining consensus phosphorylation sequences for protein kinases. on the one hand, peptide macroarrays featuring combinatorial peptide libraries or random peptide libraries such as those on the spot cellulose membranes were indispensable tools for elucidating the recognition sequences targeted by the kinases for which little information on their physiological substrates is available. the availability of expanding collections of recombinant active protein kinases in the past several years has facilitated the effort in this front. on the other hand, incorporation of increasing numbers of physiological phosphorylation sites uncovered through recent large-scale mass spectrometry-based phosphoproteomics studies into peptide microarrays has also significantly improved the efficiency of the substrate peptide screening process. currently, many large-scale peptide microarrays comprising a large number of experimentally verified phosphosites as well as those identified and optimized using the peptide library approach are readily available through various commercial sources such as pepstar™ from jpt peptide technologies (jpt peptide technologies gmbh, berlin, germany, http://www.jpt.com), pamchip® from pamgene (pamgene international b.v., hertogenbosch, the netherlands, http:// www.pamgene.com), and pepchip™ from pepscan presto (pepscan presto, lelystad, the netherlands, http://www.pepscanpresto.com), either as products or services, for profiling kinase activities in biological samples. various experimental protocols based on the same approach have been developed (schutkowski et al. ; thiele et al. ) . the approach was also adapted to kineome activity profiling in bovine samples by utilizing information gathered through bioinformatics analysis of the phosphorylation sites conserved in evolution (jalal et al. ). however, inferring endogenous kinase activities based on the data from such peptide microarrays is less straightforward than initially thought. one of the main issues associated with the current approach is the overlapping specificity among protein kinases dictated by the promiscuity in substrate recognition, especially for the kinases from the same or related families. phosphorylation of a peptide on each spot may represent the sum of activity of all the kinases targeting this particular peptide. indeed, a specific phosphosite sequence may be optimized through evolution to be recognized by a panel of kinases and phosphatases and not be optimized for an individual kinase or phosphatase. thus, under most circumstances, it is impossible to directly correlate the level of peptide phosphorylation on the array with the activity of a specific kinase. it is even more challenging when the activity of kinases in crude cell or tissue lysates is to be assessed, where the cell compartmentalization has been destroyed and the proper subcellular localization of proteins cannot be maintained. in light of these challenges, we set out to identify the optimal peptide substrate sequences unique to each kinase by combining the high-throughput capability of peptide microarrays with the power of a proprietary kinase-substrate prediction algorithm developed at kinexus (fig. . ). the algorithm was built based on the information gathered through manual analysis of close to , confirmed kinase-substrate pairs for typical kinases. coupling with the alignment of the primary amino acid sequences of the catalytic domains of protein kinases, the specificity-determining residues (sdrs) were identified, and the position-specific scoring matrix (pssm) was generated for each of the kinases for predicting their respective recognition sequences around the phosphorylation site. the pssms were then used to derive the optimal substrate peptide sequences. in total, -mer peptides corresponding to the predicted sequences with a single phosphorylatable residue (ser, thr, or tyr) in the middle were synthesized and immobilized onto an epoxysilane-coated glass microarray surface. the resulting peptide microarray was made up of four identical subfields to allowing four kinase assays to be run in parallel. phosphorylation of the peptides on the array was carried out by applying active protein kinases individually into each field under their respective assay conditions, and the extent of peptide phosphorylation was then detected with pro-q diamond (life technologies), a fluorescent dye that had been validated to bind specifically to phosphorylated residues including ser, thr, and tyr, regardless the context of sequences they are in. so far, over protein kinases have been assayed. many highly reactive and selective peptides have been identified as substrates for the kinases tested. while most of the sequences conformed to those reported previously, some novel motifs were also uncovered. detailed analysis of the "hit" peptide sequences is expected to reveal the prototype optimal substrate peptides unique to each kinase, which will then be further optimized for their reactivity and selectivity using an oriented peptide library approach. it is expected in the near future a peptide microarray spotted with substrate peptides that are preoptimized to each of the kinases will become available from kinexus for kineome profiling in complex biological samples. compared to protein kinases, protein phosphatases have been less well characterized with respect to their regulation and physiological substrates. this can be attributed to the misconception that phosphatases are promiscuous in substrate recognition and a b c d fig. . a bioinformatics algorithm-guided identification of the optimal peptide substrates unique to each kinase on peptide microarrays. panel a. schematic description of the workflow from peptide substrate sequence prediction using the kinase predictor . algorithm developed by kinexus to select test peptides for phosphorylation by kinases on the peptide microarray and, finally, to the deduction of the optimal substrate sequences for individual kinases. panel b. scanned image of the full kinex™ kinase substrate peptide microarray phosphorylated with three different kinases. the second field was incubated with atp in the absence of added kinase as a control. each peptide featured a phosphorylatable residue (ser, thr, or tyr) in the middle. the strong spots common among all four fields are the orientation markers designed for easy peptide localization. panel c. close-up scanned image of one field of the kinex™ kinase substrate peptide microarray. panel d. alignment of the top phosphorylated peptides detected following incubation with amp-dependent protein kinase alpha . peptides were ranked according to their respective phosphorylation signal intensity, and an optimal substrate peptide sequence is shown in the bottom row regulated in less stringent fashion in vivo, which might have arisen from that observation that a relatively small number of protein-serine/threonine-(ser/thr-) specific phosphatases are able to catalyze a myriad of dephosphorylation events, and that most protein phosphatases have not been found to recognize well-defined linear sequences or consensus motifs within their substrates so far. despite prevailing evidence that short synthetic phosphopeptides are poor phosphatase substrates compared to their parent proteins (zhao and lee ) , as supported by the role of regulatory subunits in forming the substrate-binding sites required for substrate recognition according to crystallography studies (virshup and shenolikar ) , several phosphopeptide-based studies have been reported that aimed at the delineation of substrate preferences using either activity-or interaction-based approaches (sun et al. ; wang et al. ) . among the two main classes of protein phosphatases, protein-tyrosine (tyr-) phosphatases (ptps), not protein-ser/thr phosphatases, had been the focus of early studies on substrate specificities, due to the availability of better characterized phospho-tyr antibodies than phospho-ser/thr antibodies. in those studies, phosphatase substrate specificities were commonly delineated using individually synthesized phosphopeptides (cho et al. ; zhang et al. ) . in recent years, peptide arrays, peptide microarrays in particular, have been demonstrated for their utility in protein phosphatase specificity mapping and activity profiling. in , waldmann's and yao's groups independently used phosphopeptide microarrays for large-scale, high-throughput characterization of ptp and protein-ser/thr phosphatase substrate specificities, respectively (k€ ohn et al. ; sun, et al. ) , for the first time. while a fluorescently labeled phospho-tyr antibody was employed to monitor dephosphorylation of tyrosine in the peptides in waldman's study, yao and coworkers used pro-q diamond dye to detect dephosphorylation of ser/thr, circumventing the detection problem as a result of the lack of well-characterized generic antibodies for phospho-ser/thr. the dye has recently extended to the detection of dephosphorylation of tyrosine in place of anti-phospho-tyr antibodies on peptide microarray by the same group (gao et al. ) . a phosphopeptide microarray featuring the most evolutionarily conserved human phosphorylation sites is now being explored for its potential for the determination of phosphatase specificities and activity profiling in our laboratory. in addition to protein kinases and phosphatases, peptide microarrays have also been successfully used to characterize protease specificity, based on the notion that proteolytic cleavage can be monitored by the changes in fluorescent signals on fluorogenic peptides immobilized on the array upon the action of proteases. salisbury et al. ( ) used a fluorogenic peptide substrate array with spatially addressable peptides to decipher the specificity of thrombin. gosalia and colleagues employed a solution-phase fluorogenic peptide microarray, in which peptides were spotted as spatially separate nanodroplets, to reveal the evolutionary conservation of substrate specificity of thrombin from human, bovine, and salmon (gosalia and diamond ) . the approach was also applied to determine substrate preferences of serine and cysteine proteases (gosalia et al. ) . winssinger et al. ( ) generated a library of peptides tagged with peptide nucleic acid (pna) molecules and incubated it with protease in solution, followed by spatial deconvolution on a dna microarray to profile the substrate specificities of thrombin, plasmin, and caspase- . the peptide array-based protease specificity profiling approach has now become an essential part of protease characterization platform, complementary and synergistic to other proteomic approaches used to detect alterations of substrate abundance and to identify and quantitate proteolytically generated neo amino-or carboxy-termini (auf dem keller and schilling ). the application of peptide arrays for protein-protein interaction characterization has been well documented since the advent of spot peptide synthesis. it is applicable to characterizing protein-protein interactions where the interface between the two interacting proteins can be recapitulated by linear peptide sequences derived from the parent proteins. it is even more advantageous compared to other proteomics techniques such as protein arrays when the protein-protein interactions mediated by ptms such as phosphorylation are concerned, as amino acids carrying corresponding ptms can be readily incorporated into specific sites during peptide synthesis. not only can the peptide array-based approach be used to map the consensus sequences recognized by these domains, it can also provide dynamic information on signal-dependent change in molecular networks for proteins defined by the peptides on the array and the proteins for which the binding is monitored (sinzinger and brock ) . intracellular signal networks are organized through the interactions of proteins, which are often mediated by a group of diverse modular protein interaction domains (pids) with defined specificity. among them, the src homology (sh ) domains are the largest family recognizing tyrosine phosphorylated sequences, and thus play pivotal roles in relaying information flow emanating from receptor protein-tyr kinases (pawson ) . a phospho-tyr-oriented peptide library with only one amino acid introduced at the defined positions at a time, and a mixture of amino acids at the randomized position, was spotted on the array and was interrogated with bacterially expressed human sh domains, and the phosphotyrosinecontaining peptide sequence motifs for of them were defined (huang et al. ) . combining the power of phage-displayed libraries, spot technology, and bioinformatics, the peptide array-based approach was also successfully used to deduce the consensus sequences of yeast sh domains (tonikian et al. (tonikian et al. , . a peptide microarray featuring peptides with inverted configuration representing , c-terminal sequences of human proteins was probed with a pdz domain to screen for putative interaction partners (boisguerin et al. ). with the knowledge of consensus sequences for the pids, peptide microarrays that carry peptides corresponding to known sequences recognized by sh , sh , pdz, and other pids have been employed to profile the binding of proteins from complex biological samples to detect the differences in molecular interactions between different physiological states (stoevesandt et al. ; sinzinger and brock ) . a peptide microarray populated with peptides, in which kinase consensus sequences and caspase cleavage recognition motifs (identified through a search of the human proteome) are overlapping, was employed in a study to investigate the role of phosphorylation in the regulation of caspase signaling pathways. protein kinase ck emerged as the kinase with the most number of substrates that contained kinase consensus sequences that overlapped with caspase- cleavage motifs, indicating a role of phosphorylation in the inhibition of caspase-mediated apoptosis signaling pathways (duncan et al. ) . as a natural extension to their classical application in antigenic epitope mapping, peptide arrays displaying a collection of biologically active synthetic peptides have been demonstrated in recent years to be a very versatile tool for profiling the antibody repertoire in complex biological samples such as serum, urine, saliva, and other types of body fluids for the diagnosis of pathogen infections, allergy reactions, and autoimmunity, based on the notion that the immune response to pathogens, allergens, or autoantigens can be captured by the presence or absence of specific populations of antibodies. hence, serological mapping has become one of the most sought after applications of the peptide array technology, as it appears to have the greatest clinical potential. peptide libraries featuring fragments derived from autoantigens, allergens, or viral proteins presented on either the spot membrane-based peptide macroarrays or glass slide-based peptide microarrays have been used for antibody profiling in clinical samples. the clinical potential of such analyses have been shown by their use for antibody spectrum profiling in the sera from patients infected with hepatitis b and c, with a simian-human immunodeficiency virus (shiv), the severe acute respiratory syndrome (sars) corona virus, and with herpes virus. this provides crucial information not only for infection diagnostics but also for the development of vaccines (neuman de vegvar et al. ; duburcq et al. ; guo et al. ; andresen and gr€ otzinger ) . the use of peptide arrays in kineome profiling has also inspired the exploration of their application in the studies of human diseases. as increasing numbers of kinase substrate peptides have been identified in recent years, peptide microarrays with the capability of screening a broad range of protein kinases have been established and used to profile the aberrant kinase activity in clinical samples as well as for monitoring the response to kinase inhibitory compounds in a highthroughput manner. this underscores the potential of peptide arrays in disease diagnosis and drug discovery (piersma et al. ). among the handful of studies reported so far in this area, schrage et al. ( ) recently reported the activation of multiple pathways in relation to akt/gsk b, pdgfrb, and src protein kinases in chondrosarcoma cells on a kinase substrate peptide array containing , peptides. supplemented with the cell viability data in vitro, the study indicated that the src inhibitor dasatinib is a potential treatment option for patients who are inoperable (schrage et al. ). tuynman and colleagues investigated the molecular mechanism underlying anticarcinogenic activity of celecoxib (celebrex), a selective cyclooxygenase- (cox- ) inhibitor, against colorectal cancer (crc) using a kinase substrate peptide array with , different kinase substrate consensus sequences and found that celecoxib represses c-met-dependent signaling, which in turn led to downregulation of oncogenic wnt signaling in crc, supporting the potential of targeting c-met and wnt signaling in crc therapy (tuynman et al. ) . recently, a cellulose membrane-based peptide array of peptides derived from p peptide, a cancer cell targeting peptide identified by phage display, was employed to optimize the affinity of the peptides for human cancer cells using peptide-whole cell interaction assay (ahmed et al. ) . the binding of the three peptides with the highest affinity and selectivity for cancer cells was further confirmed using fluorescence imaging and flow cytometry. the study revealed the potential of the peptide array-based whole cell binding assay for screening and identifying cancer cell targeting peptides for cancer diagnosis and drug targeted delivery. peptide microarrays broaden a new field of research and applications often referred to as functional proteomics (thiele et al. ) . while dna and protein arrays mostly focus on determination of abundance of rna or protein molecules, peptide arrays allow the functional analysis of multiple proteins or protein families (fig. . ) . by functional analysis, we mean the detection of protein activity. clear examples are the detection of enzymatic activities, for example, of kinases, phosphatases, and proteases in lysates from cells or tissues. however, nonenzymatic functions, like the responses to hormone binding of nuclear receptors in terms of specific coregulator protein recruitment, are also currently being studied on peptide arrays (heneweer et al. ; koppen et al. ). peptide arrays enable the miniaturization and multiplexing of activity-based assays. in the context of pharmaceutical research, and in the field of translational medicine in particular, such array-based approaches are emerging. this makes sense since the majority of the drugs being developed target protein activity and function. these new and more targeted drugs act by effecting protein function rather than targeting dna or rna or interfering with the modulation of protein levels. because functional profiling of the interaction of drugs with cellular or tissue samples is of specific interest in pharmaceutical research, peptide microarrays are proving to be very useful with their ability to profile protein activity and its modulation by drugs. we focus here on the drug class of kinase inhibitors which have been reshaping the oncology field due to their high success rate. these molecules inhibit kinase function by reducing kinase activity, which can be monitored on a peptide array. kinases play a pivotal role in cellular biology by being the key regulators of signal transduction. signals being detected by a membrane-bound receptor are transduced to the inner parts of the cell to result in an appropriate response. this happens via highly complex cascades of events in which the signal is received and propagated using transphosphorylation reactions (fig. . ) . these reactions are catalyzed by protein kinases together with the crucial atp molecule. atp is important as not only does it provide the kinase's energy source, it also supplies the phosphate moiety, vital to the whole signal transduction cascade. a kinase becomes activated and places this phosphate group on a substrate protein; this being the subsequent link in the signal transduction pathway. often this substrate protein is a kinase as well. a signal transduction event can be compared to a relay in athletics, where each kinase gets activated by an upstream event and subsequently passes on the baton to the next member downstream in the pathway. most protein kinases have distinct preferences for the aromatic hydroxyl groups of tyrosine residues or for the aliphatic serines or threonines. it is this characteristic which divides this family of more than members into two kinase subfamilies: proteintyrosine kinases (ptks) and protein-serine/threonine kinases (stks). while in classic kinase assays the activity is detected by the phosphorylation of a single substrate, multiple substrates can now be immobilized and monitored on a microarray. instead of placing multiple protein substrates on a chip, only the phosphosites (the sites within the protein which become phosphorylated) are immobilized in a peptide microarray. thus, the peptides represent the protein substrates. as has been discussed in the previous chapters, this can be done in a variety of ways, but all are based on a solid support. in most cases, the sequences are derived from known phosphorylation sites in the human proteome. as the human proteome is estimated to comprise more than a million proteins, of which more than two-thirds can be phosphorylated, this indicates the huge amount of different phosphosites that can be investigated by peptide arrays. the principle of the assay is that the kinase activities in the sample of interest phosphorylate the peptides. the phosphorylation event is detected by either radiography or fluorescence imaging of the array. in radio assays, the peptide is phosphorylated using radioactive atp as the phosphate source. this approach is increasingly being replaced by the use of fluorescence assays. in the latter case, the phosphorylation of the peptide is detected by a fluorescently labeled molecule which is either a chelator (e.g., phosphotag) or an antibody. ideally, the antibody needs to detect the phosphoamino acid in all the available peptide sequences on the chip equally well and independently of the adjacent amino acids. antibodies like py work very well in detecting tyrosine phosphorylated peptides, but for serine/ threonine phosphorylated peptides, cocktails are needed for full coverage of detection. the first peptide microarray applications for kinase profiling used glass as a solid support and radioactivity for readout at a single time point. later, protocols were developed based on the fluorescent readout of labeled antibodies (or cocktails of antibodies) binding to phosphorylated peptides. a second generation of this technology was developed by researchers in the netherlands and is referred to as the pamchip® technology (lemeer et al. ; hilhorst et al. ; versele et al. ) (fig. . ) . with this technology, antibody-based fluorescence detection has been combined with a change of solid support from glass slides to a porous ceramic. in this format, the sample is pumped up and down through the porous aluminum oxide ceramic sheets, in which the peptides are immobilized at designated spots. each spot comprises thousands of separated pores with diameters of . mm in which the peptides are site-specifically immobilized. each time the sample is below the solid support, the degree of phosphorylation is monitored by imaging the fluorescence intensities caused by the antibody binding to the phosphorylated peptides alone. these time curves, or kinetic readouts, appear to be instrumental in the enzymatic studies; a kinase is after all an enzyme which catalyzes the rate (the kinetics) of a phosphorylation reaction. in addition, the kinetic and multi(time) step readout for each of the or peptides on each single array allows much more comprehensive statistical analysis of the signals than data from a single time point per peptide spot on a glass array (thilakarathne et al. ) . the application of peptide arrays in biological, pharmaceutical, and medical studies often requires the analysis of many samples under variable conditions. for example, lysates from cells should be analyzed using a range of time points, varying concentrations, and with multiple different drugs. for this reason, a system has been developed which has the capability of analyzing arrays at once. this latest technology for kinase activity profiling is based on a -well plate format, in which each well comprises a peptide microarray. bioinformatics for analysis of the vast datasets from such studies has been evolving in parallel. thilakarathne et al. ( ) developed a new method based on semiparametric mixed linear models to further enhance the amount of information that can be obtained from the multiparallel kinetic readouts from each microarray. a straightforward application is substrate identification using recombinant kinases. such studies have indicated that different kinases have their own preferences for the peptide sequences they phosphorylate. clear differentiation between the ptks and stks has been confirmed, although dual specificity kinases have also been found. in addition, it has become apparent that although each kinase has a preference for particular peptide sequences, they can also be promiscuous, resulting in multiple peptides being phosphorylated to different degrees in diverse peptide sets. in short, the degree of phosphorylation by purified kinases varies from peptide to peptide and can be profiled in hundreds per array, resulting in phosphorylation fingerprints. substrate profiling studies have revealed important biological information as described in the paper by schutkowski et al. where they showed that for optimal recognition by gsk b, a peptide substrate should be prephosphorylated or primed . due to this porosity, the sample can be pumped up and down through this solid support. every time when the sample is positioned below the microarray, an image is taken of the microarray by a ccd camera (middle panel). via this realtime imaging of the microarray, the signal, developing in the peptide spots due to binding of fluorescent antibody detecting peptides phosphorylated by the kinases, can be monitored (schutkowski et al. ). another interesting application was explored by poot et al. who identified an optimal substrate for pkc isozymes and coupled this peptide to an atp-binding site inhibitor to generate a bisubstrate (poot et al. ; van ameijde et al. ) . the peptide microarrays were subsequently used to evaluate the resulting inhibitors, which were potent and selective toward the theta isozyme. with the development of protocols for profiling cell lysates of tissue homogenates, the application area has been broadened to signal transduction and pathway studies [well reviewed by the group of schutkowski (thiele et al. )] . the effect of a stimulus or a kinase-inhibiting drug on cultured cells can now be investigated at the complexity level of a cell, where multiple kinases can be active in the context of the interacting networks that exist. at this point, peptide arrays provide a welcome extension to classical methods like (phospho) western blots and elisas, which monitor drug effects on a (single) kinase by detecting the variation in abundance of the downstream phosphorylated substrate. the peptide arrays monitor the enzyme activity of multiple kinases at once and not only the end result of this activity. an interesting feature of functional proteomics is found in the ability to study direct effects of the investigative drug. because activities of kinases can be monitored in cell lysates or tissues, drugs can be characterized in a complex, and probably more realistic, context than in classical single readout (singleplex) assays. this latter type of assay is limited as it can only investigate the activity of the isolated drug target. drug selectivity profiling is a clear example of an application which benefits from the combination of multiplexing and miniaturization (fig. . ) . another example of an application area is the unraveling of a drug's cellular mechanism of action. in this application, the peptides on the chip represent multiple different proteins involved in complex cellular pathways and signal transduction networks. in a small lysate sample, derived from just , to , cells or less than a tenth of a cubic millimeter of tissue, multiple diverse interactions can be studied at once. a recent development of peptide microarrays has been the application of new drugs in pharmaceutical research and clinical development. in the field of oncology in particular, fundamental progress has been made by so-called targeted medicine. previously, anticancer drugs were targeting cellular processes, like cell division, more globally. new insights into cell signaling and signal transduction cascades have changed the way novel oncological drugs are being developed, and it is the kinase enzyme class which is playing a crucial role in this progress. many of its members play a pivotal role in the mechanisms of tumor genesis, and some kinases are even the active protein products of oncogenes. examples of successful cancer drugs targeting protein kinases-or the signaling pathways they are involved with-are imatinib, erlotinib, gefitinib, and the previously mentioned sunitinib and sorafenib. these are all molecules that block the catalytic activity of protein kinases. a related class of therapeutics is antibodies, which intervene in a different way with cellular signaling: they act by blocking the initiation of receptor signaling. examples of the latter are trastuzumab and bevacizumab, which block egfr kinase and vegfr signaling, respectively. these drugs can be studied comprehensively with peptide arrays. in these studies, two formats are currently being used. using cell line models, the cells are either treated with the drug in culture or on the chip. in the first case, lysates are prepared from the cells before and after treatment and profiled for activities on the chip. in the second case, lysates can be treated directly by spiking the drug into the solution just before application onto the chip. in the latter instance, cell lines, tissue homogenates from animal models, or even clinical samples can all be used. the effects of the inhibitors on the kinase activities in these samples can all be directly assessed. although the highly important context of the cellular architecture is lost, which is surely a downside, the potential to profile all detectable, full-length kinases-with their relevant posttranslational modifications-in the same sample, opens up vast new fields of applications. in drug discovery, researchers screen for kinase-inhibiting compounds in chemical libraries. during such studies, they often use an abstracted model, the purified protein, but this protein is frequently truncated to its domain only. a major disadvantage of this approach is the absence of other domains, including those with a inhibition / activation sorafenib sunitinib fig. . selectivity profiling of kinase inhibitor drugs using peptide microarrays. here sorafenib inhibition profiles are compared with sunitinib in extracts from both normal and tumor tissue from a renal cell carcinoma patient regulatory function. in the recently developed protocols for peptide array analysis of kinases in cell and tissue lysates, the drug target can now be studied more naturally as a full-length protein, in the way it is actually expressed in cells or tissues. at first, this was shown in a model system, but interestingly, this approach appears to be translatable to patient-derived tissues. this means that the kinase drug targets can now be studied in the same form as they are expressed in a patient's tumor, thus full length, fully decorated with all relevant posttranslational modifications and in the presence of stabilizing or activating cofactors (e.g., heatshock proteins). in addition, they can be studied in the presence of all other kinases expressed in the cell or tissue being investigated. such analysis of patient-derived tumor samples can result in the identification of tumor-specific kinase activities. when linked to pathological, diagnostic, and/or clinical data, this can lead to the identification of diagnostic or prognostic biomarkers. while the on-target effects are being monitored, the researchers can also obtain insights into the drug's effect(s) on other kinase targets, which are either intended-in the case of multitarget inhibitors-or unintended and can putatively cause side effects. the latter opens up new opportunities for the toxicologist in investigating and understanding adverse drug reactions leading to toxicological biomarkers. a very typical feature of activity-based assays is the capability of drug testing. with peptide microarray-based kinase assays, not only can multiple kinases in a patient sample be studied at once, their response to their inhibiting drugs can also be studied. this possibility links the presence and activity of the kinase drug targets to their responsiveness to the drug. drug response is a leading parameter in the clinical development of a drug. in the development of kinase inhibitors in cancer, the response rates are often very low, even in case of effective drugs. these drugs are developed against specific kinase targets, but these targets are not always equally present or active in the whole treated population. furthermore, in a subset of nonsensitive or resistant patients, the role of this target in tumorigenesis and growth or metastasis is not essential and can be overruled by other mechanisms. in order to identify the patient subpopulation that is likely to respond, tests need to be developed that match the right patient to the right drug and vice versa as more drugs are being developed. there are already examples of such companion diagnostic tests. for the prediction of response to trastuzumab (moelans et al. ) , targeting the receptor tyrosine kinase her /neu, patients are tested for the presence of this target on their tumor cells, before they receive this breast cancer therapy. a recent example is the test for the elm -alk translocation to select patients for pharmacotherapy with crizotinib (kwak et al. ) , an alk kinase inhibitor. if the whole lung cancer patient population would have been treated, only an extremely low percentage would have shown a clinical response because only % have this mutation. the availability of the companion diagnostic test was therefore essential for the success of the clinical trial. the identification of predictive biomarkers appears to become essential in many drug development programs. the classical technologies for biomarker discovery are based on testing for dna mutation or rna or protein expression levels. molecular data are obtained in biopsies taken before the patient is treated. if these data can be correlated or associated to the therapy response, this can be the start of generating a companion diagnostic test. peptide microarrays are also currently being used in this effort. while classical methods cannot involve the drug of interest in predose tissue samples, kinase microarrays can, as discussed above. in addition, the drug can actually be used in the test. this means that drugspecific data and information can be generated using predose biopsies. proof of concept of this approach was shown by versele et al. in a multiple cell line study. analogous to the way it is aimed to work in a clinical setting, they profiled the lysate of a cell line on a peptide microarray in the presence and absence of their drug candidate. the inhibition profiles were used to predict the response of the cell line to drug treatment in culture. from these profiles, they could identify a set of peptide phosphorylations of which the response (inhibition) on the chip was predictive for the tumor cell proliferation (versele et al. ). this concept (fig. . ) is now being explored in clinical studies by my research group in collaboration with the netherlands cancer institute (nki), the vu medical center, and other cancer centers in both the usa and japan. in a study presented at asco in on neoadjuvant treatment of non-small-cell lung cancer with the egfr kinase inhibitor erlotinib, we showed that candidate biomarkers could be identified. on-chip peptide phosphorylations and inhibitions were correlated to clinical responses. with no information on the pathological assessment of the resection tissues available to the testers, a model built on those profiles could still predict the pathological response (hilhorst et al. ) . it should be noted that resection tissue was used and not pretreatment biopsies which is needed to make this into a it could be possible to apply this principle of drug testing on patient-derived tissues to other targeted therapies as well. in addition, if other protein classes are targeted, for example, phosphatases, proteases, nuclear receptors, acetyltransferases, histone deacetylases, and methyltransferases, the target responses in patient-derived samples could be tested using a peptide microarray. the nonfocused, nonbiased, and global profiling nature of the arrays allows parallel monitoring of drug targets and class-related nontargets. these nontargets can be functional proteins involved in the mechanisms of resistance and are therefore possibly very useful markers for predicting resistance to targeted therapies. finally, nontargeted therapies such as chemoradiation could also be accompanied in the future by such testing methods, as was shown in a recent publication by a norwegian group. they generated kinase activity profiles of tens of biopsies taken before patients were treated and could identify peptide phosphorylation patterns that correlated to the tumor regression grade after therapy. they generated a response prediction model that could predict the responses of a newly tested set of patients with promising accuracy (folkvord et al. ) . thomas felgenhauer, ralf bischoff, frank breitling, and volker stadler several sophisticated methods are in use worldwide to produce peptide microarrays. each of these methods has its special advantages and drawbacks. high amounts of identical oligomers are achievable on cellulose supports via spot synthesis (frank ; dikmans et al. ) , but spot densities are very low due to droplet handling. with photochemical methods where chain growth is induced by a laser beam, very small spot sizes and high spot densities are possible (fodor et al. a, b; lipshutz et al. ) . in this case, the drawback is in the sequential use of monomer solutions which might be acceptable in dna synthesis (four monomers), but the yield is dramatically reduced when the number of needed coupling cycles increases as in the case of standard peptide synthesis where minimum individual cycles are needed to complete a fully combinatorial layer. the use of a laser printer as synthesis machine makes it possible to overcome the obstacles of the methods described above. solid particles (toners) carrying the reactive building blocks are printed in parallel in high resolution to a desired support. a full combinatorial layer is developed-like a color picture printoutat once, and the coupling cycles are reduced from to a single one per layer (stadler et al. ) . a commercial laser printer uses small solid toner particles (~ mm) that are triboelectrically charged by friction inside a toner cartridge drum system. because of the materials involved, this procedure leads to strong electrical charges on the particle surfaces, which enables the directional movement of the particles within electrical fields. a laser beam or an led row translates d light patterns into electrical patterns on top of an organic photoconductor drum. these images are developed with the charged toner particles that are finally transferred to the support. at office applications, a color laser printer system delivers four different color toners (black, cyan, yellow, and magenta) on a sheet of paper with a resolution of , up to , dpi. the polymer-based toner particles are fixed to the cellulose support by heat. the main challenge in combinatorial synthesis is to deliver different kinds of monomers with high accuracy to their designated reaction partner or reaction site. whereas a color laser printer delivers only four toners, a peptide synthesizer based on the xerographic technique should be able to handle at least different building blocks for basic peptide synthesis or other feasible monomers for the production of peptide mimetics (amino acids in d-form, methylated, phosphorylated derivatives, nonnatural versions). in addition to the great flexibility of the synthesizer, an exact positioning of consecutively printed layers is the basic requirement for the parallel elongation of combinatorial assembled oligomer chains. with increasingly better printing accuracy, the spot density also increases, as well as the diversity of synthesized peptides. to benefit from the laser printer as delivery machine for monomers in combinatorial chemistry, the toner particles (delivery packages) have to be modified for this chemical purpose. in addition to their properties as solid, electrically charged particles, they also need the attributes of a solvent once melted. this change of properties happens after the particles have been addressed to their designated reaction site, where they are transformed into a liquid sphere simply by melting. thereby, activated monomers are mobilized, which allows them to diffuse to their reaction partner for chain elongation. these very special solid/liquid characteristics of the toner particle depend on the choice of the appropriate matrix material. on the one hand, this material should withstand the harsh mechanical treatment inside the printer (e.g., friction, charging, transport); on the other hand, the liquefaction at moderate temperatures (< c) is fundamental in order to perform as a solvent for a chemical reaction. in addition, the matrix material should protect the reactive monomers from ambient conditions during long-term storage in cartridges, and finally, the material itself must be inert toward the components inside. since all the different monomer particles are addressed in parallel by the printer, all the different activated amino acid derivatives within a completed layer of amino acid particles are activated at once in a single melting step. this feature is the main advantage of our technique. washing and deprotection steps that follow after the coupling step finish the cycle, and result, if repeated, in the combinatorial synthesis of a peptide array (fig. . ) . our method uses conventional fmoc chemistry (chan and white ) and differs from the spot synthesis only in the solvent we employ: it is solid at room temperature, which allows for the intermittent immobilization of chemically highly activated amino acid derivatives within particles. this activation is due to a c-terminal pentafluorophenyl ester in combination with n-terminal fmoc protection and standard side chain protecting groups. surprisingly, when embedded in particle matrix, these very reactive chemicals proved to be stable for months at room temperature, an exception being fmoc-arginine-opfp that shows a decay of % per month. however, if compared to the much faster decay of activated arginine esters in solution, this decay is negligible. the surface-coated solid support must provide free amino groups that react with preactivated amino acid derivatives. it must stand harsh conditions during peptide synthesis (solvents, bases, strong acids during final cleavage of side chain protecting groups) and postsynthesis; it must allow for the incubation of arrays with an analyte, for example, an antibody solution. we employ - nm thick d polymer coatings that have a high loading capacity (high density of amino groups). alternatively, essentially d layers are used as solid supports for peptide synthesis. these are generated from functionalized silanes that also stand the conditions during peptide synthesis and, dependent on the used assay system, sometimes perform better when compared to the polymer coating. such surfaces are described in detail elsewhere (beyer et al. ; stadler et al. ). the peptide laser printer at pepperprint gmbh (see fig. . a, b) has printing units assembled in a row. twenty of these toner cartridges that are based on the oki system are equipped with fmoc-amino acid esters; the four remaining cartridges are used for nonstandard amino acid particles. the printer works with micron resolution which currently allows for the synthesis of , peptides on a ( Â ) cm glass substrate. this corresponds to a spot density of~ spots per cm . currently, the machine is used for the synthesis of up to meric peptides. our particle-based synthesis method makes available to the scientific community, for the first time, very high-density peptide arrays. this is, in technical terms, the main novelty of this method (table . ). as such, this method certainly soon will approach the number of different molecules that nature's screening systems employ. these use, for example, millions of randomly generated antibodies to screen for binders against virtually any target molecule, among them nonnatural molecules that have never been encountered by evolution. peptide arrays with natural amino acids (l-form) have been used in the past mainly as protein fragment libraries in proteome research, or as diagnostic tools for serum screening and for antibody profiling. for the development of novel therapeutics, often nonnatural amino acids (e.g., d-amino acids) were integrated at critical positions within the peptide sequence in order to increase the metabolic stability of the peptides. however, due to the use of only low-density peptide arrays, up to now, such screens usually had to rely on extensive previously available knowledge. typically, a peptide sequence that was already known to bind to the target was then modified to improve the stability or the binding affinity, or an already known antigenic protein sequence was used to generate many overlapping peptides in order to narrow down an antibody's binding epitope. in the near future, we will certainly see that very high-density and affordable peptide arrays will be used to find binders without extensive previous knowledge about the sequence of a potential binder. similar to nature's screening systems, a vast number of different peptides should be sufficient to find binders against nearly any target molecule, and different from surface display techniques, the array format will allow for an easy and unequivocal discrimination of specific from nonspecific binders. very high-density peptide arrays will be used for such screens that have been cleared from all those peptides that were found to bind to more than a few different target proteins. it is practically impossible to avoid such nonspecific binders in all the surface display methods. thus, the high combinatorial diversity given by the laser printer method should increase the possibility to discover potent low-density peptide arrays have been used previously to narrow down the binding epitope(s) of binding antibodies by synthesizing many overlapping peptides derived from the sequence of a known antigen that has been used, for example, to immunize an animal; however, such experiments used to be prohibitively expensive. highdensity peptide arrays are cheaper-they simply allow more of these experiments. it should be feasible in the future, for example, to routinely monitor the kind of antibodies that evolved in a mouse that was immunized with a protein. the experimenter could then use only those mice that evolved an antibody specific for an especially interesting epitope within the protein that was used for immunization, thus saving a lot of time and money by using only selected mice for the generation of hybridomas. shown as an example for this statement are the results obtained when we used four different rabbit sera that were immunized with protein a and closely related protein b (fig. . ). only one of two rabbits immunized with protein a or with protein b revealed that specific antibodies have been generated, while the other two rabbits did not generate protein a-or protein b-specific antibodies. the staining pattern revealed that rabbit developed several antibodies that were specific for the c-terminal region of protein a, while rabbit developed antibodies that targeted the n-terminal region of protein b, and when scrutinizing the peptide sequences, both productive sera did not reveal cross-reacting antibodies against determinants from closely related proteins #a and #b. a biomarker is a substance that is used as an indicator of a biological state. the biomarker serves as an indicator to measure and evaluate normal or pathogenic biological processes, or pharmacologic responses to a therapeutic intervention. especially useful are biomarkers that are found in human sera. these are a rich and accessible source for the detection of diagnostic markers in human diseases. serum antibodies have been used extensively for diagnosis, for example, of flu antibodies in a patient. such antibodies often are found in a patient decades after infection and can easily be analyzed with peptide arrays. especially interesting are those (peptide-)antigens (and their corresponding antibodies) that are targeted by an immune response. these are useful as biomarkers for drug discovery and diagnostics. however, the most interesting scientific question in biomarker discovery is as follows: can we find novel antigens and their corresponding antibodies without previous knowledge about the antigen? figure . a shows that such a scientific question could be answered with very high-density peptide arrays. a randomized high-density array of meric stochastically chosen peptides (only a detail view of~ , structures is shown) was used to find peptide binders for the flag m antibody with its known binding epitope nnndyknnnd/ennn. indeed, we could find six weak binders in a first screen. sequences from all of these initial hits were then used in a follow-up screen that stained the completely permutated sequences from these initial binders. figure . b shows such a permutation screen that started with the sequence "ecwgdyksmecadwh" found as an initial hit. this sequence, and all the other five hits from the initial screen, then revealed either the sequence nnndyknnnennn or nnndyknnndnnn, i.e., the flag m epitope. all amino acid positions depicted by "n" could be exchanged by other amino acids. it remains to be seen if such a screen could be employed to also find novel biomarkers when staining stochastically chosen very high-density peptide arrays with serum antibodies derived from patients with enigmatic diseases. many protein-protein interactions are mediated by short linear motifs. some of these interactions are deregulated in diseases and thus are potential targets for modulating peptide-based drugs. these peptides should interfere with the protein-protein interactions by binding to one of the partners, either activating or inhibiting the signals that depend on the respective proteins. historically, and as stated above, peptide drugs have been based, for example, upon the optimization of natural peptide hormones but, more recently, novel peptides are being developed that have been isolated from combinatorial recombinant libraries. the idea is to offer such a large number of different potential peptide-based binders that simply by chance any protein would "find" at least one binder among a plurality of different peptides. however, the current size limits of peptide arrays and the associated costs had made it, until now, unrealistic to conduct such comprehensive profiling screens. the production of microarrays by laser printing uses a novel chemical concept where an activated monomer is encapsulated within solid particles that are sent to different "addresses" on a surface displaying reactive groups. thereby, an established technology is used for the rapid construction of a densely spaced pattern of different kinds of particles. these comprise different building blocks that are wild-type residues are highlighted by a red circle initially "frozen" at room temperature within solid particles. thawing these particles at once leads to a single coupling step per layer, which is the main advantage of this method when compared to sequential coupling, for example, in lithographic methods. our particle-based method is particularly well suited for automation and, thereby, results into drastically reduced cost per peptide spot. it brings affordable high-density peptide arrays within reach of normal laboratories and may have an impact similar to the one that high-density oligonucleotide arrays had in the field of genomics. peptide arrays for screening cancer specific peptides deciphering the antibodyome-peptide arrays for serum antibody biomarker diagnostics functional peptide microarrays for specific and sensitive antibody diagnostics proteomic techniques and activity-based probes for the system-wide study of proteolysis a novel glass slide-based peptide array support with high functionality resisting non-specific protein adsorption light-directed maskless synthesis of peptide arrays using photolabile amino acid monomers an improved method for the synthesis of cellulose membrane-bound peptides with free c termini is useful for pdz domain binding studies protein kinase signaling networks in cancer fmoc solid phase peptide synthesis-a practical approach substrate specificities of catalytic fragments of protein tyrosine phosphatases (hptp beta, lar, and cd ) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors peptide-protein microarrays for the simultaneous detection of pathogen infections a peptide-based target screen implicates the protein kinase ck in the global regulation of caspase signaling solas d ( a) light-directed, spatially addressable parallel chemical synthesis prediction of response to preoperative chemoradiotherapy in rectal cancer by multiplex kinase activity profiling spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support bl€ ocker h ( ) a new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports light directed massively parallel on-chip synthesis of peptide arrays with t-boc chemistry activity-based high-throughput determination of ptps substrate specificity using a phosphopeptide microarray use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid printing chemical libraries on microarrays for fluid phase nanoliter reactions high throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays sars corona virus peptides recognized by antibodies in the sera of convalescent cases estrogenic effects in the immature rat uterus after dietary exposure to ethinylestradiol and zearalenone using a systems biology approach peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase a peptide arrays on cellulose support: spot synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion defining the specificity space of the human src homology domain optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis genome to kinome: species-specific peptide arrays for kinome analysis a microarray strategy for mapping the substrate specificity of protein tyrosine phosphatase nuclear receptor-coregulator interaction profiling identifies trip as a novel peroxisome proliferatoractivated receptor gamma cofactor anaplastic lymphoma kinase inhibition in non-small-cell lung cancer proteintyrosine kinase activity profiling in knock down zebrafish embryos developing site-specific immobilization strategies of peptides in a microarray high density synthetic oligonucleotide arrays the specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library a study to assess the cross-reactivity of cellulose membrane-bound peptides with detection systems: an analysis at the amino acid level photolithographic synthesis of high-density oligonucleotide arrays automated synthesis of peptides solid-phase peptide syntheses current technologies for her testing in breast cancer microarray profiling of antibody responses against simian-human immunodeficiency virus: postchallenge convergence of reactivities independent of host histocompatibility type and vaccine regimen dynamic control of signaling by modular adaptor proteins light-generated oligonucleotide arrays for rapid dna sequence analysis strategies for kinome profiling in cancer and potential clinical applications: chemical proteomics and array-based methods development of selective bisubstrate-based inhibitors against protein kinase c (pkc) isozymes by using dynamic peptide microarrays blind prediction of response to erlotinib in early-stage non-small cell lung cancer (nsclc) in a neoadjuvant setting based on kinase activity profiles peptide microarrays for the determination of protease substrate specificity kinome profiling of chondrosarcoma reveals src-pathway activity and dasatinib as option for treatment high-content peptide microarrays for deciphering kinase specificity and biology peptide arrays for kinase profiling automated maskless photolithography system for peptide microarray synthesis on a chip maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array peptide microarrays for a network analysis of changes in molecular interactions in cellular signalling multifunctional cmos microchip coatings for protein and peptide arrays combinatorial synthesis of peptide arrays with a laser printer peptide microarrays for the detection of molecular interactions in cellular signal transduction peptide microarray for highthroughput determination of phosphatase specificity and biology high-throughput screening of catalytically inactive mutants of protein tyrosine phosphatases (ptps) in a phosphopeptide microarray high density peptide microarrays for proteome-wide fingerprinting of kinase activities in cell lysates deciphering enzyme function using peptide arrays the use of semi-parametric mixed models to analyze pamchip peptide array data: an application to an oncology experiment identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries bayesian modeling of the yeast sh domain interactome predicts spatiotemporal dynamics of endocytosis proteins cyclooxygenase- inhibition inhibits c-met kinase activity and wnt activity in colon cancer preparation of novel alkylated arginine derivatives suitable for click-cycloaddition chemistry and their incorporation into pseudosubstrate-and bisubstrate-based kinase inhibitors response prediction to a multitargeted kinase inhibitor in cancer cell lines and xenograft tumors using high-content tyrosine peptide arrays with a kinetic readout from promiscuity to precision: protein phosphatases get a makeover screening combinatorial libraries by mass spectrometry. . identification of optimal substrates of protein tyrosine phosphatase shp- pnaencoded protease substrate microarrays a special cellulose membrane support for the combinatorial and parallel synthesis of peptide libraries suitable for the sc -type manufacturing of high density multi-purpose chemical micro-arrays protein tyrosine phosphatase substrate specificity: size and phosphotyrosine positioning requirements in peptide substrates a protein phosphatase- -binding motif identified by the panning of a random peptide display library key: cord- -w gc nx authors: nan title: poster presentation abstracts date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: w gc nx nan background and aims: homodimerization of myd adapter protein is essential for nf-kb activation in the inflammatory pathway triggered by il- and tlr [ ] . we designed a peptidomimetic of the myd tir domain consensus peptide arg-asp-val-leu-pro-gly-thr [ ] , named st . here, we report its synthesis and biological activity. we also report the synthesis and biological activity of its enantiomer, st , and its diastereoisomers, st and st . methods: the structure of the myd tir domain consensus peptide is subdivided into three distinct portions, the most important of which is a b-turn. in the peptidomimetic design we changed the b-turn with a tricyclic spirolactam [ ] , already known [ ] . we synthesized this building block, its enantiomer and two of possible diastereoisomers by "in solution" synthesis. based on semiempirical calculation of heat of formation [ ] , we could predict the right stereochemistry of the products selectively obtained in the last cyclization step. results: these four compounds were tested for their biological activity by reporter gene assay (rga). some coimmunoprecipitation experiments were also carried out and we report their results. conclusions: the results show the activity of st and its isomers on our target, with limited specificity towards their stereostructure. introduction of a methylene bridge between the cα(i+ ) and the n(i+ ) atoms in an open peptide (i) to mimic simultaneously the cαh(i+ ) and hn(i+ ) protons (β-lactam scaffold assisted design -β-lsad) has proven to be a practical tool for the preparation of monotopic β-turn peptidomimetics (ii, r = r = h), according to the principle of separation of constraint and recognition elements . in this work we report a short, general, and stereocontrolled synthesis of multitopic β-lactam scaffolds of type vi. α-alkyl serinates iii are converted into the corresponding enantiopure nnosyl-aziridines iv which undergo "in situ" ring-opening with amino acids v. subsequent base-promoted cyclization affords the n-protected α-alkyl-α-amino-β-lactams vii. incorporation of the novel scaffolds into linear and cyclic peptides and their conformational features are also presented, most of them showing stabilized β-and γ-turn conformations. poly(amino acids) are emerging as promising therapeutic carriers finding widespread application in the field of drug delivery. in this context, polyproline polymers have been used to solubilize poorly water-soluble proteins, in affinity chromatography for the purification of platelet profilin, and more recently, in the design of dendrimers. poly(amino acids) are most conveniently synthesized by polymerization of the corresponding amino acid n-carboxyanhydride (nca). in spite of the interest of polyproline, the preparation of proline n-carboxyanhydride (pro-nca) renders poor synthetic yields. in this work a new method for the preparation of pro-nca in high yields and purities is described. amino acid n-carboxyanhydrides are obtained by the method described by fuchs. but, in the case of proline, the n-carbamoyl chloride does not cyclise spontaneously as it takes place with other amino acids, and the use of a non-nucleophilic base is required for the cyclisation. a tertiary amine, such as triethylamine, is commonly used but it renders a low conversion of the n-carbamoyl chloride to the expected pro-nca, together with the presence of the pro-pro diketopiperazine byproduct. in the present work, polymer-supported bases have been used instead of triethylamine. higher yields of pro-nca, and very low percentages of diketopiperazine have been obtained. in addition, no tertiary amine contamination was observed. polymer-supported bases could also be recycled and pro-nca yields were reproducible. in conclusion, we have developed an efficient method for pro-nca preparation with polymer-supported bases. the introduction of novel nonproteinaceous heterocyclic amino acids into peptides results in new compounds with interesting structural, physicochemical and biological properties. the transformation of amino acid side chains after the peptide assembly is a convenient method of generating such modified peptides. taking into account the biological activity and complexing abilities of nitrogen-containing heterocycles, we investigated the formation of imidazole, benzimidazole and quinoxaline moieties using condensation with various aldehydes and α-dicarbonyl compounds after classical peptide synthesis on solid support. the imidazole synthesis utilizes the n-terminal or side chain amino group of amino acids, whereas a derivative of phenylalanine, β-( -amino- -nitrophenyl)alanine, was developed for benzimidazole and quinoxaline synthesis. the modified peptides were purified by preparative hplc and characterized by esi-ms, uv and nmr. in conclusion, we developed a straightforward method of synthesis of peptides with specific ion affinity and spectral characteristic. the broad range of commercially available aromatic aldehydes and dicarbonyl compounds makes possible the synthesis of combinatorial libraries of modified amino acids and peptides. part of this work was supported by a grant no. t a from the ministry of education and science. nmda receptors belong to the ionotropic group of glutamate receptors. the activity of the receptor can be altered by compounds acting at binding sites. the (r,s)-(tetrazol- -yl)glycine (tg) has been shown to be a highly potent nmda (n-methyl-d-aspartic acid) receptor agonist with exitotoxic effects [ ] . the aim of our studies was to investigate the chelating ability of tg towards copper(ii) ions. copper is widely distributed throughout the body with a distinct concentration in the brain. copper enters cells as complex and seeks out targets requiring it to function. for these reasons it was interesting to evaluate stability and structure of tg -copper(ii) complexes. the equilibrium and structural properties of complex species were characterized by ph-metric and spectroscopic (uv-vis and epr) methods. in the system, polymeric species are dominant at acidic ph range having { nh , coo-} coordination with possible ntetr bridging elements. monomeric complexes were found at physiological ph. the two tg molecules are bound to copper ion via four nitrogen donors. the formation of two {nh , ntetr} donor sets results in very strong metal-ligand interactions and the complex species are very stable over a wide ph region. we have also performed an investigation on similar tetrazole compounds in order to compare the chelating ability of the tetrazole moiety . the targets of our studies were , -diamino- h- , , , -tetrazole [ ] and tetrazole aspartic acid. references continuing work in that field, we synthesized oxytocins containing tetrazole analogues of amino acids. the -tetrazolyl group is widely used in medicinal chemistry as an isostere of the carboxyl group. compounds containing tetrazole ring appear to be metabolically more stable than their carboxylic analogues and have comparable acidity. we synthesized derivatives of aspartic, glutamic, and alpha-aminoadipic acids containing h-tetrazole ring in side chains. these derivatives were then used for syntheses of oxytocin analogues substituted in position . apart from above we also obtained two analogues with tetrazole analogue of glycine in position . the first one contains h-tetrazole ring, the second one has tetrazole ring substituted with methyl group in position . oxytocin analogues possessing amino acids with tetrazole ring in side chains were synthesized on amide resin using fmoc methodology. in the case of analogues with c-terminal tetrazole ring, fragments - were synthesized on resin and then coupled with suitable dipeptides in solution. all obtained peptides show no pressor and rather low uteronic activity. however, for some analogues the uterotonic activity when measured in the presence of magnesium ions was several times higher. in humans, two classes of defensins, α-defensin and β-defensin, have been identified on the basis of tissue specificities and structural features including their modes of disulfide pairing. in general, particular combinations with disulfide bonding in cysteine-containing peptides are critical for expressing their intrinsic biological activities. in the case of human α-and β-defensins, however, disulfide isomers without the native pairing were demonstrated to exhibit similar antimicrobial activity to that of the native defensins. therefore, to assess the biological activities of defensins as well as defensin-based therapeutics, extreme care is required in the chemical synthesis of these peptides to avoid ambiguity in quality. in the present study, we synthesized human α-defensin- , - and - , and human β-defensin- , - , - and - by employing boc chemistry, and determined the optimal conditions for folding the respective reduced peptides preferentially into a native conformation. among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione, the buffer concentration and reaction temperature were essential. all the synthetic human α-and β-defensins were confirmed to have the respective native disulfide pairing by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. all the human α-and β-defensins could be efficiently oxidized to the α-and β-defensin-type disulfide structure, respectively, under several conditions determined in the present study. these synthetic peptides of high homogeneity were used to accurately assess the antimicrobial activity. native chemical ligation is based on the reaction of a peptide bearing a c-terminal thioester group with an n-terminal cysteinyl peptide, leading to the formation of an amide bond at the aa-cys junction. the key starting materials for native chemical ligation are unprotected c-terminal thioester peptides. thioester peptides are often prepared using boc/benzyl solidphase peptide chemistry. however, the widespread use of the fmoc/tert-butyl chemistry for peptide synthesis, over the boc/benzyl method, has stimulated the development of methods allowing the preparation of thioester peptides that are compatible with the basic treatments used to remove the fmoc alpha-amino protecting group. we report here a novel method for thioester peptide synthesis that is based on the use of the sulfonamide safety-catch linker. once the peptidyl chain is assembled by fmoc/tert-butyl chemistry, the thioester function is generated on the solid-phase through an intramolecular n,s-acyl shift. the procedure seems to be insensitive to the bulkiness of the amino acid directly attached to the sulfonamide linker. the thioesters were successfully used for native chemical ligations in solution or on the solid support. we optimized the recognition sequence of the substrate and the reaction conditions with respect to the yield. the sortase-mediated ligation was successfully applied to the synthesis of cellpenetrating peptide-pna conjugates which showed enhanced activity in antisense experiments compared to pna alone. this ligation strategy was also employed for the coupling of a chemically synthesized construct of the extracellular loops of the crf-receptor with the corresponding n-terminal receptor domain, which was expressed in e. coli. this kda protein behaves like an artificial receptor, binding specifically natural ligands. linear gramicidins represent the most investigated family of antibiotic peptides forming ionic channels. gramicidins produced by bacillus brevis are hydrophobic peptides composed of amino-acids with d and l configuration strictly alternate. the presence of d-amino acids in the sequence of gramicidin a (hco-val-gly-ala-dleu-ala-dval-val-dval-trp-dleu-trp-dleu-trp-dleu-trp-nhch ch oh) should possible make the peptide highly resistant to proteolysis [ ] . striking features like ethanolamine group in c-terminus, the n-terminal n-formylated valine and the high hydrophobicity of the peptide sequence, make the solid-phase synthesis of gramicidin a very tricky. therefore, we followed a new synthetic strategy for peptide chain elongation assisted by microwave energy. in fact, microwave energy has been demonstrated to produce highpurity compounds with more rapid reaction times, enhancing coupling rates and efficiency in difficult syntheses [ ] . however, microwave-assisted solid phase peptide synthesis (mw-spps) has not been yet extensively investigated. in this context, we synthesized gramicidin a by mw-spps in high yield and purity, enhancing reaction rate compared to the traditional spps. thermal disruption of peptide aggregation, induced by microwaves, is possible favorable for obtaining this particularly difficult sequence. gramicidin a was incorporated in synthetic lipid bilayers, self-assembled on mercury electrodes, characterized by hydrophilic spacers interposed between the metal and the lipid bilayer. we tested the behaviour of gramicidin a in biomimetic membranes using electrochemical impedance spectroscopy (eis), ac voltammetry and other electrochemical techniques [ ] . [ csf (glc) is an n-glucosylated peptide to be produced in large scale by peptlab because it is the active molecule of the first specific diagnostic/prognostic test for monitoring disease activity and guiding therapeutic treatments of multiple sclerosis patients [ ] . in order to develop a synthetic protocol by an automated instrumentation, increasing yield, purity of the crude, and reaction time, a microwave-assisted solid phase peptide synthesis was validated comparing the use of the new generation of triazine-based coupling reagents (tbcrs) with a series of commonly used ones. activation of carboxylic acids by tbcrs is particularly effective because of formation of triazine "superactive esters". the usefulness of tbcrs as coupling reagents has been recently confirmed in the synthesis of z-, boc-, and fmoc-protected dipeptides, sterically hindered amino acids, in the synthesis of esters, in manual and automated spps of difficult peptide sequences, and head-to-tail constrained cyclopeptide analogues [ ] . moreover, we also demonstrated tbcrs efficient in a microwave-assisted solution synthesis of the n-glucosylated building block fmoc-asn(glcoac )-oh using a manual monomode microwave instrument [ ] . this building block was used to obtain csf (glc) comparing the efficacy of a monomode microwave automatic instrument with the traditional solid-phase peptide synthesizers such as the manual and automatic in batch systems, as well as the continuous-flow one. it is known that enzymatic peptide synthesis is more advantageous than chemical synthesis in many aspects; it is highly stereoselective, racemization-free and requires minimal side-chain protection. the method is, however, limited to the use of amino acid derivatives which meet the enzymatic specificity as a coupling component. this problem may be solved using enzymes which have wide specificity of substrate. but in this case, secondary hydrolysis of the resulting peptide may arise from the inherent nature of the protease. in this matter, ficin and ficin-like enzymes were used as cysteine protease to analyze the diminishment of specificity for the substrate. the cysteine protease-catalyzed peptide coupling reaction has been studied by using synthetic fourteen boc-amino acid phenyl and naphthyl esters as acyl donor. the reaction conditions were optimized for organic solvent, ph, and concentration of acceptor. the coupling reaction was carried out by incubating an acyl donor ( mm) with an acyl acceptor (ala p-nitroanilide, mm) and enzyme ( . u) in a mixture of gta buffer ( mm, ph . ) and dmso ( : ) at ْc. the progress of the coupling reaction was monitored by rp-hplc. the products were obtained in satisfactory yields. non-enzymatic glycosylation, also called glycation, is a common modification in living organisms formed by the reaction of carbohydrates with free amino groups of peptides and proteins. it is a slow chemical reaction yielding amadori products undergoing further oxidation and degradation reactions finally leading to advanced glycation end-products (age). amadori products are early markers for ageing, diabetes mellitus and alzheimer's disease. despite the clinical importance of these amadori products, universal protocols to synthesize amadori modified peptides are still missing. here we describe a solid phase strategy for the glycation of specific amino groups on partially protected resin bound peptides using a global post synthetic approach. the peptides were synthesized by standard fmoc/tbu-chemistry using carbodiimide activation. the lysine position to be modified was incorporated with a methyltrityl protected ε-amino group, which can be selectively cleaved after completion of the peptide synthesis with % tfa in dichloromethane. the partly deprotected peptide was glycated in methanol using a ten-fold molar excess of , - , -di-o-isopropylidene-aldehydo-β-d-arabino-hexos- -ulo- , -pyranose and nabh cn for h at °c. after cleavage the overall yields were in the range of - % for the tested octapeptides. all byproducts were well separated by rp-hplc allowing a simple purification strategy even for medium-sized peptides. thus the general strategy presented here allows routine synthesis of amadori peptides at reasonable yields and purities using standard protocols established in most laboratories synthesizing peptides. -chlorotrityl chloride resins are recommended for the synthesis of c-terminal proline peptide acids to overcome diketopiperazine formation during chain assembly. however, we have found these (and similar) resins to be unsuitable for the synthesis of peptides greater than residues. for example, the chemokine guinea pig eotaxin, ( residues c-terminal proline) assembles poorly if not at all on a -chlorotrityl resin. we sought to circumvent these problems in the chemical synthesis of peptides and proteins, through the development of a resin-swap procedure. whereby the initial c-terminal protected tripeptide is assembled on a -chlorotrityl resin, liberated from the solid-support, then reattached to a resin that is suited for long chain peptide / protein synthesis. using this approach, the synthesis of guinea pig eotaxin is reported. the tripeptide fmoc-thr(but)-lys(boc)-pro-oh was assemble on -chlorotrityl resin, cleaved with % tfe in dcm and attached to wang resin using standard protocols. peptide assembly gave the gp eotaxin in % overall yield (as determined by uv monitoring). fmoc-on cleavage, purification and tag removal followed by folding gave the native chemokine in good yield. choice of resin is one of the most critical factors in ensuring a successful peptide synthesis, we have shown the superiority of wang resin over chlorotrityl resin in the synthesis of medium and long peptides and developed a method for the synthesis of c-terminal proline containing peptides which overcomes the problem of diketopiperazine formation. the technique is being applied to the synthesis of other c-terminal proline peptides e.g. human eotaxin and ip . dimerization of cell receptors, involved in antigen presentation, is an essential step in several cellular signal transduction processes, therefore substances that are able to modulate such a process are of potential therapeutic value. dimeric peptide ligands could represent useful tools to cause dimerization of such receptors. a similar strategy applies dimerization of ligands, interacting with dimeric proteins or proteins with multiple binding sites, to design molecules with enhanced affinity. dimeric analogs of the immunosuppressory hla class ii fragments were synthesized using suitably modified, standard fmoc solid-phase protocols and mbha-resin. the dimerization was achieved by crosslinking n-terminal amino groups of the peptides with the commercially available mixture of poly(ethyleneglycol)biscarboxylic acid (average mw , length range - Å), activated by esterification with pentafluorophenol. the same procedure was applied to synthesize a series of dimeric analogs of c-terminal fragments of plexin-b, consisting of two undecapeptides, linked by the polyethyleneglycol spacers. other biand polyvalent linkers were also investigated. our results demonstrated that the amino-terminal dimerizations of the tested hla-fragments resulted in enhanced immunosuppressive activities, whereas interaction of pdz dimer with the plexin fragments led to about -fold increase in affinity, as compared to their monomeric counterparts. [background and aims] elucidation of alzheimer's disease (ad)-related aß - dynamic events is a difficult issue due to uncontrolled polymerization. [methods] based on the "o-acyl isopeptide method" (chem. commun. , ; j. am. chem. soc. , , ), we have developed a novel photo-triggered "click peptide" of aß - ( ), e.g., " -n-nvoc- -aiaß ( )", in which a -nitroveratryloxycarbonyl (nvoc) group was introduced at ser in -o-acyl isoaß - ( -aiaß , ). [results] i) the click peptide did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; ii) photo-irradiation of the click peptide and subsequent o-n intramolecular acyl migration afforded the intact aß - ( ) with a quick and one-way conversion (so-called "click"); and iii) no additional fibril inhibitory auxiliaries were released during conversion to aß - ( ). [conclusions] this method provides a novel system useful for investigating the dynamic biological functions of aß - , such as the self-assembly and aggregation processes in ad. several insulin analogues have recently been introduced clinically for improved treatment of diabetes. industrial productions of such insulins are based on microbial expression systems, which are highly efficient, but generally limited to the proteogenic amino acids. also, some sequences form inclusion bodies or fail to express. the total chemical synthesis of insulin in research scale was a landmark achievement in peptide science. however, the most commonly used method relies on recombination of a-and b-chains under "random" folding and pairing of the three disulfide bridges. this folding/oxidation step is difficult and low yielding. a general approach using a removable auxiliary which can direct correct formation of disulfide bridges is highly desirable. in the pancreas as well as in microbial expression systems, insulins are prepared and folded as single chain precursors, with a c-peptide connecting the a and bchains. the c-peptide helps direct the orientation of a and b-chains in obtaining the correct disulfide pairing and overall peptide folding. upon folding, the c-peptide is removed enzymatically. we report here a new method for total chemical synthesis of insulin by use of fmoc-based step-wise solid-phase synthesis of single-chain precursors followed by cpeptide directed folding and cleavage of c-peptide, thereby allowing total chemical synthesis of novel insulins with unnatural substitutions. -chloro- -methoxy- , , -triazines a-c anchored on cellulose, silica or wang resin were prepared by the treatment of , -dichloro- -methoxy- , , -triazine with appropriate solid support in the presence of a base. immobilized, environmentally friendly triazine coupling reagents a-c were obtained by treatment of a-c with n-methylmorpholinium p-toluenesulfonates in the presence of hcl acceptor. the loading of the solid carriers were calculated from n, s contents, determined by microanalysis. all prepared immobilized n-triazynylammonium toluenosulfonates a-c have been found stable at room temperatures. activation of carboxylic components afforded triazine activate esters a-c connected to the support. treatment of a-c with appropriate amino components gave amides or peptides. the final products, chromatographically homogenous amides and peptides, were isolated by filtration or extraction from the solid support. mutter's pseudoproline dipeptides and sheppard's hmb derivatives are powerful tools for enhancing synthetic efficiency in fmoc spps. they work by exploiting the natural propensity of n-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. however, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the hmb residue can be extremely difficult. to alleviate some of these shortcomings, we have prepared fmoc-ala-(dmb)gly-oh and fmoc-gly-(dmb)gly-oh. these dmb-dipeptides can be incorporated into peptides in place of ala-gly and gly-gly, resulting in peptides containing structure breaking (dmb)gly residues. by introducing the (dmb)gly residue as part of a dipeptide unit, the need to acylate the highly hindered secondary amino group of (dmb)gly is avoided. on treatment with tfa the dmb group is cleaved regenerating gly. to test the efficacy of our new derivatives in expediting the synthesis of hydrophobic peptides, we undertook the preparation of the challenging neurotoxic prion peptide - <b> </b>; this peptide reportedly can not be made using fmoc spps methods. the dipeptides marked in bold were systematically substituted with the appropriate dmb peptides. the effects of the substitution were evaluated using conductivity monitoring and lc-ms analysis of the crude peptides. h-lys-thr-asn-met-lys-his-met-<b>ala-gly</b>-ala-ala-ala-<b>ala-gly</b>-ala-val-val-<b>gly-gly</b>-leu-gly-oh <b> </b> th efficient dipeptide production form unprotected l-amino acids with the novel enzyme l-amino acid α-ligase. k. tabata , h. ikeda , m. yagasaki , s. hashimoto background and aims: application of α-dipeptides has been limited due to the lack of cost-effective manufacturing methods. the known methods require the protection of amino acid(s) to fix the order of the amino acids ( fig. ) . furthermore, they usually accompany the formation of longer peptides. to establish the costeffective manufacturing method, a novel activity which synthesizes α-dipeptides from two unprotected l-amino acids was screened. methods and results: a gene was found in the genome of bacillus subtilis by in silico screening based on a putative reaction mechanism. the purified protein coded on the gene, i) catalyses α-dipeptide formation from unmodified l-amino acids with a specific order in an atp-dependent manner, ii) never forms tri-or longer peptides, and iii) takes a wide variety of l-amino acids but no d-amino acids. the enzyme was tentatively named l-amino acid α-ligase (lal). the whole cell reaction of a recombinant e. coli strain expressing lal and polyphosphate kinase (ppk) with two l-amino acids and polyphosphate (polyp) enable the efficient production of many dipeptides with a certain order of the constituent amino acids through the coupling reaction of lal and ppk (fig. ) . conclusion: a novel enzyme, lal, enables to synthesize dipeptides cost-effectively directly from unmodified l-amino acids. t. ye marine organisms continue to provide rich sources of structurally unique and pharmaceutically active compounds. due to the difficulties in the isolation of significant quantities of these natural products, synthetic chemistry serves an important role in their structural assignment and biological evaluation. antifungal agents have received considerable attention recently since the spread of hiv has left many people open to fungal infections, and there is a rapidly growing number of drug resistant strains of fungus emerging. ll- g gamma is a cyclodepsipeptide isolated from the marine fungus hypoxylon oceanicum and structurally assigned in by schlingmann. the structure of ll- g gamma was determined by a combination of chemical degradation, chiral chromatography and spectroscopic analysis. ll- g gamma uniquely combines a beta-ketotryptophan and a polyketide portion within a macrolactone ring. ll- g gamma has exhibited potent activity against fungal strains and as such, is an attractive compound to develop as a future therapeutic agent. to date, there have been no reported studies towards the synthesis of ll- g gamma. we have completed the total synthesis of ll- g gamma by employing the macrolactamization followed by a c-h oxidation as the key step. aspartimide (aminosuccinimide, asu) formation is the first step in the degradation of asp/asn containing peptides and proteins. the reaction is especially prevalent at asx-gly sites and results in a variety of rearranged and racemized products. the bases used in fmoc-tert-based spps promote the formation of asu and related products. we recently found that the dmb backbone protection efficiently prevents secondary structure formation at gg sites and is orthogonal with respect to standard fmoc spps. here we explore the use of dmb, tmb and nbzl groups (z) for the synthesis of "difficult"/asu-prone peptides, in three different schemes: a) fmoc-asx-(z)gly-oh dipeptide building blocks; b) fmoc-(z)gly-oh monomer building blocks, and c) two steps "submonomeric" approach for synthesis of substituted n-benzyl glycines on the resin. we tested the new methods on two model peptides vkd/ngyi and ha - hiv-tat - (h-g lfgaiagfi engwegmidg grkkrrqrrr -oh) fusion peptide. the yield and purity of the products reach and even exceed the level in control experiments obtained with hmb protection and the peptides were found free of asu/piperidides. the acid removal of the dmb protection is ~ % faster than that of hmb. the submonomeric route (strategy c) is especially simple, efficient, cost effective and it allows the use of different amines for halogen-displacement. the backbone protecting groups used were in many respects superior to the commercial reagents and applicable for synthesis of both peptide acids and peptide amides. the use of nbzl-nh for halogen displacement represents a new method for preparation of backbone-caged peptides. alkyl bonded silica gels historically have been the standard in reversed phase (rp) purification of biomolecules such as synthetic peptides, small proteins, and oligonucleotides. silica gels provided the resolving power needed for challenging separations and the mechanical stability required to be operated industrially under high pressure conditions. the chief disadvantage of silica gels is poor chemical stability under alkaline conditions, which limits their capability to withstand rigorous clean and sanitization -in-place (cip/sip) protocols. as a result, polymeric media have gained recent market attention because of their excellent chemical stability, which enables full compatibility with modern cip/sip protocols. however, first generation polymeric gels lacked both the resolving power and the mechanical stability to be compatible with industrial high pressure dynamic axial compression (dac) hardware. rohm and haas' advanced biosciences division recently introduced a new, monospheric, micron, high performance polymeric rp material. unlike existing softer polymeric gels, this product has higher mechanical stability which enables it to be used effectively with industrial dac / hplc hardware. in addition, this material provides high resolving power for the most challenging industrial separations, because of its unique and selective pore structure, as well as its small monospheric particle size. finally, because of its excellent chemical stability, the media is not limited in the range of ph that can be used. the combination of mechanical stability for high throughput, chemical stability for long lifetime in use, and high resolution for high yield, together translate to an effective cost-in-use solution for industrial polishing processes. we have developed new types of peptide nucleic acids with improved water solubility by introducing ether linkages and pyrrolidine rings in the main chain; pyrrolidine-based oxy-pnas (popnas). in this work, cellular uptake and endosomal release of the trans-l-popna oligomers, one of stereoisomes of the popna, were investigated. the cellular uptake was achieved by combining the popna oligomer with an n-terminal -mer peptide of an influenza virus hemagglutinin protein (ha ) that is labeled with a rhodamine fluorophore at the n-terminal and covalently linked with a hepta-arginine unit at the c-terminal (rho-ha -r ). the fluorescence images of the cho cells after incubation with fam-po( ) [fam-o-cag tta ggg tta g-gly-nh ] in the absence and presence of rho-ha -r were observed with confocal laser-scanning microscopy. incubation with fam-po( ) alone, no internalization of the oligomer was observed. in the presence of rho-ha -r , however, fam-po( ) was successfully internalized into cho cells and, more importantly, the fluorescence spread over the whole cell. the fluorescence image indicates that the popna oligomer in combination with the ha -r peptide was transferred into cytoplasm within h. since both the red (rho) and green (fam) fluorescence spread over the cytoplasm, the popna oligomers that were taken up into endosomes together with the rho-ha -r were released into cytoplasm as the disruption of the endosomes by the ha peptide. in summary, the popna oligomers were readily taken up into cytoplasm of cho cells, when combined with a ha -r peptide. most of functional rnas have post-transcriptional modifications, some of which are quite important for their structure and function. thus, for studying such rnas, it is necessary to use purified raw rnas obtained from living organisms. isolation of native rna is necessary also in the case of analyzing the sequence and modifications of mature rna, which may be different from simple transcript of its gene. therefore, rna isolation method is required. many previous reports demonstrated isolation of rnas, especially trnas. most common and traditional purification methods are based on successive column chromatographies. it seems difficult to apply such method to every trna because effective combination of columns varies among individual trnas. to overcome the difficulty, a sequence-specific selection method using a solid-phase dna has been devised. in this method, a trna can be purified from rna mixture by a single step. however, this method needs high temperature treatment, which might assist hydrolysis of rna strand and might impair heat labile modifications. pna-rna hybrid has been known to be much more stable than dna-rna hybrid. thus pna-based rna purification method seems to be possible for wider variety of rnas in lower temperature, in comparison with dna-based method. in this study, we attempted to purify a single rna, such as a trna and a noncoding rna, from rna mixture by using immobilized pna. r. pipkorn , w. waldeck , h. spring , j. jenne , k. braun background and aims: safe drug delivery technologies are pivotal for genetic interventions, but viral vectors baer the risk of inflammatory reaction. questions concerning the efficacy of delivery of the genetic substances, the desired topical gene activation and targeting must be answered. therefore we attempted to develop a membrane non-perturbing delivery system for transport of inactive functional genes into cells and tissues. genes can be subsequently activated at the target site. our concept bases on the use of peptide-nucleic-acids (pnas) resistant against proteases and nucleases, oligonucleotide derivatives, in which the phosphate-backbone has been replaced with ethylen-amin connected alpha-amino-ethylglycine-units. methods: peptides conjugates were composed and synthesized according to the solid phase synthesis and protecting group chemistry strategies. pna sequences were conjugated covalently, non cleavable, with a capronic acid spacer to the nls, pkkkrkv. pnas have gained broad attention in antisense/antigene experiments and as diagnostic tools. in principal, they can be synthesised with several activating reagents known from peptide synthesis. namely, hatu or pybop are often used. synthesis with hatu is more laborious, because preactivation is needed in order to avoid guadinylation of the n-terminus of the growing pna-chain. we wanted to use pybop, because preactivation should not be needed in this case, which is especially useful in automated synthesis. surprisingly, in the pybop-mediated syntheses of mer pnas we obtained products showing molecular masses approx. da above the expected ones. detailed analysis revealed, that the modification occurred at the only guanine residue in the sequence. in order to further characterise the side reaction, a short pna fragment was synthesised using hatu and pybop activation, respectively, and cleaved from the resin with and without the n-terminal fmoc-group. while synthesis with hatu gave the desired products, pybop partly activates the aromatic carboxy group of the guanine residue, which is substituted by piperidine during subsequent fmoc cleavage. the modified sequences could be further characterised by ms/ms-fragmentation. our results show that care must be taken when synthesising pnas with pybop activation. on the other hand, this reaction possibly opens an opportunity to synthesise guanine derivatives. the opioid receptor system in the central nervous system (cns) controls a number of physiological processes including pain, reward, gastrointestinal and cardiovascular functions. as a consequence, most pain modulating compounds currently available cause a variety of side-effects. the endogenous ligands for the opioid receptors are a series of peptides that includes endomorphin- . endomorphin- has been shown to elicit potent anti-nociception through the highly selective activation of µ-opioid receptors. it is this receptor that mediates supraspinal analgesia and thus, selectivity for this receptor results in analgesia without affecting other processes. therefore, endomorphin- is considered a promising lead compound for the development of a new, safer pain medication. we have synthesized a large number of lipid-and carbohydrate-modified endomorphin- analogues and screened these compounds for their binding and activation of µ-and δ-opioid receptors in sh-sy y cells as well as caco- cell monolayer permeability and plasma stability. compounds conjugated with either a lipoamino acid or sugar moiety on the c-terminus lost binding affinity by several orders of magnitude, whilst n-terminal conjugations resulted in minimal loss of binding affinity. a number of analogues showed pm binding affinity and high apparent permeability, and of these compounds, one has been selected for assessment in nociceptive and neuropathic pain models. in addition to these pre-clinical studies, internalization and tolerance formation of these compounds has also been measured in an effort to synthesise a non-tolerant opioid agonist. endomorphin- analogues with a high degree of amphiphilicity cause increased receptor internalization and subsequently less tolerance formation. a. marcinkowska , l. borovičkova , j. slaninowá , z. grzonka carbohydrate moieties of glycopeptides and glycoproteins play different decisive roles in various biological phenomena. conformation and solubility of proteins are influenced by the oligosaccharide chains, which can also inhibit the proteolytic degradation. as a result, the synthesis of glycopeptides is an attractive field that contributes to understanding of mutual interactions between both moieties and for their biological interest. the synthesis of glycopeptides requires a combination of synthetic methods from both carbohydrate and peptide chemistry. moreover, this synthesis needs stereoselective formation of the glycosyl bond between a carbohydrate and a peptide (amino acid) part, and also an appropriate protecting group methodology that allows selective deblocking of only one functional group in these polyfunctional molecules. in the present work we modified the oxytocin and vasopressin structure with glycoamino acids. transformations of fmoc-protected serine and threonine derivatives into appropriate o-glycosylated precursors suitable for solid phase peptide synthesis were worked out. the -and -o-glycosides were synthesized from fmocserine and fmoc-threonine allyl esters and appropriate glycosyl bromide using hanessian's modification of the koenigs -knorr reaction. these n--fmoc-protected glycosides were used in synthesis of glycopeptides. eight analogues of oxytocin modified in position were obtained. we have also prepared two types of lysin-vasopressin analogues modified with glycoamino acid, in which the glucuronic acid was attached to the ω-amino group of lysine in position through the amide bond. glycosylated analogues of oxytocin and vasopressin display an increased stability towards enzymatic degradation, and retain some hormonal activities. supported by grants: ds/ - - - (zg) and z (js) according to many authors the formation of amadori products is a key stage in the glycation process. glycated proteins may show allergenic properties and potentially initiate autoimmunological processes. they may also serve as the markers of diabetes. to our best knowledge, all procedures concerning the synthesis of peptide-derived amadori products reported in literature are based on "in solution" approach which makes them tedious and time consuming. a modified method of the solid phase synthesis of peptide-derived amadori products based on direct alkylation of the deprotected ε-amino groups with , : , -di-oisopropylidene-β-d-arabino-hexos- -ulo- , -pyranose in the presence of sodium cyanoborohydride was proposed. isopropylidene groups, protecting the sugar moiety in the obtained conjugate, were removed with trifluoroacetic acid containing % water. studies on optimization of the reaction performed on the model peptide attached to a wang resin, fmoc-lys-leu-leu-phe-(resin), showed that the best yield of the product is attained with a two-fold excess of , : , -di-oisopropylidene-β-d-arabino-hexos- -ulo- , -pyranose and a five-fold excess of sodium cyanoborohydride. the identity of the product was confirmed by high resolution ms. the several side products were isolated and their structures will be discussed. our results prove that the synthesis of glycated peptides in the solid phase is feasible. the lack of homogeneous glycoproteins in sufficient quantities is an ongoing challenge in glycobiology. in order to solve this problem researchers have turned to a variety of approaches ranging from mutant eukaryotic strains to the highly demanding total synthesis of glycoproteins. [ ] using rnase b as a model nglycoprotein [ ] we have searched a path to assemble this enzyme employing a combination of chemical and recombinant methods. native chemical ligation [ ] allows the coupling of protein segments of unrestricted size in a chemoselective manner. we have developed solid phase methods to produce the required thioester building blocks - -sr (a) and glycopeptide thioester - -sr (b) containing an n-glycan at asn on a dual linker pega resin. [ ] the remaining segment - (c) was expressed in e. coli as a fusion protein and released by intein mediated protein cleavage. [ ] sequential coupling of the three rnase segments requires the use of a protective group at the n-terminus of segment b compatible with the oligosaccharide part. dysfunctional mutations of antitrypsin can result in a loss of elastase inhibitory activity or allow self-aggregation to occur and cause emphysema and cirrhosis, respectively. insights of the mechanism of disease provide strategy to cope with the aberrant protein aggregation and may bring potential therapeutic agents. in the present work, we describe our effort to identify effective anti-protein polymerization ligands by the employment of combinatorial technology. antitrypsin from human plasma was purified by glutathione sepharose and mono q-sepharose column chromatography. both ala-scanning and peptide shortening were carried out systematically to explore the structural requirements necessary for binding. combinatorial chemistry was then employed to conduct the library screening experiments. assessment of peptide binding was achieved through an unique gel electrophoresis assay. the structural requirements and the minimal peptide length required for binding were revealed by our systematic approach. this information was critical for the design of combinatorial library and the discovery of antitrypsin binding peptides with much improved affinity and specificity. there is currently no effective cure for z antitrypsin related cirrhosis and emphysema. the synthesis and screening of combinatorial libraries offer avenues to increase throughput and ultimately lead to the discovery of inhibitory peptides to the polymerization of pathogenic antitrypsin. with the rapidly increasing number of biopharmaceuticals in the industrial pipeline the need for efficient and expedient purification procedures is growing ever greater. affinity chromatography is one of the most promising technologies in this regard, as it offers very high selectivity and can often replace lengthy and expensive traditional chromatographic procedures. the use of combinatorial split-and-mix libraries is a powerful tool for discovering new affinity ligands but the technique has been limited by the laborious spectroscopic and chemical analysis needed to identify the binding ligand. we have previously introduced a novel bead encoding technology based on a -dimensional image recognition of patterns made by fluorescent particles randomly distributed inside larger beads. [ ] the beads are read prior to each chemical transformation by an instrument featuring three fluorescence microscopes at a rate of , beads per hour. we here present the development of small peptidomimetic affinity ligands for the human growth hormone (hgh) by the use of this technology. the library was sought enriched prior to synthesis by in silico screening of a virtual combinatorial library using a large number of diverse building blocks. binding ligands were identified by incubation with fluorescence tagged hgh. [ the cinnamic acids and their derivatives have been found to possess a variety of biological effects, including antiviral, antimicrobial, antitumor and antioxidant activity. for example, several hydroxycinnamic acid conjugates with amino acids, isolated from plant sources showed enhanced antioxidant activity. the synthesis of cinnamic acid amides and their opioid activity was also cited in the literature. however the synthesis and pharmacological properties of sinapoyl-peptide amides continues to be virtually unexplored. on the other hand, the synthesis and opioid activity of analogs of tyr-mif- has been well documented by us. herein we present a synthesis of a series of sinapoyl -peptide amides where sinapic acid were attached consecutively to both c-and n-end of the tyr-mif- peptide chain: sa-pro-xaa-gly-nh ; sa-tyr-pro-xaa-gly-nh ; pro-leu-gly-nh(ch )nnh-sa sa=sinapic acid; xaa=leu, unusual aminoacid; n= , to obtain the sinapoyl-peptide-amides, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the randall-sellitto paw-pressure test. the antioxidant effects were examined by dpph test as well. studies to establish the importance of introducing the sinapoyl moiety in the tyr-mif- molecule for the antioxidant and opioid activities are underway. several proteins are involved in the transcription of dna to mrna, among which the basic leucine zipper (bzip) proteins. these transcription factors bind specific dna sequences by dimerization and inserting short alpha-helices into the dna major groove. because the dimerization domain is only required to obtain the correct geometrical positioning of the alpha-helices, we will replace it by a dipodal steroid scaffold with defined stereochemistry. due to orthogonal protecting groups, a unique feature of this scaffold is the possibility to design not only homodimers, but also heterodimers. therefore this strategy allows for the construction of both major/major groove and major/minor groove binding peptides, either mimicking naturally occurring proteins or designing peptides with new binding properties. native chemical ligation and staudinger ligation are both suitable for the construction of these peptide dimers. moreover, a combination of solution-and solid-phase chemistry allows for the generation of combinatorial libraries. the increasing number of antibiotic-resistant bacteria is a global health problem. therefore the development of new highly efficient drugs is one of the major tasks of this century. as an example of peptides, which inhibit the growth of e. coli, we demonstrate an easy and rapid method for finding peptides with optimized antimicrobial properties. as a first step we built a modular construct. this construct consists of a constant cationic and a variable module. the cationic module was choosen to achieve cellpenetrating properties. the variable module was expected to act as the virtual active part of the peptide. to increase the proteolytic stability of the peptide we synthesized them in cyclic form. in the first step we used the combinatorial approach to screen approximately . . peptide sequences in the variable region in order to find highly active peptides against e. coli. to optimize the identified sequence, we substituted all amino acids of the sequence with other amino acids and building blocks. additionally, in order to increase stability we modified the bridging. in this way we were able to uncover peptides with high antimicrobial activity as well as proteolytic stability and reasonable solubility. a series of melanocortin active core tetrapeptide hfrw nonpeptide imitations has been prepared using a combination of solution and solid phase synthesis. most of them included residue of -( -imidazolyl) propylamine or histamine as substitutes of histidine. phenylalanine residue, which is included in melanocortins was replaced by residues of derivatives of , '-disubstituted isopropylidenedicyclohexane, , '-disubstituted bicyclohexane, , -disubstituted cyclohexane, , -disubstituted cyclooctane, and , -, , -or , disubstituted benzenes. instead of arginine, residues of oligomethylene diamines, -butyl- -ethyl- , -pentanediamine, , '-methylene-bis(cyclohexylamine), and , '-diaminodiphenylmethane were introduced. -naphtyloxyacetyl-, ( - h-indol- -yl)-butyryl-, -phenyl-ethanesulfonyl-and naphthalene- -sulfonyl-groups served as replacement of tryptophan residue. tested on binding assay on melanocortin receptors, active core imitations exhibited a micromolar affinity to them. isopropylidenedicyclohexane and bicyclohexane derivatives showed about fold higher affinity compared with corresponding derivatives of cyclohexane, cyclooctane or disubstituted benzene. obestatin is a novel endogenous ghrelin-associate peptide, which is involved in the regulation of food intake and weight gain. it was shown to be anorexigenic, able to decrease food intake, gastric emptying and jejunal motility. although obestatin and ghrelin originate from a common prepropeptide of residues, they are reported to exert opposing physiological roles, by binding distinct receptors belonging to the subgroup of type a gpcrs [ ] . obestatin was found to be the natural ligand of the orphan gpr receptor, a gpcr, expressed in jejunum, duodenum, stomach, pituitary, ileum, liver and hypothalamus. as many other peptides involved in the obesity process, it is a new and interesting drug target for the discovery of new anti-obesity molecules. in particular, the first step for the design of new molecules with potential improved anti-obesity activity, is the elucidation of the obestatin conformational features. here, we present the synthesis and the conformational analysis by nmr and cd spectroscopies of obestatin and its related -mer c-terminal sub-fragment, in aqueous solution and in membrane mimicking environment. the data outline the obestatin c-terminal portion as the region characterized by significant conformational features potentially opened to interesting future developments. [ a total of isolates of rhizobium were collected from root nodules of medicago sativa and melilotus officialis plants in different regions of isfahan province .all of isolates on ty medium formed white ,slimy colonies with smooth margins and their inoculation on to roots of young alfalfa plants produced spindly nodules . the nodules developed with some of the isolates were big and pinkish ,although the rest of isolates produced small and white nodules .the speed of nodulation for all the isolates was almost similar and the related nodules were appeared within two weeks . the production of brown pigments on aged colonies of some isolates on ty or ty supplemented with l_tyrosine and copper sulfate revealed that these isolates of s. meliloti are melanin-producing rhizobia.based on the motility and sensitivity to antibiotics tests ,all of the isolates formed a reasonably homogenous group .however a few of them were able to produce an anti-microbial compound which was found to inhibit a number of isolates of s. meliloti .the compound did not suppress the growth of other bacteria . partial purification and spectrophotometery of the compound suggested that it likely belong to the antimicrobial polypeptides .considering on their physiological and biochemical properties ,none of the isolates were selected as a superior and competitive strain ,although based on nodulation efficiency , melanin and antimicrobial compounds production capability the isolate s. meliloti sm and sa were nominated to investigate in details. cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. this conserved structural architecture, termed the cyclic cystine knot, is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. cyclotides have a variety of biological activities but their insecticidal activities suggest that their primary function is in plant defense. in this study we determined the cyclotide content of the sweet violet viola odorata, a member of the violaceae family. we identified cyclotides from the aerial parts and roots of this plant, of which are novel sequences. the new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. as many of the biological activities of cyclotides appear to be associated with membrane interactions, we used hemolytic activity as a marker of bioactivity for a selection of the new cyclotides. the new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. the results show that while biological activity varies with the sequence the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. the structure of one of the new cyclotides, cycloviolacin o , was determined and shown to contain the cyclic cystine knot motif. this study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens. furthermore, the inherent stability of the framework makes it an excellent scaffold for protein engineering applications. warfarin is the most widely prescribed anticoagulant drug for the prevention and treatment of arterial and venous thromboembolic disorders.because of large interpatient variability in the dose-anticoagulant effect relationship and a narrow therapeutic index careful dosage adjustment based on inr is essential. warfarin is available as a racemic mixture of two enantiomers,(s)-and (r)-warfarin. in contrast to (r)-warfarin, which is metabolized by multiple cytochrome p s(cyps), including cyp a and cyp a ,(s)-warfarin, is predominantly metabolized to -hydroxywarfarin by polymorphic cyp c . since the potency of (s)-warfarin is much higher than that of (r)-warfarin, about -to -fold,any change in the activity of cyp c gene is likely to have a significant influence on the anticoagulant response. previous in vitro findings revealed that certain variants in the cyp c gene are associated with large interindividual differences in the pharmacokinetic and pharmacodynamic outcomes of warfarin therapy. three major alleles have been found to date in humans:arg /ile , and cys /ile and arg /leu , and arg /leu , which have been designated cyp c * (wild-type), cyp c * , and cyp c * , respectively. we have investigated this polymorphism in iranians that has not been described previously. genomic dna was isolated from whole blood. for detection of cyp c * , and cyp c * variants, a protocol based on pcr technique and endonuclease digestion with kpni, ava ii was used. in this research work, we have studied a group of patients, in which warfarin therapy was initiated. recently new -residue antimicrobial peptides -arenicins were isolated from coelomocytes of marine polychaeta arenicola marina and their sequences were determined [ ] . there are two isoforms of arenicins which differ only with single amino acid. these peptides have no structure similarity to any previously identified antimicrobial peptides. we have synthesized and estimated the antibacterial properties of arenicin- : rwcvyayvrvrgvlvryrrcw. the linear peptide was prepared by solid phase method using boc-technology without any problem. however the cyclization caused the appreciable difficulties. the following methods of oxidation were used: oxygen of air, k fe(cn) and hydrogen peroxide in aqueous or organic media. the best results were obtained by using hydrogen peroxide in methanol, but and in this case the yield of the aim peptide did not exceed %. synthetic arenicin had the same hplc profile and maldi-tof spectra as a natural molecule. the peptide showed an antimicrobial activity against gram-positive bacteria: peptidergic hormones and neurotransmitters are known to be produced by the specific cleavage of their precursor proteins that per se have no biological functions. the neutrophil-activating peptides we recently identified, however, are the peptides cleaved from mitochondrial proteins by proteolysis. therefore, we named them "functional cryptic peptides" because they are hidden in protein sequences. some of these peptides activate gi type of g proteins directly, and neutrophils are suggested to be stimulated by the direct (i.e., not via gpcrs) activation of g proteins. these peptides had features, in common, in their distributions of charged and hydrophobic amino acid residues, but homologies in their primary structures were not apparent. in the present study, we predicted functional cryptic peptides that activate g proteins, based on the distribution of charged and hydrophobic residues. receptors for these peptides were also investigated by the direct cross-linking experiments between peptides and their targeted proteins. the finding of functional cryptic peptides is expected to lead to the identification of novel signaling mechanisms where such peptides are involved in the regulation of bio-functions. the fragment - of the precursor of human interleukin- alpha (pil- α) (gk-vlkkrr) appeared to have more than % homology with corticotropin fragment - (gkpvgkkrr). we have previously synthesized the octapeptide gkvlkkrr (referred to as leucocorticotropin, lct) and found its high affinity binding to corticotropin receptors on various immunocompetent cells in human and mouse. in this study we investigated the interaction of lct with rat adrenal cortex membranes and the effects of lct on the level of -oxycorticosteroids (cs) in rat adrenal glands and plasma in vivo. lct was labeled with tritium by the high-temperature solid-state catalytic isotope exchange reaction to specific activity of ci/mmol. receptor binding studies revealed that tritium-labeled lct bound with high affinity and specificity to corticotropin receptor on rat adrenal cortex membranes (kd = . nm). lct at concentrations of . - nМ was found to have no influence on the adenylate cyclase activity in adrenal cortex membranes, while intranasal injection of lct to rats at doses of - microg/kg was found to inhibit the secretion of cs from the adrenals to the bloodstream. thus, lct is an antagonist of corticotropin receptor. comarin derivatives such as warfarin are prescribed widely for treatment and prevention of thrombosis. warfarin is the widespread oral anticoagulant drug employed, but its required dose is highly variable both inter-individually and inter-ethnically. so it is desirable to develop strategies to predict the warfarin dose response in patients before initiation of anticoagulation. the vitamin k-dependent γ-carboxilation system, consists of the vitamin k-dependent γ-carboxylase, which requires the reduced hydroquinone form of vitamin k as a cofactor and the warfarin sensitive enzyme vitamin k , -epoxide reductase (vkor), which produces the cofactor. warfarin exerts its anticoagulant effect by inhibiting the vitamin k epoxid reductase enzyme complex (vkor) that recycles vitamin k , -epoxide to vitamin k hydroquinone. a component of the vkor termed vkorc , has now been identified as a therapeutic target site of warfarin. point mutations were identified within the gene encoding vkorc in individuals who required large doses of wafarin to maintain therapeutic anticoagulation. however the relationship between the primary structure of vkorc and the mechanism of action of warfarin is poorly understood. in previous works we have shown that naturally occurring functional protein fragments affect cell proliferation [ ] . their mechanisms of action involve receptors of "classical" regulatory peptides, or are non-receptoric [ ] . among protein kinases involved are pka, camkii, mapk [ ] . in organism bioactive functional protein fragments could participate in maintenance of tissue homeostasis. in present work, homeostatic potential of functional protein fragments was studied in compare with classical regulatory peptides. the panel of test substances was formed, including signal transduction modulators (pka, pkc, ca +-channel activators), classical regulatory peptides (bradykinin, somatostatin, met-enkephalin, endothelin, neurotensin) and fragments of functional proteins (β-actin fragments from ( - ) and ( - ) segments; valorphin (β-globin ( - )); neokyotorphin (α-globin ( - ); short acidic peptides from multiple precursors). their activity was tested in cultures derived from similar sources but differing in transformation degree (e.g., mouse embryonic vs. tumor fibroblasts) and/or culturing conditions. the factors most affecting cell sensitivity to the test substances were (in order of the importance decrease): ( ) cell type; ( ) transformed vs. normal phenotype; ( ) cell density; ( ) serum supply. activity of fragments of functional proteins, showing general correlation with other test substances, was more influenced by the culturing conditions (i.e., cell population status). thus, fragments of functional proteins could be regarded as partners of "classical" homeostasis regulators, playing role of finer tuners of tissue proliferative status. the study was supported by ras presidium programme "molecular and cell biology" histone-like proteins in bacteria are small basic proteins that contribute to the control of gene expression, recombination and dna replication. they are also an important factor in compressing the bacterial dna in the nucleoid. among the hlps, hu protein is attracted to dna containing structural aberrations such as four way junctions or single stranded lesions. this protein plays an important role in binding as a dimer and bending dna. it also contributes to the beginning of the dna replication. in this study we showed that a -kda protein, probably hu exists in halobacillus karajiensis which is a novel gram positive moderate halophile bacteria that was recently isolated from surface saline soil of the karaj region, iran. since hu is purified and characterized in e.coli we used this bacteria as the control in this study. the -kda protein extraction was carried out by using pca % which is normally used for extracting histones from eukaryotic cells. the results of running the protein extracts on sds-page demonstrated a band around -kda which was seen in protein extracts of this protocol. these results supported the hypothesis of the existence of a -kda protein in halobacillus karajiensis. sufficient oxygen and nutrient delivery is a necessity for tumors. when oxygen supply decreases, tumors initiate growth of new blood vessels. low grade astrocytomas, a class of malignant brain tumors, grow along the existing vessels in a process called co-option. hypoxia is induced in the progression from grade iii to grade iv astrocytomas (glioblastoma multiforme, gbm) which in turn triggers the formation of a new and distinct tumor vasculature. the new vessels formed by tumor-triggered angiogenesis differ by molecular composition from their normal vascular counterparts. we are utilizing phage displayed peptide libraries to identify peptides that specifically home to either co-opted or angiogenic brain tumor vessels. furthermore, we aim at characterizing differentially expressed endothelial markers (receptor molecules) to get a better understanding of the molecular changes in the vasculature. several rounds of in vivo biopanning was performed in mouse models of astrocytomas to isolate a phage pool that has up to a hundred-fold homing to low grade tumor lesions. out of the selected pool we discovered peptides capable of homing and accumulating to the tumor islets and co-opted vasculature. the homing potential of our newly identified peptides has shown to be highly specific for clusters formed by the tumor cells and co-opted "early" vessels within these palisades. these homing peptides represent promising candidates to selectively target co-opted vessels and tumor lesions in the brain and act as lead compounds in identification of surface molecules (receptors) differentially expressed by co-opted tumor vessels. the αvβ integrin receptors play an important role in human tumor metastasis and growth. the inhibition of these receptors by antibodies or by cyclic peptides containing the arg-gly-asp( rgd) sequence may be used as selectively treatment to suppress the disease. our research group has previously described that the formal introduction of a single carbon atom to bridge the cα (i) and n(i+ ) contiguous residues of a linear or cyclic peptide leads to α-amino-β-lactam peptidomimetics containing predictably placed β-turn and γ-turn motifs, respectively. the combination of these results with the well-known capacity of rgd tripeptide for inhibition of the biological answer in integrin led us to the design of the following cyclic peptide. the adhesion and cell-growth "in vitro" assays using human umbilical vein endothelial cells (huvec), as well as "in vivo" assays with xenograph mice revealed that the rgd peptidomimetic was active to micromolar concentrations, slightly better than the reference compound in this field: cilengitide®. whole saliva is composed of secretions from parotid, submandibular and sublingual glands, and smaller ones from saliva of minor salivary glands (e.g. palatal and labial). saliva contains a variety of proteins and polypeptides. one of them is statherin, a multifunctional -amino acid residue, phosphominiprotein. this peptide is present in human parotid and submandibular saliva . the aim of our study was to investigate the stability of statherin in extracts of the major salivary glands. submandibular, sublingual and parotid gland tissues were obtained at autopsy h after death. samples of the gland tissues were homogenized, centrifugated ( , g, min., c) and the supernatants were frozen and stored at - c prior to analysis. synthetic statherin was added to the supernatants before analysis ( microgram/ml). the samples were analysed for the presence of the peptide by the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (maldi-tof ms}technique and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds page). statherin has been found to be decomposed in extracts of parotid and submandibular glands and also in the extract of sublingual glands. the dramatic increase in research for new anthrax therapeutic approach was prompted by potential use of the causative agent of anthrax bacillus anthracis as a biological weapon. anthrax toxin consists of three proteins, the protective antigen (pa) and the two enzymes lethal factor (lf) and edema factor (ef) that are carried through the membrane of the target cell upon binding to specific site on the membrane receptor-bound pa. lethal factor and edema factor were found to cooperate to promote immune evasion of the bacterium. here we describe the production of peptide inhibitors of pa-lf binding, obtained by selecting pa-binding peptide by a competitive panning of a phage peptide library, using recombinant lf. we selected several mer peptides, which were synthesized in tetra-branched multiple antigen peptide (map) form, inducing resistance to proteolytic degradation ( ) and maintaining biological activity of phage peptides. lead tetra-branched peptides were systematically modified by progressive shortening and residue randomization, to obtain an increase of peptide affinity and inhibitory efficiency. affinity maturation of lead sequences enabled selection of a peptide which has an ic at least one log lower that any other lethal-toxin-inhibiting peptide described so far and is effective for in vivo neutralization of anthrax toxin activity ( ) . the same peptide can also efficiently inhibit the binding of ef to pa and ef-induced camp increase in different cell lines. microtubules are dynamic polymers that have important roles in eukaryotic cellular processes such as signal transduction, cell polarity, vesicular transport and chromosomal movement. the dynamic behavior of microtubules has been studied both in vivo and in vitro. the effect of arsenic trioxide on microtubule polymerization has been studied under in vivo experimentation shown that it inhibits formation of mitotic bundles. we studied the mechanism of arsenic trioxide effect on polymerization of microtubule protein purified from sheep brain in vitro. microtubule polymerization has been conducted by adding mm gtp to purified tubuline in pem buffer at oc for minutes and simultaneously followed by measuring turbidity ( nm). the results shown that lag time of polymerization (nucleation step) is affected by increasing concentrations of arsenic trioxide from - micromolar. moreover the rate of elongation step was decreased exponentially by increasing arsenic trioxide concentration. electron micrographs also showed microtubules length decrement due to arsenic trioxide. the results have shown the inhibitory effect of arsenic trioxide on microtubule polymerization via its effect on nucleation step as well as elongation rate. background and aims: alzheimer's disease (ad) is the major cause of dementia among the elderly. the increase in life expectancy worldwide demands new therapies for ad urgently. self-association of the amyloid ß-protein (aß) into neurotoxic assemblies, a seminal event in the etiology of ad, is considered to follow interactions of the c-terminus of the -residue form of aß (aß ). we hypothesized that molecules with high affinity for the c-terminus of aß will disrupt aß oligomerization. a series of c-terminal fragments (ctfs) of aß , aß(x- ) with x = - , has been prepared to study their potential to inhibit aß oligomerization and neurotoxicity. methods: attenuation of aß assembly by ctfs was studied by quantitative analysis of oligomer size distributions using a photo-cross-linking assay followed by sds-page. biological activity of ctfs themselves and as inhibitors of aß -induced neurotoxicity was assessed by mtt reduction assay using differentiated pc- cells. the structure of the ctfs was studied by circular dichroism (cd) spectroscopy and ion mobility spectrometry-mass spectrometry (ims-ms) coupled with molecular dynamics (md) simulations. results: ctfs were found to inhibit aß oligomerization in a length dependent manner with minimal or no toxicity of the ctfs themselves. certain ctfs were found to inhibit aß -induced neurotoxicity. cd spectra indicate that increasing peptide length results in growing ß-sheet content. structures based on experimentally determined cross-sections support the existence of a previously proposed turn around residues gly -gly . the data suggest that aß ctfs can serve as lead compounds for development of peptidomimetic drugs for treatment and prevention of ad. background and aims: human kallikrein hk is a prostate specific serine protease, which expression level is elevated in aggressive human prostate cancer suggesting a possible role in a tumour growth and spreading. since hk protease is highly prostate specific, inhibition of its activity is a possible method to prevent tumour growth without interfering the function of the other proteases. we have identified hk specific linear peptide inhibitor by using phage display techniques. in order to design peptide for in vivo studies we tested the protease stability of the linear and the cyclic forms of the peptide. methods: the prerequisite of the binding was studied by using conventional ala-replacement method and the most optimal sequence was selected for further studies. the stability of the original linear form, acetylated form, peptide with cystein bridge and head-to-tail cyclic peptide was tested with modified trypsin (sequencing grade) and with human plasma. results: both linear versions and peptide with cystein bridge were unstable and were degraded during the first minutes in both stability tests. head-to-tail form of the peptide was stable in both tests during the first minutes. conclusions: since our peptide contains arginine there was a possibility that our peptide is sensitive to trypsin and other serum proteases. indeed both linear and one cyclic from degraded in our tests. only head-to-tail peptide was stable during the first hours suggesting protease resistant folding. background and aims: a large number of anticancer agents has been developed in recent years. however, these agents have very little or no specificity which leads to systemic toxicity. among them paclitaxel is considered to be one of the most important drugs in cancer chemotherapy; however, this agent has also lack of selectivity to the tumor tissue. therefore, development of tumor-targeting prodrug is highly promising. methods: to activate cytotoxic agent specifically at the tumor tissue, we developed a new prodrug strategy based on o-n intramolecular acyl migration, which is a well-known reaction in peptide chemistry, and photodynamic therapy. results: we synthesized a prodrug which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (o-acyl isoform of paclitaxel). the prodrug was selectively activated by visible light irradiation ( nm) leading to cleavage of coumarin. finally, paclitaxel was released by subsequent o-n intramolecular acyl migration. conclusion: we synthesized and evaluated a novel type of paclitaxel prodrug. this prodrug showed promising kinetic data. therefore, we believe that photoactivation can be promising novel strategy for design of tumor-targeting prodrugs. the search of new immunosupressants, exhibiting the mechanism of action characteristic for cyclosporine a (csa) and fk- is an important challenge for medicinal chemistry. cyclolinopeptide a (cla) natural cyclic nonapeptide [cyclo(leu-ile-ile-leu-val-pro-pro-phe-phe)] possesses a strong immunosuppressive activity comparable with that of csa in low doses. the possibility of practical application of cla as a therapeutic agent is limited due to its high hydrophobicity. it has been suggested that the tetrapeptide sequence pro( )-pro( )-phe( )-phe( ) is responsible for the interaction of the cla molecule with the proper cellular receptor. in order to evaluate the role of this tetrapeptide unit for biological activity of native peptide, we decided to modified this fragment. in this communication we present linear and cyclic cla analogues in which phenylalanine residues in position and/or have been replaced with amphiphilic; alphahydroxmethylphenylalnine or homophenylalanine . the synthetic strategy and biological activity will be evaluated. resistance to currently used small molecule antibiotics develops at an alarming rate. while resistance to β-lactams in clinical isolates is primarily due to hydrolysis of the ring by β-lactamases, when bacteria develop resistance to fluoroquinolones or aminoglycosides, the sequences of the target biopolymers are altered. earlier we developed a family of antibacterial peptide derivatives that kill bacteria by inhibiting protein folding and are active in animal models of infection. in the current study we examined the synergy between antibiotics acting by different modes of action. inhibition of properly folded active resistance enzymes was completely efficacious to recover the activity of amoxicillin, a β-lactam antibiotic against strains that were originally resistant to this molecule. some activity of ciprofloxacin was also recovered by reducing the load of the induced self-defense dnak protein, but the synergy between the antibacterial peptide and the fluoroquinolone did not yield full bacterial killing. the mode of action of the synergy is indeed inhibition of protein folding because no such effect could be observed with kanamycin where resistance involves changes in the target protein sequence. as opposed to current β-lactamase inhibitors and combination therapies that work against only a limited number of strains, inhibition of all protein folding in bacteria is a universally applicable treatment option. elimination of resistance to β-lactams by proline-rich peptide derivatives may represent a viable avenue to give second life to these antibiotics for which large stockpiles are available for pharmaceutical companies in both patented and generic forms. the integrin αiibβ is the major integrin-adhesion receptor on platelets. in unstimulated platelets αiibβ is present in a resting conformation state. upon platelet activation by agonists, αiibβ receives intracellular signals (inside-out signaling) that allow its rapid conversion to a high-affinity state capable of binding soluble ligands, resulting in platelet aggregation. the intracellular signals include proteins that bind to the cytoplasmic tails of the two subunits α and β of the integrin, or integrin-associated membrane proteins. in vivo charge swapping mutation studies suggested that αiib and β tails have a direct site of interaction between αiib (r ) and β (d ). peptides derived from the cytoplasmic tail sequences can specifically induce or block αiibβ activation in platelets. the aim of this study is to develop peptide analogues based on the cytoplasmic tail sequences of both αiib and β subunits that could inhibit platelet thrombus formation by specifically disrupting the inside-out signaling pathway. peptide analogues of the αiib and β subunits spanning the sequences αiib- - , αiib - - , αiib - - , αiib- - and β - - , β - - , β - - were synthesized in their free state, palmitoylated and/or tagged with the tat fragment - and carboxyfluorescein-labeled, in order to investigate their membrane permeability, as well as their inhibition of the platelet aggregation. inflammatory pain begins when noxious stimuli (thermal, chemical or mechanical) excite sensorial neurons called nociceptors. the activation of nociceptors leads to the opening of some ionic channels and depolarization of the cell membrane. one of these channels is trpv , which is directly implied in thermal hyperalgesia associated to inflammation. in previous work it has been found that peptoid h-arg- - c ( fig. ) inhibits the activation of trpv by blocking the pore entrance. however, this compound showed toxicity in vivo. the aim of our work is the design and synthesis of new compounds, based on the structure of h-arg- - c, with better therapeutic properties. we synthesized some new non-competitive antagonists of trpv that exhibit notable anti-inflammatory and analgesic activity in vivo. th. skarlas, e. panou-pomonis, d. krikorian, m. sakarellos-daitsiotis, c. sakarellos erf is a transcriptional repressor with tumor suppressor activity regulated by the ras/erk signaling pathway. it has been shown that erf interacts with, and is phosphorylated by erks in vitro and in vivo. this phosphorylation determines its subcellular localization and biological function. erf exhibits a high degree of specificity and sensitivity for erks. the major objective of our study is to provide proof of principal for a specific anti-cancer approach targeting the ras-pathway, which is commonly activated in human tumors, via the stimulus of the downstream effector erf. this will be attained by modeling specific peptide inhibitors that block the erf phosphorylation and inactivation by the ras/erk signaling pathway. we present the design and synthesis of peptide inhibitors incorporating the fsf and fkf motifs, known to play a critical role in the erf/erk interaction, in their free forms or conjugated to a carrier. ubiquitinium is a well known mechanism in protein degredation of eukaryotic cells ,in which many obsolte and corrupted three dimentional structure protein ,become marked by covalent attachment of ubuquitin through a multi-step enzymatic pathway.ubiquitin is a small , . kda peptide of amino acid residues that targets such substrtes for proteolysis in proteasome .recnt studies showed that an extra cellular ubiquitination process also taking place in the epididymes of humans and other animals marks protein on the surface of the defective sperm .it appears that structurally and functionally defective sperm become surface ubiquitinated by epididymal epithelial cells. a certain portion of ubiquitin -labeled sperm is phagocytosed and the remaining is ejaculated .hence ubiquitin on the sperm surface could be a good marker of semen quality control in men. the aim of present study is to purify ubiquitin from packed blood cells , to produce and purify antiubiquitin antibodies,to design an immunofluorescence assy for detection of defective sperm, to compare the percentage of ubiquitinated sperm in oligoasthenotertozoospermia and normozoospermia and finally to determine correlations between sperm parameters and sperm ubiquitination. p. vakalopoulou, ch. anastasopoulos, g. stavropoulos, s. yiannis c-terminal analogues of substance p (sp) have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. the incorporation of damino acids into the sequence of sp and n-methylation of peptide bonds have shown to protect sp from the action of plasma and tissue peptidases. aiming to design and prepare more potential antagonist of cancer cells proliferation and taking into account that all the metabolites of the c-terminal hexapeptide analogue [arg , d-trp , , mephe ]sp - (antagonist g) possess the n-me group and d-trp residue, we proceeded to the synthesis of peptoid-peptide hybrids. they are oligomeric peptido-mimetics containing the residue [-Ν(bzl)-ch -co-]=(nphe). the incorporation of n-substituted glycine in peptide chains has been proved to improve their stability against proteases and give biologically active peptides. thus, the tetrapeptoid-peptide hybrids h-arg -d-trp -nphe -d-trp -oh and h-d-trp -nphe -d-trp -leu -oh, corresponding to metabolites of antagonist g and also the hexapeptoid-peptide hybrids glp -d-trp -Νphe -d-trp -leu -glu(obzl) -ΝΗ and glp -d-trp -Νphe -d-trp -leu -glu(obzl) -oh have been synthesized. the latter have incorporated the amino acid residues glp at the n-terminal and glu(obzl) at the c-terminal of the analogue, which have shown to give to the analogues increased resistance and biological activity. all the products were purified (hplc), identified (esi-ms) and set about for study their biological properties and activity against cancer cells proliferation. a chemokine receptor cxcr has multiple critical functions in normal and pathologic physiology. cxcr is a gpcr that transduces signals of its endogenous ligand, cxcl (stromal cell-derived factor- , sdf- ). the cxcl -cxcr axis plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems and so on. this axis has recently been proven to be involved in several problematic diseases, including hiv infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (ra) and pulmonary fibrosis. thus, cxcr is a great therapeutic target to overcome the above diseases. fourteen-mer peptides, t and its analogs, were previously found to be specific cxcr antagonists that were identified as hiv-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents and anti-ra agents. cyclic pentapeptides, such as fc [cyclo(d-tyr-arg-arg-l- -( -naphthyl)alanine-gly)], were previously found as cxcr antagonist leads based on pharmacophores of t . in this symposium, we would like to report the development of low molecular weight cxcr antagonists involving fc analogs and other compounds with different scaffolds including leaner-type structures. erythropoietin (epo) controls the proliferation and differentiation of red blood cells. it activates epor by inducing dimerization and reorientation of two receptor chains. peptides mimicking the action of epo, epo mimetic peptides (emp), have been discovered by phage display, interacting with the receptor on the active site and competing with the hormone [ ] . another peptide, epor derived peptide (erp), was reported to activate the receptor through an alternative site distant from the hormone binding site, and to have synergic action with epo [ ] . we report the design of new synthetic epo-r agonists by dimerization of active peptides. pegbased polyamide linkers of precise length were used to link the molecules, using oxime chemistry [ ] . these peptides include emps that have been homo-dimerized through their n-or c-terminus. a hetero-dimer of one emp and one erp peptides was also created. biological characterization of the molecules is currently under investigation. [ the envelope spike (s) glycoprotein of the severe acute respiratory syndrome associated coronavirus (sars-cov) mediates the entry of the virus into target cells. recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as hiv- . as it happens with other viruses peptidic fusion inhibitors, sars-cov s protein hr derived peptides are potential therapeutic drugs against the virus. it is believed that hr peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the hr region. it is a matter of discussion if the hiv- gp hr derived peptide t (enfuvirtide) could be a possible sars-cov inhibitor given the similarities between the two viruses. we used fluorescence spectroscopy techniques to test the possibility of interaction between both t (hiv- gp hr derived peptide) and t- with s protein hr and hr derived peptides. our biophysical data show a significant interaction between a sars-cov hr derived peptide and t . however the interaction is only moderate (kb = ( . ± . ) × m- ). this finding shows that the reasoning behind the hypothesis that t , already approved for clinical application in aids treatment, could inhibit the fusion of sars-cov with target cells is correct but the effect may not be strong enough for application. [ ] were used to investigate the structure, dynamics and thermodynamics of the known complex between erythropoietin mimetic peptides (emp) and erythropoietin receptor (epor). with gromacs . bioinformatics software, we have obtained from the known emps about the key functional amino acids required for effective epo mimetic action. then we systematically altered the amino acids in those peptides, and simulated the complex to observe the differences between the altered peptides with the original ones. based on these results, we designed new emps of potential significance. in order to fast identify the mimetic action of these new peptides, we synthesized these peptides and labeled the epor binding peptide (ebp) with quantum dots [ ] , to study the binding of these new emps to epor. our results illustrate a principle for fast identifying receptor-specific sites importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists. blood vessel formation largely contributes to the pathogenesis of numerous diseases, including ischemia and cancer [ ] [ ] . in this regard therapeutic strategies aim to stimulate vascular growth in ischemic tissues and suppress their formation in pathologies like in tumour and diabetic retinopathy. placental growth factor (plgf), an homolog of vascular endothelial growth factor (vegf), ( % amino acid sequence identity), stimulates angiogenesis and collateral growth in ischemic heart and limb. whereas vegf exerts it biological function through the binding to both vegf receptor- (vegfr- or flt ) and vegfr- (or kdr) plgf binds specifically to flt . the complex plgf/flt constitutes a potential candidate for therapeutic modulation of angiogenesis and inflammation [ ] . the binding between plgf and flt- has multipunctual features [ ] and potential antagonist must have a sufficient molecular surface to spatially distant contact points. we have used an elisa-like screening assay to select antagonists of plgf/flt- complex from a large random library of tetrameric unnatural peptides (complexity: ^ = . molecules) identifying two active molecules with an about m ic . the relative stability of identified peptides were assessed in human serum and their inhibitory properties were tested in a capillary-like tube formation assay performed with human umbilical vein endothelial cells (huvec the αvβ integrin is a cell adhesion receptor involved in angiogenesis and tumor cell invasion. the tripeptide motif rgd is the αvβ minimal recognition sequence and many rgd-containing peptides have been investigated as radiopharmaceuticals for targeting angiogenesis and tumor metastatic phenotype. since rgd sequence binds also to other integrins, the aim of the present study was to develop and characterize a selective αvβ ligand suitable for imaging. a novel peptide containing the rgd loop covalently linked to an echistatin domain (crgdechi) was designed, synthesized and then tested for selective binding to αvβ integrin. a panel of peptides were used for comparison. adhesion assays showed that the novel peptide was able to inhibit adhesion of αvβ overexpressing cells but not αiibβ and αvβ overexpressing clones. in conclusion the novel peptide showed a high affinity and specificity for αvβ integrin. the design of new molecules, based on the lead compound presented here, is currently ongoing with the aim at developing novel anticancer drugs and/or new class of diagnostic noninvasive tracers as suitable tools for αvβ -targeted therapy and imaging. background and aims: short peptides like leu-pro-phe-phe-asp (lpffd) and leu-pro-tyr-phe-asp-amide (lpyfda) can influence the structure and aggregation of ß-amyloid peptides. soto's pentapeptide lpffd has been published as a ß-sheet breaker (bsb). it is necessary to gain more information about the nature of the interaction of aß and the pentapeptides mentioned above for the understanding of their action and for the possible development of future therapeutic agents. methods: in this study radioligand binding assay, diffusion ordered nmr spectroscopy, dynamic light scattering, circular dichroism and ft-infrared spectroscopy was used. results: it was shown by radioligand binding assay and diffusion ordered nmr spectroscopy that both pentapeptides bind to aggregated aß. dynamic light scattering, circular dichroism and ft-infrared spectroscopy revealed, that after the treatment of the aß with the pentapeptides aß fibrils are still present. conclusion: both peptide can bind to aß and can cause small conformational changes of aß, however, they cannot prevent completely the formation of aß fibrils in - micromolar concentration using : molar ratio of aß and the bsb peptide. peptide arrays are convenient tools for the analysis of antibodies, protein binding domains and to address other biological questions. here we present a new method to produce identical copies of arrays on microscope slides. the peptides are synthesized on modified cellulose-discs, using a variation of the spot-method introduced by ronald frank more than years ago [ ] . the new array format overcomes several limitations of the spot-method, e.g. the low throughput with only one copy of the library and the large sample volumes that are needed for membrane incubations. for the presented arrays modified cellulose discs with covalently bound peptides are dissolved after synthesis. the resulting solutions can be spotted onto glass slides by conventional spotting techniques. three dimensional layers of cellulosepeptide molecules are formed on the surface of the supports used for spotting. a virtually unlimited number of identical arrays can be printed and assays are performed with a sample volume of µl or less. as application example we show mapping experiments of the streptavidin recognition site with a peptide library containing histidine-proline motives. because of the much higher peptide loading compared to conventional arrays, the formed -dimensional structure might be superior for protein-interaction studies with even low binding constants. [ the interaction between the cap binding protein, eukaryotic initiation factor (eif) e, and the scaffolding protein eif g is critical for the formation of the heterotrimeric eif f translation initiation (ti) complex (eif e/eif g/eif a). elevated levels of eif e and eif g found in several human solid tumor cancers and the induction of malignant transformation in animal models by overexpression of eif e and the reversal of this phenotype by treatment with anti-sense rna, suggest the importance of the eif e/eif g interaction in the excessive translation of oncogenic proteins. eif g binds to eif e through a conserved eif e-binding motif yx l (non-specified (x) and hydrophobic ( ) amino acids) that interacts with an hydrophobic hot spot on eif e. we report here the identification of a putative eif e anionic exosite that is distinct from the hot spot and contributes to the binding of eif g-derived ligands. our strategy focuses on in situ eif e-templated click reaction-mediated assembly of hybrids comprised from an anchoring minimal eif g-derived peptide fragment, which binds to the hot spot, and a series of complementing positively charged fragments targeting the anionic exosite. we synthesized a training set of [ , , ] triazole-containing hybrid peptides that are potent inhibitors of eif e/eif g interaction. moreover, we achieved in situ eif e-templated assembly of these hybrids from the corresponding fragments via click reaction in the absence of cu(i) catalysis. as such, we demonstrate a proof-of-concept for a new paradigm in the development of inhibitors of protein-protein interaction merging click reaction with fragment-based and in situ target-templated approaches. goodpasture disease is an autoimmune pathology caused by the accumulation of reactive autoantibodies against the alpha- of collagen iv. goodpasture antigenbinding protein (gpbp) is a ser/thr protein kinase that phosphorylates the alpha- chain and might be important in human autoimmune pathogenesis [ ] . we are carrying out in our laboratories the biophysical and functional characterization of gpbp protein. in the presence of some proteins and at specific experimental conditions, gpbp participates in structurally ordered intra-and inter-protein aggregation processes. structure prediction programs identify four different domains for gpbp: an n-terminal domain showing pleckstrin homology (ph domain); a central domain with high tendency to form coiled-coils; a domain with ww features; and a c-terminal start domain ('star-related lipid-transfer'). using the tango algorithm [ ] , we have identified several aminoacid sequences in the gpbp start domain of vertebrates with high tendency to participate in protein aggregation. in this work we present the synthesis and structural characterization of a collection of peptides derived from the sequences described above. we recently developed a combinatorial library screening protocol to identify hpq-containing cyclopeptides that bind streptavidin more tightly than its linear analogues. the relative affinities in ic of these structurally constrained ligands and its linear counterparts were measured by a captured enzyme-linked immunosorbent assay. however, their intrinsic binding kinetics remained to be elucidated. in this work, surface plasmon resonance (spr) was employed to directly determine the kinetics and thermodynamics of the ligands binding to a streptavidin chip. solid-phase peptide synthesis was carried out using standard fmoc chemistry. spr experiments were carried out using biacore optical biosensor. streptavidin was immobilized onto a cm sensor chip using the standard amine coupling procedure. the equilibrium dissociation constants and kinetic on/off rates of n-to-side chain and n-to-c cyclopeptides were deduced by scatchard analysis and computational simulation, respectively. it was found that both cyclopeptides exhibited similar binding kinetics and bound streptavidin far more avidly than its linear form ( -fold). in addition, the reversed (qph) linear and cyclic peptidyl ligands were hardly recognized by streptavidin. not only the binding specificity was distinguished qualitatively, but also the entropic advantage acquired by the pre-organized conformation over its linear analogues was demonstrated quantitatively by spr in this study. the mutation of tumour suppressor genes in the progression of cancer is well characterized. for example, p is found to be mutated in approximately % of cancers and the loss of this proteins activity has been shown to lead to the deregulation of cell growth and apoptosis. the potential of peptide aptamers to inhibit protein/protein interactions in a highly specific manner makes them very attractive as research reagents or as target validation tools in anti-cancer drug discovery. more interestingly, these molecules have the potentially to inhibit the activity of proteins which are key regulators of cancer cell growth and therefore could act as synthetic tumour suppressor proteins. we used peptides based on known protein/protein interactions, as well as peptides isolated using display technologies, for the design of protein aptamers that were used to analyze pathways critical in controlling cancer cell growth. a range of scaffolds were used to present these peptides in an effort to optimize the peptides activities. data relating to the activity of these peptide aptamers in vitro as well as in cellular systems will be discussed. the cyclic undecapeptide urotensin ii (u-ii) is the endogenous agonist for the u-ii receptor (ut), a gq coupled gpcr. current views suggest that binding by agonist, but not antagonist, leads to induction of stabilization of an active receptor conformation. we have previously probed the interactions of urotensin ii with rat ut (rut) using a series of photolabile u-ii analogues containing p-benzoyl-l-phenylalanine (bpa). it was found that the c-jun n-terminal kinases (jnks) are important mitogen-activated protein kinases. these ser/thr protein kinases are activated by various growth factors, cytokines, and cellular stresses. jnks have been shown to play a key role in phosphorylation of proteins in signal transduction of different diseases including cancer, neurodegenerative, cardiovascular, and inflammatory diseases. therefore, these enzymes are considered as important therapeutic target proteins. the interactions of jnk with peptides are of special interest for development of novel specific atp-noncompetitive inhibitors. interactions of this kinase and its mutants with various substrates were demonstrated in vivo using yeast ras-recruitment system. bioinformatical tools have been developed to predict optimized binding peptides as well as to correlate sequence position and amino acid with binding effiency to extract binding determinants. biomolecular interaction analysis have been performed for selected peptide sequences using surface plasmon resonance (spr) technology. real time measurements of the binding of peptides to the different isoforms jnk and jnk resulted in the determination of affinities as well as kinetic constants for association and dissociation. experimental results and their bioinformatic analysis are discussed with respect to critical features of potential atpnoncompetitive inhibitors. the b-domain of is one of the five nearly homologous domains of staphylococcal protein a. this domain contains three alpha-helices which are assembled in an anti parallel three-helix bundle. the b-domain binds the fc region of mammalian immunoglobulins through the n-terminal fragment that contains two alpha-helices. the c-terminal helix does not interact with fc but it is necessary for the correct folding and immunoglobulin recognition of the b-domain. to search for new peptide analogues of the c-terminal helix that bind the n-terminal fragment, a ¨one-bead one-compound¨ library of peptides was designed based on the sequence of the c-terminal helix. active peptides were obtained after incubation of the library with the n-terminal fragment and rabbit immunoglobulin g labelled with fluorescein. new peptides were found and their sequences identified by maldi tof-tof mass spectrometry. the synthesis of the two most active peptides was carried out and the binding with the n-terminal fragment was confirmed by cd spectroscopy. the nterminal fragment peptide showed an increase in helicity when the c-terminal wild type peptide or some analogues were present in solution. the complete domains with the c-terminal fragment mutations were synthesized and structurally characterized by cd and nmr spectroscopy. the wild type and the new mutants adopt predominantly an alpha-helical structure. the interaction between rabbit immunoglobulin g and the wild type b-domain and the new analogues was investigated using surface plasmon resonance. although compared with the wild type, the mutants exhibited different kinetics, they were able to bind the immunoglobulin with high affinity. a. jaśkiewicz, e. bulak, h. miecznikowska, k. rolka sfti- , a strong trypsin inhibitor, was isolated in from seeds of sunflower. it is homodetic -amino-acid-residue peptide containing a disulfide bridge. because of its small size and strong trypsin inhibitory activity, this inhibitor became an interesting model for studying enzyme -inhibitor interactions. sfti- possesses one reactive site located at the lys -thr peptide bond and therefore is able to interact with the enzyme in a : stoichiometry. in this report we describe chemical synthesis and kinetic studies of a series of sfti- analogues containing double sequences of the wild inhibitor. their structures contain combinations of disulfide bridges and/or head-to-tail cyclization. each of these analogues contains two trypsin-specific reactive sites. we expect that kinetic studies should answer the question whether such dimeric analogues are able to interact simultaneously with more then one trypsin molecule and how this fact affects their inhibitory potency. in addition, we alsopresenttwo analogues in which we substituted the disulfide bridge with a carbonyl one. since carbonyl bridge has not been previously introduced into molecules proteinase inhibitors, we decided to check its impact on the activity and proteolytic stability of such modified analogues. ubiquitination, the covalent attachment of one or multiple polymerized ubiquitins is a post-translational modification of proteins, which has manifold functions. it mainly determines the protein for degradation, but also activation, deactivation or substrate alteration. due to its ubiquitinous distribution in all eucarionts no high-affinity antibodies could be originated. highaffinity ligand peptides are of interest to study ubiqutination. based on bioinformatical considerations and investigations of ubiquitin-interacting proteins short peptide sequences were selected. by using a peptide array specific ubiquitin binding was monitored and quantified with label free detection based on reflectometric interference spectroscopy (rifs). the results from rifs were confirmed by detection of binding in solution with fluorescence correlation spectroscopy (fcs) using carboxyfluorescein and s -labelled peptide amides. binding constants were determined by isothermal calorimetry (itc) and rifs. finally h, n-nmr chemical shift analyses of the peptides with the highest affinity were carried out, which allowed the localisation of the interaction site of ubiquitin with the peptide the results from all four methods correlated very good. they showed fast equilibria within s and binding constants down to the low micromolar range. nmr results revealed hints for discrimination possibilities between lys and lys polymerized ubiquitins. ( f is a potent neutralizing mab that binds the human respiratory syncytial virus (hrsv) f protein and is a promising candidate for clinical development. the majority of neutralizing antibodies to hrsv f protein map to two regions of the protein designated site ii and site iv,v,vi. to further characterize the f epitope, we employed a trypsin digestion of a hrsv f protein- f mab complex, followed by mass spectrometry analysis of the resulting recovered mab bound peptide. one peptide at m/z was captured by the f mab. sequence assignment was based upon mass and matched with the database from a virtual digest. this peptide was assigned as residues - [tkctasnknrgiiktfsngcdyvsnk] of the hrsv f protein which spans antigenic site iv,v,vi. to further delineate the epitope, the binding of f mab to a series of peptides corresponding to antigenic site iv,v,vi in the hrsv f protein was determined. based on the peptide elisa data, the f-binding region could be reduced to - sequence [ctasnknrgiiktfs] . as demonstrated by the substitution analysis, r and k significantly contribute to epitope binding, but another positively charged residue, k makes a minor contribution to the binding. both, the peptide elisa and proteolytic digestion of the mab-antigen complex approaches identified the same region of hrsv f protein as being critical for the binding of f. furthermore, these data confirm the results obtained using complementing genetic approaches using a panel of mutations in recombinantly expressed f protein and selection of antibody escape virus mutants (data not presented). the recently identified uracil-dna specific nuclease (ude) is the first representative of a new family of nucleases. the protein sequence has no detectable homology to other proteins except a group of sequences present in genomes of other pupating insects (vertessy et al, submitted). to analyze the physiological function of this protein, peptide conjugates were prepared to serve as synthetic antigens for the generation of antibodies against isoforms of dutpase, an enzyme inherently involved in preventing the synthesis of uracil-dna [ , ] . we used poly[lys(seri-dl-alam)] (sak) as a synthetic branched polypeptide [ ] or bovine serum albumin (bsa) as a natural macromolecular carrier. peptides were prepared by solid phase method utilizing syro (mulltisyntech gmbh, germany) peptide synthesizer, using fmoc chemistry and dipci/hobt-mediated coupling on rink-amid mbha resin. a c-terminal cys(acm) was added to the native sequences for incorporation of sh group into the peptide. in case of sak choroacetylated polypeptide was conjugated with sh-peptide to form thioether linkage. the maleimidobenzoyil-n-hydroxyszukcinimid (mbs) derivative of bsa was used to introduce the peptide into the macromolecule. antibodies have been developed as diagnostic tools and therapeutics for many different diseases. however, the isolation and preparation of intact specific antibodies is often very tedious or even unfeasible. recent studies have shown that single paratope peptides might be well capable to mimic corresponding antigen ligands [ , ] , suggesting that paratope peptides from a native antibody might have many advantages, e.g. for molecular vaccine design and targeting. we have developed a new method for identification of paratope-containing peptides by proteolytic affinity-proteome analysis in combination with high resolution fticr-mass spectrometry (fticr-ms) [ ] . in the present study we used hen eggwhite lysozyme (hel) and a polyclonal rabbit anti-lysozyme antibody (helpab) as a model system. the direct determination of paratope peptides was obtained by selective binding of a dtt-cleavage mixture of the anti-lysozyme antibody to immobilised hel, followed by proteolytic digestion of the antibody-antigen complex (paratope excision, parexprot). two specific paratope peptides were identified by maldi-fticr-ms, and the corresponding peptide sequences were identified by database search within a - ppm threshold. additionally, the identified paratope peptides were synthesised and characterised by affinity mass spectrometry, which ascertained their full binding specificity to lysozyme. the propeptide blocks the active site of inactive zymogen of cathepsin d and is cleaved off during maturation. we have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin d using kinetic activity assay. the mapping localized two segments in the propeptide involved in the inhibitory interaction with the enzyme core: n-terminus of propeptide plays a major role and the active site anchor plays a minor role according to their respective ki values. in addition, a fragment derived from the mature n-terminus of cathepsin d displayed inhibition, which supports its proposed regulatory role. the mechanism of interaction of both propeptide segments was characterized by the mode of inhibition and by spatial modeling of propeptide in cathepsin d zymogen. using fluorescence polarization measurements, kd in nanomolar range was determined for the n-terminal propeptide segment. the inhibitory potency of the active site anchor segment was modulated by ala val mutation that was reported to be associated with cathepsin d pathology. . by comparing the resulting low-energy conformations using different sets of atoms, specific conformational features common only to the high/medium affinity compounds were identified. they included the spatial arrangement of the three most important pharmacophoric side chains tyr , arg , and nal as well as the orientation of the xaa -arg amide bond, which together represent a "minimalistic" d pharmacophore model for binding of the cyclopentapeptide antagonists to cxcr . this model rationalizes the data for the cyclopentapeptides as well as for the peptidomimetic cxcr antagonist krh- . automated docking of the pharmacophore model to the d structure of the tm region of cxcr revealed that the pharmacophoric groups of the cyclopentapeptide ligands were involved in favorable interactions with their counterparts in cxcr . for instance, the hydroxyl group of tyr formed a hydrogen bond with lys , the guanidino group of arg formed a salt bridge with glu , and the backbone carbonyl of xaa -arg formed a hydrogen bond with lys . this finding gives additional support for the suggested d pharmacophore model, and also provides opportunities for rational design of cxcr mutants to map potential contacts with peptide ligands. with the successful completion of the human genome project, the next challenge is to assimilate enormous amount of genetic information generated and to assign functions to a large number of proteins encoded. although the dna chip technology to detect the abundance of mrnas has been established, it is known that the abundance of mrnas and proteins does not correlate. thus, protein detection methods for reproducible and quantitative investigation of protein networks are strongly required. we attempted to establish a novel protein detection system based on a fluorescent measurement that does not require labeling of target molecules and preparation of secondary antibodies. we focused on a steric hindrance caused by the interaction between a target protein and a specific capture agent. when a target protein interacts with a specific capture agent immobilized on solid surface, we assumed that a steric hindrance in the vicinity of a capture agent increases. in order to detect the differences in the steric hindrance, we utilized a fluorescent system with the staudinger reaction. this reaction is a chemical ligation between a phosphine and an azide group. these two functionalities are unreactive with protein surfaces under biological conditions. we incorporated an azide group into an immobilized capture agent and investigated the efficiency in the staudinger reaction between the azide and an external triphenyl phosphine derivative. it was found that a target protein bound to the capture agent immobilized onto the solid support interferes with the efficiency in the staudinger reaction. the major histocompatibility complex (mhc) has a crucial role to initiate the immune response via the binding of the peptide fragments (epitopes) of foreign antigens and their presentation to the t-cell receptors (tcr). the co-receptor molecule cd enhances the binding between tcr and mhc ii. small molecules that mimic surfaces of mhc-ii may lead to blockage of the autoimmune response and the development of drugs for immunotherapy. hla-dqa * /dqb * (dq ) and hla-dqa * /dqb * (dq ) are highly correlated to autoimmune diseases as sjogren syndrome (ss) and systemic lupus erythematosus (sle). the non polymorphic β regions of the modelled hla-dq , which are exposed to the solvent and may disrupt the interaction of dq with cd + t lymphocytes were determinated using the getarea program. it was found that the regions - (arg-asn-asp-gln-glu-glu-thr-thr) and - (glu-tyr-trp-asn-ser-gln-lys-glu) display the highest solvent accessibility. peptide analogs of these regions were synthesized, by the fmoc/otbu solid phase strategy, purified by rp-hplc and characterized by mass spectrometry esi-ms. the dimeric analogs of the peptides, designed to mimic the superdimeric nature of the immunosuppressory fragments of hla class ii molecules were also synthesized and investigated. conformational studies were performed with cd spectroscopy and biological experiments are in progress. background and aims: aggregates of β-amyloid peptide (aβ) play central role in the etiopathology of alzheimer's disease (ad). short peptides like c. soto's pentapeptide lpffd and lpyfd-amide synthesized in our laboratory are neuroprotective agents against aβ assemblies both in vitro and in vivo. however, the mechanism of their neuroprotective effect has not yet been fully understood. methods: transmission electron microscopy (tem), cd, ft-ir, diffusion ordered nmr spectroscopy, dynamic light scattering, and radioligand binding assays were used. results: all the methods applied showed that the pentapeptides mentioned above do not break the fibrillar structure of aβ, that is these molecules are not real β-sheet breakers (bsb). the pentapeptides bind to aβ fibrils and cause small structural changes by intercalating into the aβ assemblies. fibrils of aβ survive one week treatment with the pentapeptides using them in to -time molar excess. conclusion: all the results in our laboratory show that the short peptides have long-term interaction on aβ-assemblies. in the first step they bind tightly to the aβ surface and prevent further interaction of aβ fibrils with the neuronal membranes. after this step the short peptides can be built into the structure of aβ-assemblies with intercalation causing a less ordered β-conformation. proteolytic enzymes (neprilisin, ide) could cleave and hydrolyze aβ peptides after this structural change, therefore the short peptides are good drug candidates for the treatment of ad. cellular processes in normal and pathogenic cell states are regulated by external stimuli via complex networks of catalytic and non-catalytic protein-protein interactions. we have developed methodology for the synthetic variation of peptides and peptidomimetics using polymer reagents including linker reagents enabling polymer-supported cacylations. [ ] in combination with the virtual screening of protein subsites, we have demonstrated the application of the novel synthetic methods to inhibitor optimization for various proteases including plasmepsin ii, hiv protease, and sars coronavirus main protease. [ , ] moreover, multivalent peptide polymers have been developed for the intracellular targeting of proteins. [ ] this methodology was now extended to the inhibition of peptide-protein interactions by small molecules. for this purpose, we have composed a library of , small molecules by algorithmic searching of a database of bioactive molecules with virtually designed substructures (fragments). high throughput assays were developed on the basis of fluorescence and fluorescence polarization detection. despite the scepticism regarding the inhibition of protein-protein interactions with small molecules, efficient hit molecules have been developed for several intracellular targets and were subjected to synthetic variation and cellular follow-up assays. the essential event in platelet adhesion to the blood vessel wall after injury or in thrombosis is the binding to sub-endothelial collagen of plasma von willebrand factor (vwf), a protein which interacts transiently with platelet glycoprotein (gp) ibalpha , slowing circulating platelets to facilitate their firm adhesion through other collagen receptors, e.g. integrin alpha beta and gpvi. to locate thevwf-binding site in collagen iii; we synthesized overlapping triple-helical peptides which comprise the whole native sequence of collagen iii . peptide # (gpogpsgprgqogvmgfogpkgnd (o is hydroxyproline)) alone bound vwf, with an affinity comparable to that of native collagen iii. immobilized peptide # supported platelet adhesion under static and flow conditions, processes blocked by an antibody which prevents the vwf a domain from binding full-length collagen. truncated and alaninesubstituted triple-helical peptides derived from # either strongly interacted with both vwf and platelets, or lacked both vwf and platelet binding. thus, we identified the sequence rgqogvmgf as the minimal vwf-binding sequence in collagen iii. the present work completes our understanding of the collagen-vwf interaction, providing information on crucial sequences in collagen that perfectly complements our existing knowledge of the collagen-binding site in vwf and may assist in targeting the collagen-vwf interaction for therapeutic purposes solid phase assay systems such as enzyme-linked immunosorbent assay (elisa), surface plasmon resonance (spr), and overlay gels are used to study processes of protein-protein and protein-peptide interactions. the common principle of all these methods is that they monitor the binding between soluble and surfaceimmobilized molecules. following the use of bovine serum albumin (bsa)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, we were able to demonstrate that positively charged peptides, which would be expected to repulse each other, can interact with each other. both the elisa and spr methods showed that the binding process reached saturation with kd values ranging between and nm. no interaction was observed between bsa conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure bsa molecules, strengthening the view that interaction occurs only between positively charged peptides. however, interactions between the same peptides were not observed in solution when was monitored by nuclear magnetic resonance (nmr) or by native gel electrophoresis. thus, it appears that for positively charged molecules to interact one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. the relevance of our findings for the use of solid phase assay systems to study interactions between biomolecules will be discussed. the hematopoietic progenitor kinase (hpk ), a mammalian hematopoiesis-specific ste kinase, contains a cluster of four proline-rich sequences called p , p , p and p located after the kinase domain. these pro-rich regions play an important role in the interactions of this kinase with different adapter proteins. previous studies showed that p , which contains the canonical pxxpxr motif, and p and p with the canonical pxxpxk motif interact with the c-terminal sh domain of hematopoietic lineage cell-specific protein (hs ) even if with different affinity. hs protein shares a high amino acid sequence and structural similarity to cortactin although their functions differ considerably. here we report the results of our investigation on the interaction between the c-terminal sh domain of cortactin and the four proline-rich motifs of hpk . these interactions were analyzed by non-immobilized ligand interaction assay by circular dichroism (nilia-cd). upon peptide addition, the binding was monitored by the cd changes of the trp side-chains of the conjugate gst-sh cort. the dissociation constants kd were determined analyzing the cd data at nm using a nonlinear regression method. the results demonstrate that gst-sh cort displays an affinity binding higher than that found with the corresponding hs domain and that the four hpk pro-rich regions are not equivalent. p appears to bind with the highest affinity (kd= . µm), followed by p (kd= µm) and p (kd= µm), whilst p does not interact at all. the generation of a fibrin clot is mediated by the regulated activation of a series of serine proteases and their cofactors. factor viii in its activated form, fviiia, acts as a cofactor to the serine protease fixa, in the conversion of the zymogen fx to the active enzyme fxa. both fviii and fix are essential for normal coagulation, deficiencies of either are associated with the bleeding diatheses hemophilia a and b, respectively. the role of fviiia is to bind factor ixa, generating the phospholipid-dependent intrinsic factor xase complex. at least two interactive sites have been identified for the enzyme-cofactor interaction. the ser -gln region within the a subunit has been shown to be crucial for viiia-ixa interaction. in an attempt to study this interaction, we synthesized a series of peptides of - loop of the a subunit. the syntheses of these peptides were carried out by using spps and fmoc/but methodology. the synthesized compounds were purified by rp-hplc and lyophilized to give fluffy solid, identified by ft-ir, nmr and es-ms spectra. these compounds were tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents to citrated platelet rich plasma (prp). the aggregation was determined using a dual channel electronic aggregometer by recording the light transmission. and ci/mmol, respectively. both tritiated aβ peptides were used in cat brain( in vivo) experiments and it was found that the peptide aggregates enter the neurons within min (electronmicroscopic autoradiography). this transport is most probably an endocytotic pathway. aβ aggregates could interact also with cytoplasmic proteins such as -phosphoglyceraldehyde dehydrogenase etc. we suppose that aβ assemblies can interact both with membrane receptors (nmda, ampa, ach) and with cytoplasmic proteins triggering neuronal dysfunction and death. background and aims: ww domains are the shortest known protein domains and contain a stable three-stranded b-sheet, which presents the binding site for prolinerich ligands. this interaction is mediated by hydrophobic interactions between aromatic and hydrophobic residues of the domain, and the polyproline core of the ligand. as part of our ongoing efforts aimed at synthetically mimicking conformationally defined protein binding sites ( ), we have designed and synthesized linear and cyclic peptides covering the binding site of the ww domain of human yes-associated protein (hyap-ww), whose structure in complex with a proline-rich ligand had been solved by nmr spectroscopy ( ) . methods: peptides were synthesized by spps, purified by hplc, and characterized by d-nmr spectroscopy, as well as by molecular dynamics calculations. affinities of the peptides to a hyap-ww ligand were determined in direct and competitive binding assays. results and conclusions: a cyclic peptide covering the sequence stretch of hyap-ww that contains its primary contact residues for proline-rich ligands, was found to compete with the domain for binding to a hyap-ww ligand. long-ranging noes identified in the nmr spectra of this peptide indicate a conformation, in which sequentially distant residues are brought into spatial proximity, likely through formation of a beta-sheet. these result demonstrate the feasibility of functional, as well as structural, mimicry of conformationally defined protein binding sites through synthetic peptides. the rockefeller university, new york, ny, usa integrins constitute a family of transmembrane cell surface receptors. they are involved in cell-cell and cell-extracellular matrix interactions. thus, they participate in many physiological and pathophysiological processes and are of crucial importance for the living organism. integrins possess two non-covalently bound subunits, &alpha; and &beta;, that jointly participate in ligand binding. these dimeric proteins show very high specificity in recognition of natural ligands. for example, α β integrin recognizes vcam- (vascular cell adhesion molecule ) and fibronectin through binding amino acid motifs tqidspln and ldv, respectively. on the other hand, fibronectin is a classical ligand for α β integrin with the recognition motif rgd. as shown, identification of the integrin ligands occurs through small recognition amino acid sequences (mostly tripeptides). thus, small cyclic peptides possessing a recognition motif in the appropriate three-dimensional conformation are able to interfere with the integrin-ligand interactions and act as inhibitors. the aim of this investigation is the characterisation of small cyclic peptides containing the rgd motif and the determination of the selectivity and specificity of these inhibitors. two new pentapeptides with -amino-cyclopropane- , -dicarboxylic acid monomethyl ester ((+/-)acc) were synthesized and tested. peptides were characterized in biological assays with living cells (k and wm ) and in surface plasmon resonance binding studies. experiments have shown that cyclic peptide cyclo-(arg-gly-asp-(+)acc-val) is a very potent inhibitor (ic -value in nm range) of interaction between vitronectin and αvβ or αvβ integrins. when preparing biotin-labelled peptides as ligands for avidin-based assays, it is chemically most expedient to locate the biotin label on the n-terminal group of the peptide. this is done without any regard to how this may affect peptide-target interactions, biotin-avidin binding, and the solubility properties of the resultant peptide. in many instances, the products are poorly soluble, have little biological activity, and poor affinity for avidin. problems can also arise during the synthesis of such nterminally biotinylated peptides due to the poor solubility and reactivity of many of the reagents used for biotin introduction. to overcome these limitations, we have developed an extremely simple method for synthesising peptides c-terminally with biotin. peptides can now be easily prepared by standard solid phase techniques either n-or c-terminally labelled, and screened to determine the optimum presentation for the biotin. in cases studies using protein-protein interaction and kinase assays, peptides c-terminally labelled with biotin gave better sensitivity. y. yang , j. eble , n. sewald many bacterial pathogens bind and enter eukaryotic cells to establish infection. invasin is an outer membrane protein required for efficient uptake of yersinia into m cells. invasin mediates its entry into eukaryotic cells by binding to members of β integrin family that lack insertion domains (i domains), such as α β ,α β ,α β ,α β , and αvβ . this type of peptide-protein interaction is an ideal subject for the rational design of inhibitors. the integrin binding motif consists of one loop region with a conservative asp residue and two synergistic regions. the aim of this project is to synthesize cyclic peptides based on the invasin binding epitope sdms. this sequence has to be positioned in a β-turn with asp in i+ position for optimal activity of the peptide. also the arg and asp residue, which are about . Å and . Å respectively away from the asp residue of the sdms loop in invasin, should be investigated. peptides that mimic these recognition sites have been synthesized and tested as ligands for the integrin peptide-dna cross-linking is a very powerful tool for studying peptide-dna complexes. it transforms non-covalent complexes into covalent complexes, which renders characterization of the adduct by classical techniques (mass spectroscopy, nmr,…) much easier. the aim of our research is to develop a new method for peptide-dna cross-linking involving the incorporation of a furan moiety. the strategy is inspired by the naturally occurring process of oxidative furan ring opening by cytochrome p . the resulting cis-butene- , -dial has been shown to react with amino-and sulfhydryl groups of macromolecules such as proteins and dna. in our research, dna binding peptides are modified with a furan moiety and then chemically oxidized into a reactive enal. this enal can react with dna to form a covalent peptide-dna complex. previous attempts to selectively oxidize furan modified minor groove binding peptides consisting of n-methylpyrrole building blocks failed. we are now applying the same strategy on major groove binding peptides consisting of natural amino acids. currently the oxidation conditions are being optimized so that the furan moiety undergoes selective oxidation. these optimized conditions will be applied to known dna binding peptides, in order to obtain a peptide-dna cross-link. we coupled octanoyl or palmitoyl group to the n-terminus of an analogue of sv nuclear localization signal (nls) peptide, sv - (ser ) to investigate the effect of fatty acid chain length on the conformation of the lipopeptide-antisense oligodeoxynucleotide (odn) complexes and to establish the optimal peptide/odn molar ratio (rm) for the effective delivery of odn into the cells. the odns used in this study were targeted towards either the green fluorescent protein (gfp) mrna and the junction sequence between ews and fli genes. the conformational change of odn at different rm values was followed by circular dichroism (cd), attenuated total reflection-fourier transform infrared (atr-ftir) spectroscopy and atomic force microscopy (afm). the sv peptide-mediated odn transfer into nih/ t cells was studied by epifluorescence microscopy. the interaction between the hiv- regulatory protein rev and rev responsive element (rre) of hiv- mrna has emerged in the last decade as an important target in antiviral therapy. the rev-rre interaction is essential for the replication of hiv. the rev protein binds to the rre site located in the env coding region of the full length viral mrna and facilities the export of the rna from the nucleus, while protecting it from the cell's splicing machinery. in the published nmr structure of the rre/rev-derived peptide complex, an -helical segment of rev binding domain recognises a specific region of rre. an approach is described to design a new class of -hairpin peptidomimetic ligands for hiv- rev protein, which inhibit its binding to the rre rna. a model -hairpin peptide served as a scaffold to pre-organise side chains into a geometry similar to that seen in a helical peptide. a library of peptidomimetics was prepared by grafting sequences related to the rna recognition element in rev onto a hairpin-inducing d-pro-l-pro template. the electrophoretic mobility shift assay (emsa) revealed that all of the designed peptidomimetics bind to rre and the best examples show affinities (kd) in a nanomolar range. these new ligands show a novel approach to designing rev peptidomimetics, represent interesting leads for the development of more potent hiv rre/rev inhibitors and permit more detailed studies of the mechanism of binding to rna. a. napiorkowska, a. sawula, m. olkowicz, p. mucha, p. rekowski tat (trans-activator of transcription) is the protein which controls the early phase of hiv- replication cycle. it is a potent viral trans-activator containing from to amino acid residues which binds to tar rna. the fundamental role of tat is promoting effective elongation of viral mrna (vmrna). binding of tat to tar is mediated by a -amino-acid, highly basic arg -lys-lys-arg -arg-gln-arg-arg-arg sequence of the arm (arginine rich motif); the key role in these interactions is played by arg . the goal of our research was to investigate the interaction of -nucleotide tar rna with synthesized tat peptide analogues using capillary electrophoresis (ce), a powerful analytical technique of biochemical studies. changes in electrophoretic mobility of the tar peak are employed for monitoring tar-tat complex formation. ce experiments were performed using lpa-coated capillary and a physical gel containing buffer. native arm fragment tat( - )nh , its analogues ac-tat( - )nh and ac-[lys ]tat( - )nh and analogues substituted in position with alanine-, homoalanine and lysine-derived amino acids containing nucleobases (adenine, guanine, cytosine, uracil, thymine) and nucleosides (adenosine, guanosine, uridine and cytidine) in the side chain were studied. specific interactions and complex formation were observed for both the native arm peptide fragment and selected tat analogues. the research is aimed at improving our understanding of the molecular mechanism of peptide-nucleic acid interaction, as well as evaluating the usefulness of selected nucleobase-containing amino acids as point probes for investigating peptide-rna interactions. interactions between proteins and dna are important to all living organisms. the goal is to investigate the molecular recognition between dna and the transcription factor phob of e. coli on the single molecule level and to identify amino acids required for dna binding. phob is composed of a transactivation domain (amino acids - ) and a dna binding domain (amino acids - ) that binds to specific dna sequences (pho boxes) containing a tgtca sequence. [ ] chemical synthesis of peptide epitopes present in the dna binding domain of phob and isolation of the whole dna binding domain of phob was performed. the protein was purified using intein mediated protein purification. an additional cysteine residue was ligated to the protein using intein mediated ligation reactions and will be used for immobilization and labeling. in single molecule force spectroscopy (afm) experiments it has been shown that both a peptide with a native phob-sequence and the recombinant protein bind to dna. competition experiments were performed to prove specific dna binding. [ ] mutated peptides and proteins where strategic amino acids were replaced by alanine have also been examined to reveal the contributions of single residues to molecular recognition. the binding contribution of the proteins is determined by surface plasmon resonance, electrophoretic mobility shift assays and fluorescence correlation spectroscopy. we investigated the biophysical characteristics and the pore formation dynamics of naturally occurring and synthetic peptides forming membrane-spanning channels by using isolated rod outer segments (os) of reptilia and amphibia recorded in the whole-cell configuration. once blocked the two os endogenous conductances (the cgmp channels by light and the retinal exchanger by removing one of the transported ion species from both sides of the membrane, i.e. k+, na+ or ca +), the os membrane resistance (rm) could be > gΏ. therefore, any exogenous current can be studied down to the single channel level. macroscopic currents of amplitude of ~ pa were recorded in symmetric k+ or na+ (> mm) and ca + ( mm) from the commercially available alamethicin mixture, the synthetic alamethicin f / (a major component of the natural mixture), and selected analogues applied at µm concentration at - mv. once applied and removed the peptide, the current activates and deactivates with a time constant of about ms. the synthetic analogues [glu(ome) , , ] and [glu(ome) , ] produce a current of about pa at µm concentration, and they show a strong activation by hyperpolarization as alamethicin f / itself. clear single channel events were observed when the concentration of all of the alamethicin peptides is reduced to < nm.<br> these results indicate that the three gln residues at positions , and of alamethicin f / are not a key factor for pore formation and its conduction properties. in general, the pore assembly and disassembly are very fast and cooperative events. the translocation mechanism of penetratin (rqikiwfqnrrmkwkk) is not clear, but the involvement of cell membrane was supposed. recent studies with phospholipid model membranes have shown that penetratin interacts only with negatively charged liposomes. we aimed to analyse the effect of penetratin on liposomes composed of different phospholipids (dppc/dppg : - : ) by fluorescence spectroscopy. in the first set of experiments, liposomes labelled with fluorescent markers (dph, ans and tma-dph) were incubated with penetratin and the fluorescence polarisation was determined as a function of the temperature. in the range of - mol/mol phospholipid/penetratin ratio, no change in the transition temperature was observed indicating that penetratin has no influence on the membrane structure. next, we have analysed the interactions between phospholipids and penetratin through changes in the intrinsic fluorescence of the peptide due to the presence of two w residues in its sequence. comparing the emission spectra corresponding to penetratin in aqueous media or in presence of vesicles one can clearly appreciate a blue shift. this indicates that that tryptophan residues are mainly exposed to a hydrophobic environment. analysis of the main band shows low values of polarization suggesting a free motion of the peptide chain. on the contrary polarization measured for penetratin mixed with liposomes results in higher values. this indicates that hydrophobic residues, like trp, are inserted into the bilayer and their motion is restricted. these data suggest the presence of interation sensed strongly by trp properties. cyclopeptide antibiotic gramicidin s (gs) has antimicrobial activity against gram-positive and gram-negative bacteria and some fungi. but non-specific action of gs and its high lytic potential limits therapeutic application of gs. we attempted to elucidate in which way gs molecule could be modified to lose its haemolytic side effects. gs molecule interacts directly with membrane phospholipids due to electrostatic and hydrophobic interaction. naturally, changes in the state of a lipid bilayer cause changes in the gs molecule binding to a bilayer. we studied the effect of gs on human blood platelets and the effect of platelet membrane state on the gs-induced disaggregation of cells with the help of turbidimetric and microscopy techniques. we modified the membrane state by temperature, osmotic stress, ionizing irradiation, lipid oxidation. depending on concentration gs causes platelet shape change and activation. when added to preliminary aggregated (in response to physiological agonists -thrombin, epinephrine, adp) platelets, gs causes crumble of cells aggregates. the rate and extent of platelet disaggregation under the effect of gs non monotonously depends on temperature (range of - °c) and irradiation dose (up to gy). parameters of the gs interaction with membranes are determined by the mobility of membrane lipids. factors modifying the lipid bilayer change the degree and the speed of the gs interaction with platelet membranes. results obtained permit to use gs for testing the state of membrane lipids and on the other hand allow to suppose ways of gs molecule modifications to achieve its tolerance to blood cells. g. bai , p. gomes , r. seixas , m. hicks , m. prieto , m. bastos eukaryotic antibiotic peptides (eaps) have been widely studied for the past years as an alternative to conventional antibiotics due to emergence of multi-resistant microbial strains, and significant efforts targeting increasingly potent and specific antimicrobial peptides are being made. one interesting approach in peptide antibiotics is based on hybrid sequences derived from natural eaps, with ca ( resistance to conventional antibiotics has stimulated a search for alternative therapeutics for microbial infections, a possible source that has gained much interest in recent years are antimicrobial peptides. antimicrobial peptides target the cell membrane directly, which is a key feature as evolution has shown bacteria have had difficulty in altering their membrane composition and organization to mount a suitable defence against these peptides. a common theory is that peptides that bind strongly exhibit high biological activity, but our real-time quantitative binding studies via surface plasmon resonance (spr) have shown that this correlation does not always hold. as more information on the molecular details of membrane disruption is required, we have used atomic force microscopy (afm) to visualise peptide insertion and changes in membrane morphology by a range of antimicrobial peptides in situ. interaction studies were performed with a series of phospholipid mixtures that mimic either mammalian cells (high in phosphatidylcholine and cholesterol) or microbial cells (high in phosphatidylethanolamine and phosphatidylglycerol). the present study may assist in the design of new specific antimicrobial peptides with high antimicrobial activity and low host toxicity. proportions of popc and popg as models. very high molar ratio partition constants (( . +- . )x ^ and ( . +- . )x ^ ) were obtained for the bacterial models (popg:popc : and : , respectively), these being about one order of magnitude greater than the partition constants obtained for the less anionic mammalian model systems (( . +- . )x ^ for the % popc system). at low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially in the case of the most anionic systems. membrane saturation was shown to be related to such observations and mathematical models were derived to further characterize the peptide-lipid interaction under these conditions. the calculated peptide-to-lipid saturation proportions, together with the determined partition constants, suggest that the minimal inhibitory concentrations of omiganan pentahydrochloride could represent the conditions required for bacterial membrane saturation to occur. the hemolytic pore-forming toxin sticholysin ii (stii) produced by the sea anemone stichodactyla heliantus belongs to the actinoporin protein family. the n-terminal domain of these proteins is required for interaction with membranes. to investigate the role of stii´s n-terminal domain in membrane binding and in the molecular mechanism of hemolysis, peptides corresponding to residues to , or shorter fragments from this region, were synthesized. in some peptides leu was replaced by trp. all peptides exhibited hemolytic activity, albeit to a lesser extent than the whole protein. moreover, peptides lacking the - hydrophobic stretch were less active. the longer peptides were also able to permeabilize phospholipid vesicles. conformational studies were performed in aqueous solution and in membranemimicking environments. cd spectra showed that, while the shorter, more hydrophilic peptides, displayed a random conformation, the longer peptides underwent aggregation with increasing concentration, ph, and ionic strength. in the presence of trifluoroethanol and upon binding to detergent micelles and phospholipid bilayers, the peptides showed a propensity to acquire -helical conformation, as expected for the sequence comprising residues to . fluorescence spectra demonstrated that the first residues of stii´s n-terminus penetrate more deeply into the bilayer, whereas residues - are located more superficially. this is in agreement with the predicted amphipathic nature of the helix formed by these residues and corroborates the existing hypotheses for the role of the n-terminal domain in the process of membrane insertion and pore formation. among a great number of antibacterial peptides a group of trp-rich peptides is of special interest. taking into consideration, that in most of proteins tryptophan is not frequently occurring amino acid, the biological meaning of a high content of tryptophan in structure of these antimicrobial peptides is particularly interesting. in the present study we carried out the investigation of antimicrobial and hemolytic activities of selected trp-rich peptides and their action on microbial membrane: ilpwkwpwwpwrr-nh - indolicidin (i) pitwpwkwwkgg-nh - b (ii) plswffprtwgkr-nh - gsp- a (iii) fpvtwrwwkwwkg-nh - puroindoline (iv) vrrfpwwwpflrr-nh - tritrypticin (v). sunflower trypsin inhibitor sfti- is the smallest and the most potent known peptidic trypsin inhibitor from the bowman-birk class of proteins [ ] . this head-to-tailcyclized -amino-acid peptide contains one disulfide bridge and a lysine residue (lys ) present in the p position, which is responsible for inhibitor specificity.as was reported by us and other groups, sfti- analogues with one cycle only retain trypsin inhibitory activity. very recently we have shown [ ] that introduction of nsubstituted glycine residues mimicking lys and phe (denoted as nlys and nphe) in the p position of monocyclic sfti- with disulfide bridge yielded potent trypsin and chymotrypsin inhibitors, respectively. in this novel class of proteinase inhibitors contains completely proteolytic resistant p -p ' reactive site. in the present communication we report chemical synthesis and determination of trypsin and chymotrypsin inhibitory activity of a series of ten sfti- analogues modified in the p position by these peptoid monomers (nlys and nphe). each of the synthesized peptomeric (peptide-peptoid hybrid polymer) sfti- analogues contains one of the following cycles: head-to-tail, disulfide bridge formed by cys, by pen and by cys/pen residues. the impact of the different cycles introduced into sfti- structure on proteinase inhibitory activity will be discussed. s-protein contains a proteolytic processing site and two interacting heptad repeat regions denoted as hr-n and hr-c. following processing of s-protein mediated by host cellular protease/s, the c-terminal s -fragment fuse with host cell membranes via its hr-n and hr-c domains that form coiled coil -helix bundle (trimeric of dimers)-crucial for its receptor-mediated viral fusion. our objective in this work is to study the proteolytic site using model peptides and also to examine the interaction of hr-n and hr-c domains using fluorescence microscopy and other techniques. thus we synthesized an intramolecularly quenched fluorogenic peptide containing the proposed cleavage site [abz-eqdrntr evfatyx, abz= -amino benzoic acid and tyx= -nitro tyrosine] and showed by kinetic measurements that this cleavage is mediated most efficiently by furin, followed pc and pc . other potential substrates were also tested and compared. above cleavage can be blocked by specific-pc-inhibitors in a dose-dependent manner. in addition using fluorescent-labeled peptides derived from hr-n and hr-c domains, circular dichroism spectra and surface assisted laser desorption mass spectral interest has grown to develop specific and potent inhibitors of this enzyme. our objectives in this study are to generate soluble recombinant human (h)ski- enzyme, design potent inhibitors and study its dmodel structure. we have successfully expressed hski- enzyme lacking its transmembrane domain in hek- cells and purified the enzyme via chromatography. in addition we developed ski- inhibitors by using pseudo-and multi-branch peptide approaches. in first approach we inserted dipeptide isosteres amino oxy acetic acid (aoaa) or -amino , dioxa octanoic acid (adoa) at scissile p -p ' position ((r l) of hski- . a typical example is gryssrrl(adoa)aip . other dipeptide isosters were also incorporated at the cleavage site of either ski- prodomain or lassa virus glycoprotein. in second approach we prepared and -branch peptides containing hski- - segment. these peptides inhibit ski- in competitive manners with varying degrees ranging from low m to high nm ic . circular dichroism spectra indicated strong interactions of inhibitors with ski- consistent with observed inhibition profile. a d-model structure of catalytic domain of ski- indicated a broad catalytic pocket cysteine proteases are of great importance in biochemical processes and these enzymes are used in biotechnology, food industry and agriculture. in this connection synthesis of high selectivity and high specificity substrates for cysteine proteases is of importance. enzymatic synthesis of peptides is a good tool for obtaining different biologically active peptides. immobilized serine proteases, subtilisin carlsberg and α-chymotrypsin immobilized on poly(vinyl alcohol) cryogel (pvacryogel), proved to be a convenient biocatalyst for such kind of syntheses. the high specific chromogenic substrate for cysteine proteases assay glp-phe-ala-pna was obtained with high product yields (up to % in h) using subtilisin and chymotrypsin immobilized on pva-cryogel. the reaction was carried out according to the following scheme: glp -the residue of pyroglutamic acid, pna -p-nitroanilide. the influence of initial concentrations of components, the reaction mixture composition, the biocatalyst content and time on product yield was studied. it was shown that the optimal conditions are: dimethylformamide-acetonitrile mixture : (v/v), initial concentrations - mm, and enzyme-to-substrate ratio - : . this approach was used in order to synthesize analogous substrates, containing different fluorogenic and chromogenic groups as well as other amino acids in p position. the obtained substrates were tested for the papain assay. peptidyl-a-ketoaldehydes represent attractive lead compounds and intermediates in the development of potent protease inhibitors due to their structural similarity with peptide aldehydes, previously known to be excellent inhibitors of serine-and cysteine protease. recently, we demonstrated the application of polymer cyano methylene-and carboxylato methylene phosphoranes in the assembly of a-hydroxy-b-amino esters (norstatines), a,b-diketoesters, and a,b-unsaturated ketones. [ , , ] we now present a further development of our reagent linker approach employing peptidyl-a-ketoaldehydes and diamino propanoles . carboxylato methylen phosphoranes derived from bromo acetic esters which are readly acylated without racemization, play the key role in our synthetic concept. herein we show the oxidative cleavage to peptidyl-a-ketoaldehydes using dimethyldioxirane (dmd) in acetone as oxidant, after saponification and decarboxylation on the solid support. diamino propanoles were furnished via the reductive amination of resin-bound peptides. over the past few years nuclear magnetic resonance has emerged as a powerful means for lead molecular identification and optimization.on the other hand, the f nmr has been used succesfully in several structural studies, protein folding studies and for the identification of active compounds, using a very similar methodology that the one used in the present work. the methodology required the labeling of the substrate with a cf moiety. the enzymatic reaction is performed with the cf substrate and quenched, using an enzyme inhibitor. f nmr is then used to monitor the evolution of both substrate and product. only two peaks are observed, the starting substrate and the cleaved substrate. this nmr method has some advantages: fluorine nmr is very sensitive, , times that of the proton. there are no spectral interference from protonated solvents, buffers or detergents typically present in the enzymatic reactions.the f isotropic chemical shift is extremely sensitive to small structural perturbations resulting in different chemical shift for the signals of the substrate and product. isotopic labeling of the protein is not required. as a model, caspase- , which play a critical role in the initiation of apoptosis process and hiv- protease were chosen. two different kind of libraries were screened: one based on natural products from plant and animal extracts used in tradicional chinese medicine and a second one corresponding of a synthetic library with two sublibraries of and compounds.with this methodology it has been possible to identify some compounds with very promising inhibitory properties. background and aims: human kallikrein (hk ), a prostate specific serine protease, regulates the activity of several factors that may participate in proteolytic cascades promoting tumor growth and metastasis. thus, inhibition of its enzymatic activity is a potential way of preventing growth and metastasis of prostate cancer. moreover, specific ligands for hk have potential use for targeting and in vivo imaging of prostate cancer and for development of novel assays. methods: to find peptide ligands we panned several phage display peptide libraries against active recombinant hk captured by a monoclonal antibody exposing the active site of the enzyme. alanine scanning and amino acid deletion analyses were performed to elucidate the motifs required for hk inhibition. results: from libraries expressing and amino acid long linear peptides we isolated six different hk -binding peptides. three of these peptides are specific inhibitors of the enzymatic activity of hk . amino acid substitution and deletion studies indicated that motifs of amino acids are necessary for the inhibitory activity. conclusions: we have developed specific hk inhibitors by phage display technology. these novel hk specific peptides are potentially useful for treatment and targeting of prostate cancer. peptidylarginine deiminase iv (padiv) catalyzes the citrullination of arg residues in various peptides and proteins, such as histone, resulting in the production of citrullinated proteins in granulocytes [ , ] . the citrullination mechanism of histone subunits and its functional effects in cells are not well known yet in detail. recently, it has been reported that the protein deimination/citrullination by pad iv plays a role in rheumatoid arthritis [ ] . this implicates that the citrullination of histone may be related to rheumatoid arthritis. in order to further study the citrullination mechanism of histone, we explored the citrullination sites of histone h a and h by pad iv using a series of synthetic peptides. recently, hagiwara et. al. reported that pad iv only citrullinates the arg of histone h a as well as the arg in histone h [ , ] . in order to investigate the citrullination mechanism, the n-terminal peptides of histone h a and h were chemically synthesized and examined the citullination by pad iv. the n-terminal acetylation effect of the n-terminal synthetic peptide was also estimated on the citrullination by padiv. the velocity of each arg residues in the n-terminal peptides were estimated in vitro. the results indicated that padiv recognizes the specific arg residues in the synthetic peptide, and that the n-terminal acetylation of the histone peptides dramatically affects on the substrate recognition of padiv. in addition, the cd spectra of the n-terminal peptides were measured to elucidate the structural specificity for the recognition of pad iv. background and aims. prolyl oligopeptidase (pop) is a serine peptidase that cleaves oligopeptides after prolyl residues. it has been associated with cognitive disorders. pop inhibitors have been shown to enhance cognition in monkeys ( ) and to improve performance in verbal memory tests in humans ( ) . in the present study, the p l-prolyl residue of pop inhibitors was replaced by two l-proline mimetics, the -t-butyl-l-prolyl group and the (r)-cyclopent- -enecarbonyl group. the effect of the mimetics on in vitro potency, lipophilicity and binding kinetics were studied. methods. the l-proline mimetics were synthesized according to the published procedures ( , ) with minor modifications. the ic and ki values and the binding kinetics were determined for porcine pop. the log p values were determined with the shake-flask method. results. the replacement of the p l-prolyl residue by the l-proline mimetics gave compounds which were equipotent with their parent structures. both l-proline mimetics increased lipophilicity but the effect of the -t-butyl-l-prolyl group was more pronounced. while the -t-butyl-l-prolyl group increased the dissociation half-life of the enzymeinhibitor complex, the (r)-cyclopent- -enecarbonyl group decreased it. conclusions. both l-proline mimetics perfectly mimicked l-proline at the p position of pop inhibitors. these mimetics can be used to modify the lipophilicity and the binding kinetics of pop inhibitors. the proteasome is an essential multicatalytic protease of the ubiquitin proteasome pathway. as a prime executor of regulated proteolysis, the proteasome controls almost all aspects of cell metabolism from signal transduction to cell cycle and differentiation. pharmacological intervention into proteasome activity leads to cell apoptosis. this observation was applied to successfully treat multiple myeloma, since the cancer cells exhibit substantially higher sensitivity to competitive inhibition of proteasome than normal cells. however, the complete shutting down of the proteasome catalyzed proteolysis leads to serious side effects resulting from the disruption of proteolytic homeostasis even in noncancerous cells. here, we show an alternative approach to control the proteasome activity using peptide based noncompetitive regulators. the cathelicidins derived peptides rich in proline and arginine (pr) residues have been found to affect activity of all the proteasome complexes both in vivo and in vitro, likely by binding to the face of the enzyme. mechanism and structural constrains of the pr peptides dictating their influence on the proteasome remain elusive. our results indicate that there are three sequence related properties of the pr peptides controlling their effectiveness as proteasome regulators: length of the peptide, distribution of a set of positive charges at the peptide n-terminus, and positioning of proline residues. far uv cd spectroscopy demonstrates that these properties also correlate with the structure of pr peptides. in particular, it seems that structural propensity of the pr peptides to form beta-turns are required to bind to proteasome as regulatory competent molecules. our work is focused on the search of selective, low-molecular cathepsin b peptide inhibitors acylated with the (e)- -(benzylsulphonyl)acroyl group (bsa). the double bond, embedded in the bsa moiety is activated by two electron-withdrawing groups and may be a good target for the michael-type addition of the catalytically active -sh group. three series of peptide derivatives possessing general structures: bsa-phe-asn(r)-oh, bsa-ile-x(oh)-n(ch ) and bsa-x-pro-oh were synthesized in solution and characterized by enzyme kinetic studies against papain, cathepsins b and k. it should be noted that all the investigated compounds were competitive and reversible inhibitors of the enzymes examined. using d h nmr (tocsy, cosy, roesy) and c nmr spectroscopy along with theoretical calculations (analyse program) we determined the conformational properties of two most potent and selective cathepsin b inhibitors. this work was supported by grant ds/ - - - . background and aims: we have developed peptides inhibiting human kallikrein- (hk ) activity. as hk is overexpressed in prostate cancer tissue, these peptides are potentially useful for treatment and diagnosis of prostate cancer. two of the potential physiological substrates for hk are proform of prostate specific antigen (propsa) and insulin-like growth factor-binding protein- (igfbp- ). both of these might participate in the regulation of prostate cancer growth: igfbp- by inhibiting igf-dependent tumor growth and psa by degrading extracellular matrix. we aimed to study whether our hk -inhibiting peptides inhibit also hk activity towards natural protein substrates, i.e. activation of propsa and degradation of igfbp- . methods: the effect of the peptides on the activation of propsa by hk was studied by preincubating the peptides with hk , followed by addition of psa and specific psa substrate. igfbp- degradation was studied by two specific immunoassays, one detecting only native igfbp- , while the other one also detected proteolytically cleaved forms of the protein. results: hk -inhibiting peptides were found to inhibit propsa activation and igfbp- degradation by hk in a dose dependent fashion. conclusions: we have developed new peptides inhibiting hk activity towards natural substrates, like propsa and igfbp- . the peptides might be useful for treatment of prostate cancer and other diseases associated with increased hk activity. from the seeds of garden four-o'clock and spinach we isolated two serine proteinase inhibitors (mjti i -mirabilis jalapa trypsin inhibitor and soti i -spinacia oleracea trypsin inhibitor), which are probably representatives of a new family of inhibitors. the purification procedures of these inhibitors included affinity chromatography on immobilized methylchymotrypsin in a presence of m nacl, ion exchange chromatography and/or preparative electrophoresis and finally rp-hplc on c column. their primary structures (fig. ) differ from those of known trypsin inhibitors, but showed significant similarity to one another, as well as to the antimicrobial peptides isolated from the seeds of mirabilis jalapa (mj-amp , mj-amp ), mesembryanthemum crystallinum (amp ) and phytolacca americana (amp- and pafs-s) and from hemolymph of acrocinus longimanus (alo- , and ). the equilibrium association constants (ka) of mjti i and soti i with bovine -trypsin were found to be about - m- . mjti i and soti i have been synthesized using solid-phase method. the synthesized inhibitors and inhibitors isolated from plants have similar properties. the disulfide bridge pattern in both inhibitors was established after digestion with thermolysine, followed by the maldi-tof: cys -cys- , cys -cys and cys -cys . s. cosgrove, l. rogers, c. hewage, j.p. malthouse aspartyl proteases are required for the multiplication of the aids virus and for producing the amyloid protein which causes alzheimer's disease. hiv protease inhibitors have been highly effective in treating aids patients and it is hoped that potent inhibitors of the beta secretases will also prove effective in treating alzheimer's disease. therefore inhibitors of the aspartyl proteases have great therapeutic potential. we have shown that the peptide glyoxals are potent inhibitors of the thiol protease papain and of the serine proteases subtilisin and chymotrypsin. using c-nmr we have been able to show that glyoxal inhibitors react reversibly with an active site nucleophile in these enzymes to form a tetrahedral adduct which is tightly bound by the enzyme. in the present work we synthesise c-enriched peptide glyoxals, we assess their inhibitor potency, and use c-nmr to examine how the inhibitors interact with the aspartyl protease pepsin. z-ala-ala-[ - c]phe-glyoxal was synthesised from [ - c]phenylalanine which was converted to its methyl ester. this was then coupled with z-ala-ala to give z-ala-ala-[ - c]phe-ome which was hydrolysed to the free acid. this was converted to the diazoketone and transformed into z-ala-ala-[ - c]phe-glyoxal using dimethyldioxirane. nmr spectra at . t were recorded with a bruker avance drx standard-bore spectrometer. we show that peptide glyoxal inhibitors can be potent inhibitors of pepsin and that pepsin only binds one of the four glyoxal forms (one non-hydrated, one fully hydrated and two partially hydrated forms). alzheimer`s disease (ad) is the most common cause of dementia in older people. a major factor in the pathogenesis of ad is the cerebral deposition of amyloid fibrils, consisting of amyloid β peptides (aβ), as senile plaque. the -to amino acid long aβ is generated by the proteolysis of β-amyloid precursor protein (app) by β-and γ-secretases. since bace , a unique member of the pepsin family of aspartyl proteases initiates the pathogenic processing of app by cleaving at the n-terminus it is a molecular target for therapeutic intervention in ad proteolytic activity was found to occur, to a variable degree, in digestive organs of all studied organisms over the entire ph range. the common feature was the existence of two activity peaks, in the acid (ph . - . ) and alkaline (ph . - . ) zones, as well as a similar protease set containing e and d cathepsins, a trypsin-like enzyme, elastase, and collagenolytic proteases. proteolytic activity in the hepatopancreas of crab and sea star was found to be an order higher than in other study objects. high protease activity in crab hepatopancreas is an evolutionary mechanism compensating for a poor differentiation of digestive system, low substrate specificity of enzymes, and cold environment. trypsin activity in digestive organs of invertebrates suggests that a trypsin-like enzyme is a genetically old one, an evolutionary origin of all serine proteases. a difference of kind between vertebrates and invertebrates is that the latter have cathepsine activity (absent in vertebrates) and no pepsin activity. it is of interest to develop enzyme inhibitors containing a light activated switch that can be used to control binding and inactivation of an enzyme. several inhibitors containing the azobenzene photoswitch group have previously been developed and have shown changes in activity of around two times on photoswitching. this study aimed to improve this switching by more extensive derivatisation of azobenzene to closer resemble the peptide substrates of proteases. a series of peptidomimetics containing the azobenzene photoswitch group were synthesized and assayed against the protease alpha-chymotrypsin. these compounds contained azobenzene, linked to a known chymotrypsin inhibitory group (either a trifluoromethylketone or boronate ester), and otherwise designed to be peptide-like. in some cases both ends of the azobenzene moiety were derivatized in order to increase the impact of photoswitching on the shape of the compound and thus its enzyme binding strength. assays showed that most compounds were reversible inhibitors of chymotrypsin, with low micromolar inhibition constants (ki or ic ). up to four times increase in enzyme inhibition on light activated switching of the azobenzene group conformation was obtained. a number of peptidyl derivatives structurally based upon the inhibitory sites of cystatins has been synthesized. these compounds are prone to proteolytic degradation, are rapidly excreted and poorly bioavailable. the majority of this problems might be overcome by use of peptidomimetics with structures resembling those of previously synthesized peptidyl derivatives. among the peptidomimetics are azapeptides, in which alpha-ch group of amino-acid residue is replaced by a nitrogen atom. the azapeptides have recently been demonstrated as potent and selective inhibitors of cathepsins b and k. it was shown that azapeptide inhibitors bind along the active site cleft of cathepsin b in a bent conformation. this bent structure is likely to result from the mobility of the bonds in the vicinity of the inserted azaamino acid residue as well as from the interaction with enzyme. in our present work we have studied the peptide of a sequence: z-arg-leu-arg-gly-ile-val-ome, which is characterized by one major and three minor conformations. the replacement of alpha-ch group in the gly residue of peptide chain of z-arg-leu-arg-gly-ile-val-ome by the nitrogen atom likely results in rigid conformation. our aim was a comparison of structure of the parent peptide z-arg-leu-arg-gly-ile-val-ome and a selective cathepsin b inhibitor z-arg-leu-arg-agly-ile-val-ome by using h-nmr. severe acute respiratory syndrome corona virus associated main protease (sars cov mpro protease), alternatively known as chymotrypsin-like protease ( clpro), is a mediator of virus infection cyclus and from there a therapeutic target. a peptide aldehyde library targeting the sars corona virus main protease (sars-cov mpro, alternatively known as clpro) was designed on the basis of three different reported binding modes and supported by virtual screening. a set of peptide aldehydes were prepared by a newly developed methodology and investigated in an inhibition assay against sars-cov mpro. [ ] protected amino acid aldehydes furnished by the racemization-free oxidation of amino alcohols with dess-martin periodinane were immobilized on threonyl resins as oxazolidines. following boc-protection of the ring nitrogen yielding the n-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin releasing deprotected products under mild hydrolysis conditions. the library was tested in a new fluorimetric enzyme assay for sars cov mpro. via immobilization of the fluorophor, -( -amino- -methyl- -coumarinyl)-acetic acid, the substrate actsavlq-amca was prepared, surprisingly displaying a higher affinity than the native substrate. several potent inhibitors were found with ic values in the low micromolar range. interestingly, the most potent inhibitors seem to bind sars-cov mpro in a non-canonical binding mode. currently, the initial screen is extended towards the discovery of small molecule inhibitors of sars corona virus main protease. literature: a method of bromelain cleavage of surface glycoprotein hemagglutinin (ha) from the influenza a virions was initially employed for ha ectodomains crystallographic study [ ] . the remaining spikeless subviral particles were used by us earlier for ha c-terminal fragment extraction and mass spectrometric (ms) investigation [ ] . now sds-page analysis of the subviral particle preparations revealed several additional bands in a range of - kda together with major viral proteins comparing to intact virions (figure, m matrix protein, f -f -m protein fragments, np-nucleoprotein). maldi-tof ms analysis of the in-gel trypsin hydrolyzates has shown that the additional bands are fragments of М protein. this was confirmed by n-terminal sequencing of the protein fragments electroblotted from the bands. concentration of sh-reducing reagent in bromelain digestion reaction influenced on the m fragment bands intensity. we conclude that due to membrane destabilization during ha spikes removing, m protein localized under viral membrane inside intact virions becomes accessible to limited proteolysis by bromelain. [ dipeptidyl peptidases (dpp's) sequentially release dipeptides from polypeptides. among those enzymes, dppiv, fapα, dpp , dpp and dppii cause the release of n-terminal dipeptides containing proline or alanine at the penultimate position. they are all members of clan sc, a group of serine proteases that contains proline-specific peptidases. dipeptidyl-peptidase iv (dppiv) is the best studied member of this group of enzymes and has become a validated target for the treatment of type diabetes over the last years. the development of inhibitors for the related enzymes (i.a. dppii) has only started recently. this poster presents selected products synthesised to further elaborate the structure-activity relationship for dpp ii inhibitors with a , -diaminobutyrylpiperidine basic structure. this class of compounds was described earlier by our group as the hitherto most potent and selective inhibitors of dpp ii. starting from n -p-chlorobenzyl-substituted uamc , our lead compound, two types of modifications were proposed: • the synthesis of n -(di)alkyl-and arylalkyl analogues; • the synthesis of -methyl analogues. in our previous study, we reported potent and small-sized bace inhibitors containing phenylnorstatine [( r, s)- -amino- -hydroxy- -phenylbutyric acid; pns] at p position as a transition-state mimic. in developing more active compounds, we focused our attempts on the p position, where we replaced the pns by its thioderivative. herein, we present the synthesis of a novel phenylthionorstatine [( r, r)- -amino- -hydroxy- -(phenylthio)butyric acid; ptns] as a p moiety with hydroxymethylcarbonyl (hmc) isostere, and then an application to the bace inhibitors design. we have synthesized ptns starting from readily available n-benzyloxycarbonyl-serine and after multistep reaction (including weinreb amide formation, thiophenyl group introduction, through cyanohydrin derivative the transformation into the -hydroxy ester and then acid). purification was done by column chromatography and rp-hplc. peptides were synthesized by the fmoc based solid phase method and characterized by maldi-tof ms. the peptide inhibitors were adopted to enzyme assay using a recombinant human bace and a fluorescence-quenching substrate. bace inhibitory activity was determined based on the decrease% of the cleaved substrate by the enzyme.we have synthesized ptns and then the ( r, r)-enantiomer was applied to spps (solid phase peptide synthesis). we synthesized octa-and pentapeptide-type inhibitors of bace containing pns or ptns at the p position. these compounds were enzymatically tested and showed high bace inhibitory activity. a novel derivative of pns, ptns, was synthesized, and evaluated in comparison to corresponding pns. the inhibitors with ptns exhibited a slightly higher inhibitory activity against bace comparing to those with pns. this study suggests possibilities of the application of ptns to design other aspartyl proteases inhibitors. the αν β integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of amino acids by aza-β -amino acid analogs in cyclic rgdpeptides as αν β -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. thr rgd motif resides in position i to i+ of a regular γ-turn. we synthetized linear and cyclic aza-β rgd-peptide with the purpose to examine the effect on the conformation and the activity. are aza-β amino acids γ-turn mimetics? the preferred conformations were determined by nmr. prostaglandins are involved in a large number of biological activities mediated by their g-protein coupled receptors (gpcrs). the prostaglandins pgf alpha receptors are found specifically in uterine muscle, where they initiate parturition and labor. the pgf alpha receptor plays a key role in preterm labor, for which medical and social costs are estimated at $ billion per year in the usa (the highest per patient cost of any disorder). peptide mimics have been developed in our laboratory ( , ) , that serve as allosteric antagonists of the pgf alpha receptor. the importance of the turn geometry of the central residue in these peptide mimics has been investigated using enantiomeric indolizidin- -one beta-turn mimics which can respectively induce type ii and ii' geometry. our presentation will discuss the synthesis and biology of these novel allosteric modulators of prostaglandin pgf alpha receptor activity. it was shown that luteinising hormone -releasing hormone (lhrh) receptors are overexpressed in the most of adenocarcinoma cells in contrast to their low content in normal tissues. these data create the basis for lhrh analogues application in therapy of breast, ovary, prostate, lung, intestine, liver and kidney cancers. both agonists and antagonists utility for the targeting of cytotoxic moiety to the tumor cells is well documented. however, the number of lhrh analogues possessed their own cytotoxic activity is still very limited. we nicotianamine (na) that was first isolated from the leaves of nicotiana tabacum l [ ] , is known as a key biosynthetic precursor of phytosiderophores. various studies have proved that nicotianamine plays a significant role in plants as an iron, nickel, zinc ... transporter [ ] . the aim of our study was to synthesize unnatural analogues of na via peptide intermediates, to investigate the mechanisms of metal transport and accumulation within the plant. we found that the strategy developed for na synthesis could not be applied when the azetidine ring was changed for pyrrolidine ring and we investigated a new route to synthesize such analogue. these synthetic pathways will be discussed. the primary physiological roles of arginine vasopressin (avp), [cycle - (h-cys -tyr -phe -gln -asn -cys -pro -arg -gly -nh )], involve vasoconstriction of vascular smooth muscles, via v a receptor, and antidiuretic action in kidney (blood osmolality regulation) via v receptor. binding of avp to the v a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. in addition, activation of the v b (also known as v ) receptor causes adrenocorticotropic hormone release from the anterior pituitary. v b receptors are also present in the brain where avp functions as a neurotransmitter. in the recent years by the salivary glands of several bloodsucking animals like, teaks, leeches, vampire bats and so forth are isolated plenty of proteins and peptides with different molecular weight and well established anticoagulant activity. many of the strongest anticoagulants isolated by bloodsucking animals are found in the extract of salivary glands of different kinds of leeches. such leech is the haementeria officinalis, from which is isolated the most active inhibitor of factor xa -ats. in order to study the role of some amino acids in the process of interaction among peptides mimetics and the active site of serine proteinases, some fragment analogues of ats's active site by replacement of some amino acids with the other with similar structure or with unnatural amino acids were synthesized. in the present work the synthesis and the anticoagulant activity according to the aptt and ic of the newly synthesized peptides and structure-activity relationship will be discussed. rational design of peptides is a challenge which would benefit from a better knowledge of their rules of sequence-structure-function relationships. peptide structures can be approached by spectroscopy and nmr techniques but data from these approaches too frequently diverge. structures can also be calculated in silico from primary sequence information using three algorithms: pepstr, robetta and peplook. the most recent algorithm, peplook introduces indexes for evaluating structural polymorphism and stability. the method uses a de novo search of energy minima by an iterative boltzmann-stochastic procedure and using a combination of phi/psi couples derived from the structural alphabet for protein structures proposed by etchebest et al. for peptides with converging experimental data, calculated structures from peplook and, to a lesser extent from pepstr are close to nmr models. the peplook index for polymorphism is low and the index for stability points out possible binding sites. for peptides with divergent experimental data, calculated and nmr structures can be similar or, can be different. these differences are apparently due to polymorphism and to different conditions of structure assays and calculations. the peplook index for polymorphism maps the fragments encoding disorder and the mean force potential score indicates which residues will be most available for interactions with partners. this should provide new means for the rational design of peptides. several diseases like cancer metastasis, rheumatoid arthritis and chronic lymphocytic b-cell leukemia are linked to the interaction of the cxcr chemokine receptor to its natural ligand, the amino acid protein stromal cell-derived factor- α (sdf- α). [ ] one strategy for the treatment of these diseases could be to block the interaction between cxcr and sdf- α with small cxcr antagonists. furthermore, radiolabeling of suitable compounds with appropriate radioisotopes could provide agents for imaging of cxcr expression in vivo via pet. previous studies by fujii et al. on cxcr antagonists led to a high affinity cyclic pentapeptide with the sequence cyclo[gly-d-tyr-arg-arg-nal]. [ ] to further improve this structure, different approaches have been chosen with respect to metabolic stability, bioavailability, conformational rigidity and chemical versatility for radiolabeling. first, an n-methyl scan of the backbone amides was performed to influence conformational freedom and to increase metabolic stability and bioavailability. n-methylation of arginine residues yielded peptides with moderate affinity (ic<sub> </sub>-values: nm (n-me)arg<sup> </sup> and nm (n-me)arg<sup> </sup>, resp.) whereas n-methylation of other amino acids significantly decreased the affinity (ic<sub> </sub>> nm). by substitution of arg<sup> </sup> by ornithine, the affinity was mostly retained. [ ] the amino group of orn can be alkylated or acylated via radiolabeled groups containing short lived isotopes. moreover, the bioavailability should be improved as the high basicity of the two guanidino groups could be reduced. first ornithine-acylated derivatives showed ic<sub> </sub> values between - nm enabling for the first time <sup> </sup>f-radiolabeling of small cxcr antagonists for pet imaging in vivo. binding of ligands to integrins plays a major role in cell adhesion, migration, and signal transduction of cells. these interactions are important not only for normal cell functions, but also in pathogenic processes. the v integrin for example is involved in tumor cell adhesion and osteoporosis. the association of ligands is specific and requires minimal recognition sequences. therefore, suppression of integrin activity using competitive inhibitors bears great pharmacological potential. the tri-peptide sequence rgd is a prominent recognition sequence of integrin ligands. two new cyclic pentapeptides were synthesized containing the tripeptide sequence rgd as well as -amino-cyclopropane- , -dicarboxylic acid monomethyl ester (acc) and valine varying only with respect to the stereochemistry of acc. both the (+) (all r) and (-) (all s) isomers of acc were incorporated. acc is a cyclic -amino acid as well as a cyclopropyl analogue of aspartic acid. biological tests with cell lines expressing mainly v and v integrin show a higher inhibitory activity of cyclo-(-arg-gly-asp-(+)acc-val-). in order to derive a structure-activity relationship of these two isomers, solution structures in dmso-d were investigated by nmr spectroscopy. subsequently, structural information was obtained by applying distance restraints derived from the nmr spectra in distance geometry/simulated annealing and molecular dynamics calculations. due to the rigidity of the cyclopropyl unit in acc, the structure of the cyclopeptide is significantly influenced by the integrated propane ring, thus explaining the different biological properties. integrins are an important family of cell adhesion molecules. currently, members are known. among other functions, integrin α β is involved in inflammatory processes, leukocyte migration and tumor angiogenesis. the structure of its natural ligand vcam- , including the binding loop sequence tqidspln, has been determined by x-ray crystallography. therefore, it is possible to apply the concept of spatial screening: using small cyclic peptides with structure inducing building blocks, the binding motif is presented in different well-defined structural arrangements. for this study, a series of cyclic penta-and hexapeptides based on the tqidspln sequence has been synthesized. β-homoamino acids, i.e. β -amino acids with proteinogenic side-chains, have been incorporated as structure inducers for spatial screening. although β -amino acids are supposed to prefer the central position of Ψγ-turns, less data exist than for e.g. d-amino acids. apart from the structural characterization of potential high affinity ligands for integrin α β , a major goal of this work is to provide a better understanding of the influence of β -amino acids on the structure of cyclic peptides. the structures of the peptide library have been investigated by nmr spectroscopy, followed by dg/sa and md calculations. the results substantiate the γ-turn inducing capability of β-homoamino acids, but also prove the formation of different turn structures in certain cases. a comparison to the x-ray structure of vcam- shows that the structure of the binding sequence has been successfully approximated by some of the peptides. biological activity tests should lead to meaningful structure-affinity relationships. neuropeptide y (npy) is a -amino acid peptide amide and binds to the so-called y receptors. its most dominant element is the c-terminal alpha-helix spanning amino acid residues - . residues - form a polyproline helix with highly conserved proline residues at positions , and , followed by a loop structure. the importance of the polyproline helix strongly varies between different receptor subtypes. it obviously plays no role in ligand binding at the y receptor subtype, whereas the n-terminal segment is of importance for ligand binding at y and y . in order to further study the importance of the polyproline helix we introduced a conformationally constrained pyridone dipeptide mimetic at different single positions by solid phase peptide synthesis using fmoc/tbu strategy. the resulting peptides have been investigated in cell lines that selectively express y and y receptor, respectively. different methods including radioactive competitive binding assay, cd and nmr have been applied to investigate conformation and interaction of receptor and ligand. loss of affinity at the y receptor is independent of the position and about -, -and -fold, respectively, when introduced once, twice and thrice. introduction of the building block in position / leads to the most reduced affinity at the y receptor subtype but, surprisingly, affinity can partially be regained by introduction of the dipeptide at two additional positions. the position of the dipeptide is of greater importance at y . these novel peptides clearly indicate the importance of proline residues and the structure of the n-terminus for ligand binding. interactions of src homology (sh ) domains with phosphotyrosine (py) containing ligands is critical for regulating cellular processes. the cytosolic protein tyrosine phosphatase shp- contains two sh domains. an intramolecular interaction of the n-terminal sh domain with the catalytic (ptp) domain renders the enzyme inactive in the native state. binding of a py-ligand to shp- n-sh leads to a conformational shift and the dissociation of the sh -ptp complex [ ] . in previous studies we investigated the topographical and conformational preferences of the n-sh domain of shp- using conformationally restricted linear and cyclic peptides derived from the natural interaction partner ros py [ ] . we identified peptides that showed an increased binding affinity for the n-sh domain and partially inhibited ros-mediated shp- activity. on the basis of these results we hypothesized that an imperfect fit of the py+ and py+ side chains might be responsible for the inhibitory effect. in order to confirm this hypothesis we synthesized a new series of peptides and evaluated their biological activity. to better understand the role of each individual sh domain in the activation process we also determined the binding affinity against the c-sh domain and the activation profile of different shp- mutants. pull-down assays of the interactions of the py-ligands with full length shp- confirmed the results obtained for the binding to the individual sh domains. proteins are targets for photo-destruction due to absorption of incident light by endogenous chromophores. mass spectroscopic data presented evidence that structural modification observed upon irradiation of goat alpha-lactalbumin at nm results from tryptophan (trp) mediated cleavage of disulfide bonds [ ] . the aim of the recent studies is to define structural elements that direct the destructive influence of near-uv light on the disulfide bridges of proteins. most of the proteins of the immunoglobulin superfamily contain a so called triad, consisting of two s atoms, forming a disulfide bridge, and a single trp in their close vicinity [ ] . we have indications that this arrangement gives rise to a photolytic degradation similar to that described in our earlier studies for goat alpha-lactalbumin [ ] . we therefore investigated the influence of uv light on the single chain variable fragment (scfv) of a monoclonal antibody ( d a ) [ ] which contains two triads. the results showed that after irradiation of the wild type scfv (i) new bands (degradation products) appeared in electrophoresis experiments and (ii) the affinity for its antigen, von willebrand factor decreased. by site-directed mutagenesis, we modified the critical trp-residues to perform a parallel study on these mutants. background and aims: it is known that thrombomodulin has important function which prevents thrombus. we found kmylcvckn (m, n >= ) peptides derived from thrombomodulin had strong anti-thrombus activity in our recent studies. these peptides formed two structures, parallel and anti-parallel, as dimers, we examined the relation between structure and activity. methods: two peptides of kkkylc(acm)vckkk and kkkkylcvc(acm)kkkk were synthesized by fmoc chemistry. dimer peptides were made by removing acm with iodine, after dissolving in . m tris hcl buffer (ph . ) and oxidizing the mixture of these synthesized peptides spontaneously. then three peptides shown in figure were separated using rp-hplc. the peptide concentration in normal human pooled plasma was micro moles / l when measuring aptt (activated partial thromboplastin time). results: the anti-parallel formed peptide, peptide b, was prolonged aptt approximately . times, although two parallel formed peptide, peptide a and c, were not significantly different from the aptt of normal plasma. conclusions: these peptides have structure-activity relationship, we observed that the anti-parallel formed peptide had strong anti-thrombus activity. insect kinins share a highly conserved c-terminal pentapeptide sequence phe-xaa-xbb-trp-gly-nh , where xaa can be tyr, his, ser or asn and xbb can be ala but is generally ser or pro. they are potent diuretic peptides that stimulate the secretion of primary urine by malpighian tubules, organs involved in the regulation of salt and water balance ( ). the insect kinins preferentially form a cis-pro, type vi β-turn. insect kinin analogs containing tetrazole ( ) and -aminopyroglutamate ( ), both cis-peptide bond, type vi β-turn motifs, demonstrate significant activity in the in a cricket diuretic assay. in this study, we compare the diuretic activity of insect kinin analogs incorporating the four stereochemical variants of the -aminoglutamate (apy) motif. three of the insect kinin analogs incorporating the stereochemical variants, ( the need for new effective and to mammalian cells non-toxic antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for invasive fungal infections. in our laboratory we have produced a serie of low-molecular peptide derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch -nh-y (where x and y were acyl groups with aromatic carbocyclic system). we have found and earlier reported that some of these display high antimicrobial activities against several clinically important gram-positive pathogenic bacteria. in this study we have by solution methods synthesized a group of low-molecular compounds and investigated their antifungal activity. the study included both candida and aspergillus species. we have found that some of the compounds were highly fungicidal. we also made a conformational study in which the residues were separately replaced by selected hydrophobic amino acids and their equivalents. the conformational study showed that the desirable stable intramolecular structure could only be formed in the presence of some vital components. this work was supported by grant ds/ - - - . increased resistance of bacterial pathogens to currently employed antibiotics has resulted in efforts to develop antimicrobial compounds with new mechanisms of action. previously, we have synthesized some high potent antimicrobial compounds based upon the n-terminal binding fragment of human cystatin c. some derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch -nh-y ( ) (where x and y were acyl groups with aromatic carbocyclic system) have displayed the broad antibacterial spectrum and high activity against several clinically important gram-positive pathogens, including multi-resistant staphylococci. herein, the synthesis and structure -antibacterial properties relationship for two series of analogues of are presented. the x and y groups in were replaced by selected substituents with various geometry and distance between aromatic moieties and carbonyl. we have established the general structural features which the discussed class of peptide derivatives should possess in order to displaying the particular antimicrobial activity. this work was supported by grant ds/ - - - . we have synthesized beta-endorphine-like decapeptide immunorphin sltclvkgfy which corresponds to the - sequence of the heavy chain of human igg. immunorphin was found to be a selective agonist of non-opioid (naloxone-insensitive) beta-endorphin receptor. the purpose of this study was to prepare [ h]immunorphin and characterize by its using the non-opioid beta-endorphin receptor on mouse peritoneal macrophages and membranes isolated from various rat organs. by use of tritium-labeled immunorphin ([ h]sltclvkgfy) with specific activity of ci/mmol, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. since dehydroamino acids are quite reactive and various thiol nucleophiles are known to add to their double bonds [ , ] , we hoped that these compounds might act as alkylating inhibitors of cathepsin c (dipeptidyl-peptidase i). its main function is protein degradation in lysosymes, but it is also found to participate in the activation of neuraminidase and proenzymes of serine proteinases (leukocyte elastase, cathepsin g, granzyme a) [ , ] . it is well known, that phosphonodipeptides structural analogues of synthetic substrates of cathepsin c are the model substances in designing the new inhibitors of this enzyme. for that reason we have undertook the synthesis, theoretical and structural investigations of phosphonic analogues of dehydropeptides. gly-∆zphe-abupo(ome) gly-∆zphe-alapo(oet) gly-∆zphe-leupo(ome) gly-∆zphe-valpo(oet) gly-∆zphe-glypo(ome) gly-∆zphe-nbupo(oet) the structure and conformational preferences in this group of peptides had been investigated by mean of nmr techniques. in order to find the interactions between compounds-enzyme (cathepsin c) and interpret the results of biological test, the molecular modelling methods had been used. the interaction of v integrin receptor with its ligands is selectively implicated in various processes, like angiogenesis, bone-formation, tumor genesis and tissue-genetic migration of embryonic cells. several cyclic rgd pentapeptides are known as selective ligands for v integrin receptor. the aim of this study was to prepare a new conjugate, composed of the cyclo[rgdfc] derivative and a branched chain polycationic polypeptide, poly[lys(dl-alam)] (ak). the cyclopeptide was prepared on -cl-trityl chloride resin by fmoc/tbu strategy. the "head-to-tail" cyclisation was achieved in a diluted solution of dmf in the presence of bop and hobt coupling reagents and diea base. coupling of the cyclopeptide to ak polymer was carried out by thioether linkage. adhesion properties of soluble cyclic rgd peptides and their plate-immobilized forms were studied. free cyclopeptides evoke aggregation of cultured primary neural and cloned neural stem cells, while their plate-immobilized forms fail to support cell adhesion. on the contrary, in case the newly synthesized ak-c(rgdfc) conjugate such induction of cell aggregation was not observed. whereas immobilizing this derivative to either glass, or plastic was found to support cell-attachment in case of various cell types. in addition, all cell lines investigated -including also the primary neural cells -attached to ak-c(rgdfc) coated surface and survived, grew or differentiated even in the absence of serum. our data suggest that cyclic rgd -polypeptide conjugates represent a new tool to investigate selective cell adhesion and may provide a novel scaffold-material for directed cell-seeding. in the ph-induced channel closure in combination with the pip interactions. however, their detailed regulations are still remaining unclear. therefore, in the present study, we investigate these crucial residues with electrophysiological recordings and rationally designed mutagenesis based upon our structural analysis of kir . tetramer. lys- is located fairly close to the intracellular channel gate and protrudes its long side chain positive charge into the pore. this may interfere with the potassium flow by providing repulsion charge while ph is lowered, which pushes the channel towards its closed state. mutation to met- therefore reduces such ph-sensitivity. on the other hand, arg- is supposed to be responsible for the maintenance of channel opening in the presence of pip . loss of positive charge at this site may lead to the enhanced ph-sensitivity due to an abolished or reduced pip interaction. more interestingly, the double mutant for both sites reveals a compensation scenario. in combination with the discussion for the role of previously known r-k-r triad, our data provide very clear structural explanation for the exact functional roles of these basic residues in the regulation of ph-sensitive channel gating. mouse obese cart peptides are neurotransmitters involved in feeding, stress and endocrine regulation. leptin, a long-term adiposity signal, upregulates expression of cart in the hypothalamus. recent findings of co-localization of cart and cholecystokinin (cck)-a receptor (responsible for satiety effect of cck) in brain and gastrointestinal tract suggest a neurochemical link between cart peptides and cck. in normal fasted mice, cart( - ) peptide decreased food intake after intracerebroventricular (icv) administration in a dose-dependent manner. anorectic effect of cart peptide was enhanced by peripherally administered cck- , while cck-a receptor antagonist, devazepide blocked the effect of cart peptide on food intake. we used two mouse obesity models in this study: monosodium glutamate (msg) and diet-induced obese (dio) c bl mice. both dio and msg mice had substantially increased fat to body mass ratio compared to their controls and were hyperleptinemic. msg mice were hypophagic and neither cart peptide nor cck- and devazepide had any effect on food intake of these mice. dio mice fed high-fat diet showed slightly decreased sensitivity to central administration of cart peptide, effect of cck- on food intake was preserved. in conclusion, cart peptide and cck- showed a synergistic effect on feeding in control mice that pointed to their probably integrated action in the central nervous system. analogously, devazepide suppressed cart anorectic effect. in msg obese mice, effects of both cart peptide and cck- on food intake were diminished due to disrupted signaling in hypothalamus. in dio mice, additive effects of cart and cck- were partly preserved inspite of hyperleptinemia and increased adiposity. b. chini , s. stoev , l.l. cheng , m. manning , were subsequently shown not be selective for the rat v b receptor [ ] . peptides a-d served as excellent leads to the design of selective agonists for the rat vp v b receptor [ ] . replacement of the arg residue in a-d by lys, orn, dap and dab, led to the first potent and selective agonists for the rat v b receptor [ ] . we now report that three of these; d the aim of the de novo peptide synthesis and the incorporation of cofactors is the construction of artificial protein models. these model systems can be used for understanding the structure-function relationship of native proteins and might open a way for possible applications. protegrin- (pg- ) is an -amino acid peptide with an amidated c-terminus, which forms an antiparallel beta-sheet, constrained by two disulfide bridges. the native sequence of pg- is highly cationic, containing six positively charged arginine residues. it was found that the structural features such as amphiphilicity, charge and shape are important for the cytolitic activity of pg- . in this study we investigate the sar (structure activity relationship) of two pg- analogues: rglcycrgrfcvcvg-nh (bm- ) and rglcyrprfvcvg-nh (bm- ). our antimicrobial activity studies of these peptides show that the bm- peptide is active against microbe species as well as the native pg- , whereas the bm- is completely inactive. the bm- analoque is shorter than native pg- and contains only three arginine residues, therefore is much cheaper in the chemical synthesis, what could be an advantage of this antimicrobial peptide. the conformational studies of both analogues were performed by using d h-nmr technique (in dmso-d ) and molecular dynamics studies. the d solution structure of both analogues was established using interproton distances and torsion angles. for simulated annealing calculations the xplor program was used. our conformational studies show that the bm- forms a regular beta-hairpin structure, which is very similar to that of the native pg- peptide, whereas the bm- analogue is very flexible, what could be a reason of the antimicrobial inactivity. copper amine oxidases (ec . . . ) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, ammonia and hydrogen peroxidase. these enzymes are ubiquitous, occurring in micro-organisms, plant and animals. activity of this enzyme increases under various stress conditions including thermal and water stresses. although lsao is not a thermostable enzyme, it is in maximum stability and activity above physiological temperatures. in this study we have investigated the kinetics of thermal denaturation of lentil seedling amine oxidase (lsao) by measuring its denaturation constant (kden) at various temperatures from to degrees centigrade in mm phosphate buffer, ph . . the results of thermal inactivation curves as well as measuring of a at various temperatures were used to calculate kden. moreover, activation energy (ea) for denaturation reaction was obtained from corresponding arrhenius plot. our results showed that unfolding process started to occur at degree centigrade and ea of denaturation was changed at degree centigrade proving a dominant conformational change of the enzyme at this temperature. the results of the kinetic study are coincident with previously reported equilibrium studies denoting the optimum and melting temperature of the enzyme are and degree centigrade, respectively. development and advancing of enzymatic processes used for production and modification of natural polysaccharides are now major biochemistry challenges. the paper investigates enzymatic systems in invertebrates, in particular, an enzymatic complex obtained from the hepatopancreas of red king crab paralithodes camtschaticus, and clarifies its effect on the mechanism of chitin and chitosan hydrolysis. chitinolytic activity was estimated with spectrophotometer using -(dimemylamine)-behzaldehyde method by the concentration of n-acetyl-d-glucosamine which is educed under chitinolysis. total glycolytic activity was defined by the sum of n-acetyl-d-glucosamine and d(+)-glucosamine in the reaction with potassium hexaferricyanide (iii). content of d(+)glucosamine in the hydrolysates of chitin and chitosan was estimated by highly effective reverse-phase liquid chromatography (helc) of aminosaccharides with ortho-phthalaldehyde. the paper studies the process of chitin and chitosan glycolysis and the effects of different factors (ph, temperature and time of incubation, enzyme/substrate ratio) on the total glycolytic activity of the enzymatic complex from crab hepatopancreas, which is compared with a previously studied proteolytic and exochitinase activities. a mechanism of enzymatic hydrolysis of chitin and chitosan is suggested. study results allowed the following conclusions concerning glycolytic and deacetylase activity of ep: ) ep induces the formation of a monomer (n-acetyl-d-glucosamine) and oligomers (chitin and chitosan) with low deacetylation. thus, ep is characterised by a marked endochitinase (endochitosanase) activity; ) n-acetylglucosamine deacetylase and, apparently, exochitosanase activity was not revealed; ) it was found that chitinase and protease activities of ep are associated with different enzymes. [background] in opioids, the n-terminal amino acid ', '-dimethyl-l-tyrosine (dmt) enhances bioactivity by orders of magnitude. c-terminal modification of the dmt by a methyl group, h-dmt-nh-ch , exhibited µ-opioid receptor affinity (kiµ = . nm) equivalent to that of morphine; however, antinociception was only . - . % [ ] . dmt plays an important role in the message domain to anchor opioid ligands into the active site of opioid receptors, specifically to trigger biological activity by the µ-opioid receptor. [methods] dimerization of dmt through diaminoalkanes [ ] or , -bis-(aminoalkyl)- ( h)-pyrazinone produced potent opioidmimetics with high affinity for µ-opioid receptors (kiµ = . - . nm), agonism (gpi, ic = . - . nm), and antinociception in mice after systemic and oral administration, which verified passage through the epithelial membranes of the gastrointestinal tract and blood-brain barrier [ ] . ( -aminocycloalkane- -carboxylic acid) . in this case the ring consists of five atoms. knowing that acylation of the nterminus of several known b blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the apc substituted analogues were also synthesized in n-acylated form (with -adamantaneacetic acid (aaa)). the activity of eight new analogues was assayed in isolated rat uterus using a modified holton method in munsick solution and in rat blood pressure tests. the results clearly demonstrated the importance of the position in the peptide chain into which the sterically restricted apc residue was inserted. apc at positions led to preservation or reduction of antagonistic qualities, respectively. acc at position enhanced antagonistic qualities in blood pressure test and led to preservation of activity in antiuterotonic test.. in most cases acylation of the n-terminus led to enhancement of antagonistic potencies. our findings offer new possibilities for designing new potent and selective b blockers. background: during the course of developing opioidmimetic analgesics, data revealed that the n-terminal residue ', '-dimethyl-l-tyrosine (dmt) plays an important role in anchoring opioid ligands in the active site of opioid receptors. as a single residue c-terminally extended with an aminomethyl group exhibited µ-opioid receptor affinity (kiµ = . nm) similar to morphine; however, antinociception was only . - . % [ ] . in order to develop potent µ-opioid agonists, the dimerization of tyr or dmt through diaminoalkanes [ ] or , -bis-(aminoalkyl)- ( h)-pyrazinones [ ] resulted in production of unique opioidmimetics with high receptor affinities and potent biological activities [ ] . methods: the synthesis of opioids and opioidmimetics and the determination of their receptor binding characteristics were performed as described previously [ ] [ ] [ ] . results and conclusion: newly synthesized -(tyr-nh-butyl)- -(tyr-nh-propyl)- ( h) pyrazinone and -(tyr-nh-propyl)- -(tyr-nh-butyl)- ( h) pyrazinones (i and ii) exhibited fairly high binding affinity towards µ-opioid receptor (kiµ = . and . nm, respectively). replacement of tyr with dmt in i and ii gave opioidmimetics iii and iv (kiµ = . and . nm, respectively); they exhibited -and -fold higher binding affinity than the tyr derivatives. while iii is a dual µ-/δ-opioid agonist, iv is only a µ-opioid agonist. these findings pave the way to design additional µ-opioid receptor agonists and antagonists for therapeutic application. divalent cations have been known for a long time to influence significantly binding to receptors and biological activity of the peptide oxytocin (ot). there is very low binding of h-ot to the receptors in the absence of these ions. it has been speculated where the divalent cations work. recently an article appeared showing formation of a complex divalent cation-ot and stressing the importance of n-terminal amino group for binding and activity [ ] . however deamino analogues of ot are also very active and their binding is also influenced by divalent cations. we have studied ot, deaminooxytocin (dot) and an ot antagonist (antag) by means of electrospray ms and we have observed that all these compounds form molecular adducts with zn +, mg +, mn + and ca +. in binding experiments using -i antag, the quantity of tracer bound to membranes of hek cells having stable expressed human ot receptor strongly depends on the character and concentration of divalent ions. displacement curves using unlabelled antag do not change in the absence or presence of mm of tested divalent ions. on the other hand, displacement curves using unlabelled ot and dot are shifted to the left in the presence of mg + and mn +, and to much lesser extent by zn + and ca +. all this points to the idea that the divalent ions do work on the site of membrane receptors. biologically active peptides exhibit multiple conformations in solution. thus, the synthesis of conformationally restricted analogues is a valuable approach for determining structure -activity relationships. restrictions can be imposed e.g. through the formation of cyclic structures within the peptide framework by disulfide bridges, or by substitution of chosen amino acid residues that limit conformational freedom, thus forcing the peptide backbone and/or side chains to adopt specific orientations. in recent years, conformationally constrained analogues of bioactive peptides seem to be a feasible approach to providing useful informations concerning threedimensional structure of such compounds which, in turn, could rationalize our knowledge about structure-biological activity relationships and thus help to design peptides with desired pharmacological properties. steric restrictions can be introduced by the formation of cyclic structures within the peptide backbone or by incorporation of amino acids with limited conformational freedom, which in turn results in specific orientations of the peptide backbone and its side chains. another approach to reduce the flexibility of the analogue is substitution of chosen amino acids with various types of pseudopeptides prepared trough short-range cyclizations. the present work is a part of our studies aimed at clarifying the influence of sterical constraints in the n-terminal part of arginine vasopressin (avp) and its analogues on the pharmacological activity of the resulting peptides. we describe the synthesis of four new analogues of avp substituted at positions and or and with two diastereomers of -amino-pyroglutamic acid and four peptides in which we combined the above modification with the placement of mercaptopropionic acid (mpa) at position . all the peptides were tested for their in vitro uterotonic, pressor and antidiuretic activities in the rat. different strategies to modulate shp- activity protein tyrosine phosphatase shp- consists of two sh domains n-terminal to the catalytic (ptp) domain and a short c-terminal tail. the binding of a py-ligand to the n-sh domain is required for an efficient activation of shp- phosphatase activity. the specificity of the shp- sh domains is determined by the py-residue (position ) and residues at positions - , + and + . combinatorial peptide library methods revealed different classes of consensus sequences for both sh domains [ , ] . in addition, the importance of residues c-terminal to py+ (+ to + ), in particular for binding to the n-sh domain, has been demonstrated [ ] . together with investigations of the determinants for optimal sh domain binding and stimulation/inhibition of shp- activity [ ] , these informations were useful for the generation of different strategies for effectors of shp- activity. peptides cyclized between different positions of the general consensus py- to py+ were synthesized and evaluated with respect to n-sh domain binding and stimulation of phosphatase activity. structure-activity studies have revealed that the specificity of an integrin towards its rgd-containing ligands can be evaluated through the distances between the cβ atoms and/or the distance between the charged centers of arginine and aspartic acid as well as, the pseudo-dihedral angle (pdo), composed by the r-cζ, r-cα, d-cα and d-cγ atoms, which defines the relative orientation of the arg and asp side chains. in a previous study [ ] , the antiaggregatory activity of rgd peptide analogues, i.e. their ability to act as fibrinogen receptor αiibβ antagonists, was correlated with the above structural criteria. our results suggested that the fulfillment of the criterion - ο < pdo < + ο is a prerequisite for an analogue to exhibit activity. in the present study, we examine the above criteria to rgd-containing peptides, derived from the active sites of the ecm proteins fibrinogen, fibronectin and vitronectin, as well as, from the cryptic rgd site of von willebrand factor. the correlation of the structural data with the biological activity of compounds, are in good agreement with the previously mentioned - ο < pdo < + ο criterion. furthermore, our results show that the differences in activity of compounds, which display similar distances between the charged centers of arg and asp, can be better evaluated by the pdo structural criterion. acknwolegments : this work was supported by grants from eu and the hellenic ministry of education ( heraklitos). references : the gpiib/iiia receptor, which is a member of the integrin family, is the most abundant receptor in the surface of platelets and can interact with a variety of adhesive proteins including fibrinogen, fibronectin and von willebrand factor. fibrinogen binding on gpiib/iiia is an event essential for platelet aggregation and thrombus formation. mapping of the fibrinogen binding domains on gpiib subunit suggested the sequence - as a putative binding site [ ] . this region was restricted to sequence gpiib - (ymesradr) using synthetic octapeptides overlapping by six residues [ ] . the ymesradr octapeptide inhibits adp stimulated human platelets aggregation and binds to immobilized fibrinogen. in this study we present the conformational analysis of three synthetic analogues yaesradr (a ) ymesaadr (a ), and ymesraar (a ), using nmr spectroscopy and distance geometry calculations. common structural characteristic of peptides a and a is the interaction between the side chains of arg and glu , however in a the guanidino group of arg seems to form salt bridges with both glu and asp . peptide a is stabilized only by a week interaction between arg and glu side chains. the interactions between the residue side chains provoke different overall shape of the three molecules. the most populated structural family of a exhibits a π backbone shape, a a turn around -s a -, while a an almost extended shape. background and aims: endomorphin- (em- : h-tyr-pro-phe-phe-nh ), endogenous opioid peptide isolated from bovine and human brain, has high affinity and selectivity for the mu receptor and produces potent and prolonged analgesia in mice [ ] . in this presentation, the incorporation of ethylene-bridged phe-phe unit (eb[phe-phe]) or piperidine carboxylic aid (pic) in position was carried out to obtain more potent agonist or antagonist with stability against dipeptidyl peptidase iv (dpp iv). methods: the synthesis of eb[phe-phe] unit was achieved according to the procedure of lammek b. et al. [ ] protected peptides were synthesized by a solution method using boc-chemistry. the final products were identified by maldi-tof mass spectrometry and elemental analyses. the receptor binding affinity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from rat brain membranes or cos- cell membranes expressing each opioid receptors. muc glycoprotein, produced by the epithelium of the colon, built up mainly of repeat units of ptttpitttttvtptptptgtqt , can be underglycosylated in colon carcinoma. we have been studying the epitope structure of the muc repeat unit with the mucin peptide specific mab monoclonal antibody. this antibody recognizes the ptgtq sequence as minimal, and ptptgtq as optimal epitope. our interest lies in the modification of this epitope with maintained or enhanced specificity, and we aim to clarify the effect of different epitope modifications on mab antibody binding: a) amino acid changes in the flanking region, b) glycosylation in the epitope core and in the flank. for this we have prepared a) libraries of ax( )ptgtqaa and atptgtqx( )a peptides, and x( )ptgtqx( ) heptapeptides based on the antibody binding properties of the libraries; and b) glycopeptides pt(galnac)ptgtq, ptpt(galnac)gtq and ptptgt(galnac)q. the peptides were prepared by solid phase synthesis; after purification, esi-ms and amino acid analysis characterisation their antibody binding properties were studied by competitive elisa. our results show that a) although all amino acids in positions x( ) and x( ) resulted in antibody binding; in position x( ) hydrophobic, in x( ) aromatic residues provided stronger binding than that of the native peptide; b) glycosylation on thr( ) did not influence the binding of mab , but on thr( ) the presence of n-acetyl-galactosamine, interestingly, slightly increased the antibody recognition. these findings could be useful in designing synthetic peptide vaccines for tumour therapy. histidines play essential role in binding of biological metal ions, either in small or macromolecular chelating molecules, e.g. in metalloenzymes. therefore the low molecular weight polyhistidine type ligands are of potential importance as model substances. continuing our investigations on a novel branched oligopeptide type ligand -(his) (lys) lys-nh -prepared by solid phase peptide synthesis, we investigated the metal ion binding properties with zinc(ii) and copper(ii). the eight primary metal-binding sites are the four imidazole and four ammine groups on the ligand. phpotentiometric titrations revealed, that up to ph all these donor atoms loose their protons on increasing ph. the competition between the protons and the metal ions results the decrease of pka values to about - in the case of copper(ii) and to about - in case of zinc(ii) ion. this reflects the higher stability of the complexes formed with copper(ii) in spite of the weak axial coordination that seems to occur in zinc(ii) complexes. combined potentiometric, spectrophotometric, cd and nmr spectroscopic methods were utilized to investigate the speciation and the structure of the complexes formed in aqueous solution. the prepared cu(ii) complexes cleaved dna, but it is not known whether in oxidative or in hydrolytic manner. because of this ambiguity further studies with zn(ii) complexes will be undertaken. this work has received support through sapstclg nato collaborative linkage grant and from the hungarian science foundation (otka t ). lgr . further studies have shown that in both male and female gonads, insl and lgr represent a paracrine system important for meiosis induction in the ovary and male germ cell survival in the testis. thus insl may have clinical applications in fertility management. we undertook to determine the key structural elements responsible for its unique actions. methods: alanine-scanned analogues of human insl and mimetics of the b-chain alone were prepared by solid phase peptide synthesis. each was subjected to cd spectroscopy for secondary structure analysis and assayed for in vitro lgr binding and activation activity. the tetrapeptide h-dmt-d-arg-phe-cbp was found to be a selective µ agonist [ic (gpi) = . ± nm] with -fold lower potency than the corresponding, highly potent tiq -tetrapeptide, but with still -fold higher potency than leu-enkephalin. in conclusion, we developed selective, cbp-containing δ antagonists and µ agonists with significant potency. recently, we described the syntheses and biological activities of several opioid peptide analogues that contained the n-terminal sequence - , common to dermorphin and deltorphin. some of them showed very high agonist potency both in the gpi assay and in the mvd assay [ , ] . in this work, we designed new analogues in which the sequences were elongated at the c-terminal to obtained the full sequences of dermorphin (a) and deltorphin (b). the syntheses of compounds and their biological activity profiles will be discussed. background and aims: endomorphin- (em- : tyr-pro-phe-phe-nh ) is very potent endogenous opioid peptide, which exhibits high affinity and selectivity for the mu-opioid receptor [ ] . previously, we had reported that [ac c ]-em- containing -aminocyclopropane- -carboxylic acid (ac c) exhibited higher affinities than em- for the mu-opioid receptor [ ] . in order to clarify that the substitution of -aminocycloalkane- -carboxylic acids (acnc: n indicates the number of carbon atoms in a ring) for pro in position of em- is efficient to obtain higher affinity for the mu-receptor, we synthesized . therefore, the replacement of pro to ac c and ac c will be efficient to make these analogs adopt bioactive conformation and exhibit high affinity for mu-receptor. in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain.it was found that b gly and b phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues.it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptide fragment of the b-chain. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och , corresponding to (b -b ) insulin des gly -phe , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och , des gly were synthesized using the solid phase method. the c-terminal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administered for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. aim of this study is to develop peptides as useful tools for degradation of synthetic dyes, which are often pollutants. we focused our interest in peroxidases, a class of enzymes reported to efficiently degrade azo and anthraquinonic dyes. in particular, the fungus versatile peroxidase (vp) of pleurotus eryngii can perform this degradation. therefore, our goal is the synthesis of a peptide based on this peroxidase able to emulate its biological function. the linear and cyclic peptide sequences were derived by the theoretical model of vp [pdb: a ], which determined the amino acids fundamental for the desired function of the active sites. in particular, the residues instrumental for the coordination of the heme, the mn binding site, and the long range electron transfer pathway [ ] , were pin-pointed. moreover, we calculated the radius of the heme cavity. the next step was the synthesis of these peptides in order to verify the coordination of the heme and optimize their sequences. the syntheses were carried out by solid-phase following the fmoc/tbu strategy. because of purification difficulties of the fully-protected peptide, we undertook an alternative synthetic pathway, based on a solid phase head-to-tail cyclisation strategy, following the fmoc/tbu/allyl three-dimensional protection scheme [ ] . next steps will be to test the coordination properties of the synthetic peptides, with respect to the heme, and further computational studies based on the new model of pleurotus the calcium plays an important role in biochemical pathways. it binds to enzymes and proteins in a different process. aspartic (asp, d) and glutamic (glu, e) acid side chains are the main ligands of calcium, but the contribution of the backbone carbonyl groups in the binding is also important. generally the binding places in the proteins are an unstructured loop between two helixes ( -or alpha-helix). the common sequence is the so-called ef-hand motif, which contains amino acids [ ] . it is already known that some proteins also bind calcium with a non-ef-hand loop. for example alpha-lactalbumins have a ten amino acid long sequence for binding [ ] . it is an asp rich sequence where asps are closer to each other than in ef-hand motif (-k fldddltdd -) but only asps side chains take part in calcium coordination. we constructed a series of cyclopeptides to mimic the loop structure of alpha-lactalbumin [ ] . in this study we focus on determining the importance of conservative amino acids within the ca + binding loop of this protein, using microcalorimetry (itc). the itc measurements were performed in different organic solvents and at different temperature. the synthesis of fatty acids in adipose tissue. in this article, we present the solution structure of gip in water and tfe/water determined by nmr spectroscopy. the calculated structures are characterised by the presence of an -helical motif between residues ser -gln and phe -gln respectively. the helical conformation of gip is further supported by cd spectroscopic studies. six gip( - )ala - analogues were synthesised by replacing individual n-terminal residues with alanine. alanine scan studies of these n-terminal residues showed that the gip( - )ala was the only analogue to show insulin secreting activity similar to that of the native gip. however, when compared with glucose its insulinotropic ability was reduced. for the first time, these nmr and modelling results contribute to the understanding of the structural requirements for the biological activity of gip. a knowledge of the solution structure of gip and of the role of its individual residues will be essential in the understanding of how they interact with the gip receptor. efrapeptins are pentadecapeptides produced as a mixture of six closely related analogues (efrapeptin c-g) by the fungus tolypocladium niveum and other members of this species. they consist predominantly of the nonproteinogenic amino acids -aminoisobutyric acid (aib), isovaline (iva), -alanine ( ala) and pipecolic acid (pip), have an acetylated n-terminus and bear an unusual cationic c-terminal headgroup derived from leucinol and , -diazabicyclo[ . . ]non- -ene. efrapeptin c is a competitive inhibitor of the f -atpase and active against the malaria pathogen plasmodium falciparum. an anti-proliferative effect was also reported. conformational analysis of efrapeptin c in trifluoroethanol and dimethylsulfoxide was conducted to obtain structure-affinity relationships. the absence of amide-and -protons resulted in an imperfect assignment and unsatisfying conformational study. specific deuteration of methyl groups in aib did not simplify the assignment. cd and ft/ir spectra hint to helical or beta-turn secondary structures as main structure elements. residual dipolar couplings (rdc) were measured in a stretched cross-linked poly(dimethylsiloxane) gel in dichloromethane. the impact of the rdc on the conformational analysis led to an improved high resolution structure from simulated annealing protocols and consolidated the formation of a helical structure of efrapeptin c in nonpolar solution which is comparable with the binding pocket of the f -atpase. finally, the dynamics of the resulting structures was studied using the gromos force field in explicit solvent. serotonin selective reuptake inhibitors (ssris) are currently among the most frequently prescribed therapeutic agents of depression. their therapeutic use includes also obsessive-compulsive disorder, panic disorder, bulimia. the serotonin transporter (sert) is the target of serotonin selective reuptake inhibitors (ssris). altough the inhibition is the proximal event in antidepressant action, the clinical benefit of antidepressant medications requires weeks of continuous dosing, indicating that their mechanism of action involves events downstream from acute transporter blockade. long-term effects of ssri treatment may be due to changes in intrinsic properties of sert structure, function, or regulation. thus, understanding the mechanism of action of sert remains a primary goal in the search for developing novel treatments for diseases associated with serotonergic dysfunction. in the present study experimentally determined ligand selectivity of the buspirone analogues toward the serotonin transporter was theoretically investigated on the molecular level. the model of serotonin transporter based on the crystal structure of bacterial homologue from aquifex aeolicus (leutaa) was constructed using the traditional homology modelling approach. a series of docking experiments with ssri's were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. structural information about the serotonin transporter and its molecular interactions with ssri's is important for understanding the mechanism of action of these drugs and for development of drugs with improved potency and selectivity. the protein kinase c (prkc) is a member of a super-family of the eukaryotic receptor protein kinases. it forms dimers and is anchored in the membrane, with a cytoplasmic kinase domain and an external domain, presumably acting as a sensor. prkc enables formation of biofilms of bacillus subtilis which show a high degree of spatial organization. they colonize various surfaces and produce complex antibiotic resistant communities. prkc acts as a ser/thr kinase with features of the receptor kinase family of eukaryotic hanks kinases. our current study involved theoretical modeling of the protein kinaze prkc complexes with the modified atp. the ligands were selected from a set of molecular probes developed by k. shah and coworkers [ ] . each modified atp molecule was docked to the active site of the kinase molecule using autodock genetic algorithm procedure. the optimized structures of the complexes were submitted to the molecular dynamics simulations in the amber force field. we obtained four optimized structures of prkcc complexes in water. the results suggest the great similarity of our complexes with human cyclin-dependent kinase [ ] complexes. background and aims. indolicidin is a -residue antimicrobial peptide, which was isolated from bovine neutrophils. this molecule possesses a wide spectrum of antibacterial, antifungal and antiviral activity, furthermore it has also haemolytic effect. data derived from structural investigations led to considerably diverse conclusions regarding the secondary structure of this peptide, therefore the aim of this study was to examine the effect of cis-trans isomerization on the conformational properties of this antimicrobial peptide. methods. the conformational analysis of indolicidin containing cis or trans xxx-pro peptide bonds was performed by simulated annealing calculations with the use of amber force field. results. for the conformers of indolicidin with cis or trans xxx-pro peptide bonds, the evolving secondary structural elements were examined and poly-proline ii helix and type vi beta-turn were identified. in the case of this peptide, various intramolecular interactions may play an important role in stabilizing the structure of conformers. therefore the presence of the h-bonds between backbone atoms, the aromatic-aromatic interactions between the side-chains of trp amino acids and the proline-aromatic interactions between the side-chains of trp and the pyrrolidine rings of pro amino acids was investigated. conclusions. the conformational comparison of the peptides possessing cis or trans xxx-pro peptide bonds resulted in different secondary structural elements for both isomers, which are the poly-proline ii helix and type vi beta-turn for the trans and cis isomers of indolicidin, respectively. the occurrences of various intramolecular interactions are in agreement with the observed secondary structures. we have shown the monte carlo conformational search using macromodel is useful for conformational study of oligopeptides prepared from alpha, alphadisubstituted alpha-amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral alpha, alpha-disubstituted alphaamino acids to predict the helical screw sense of helical structures. here we report computational study on conformation of oligopeptides containing cyclic alpha, alpha-disubstituted alpha-amino acids with side-chain chiral centers. background and aims. the homopolymeric amino acids (hpaas) are polypeptides consisting of the same amino acids. some of them play a relevant role in the formation of several neurodegenerative diseases. most probably the poly-(ala) and poly-(gln) are the best representatives of these peptides because of their important biological effects. our aim was to perform conformational analysis and structural investigation of these two hpaas. methods. to explore the conformational spaces of the peptides, simulated annealing (sa) and random search (rs) calculations were carried out using amber force field. two different forms of the hpaas were modelled: either with charged n-terminal amino group and c-terminal carboxyl group, or with the n-and c-termini blocked by acetyl and n-methyl amide groups, respectively. results. for the conformers obtained by sa and rs calculations, the occurrences of various secondary structural elements like different types of beta-turns, gammaand inverse gamma-turns, alpha-helix, -helix, poly-proline ii helix and beta-strand were investigated. in the cases of various helices and beta-strand, segments with different lengths characterized by these secondary structures were determined along the entire sequence of peptides. for the conformers of the hpaas, the intramolecular h-bonds formed between the backbone atoms as well as between the backbone and side-chain atoms were identified. the vasopressin and oxytocin receptors (v ar, v r and otr) are membrane-embedded proteins belonging to the large family a g protein-coupled receptors (gpcrs). they are involved in crucial physiological functions as the regulation of water metabolism, control of blood pressure and stimulation of labor and lactation, mediated via v r, v ar and otr, respectively. as such, they are involved in a number of pathological conditions and are important drug targets. understanding their inhibition and activation mechanisms may improve design of ligands capable of selective stimulation or blockade of the respective receptors presenting the therapeutic targets. to investigate the otr, v ar and v r interactions with agonists and antagonists thirty computer models of receptor-ligand complexes have been modeled via docking and molecular dynamics (md) and analyzed in details. the receptor models were built on rd crystal structure template or using the coordinates of mii-gtα( - ), for non-active and activated models, respectively. the ligands (arginine vasopressin, oxytocin, desmopressin, atosiban ([mpa ,d-tyr(et) ,thr ,orn ]ot) and barusiban (mpa ,d-trp ,ile ,allo-ile ,asn ,abu ,mol ) were docked into the receptors. the complexes have been embedded into the hydrated popc bilayer and submitted to ns unconstrained md in the amber force field. the relaxed systems have been obtained and analyzed in details. the receptor residues responsible for agonists/antagonists binding have been identified and mechanism of binding involving the highly conserved residues has been proposed. a three-dimensional models of the neurohypophyseal hormone receptors were constructed using a multiple sequence alignment and either the crystal structure of bovine rhodopsin or the complex of activated rhodopsin with gta c-terminal peptide of transducin rd*-gt( - ) prototype to obtain nonactive or activated receptor models, respectively. analogs were docked to v ar, v r and otr, both non-active and activated models. the low-energy receptor-ligand complexes, with properly docked analogs were submitted to the constrained simulated annealing (csa), in vacuo. the relaxed receptoranalog models were obtained. the residues responsible for analogs binding to v ar, v r and otr have been identified and presumable biological activity of these compounds was determined. n-methyldehydroamino acids belong to non-standard amino acids found in nature. n-methyl-(z)-dehydrophenylalanine was found in tentoxin, a selective weed killer, having been produced by several phytopathogenic fungi of the alternaria genus. n-methyl-(z/e)-dehydrobutyrine and n-methyldehydroalanine are components of nodularins and microcystins, families of hepatoxins produced by species of freshwater cyanobacteria, primarily nodularia spumingena and microcystis aeruginosa. the simplest n-methyl dehydropeptides, ac-delta(me)xaa-nhme (where xaa = ala, (z/e)-abu, (z/e)-phe, and val) and, for comparison, the saturated ac-l-(me)ala-nhme analogue were investigated using computational methods. cis-trans b lyp/ - +g**//hf/ - g ramachandran potential energy surfaces were created. the conformers found were optimised at the b lyp/ - +g** level. the effect of the electrostatic solute/solvent (water) interaction on the solute energies was investigated within the scrf method using the polarisable continuum model (pcm) on the geometries of solutes in vacuo. it was found that for all the studied dehydropeptide molecules the lowest conformer (phi, psi = ~ - °, °) has the cis n-methyl amide bond. this feature seems to be independent of the dehydroamino acid moieties, the c-beta substituent and the z/e configuration. the pi-electron conjugation as well as the n-h···n hydrogen bond play the dominant role in the stability of this conformer (see figure) . the preliminary nmr investigations into the conformational preferences of the studied molecules in solution confirm the theoretical results obtained. the strong tendency of the n-methyl amide bond to adopt the cis configuration seems to be the reason why n-methyldehydroamino acids are found in small natural cyclic peptides, where they ensure the conformational flexibility necessary for biological action. the purpose of this study was to determine the potentials of mean force (pmf) of the interactions between models of nonpolar amino acid side chains in water. the potentials of mean force (pmf's) dependent on orientation were determined for systems forming hydrophobic and diagonal complexes composed of side-chain models of alanine, valine, leucine, proline and iso-leucine, respectively, in water. for each hydrophobic pair in water a series of umbrellasampling molecular dynamics simulations with the amber force field and explicit solvent (tip p water model) were carried out and the pmfs were calculated by using the weighted histogram analysis method (wham). in all cases a characteristic shape of pmf plots for hydrophobic association were found, which was manifested as the presence of contact minima and solvent separated minima. depths of contact minima for all systems studied were about kcal/mol. in this work we compared the ability of two theoretical methods of ph-dependent conformational calculations to reproduce experimental potentiometric-titration curves of two models of peptides: ac-k -nhme in % methanol (meoh)/ % water (h o) mixture and ac-xx(a) oo-nh (xao) (where x is diaminobutyric acid, a is alanine, and o is ornithine) in water, methanol (meoh) and dimethylsulfoxide (dmso), respectively. in theory, in all three solvents, the first pka of xao is strongly downshifted compared to the value for the reference compounds, the water and methanol curves have one, and the dmso curve has two jumps characteristic of remarkable differences in the dissociation constants of acidic groups. the predicted titration curves of ac-k -nhme are in good agreement with the experimental ones; better agreement is achieved with the md-based method. the titration curves of xao in methanol and dmso, calculated using the md-based approach, trace the shape of the experimental curves, reproducing the ph jump, while those calculated with the edmc-based approach, and the titration curve in water calculated using the md-based approach, have smooth shapes characteristic of the titration of weak multifunctional acids with small differences between the dissociation constants. quantitative agreement between theoretically predicted and experimental titration curves is not achieved in all three solvents. the poorer agreement obtained for water than for the nonaqueous solvents suggests a significant role of specific solvation in water, which cannot be accounted for by the mean-field solvation models m a. papakyriakou , g.f. vlachopoulos , g.a. spyroulias , e. manessi-zoupa , p. cordopatis angiotensin-i converting enzyme (ace) belongs to the m family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, which catalyses the proteolytic cleavage of dipeptides from the carboxy terminus of a wide variety of peptides, or as an endopeptidase, which hydrolyses peptides bearing amidated c-termini. among the former category of ace peptide substrates, the most distinguished are those involved in blood pressure regulation, such as angiotensin i (angi) and bradykinin (bk). in the latter category falls the gonadotropin-releasing hormone (gnrh) in an attempt to analyze molecular interactions at atomic level we simulated the ace-substrate complexes, using the recently determined d crystal structure of ace testis isoform and a knowledge-based docking method in order to insert the peptide substrate (angi, bk and gnrh) of ace into its catalytic cleft. in order to introduce the effect of protein mobility and gain information about enzyme-substrate recognition and interaction we have sampled the conformational space of these complexes via molecular dynamics simulations with explicit solvent representation. we have also performed molecular dynamics calculations with tace-inhibitor complexes, such as lisinopril, as well as with tace mutated at specific sites, such as the ligands of the two buried chloride ions that have been shown to affect substrate activity. our results provide new insights into the role of specific domains of tace and their implication in the enzyme activity, which is not readily apparent from the available crystal structures. two main mechanisms for the propagation of action potential in myocytes are: ) the free flow of local circuit current through gap junctions and ) the effect of electrical field. here we study effect of each mechanism and their importance during action potential propagation. method: we simulated the cardiac myocyte by the orcad software, then used the model of sinoatrial node to stimulate the myocytes model and studied the propagation of action potential with and without gap junction. result: our results show that, although gap junction solely is not able to mimic physiological condition, but it is necessary for normal cardiac functioning. on the other hand, electric field is not sufficient for successful propagation of action potential and the existence of gap junction is necessary. anthrax is a disease of animals and humans, caused by the bacterium bacillus anthracis. anthrax toxin (at) consists of three proteins, one of which is the anthrax lethal factor (alf). alf is a gluzincin zn-dependent highly specific metalloprotease (~ . kda), which belongs to the m family of the ma clan of zinc metalloproteases. alf cleaves most isoforms of mitogen-activated protein kinase (mapk)-kinases (meks) close to their amino termini, leading to the inhibition of one or more signaling pathways. no data are available on the enzyme-substrate interaction at the molecular level. therefore, we performed classical molecular dynamics simulations on the alf-mkk/mek complexes in order to probe protein-substrate interactions. the simulations pinpointed specific hydrophobic as well as electrostatic alf-peptide substrate interactions and these data were exploited in the building of virtual combinatorial libraries of di-and tri-peptides using the twenty native aminoacids. by applying docking simulations to anthrax zn-metalloprotease around . peptide substrates were virtually screened according to their binding affinity. data suggest that complexes of alf with peptides substrates bearing arg, trp, lys and phe aminoacids, exhibit the highest binding affinity providing evidence for electrostatic interactions between negatively charged residues of alf's active site and positively charged side-chains of di/tri-peptides. new libraries of substrates were built incorporating non-protein residues, organic moieties and chelating groups. alf-substrate complexes with the best score (in terms of binding energy) are further analysed. in the present studies we designed and synthesised seven new bradykinin (bk) analogues and evaluated them in the in vivo rat uterotonic assay using a modified holton method in munsick solution on a strip of rat uterus and in blood pressure test. we used [arg , hyp , thi , , d-phe ]bk, the b antagonist of vavrek and stewart as a model, when designing our analogues. in all cases, the n-terminus of our peptides is acylated with bulky substituent. we previously reported that acylation of the n-terminus of several known b antagonists with various kinds of bulky acyl groups has consistently improved their antagonistic potency in rat blood pressure assay. on the other hand, our earlier results seem to suggest that effects of acylation on the contractility of isolated rat uterus depend substantially on the chemical character and size of the acyl group, as we observed that this modification may either change the range of antagonism or even transform it into agonism. the peptides were synthesized by the solid-phase method using the fmoc-strategy the modifications proposed either preserved or increased the antagonistic potency in the rat blood pressure test. on the other hand, the seven substituents, differently influencend the interaction with the rat uterine receptors and except one led to decrease of antiuterotonic activity. in both cases acylation of the n-terminus led to enhancement of antagonistic potencies. our results may be of value in the design of new b agonists and antagonists. the formations of amyloid fibrils have been reported as for various amyloidosis. several structural models of fibrils are proposed for respective proteins so far. however, their common basic structures and universal features to induce amyloid fibril formations are not known in detail. previously, we examined intermolecular interactions among the several amino acid residues in barnase, which is known to form amyloid-like fibril. based on the experimental results using a series of mutant barnase, we discovered that the interactions between hydrophobic side-chains are the most essential driving force to form the fibrils and that both intermolecular and inter-sheet interactions in the fibril maintain highly ordered molecular packing. in the present paper, we describe a novel prediction method for core regions of various fibril-forming proteins and show the verification of the above possible structural principle. at first, we calculated the interaction's score between side-chains in the antiparallel orientation of beta-strands. next, the peptides with predicted sequences of fibril cores, a couple of high-scored regions with a designed turn moiety to induce a hairpin-like form, were chemically synthesized by spps. as a result, the formation of amyloid fibrils was confirmed for most of high-scored sequences. in addition, we also applied this method to prion protein, we could predict possible beta-strands with hetero-paired orientation. some synthetic peptides involving these strands were proved to have fibril-forming ability. thus, we have developed the novel method to predict the core regions that induce amyloid fibrils. a principal factor analysis (pfa) is a very efficient way of identifying patterns in the data sets even if the patterns are hard to find (e.g. in the high dimensional data sets). this is the reason why the pfa method can be powerful tool for analyzing molecular dynamics (md) trajectories. it is possible to reduce dramatically the trajectory size without loosing significant structural information by applying the pfa procedure. we used this tool for interpretation of results from the molecular dynamics simulations of the model of the transcription factor nf-kb. nf-kb is a protein involved in the numerous biological processes such as regulation of immune response, inflammation, various autoimmune diseases and is used by many viruses, including human immunodeficiency virus (hiv), to activate transcription of their own genes. only the trajectory of the backbone atoms of the nf-kb were subjected to the further analysis. peptides contain many basic sites such as side chains of basic amino acid residues, oxygen and nitrogen atoms of amide groups, and terminal amino groups. these parts can interact with protons. this interaction can change conformational behaviour of peptides and, consequently, their biological functions. the interaction becomes even stronger in the gas phase. in that case, the stability of the peptide chain is influenced, which may have impact on peptide fragmentation during mass spectroscopy analysis of peptide structures. in this study, we will present the interaction of proton with carbonyl oxygens in the model of alanine tripeptide. quantum chemical calculations employing density functional theory using hybrid b lyp functional and - ++g** basis set were used to describe this interaction and also to find possible pathways of proton transfer among interaction sites. two different mechanisms of proton transfer were found. the first mechanism is represented by an isomerization of the proton around the double bond of the carbonyl group. the second mechanism is based on the large conformational flexibility of the tripeptide model where all carbonyl oxygens cooperate. the later mechanism exhibits nearly half energy barrier of the rate-determining step compared to the first one. we focus our attention on situation, in which methyl groups attached to alpha atoms in tripepetide model influence the conformational behavior. results will be presented for all four possible stereochemical configurations. a. papakyriakou , p. galanakis , p. gazonis , g.a. spyroulias p protein is one of the most effective defensive weapons of human body against carcinogenesis, due to its tumor suppression properties. it has been noticed, in many types of cancer, that the functions of p are being downgraded or even suppressed and this fact is ought to the presence of mutated forms of p or to the complete absence of the protein. the suppression of p levels is being indirectly regulated by the protein itself, which activates the expression of a gene, the oncogene mdm (murine double minute ), which expresses the mdm protein, known as human-mdm or just hdm . hdmx protein is a homologue protein to hdm and is being implicated, through various biological processes, in the suppression of p . however, recent experimental evidence suggests that hdm and hdmx proteins are not the only ubiquitin ligases that negatively regulate p through ubiquitin pathway. two recently discovered e ligases, cop and pirh , are also proposed to promote p for degradation. all these proteins function as e ligases bearing a ring finger domain. these domains are characterized by their high content in cysteines and the binding of two zn(ii) ions while they catalyze the latter stage of protein signaling for proteolysis by the s proteasome, through the ubiquitin pathway. the structure variation and the stability of these ring fingers is studied through molecular dynamics simulations of - ns and structure variations are analyzed in a structure-function correlation basis. semax is a synthetic analogue of adrenocorticotropic hormone acth - . it is a nootropic agent containing seven amino acids met-glu -his-phe-pro-gly-pro without hormonal (adrenocorticotrophic) activity. semax is neuroprotective via a mechanism involving the regulation nitric oxide (no) and lipid peroxidation. semax proved to be highly effective in abating the rise in no and restoring neurologic functioning [ ] . it was found to improve intellect and memory in healthy human. it is effective in rehabilitation of people with memory and motor disorders, parkinson's and hantington's diseases, after cerebral stroke and head trauma [ ] . to study conformation dynamics in connection with in vivo activity of semax the molecular dynamics method of standard protocol was applied [ ] . semax and about twenty its analogs were studied. using cluster analysis method semax was found to be more labile among various synthesized analogous (met-gln -his-phe-pro-gly-pro; gly-glu-his-phe-pro-gly-pro; lys-glu-his-phe-pro-gly-pro; glu-his-phe-pro-gly-pro; his-phe-pro-gly-pro). because of collective degree of freedom it has one more stable configuration that is unreachable in analogs. singularities of semax and analogous were studied using -d, -d poincare maps, auto and crosscorrelation functions of special type in terms of topological structure of energy hypersurface. this work was supported by rfbr (pr. - - ), russian ministry of education and science, moscow government and crdf. rhodopsin (rd) is the only representative of g-protein coupled receptors (gpcrs) whose structure has been described with high resolution. thus, it has become the structural prototype for other gpcr. these receptors are involved in transduction of various signals into the cell and actions of many hormones and neurotransmitters. about % of all drugs act through gpcr. growing evidence that rd and related gpcrs form functional dimers/oligomers, followed by direct proof (using atomic force microscopy -afm) that in the retina rd associates into a paracristalline network of rows of dimers, need models of rd-transducin (g t -heterotrimeric protein) complex that would envision an optimal rd dimer/oligomer amenable to satisfy all well documented interactions with gt. current model includes tetramer built of two activated (metaii) and two inactive rd molecules, ligands stabilising metaii: gtα(ile -phe ) and gtγ(asp -cys )farnezy, lipid bilayer built of pc (phosphatidylocholin head groups) , ps (phosphatidyloserine) and pe (phosphatidyloetanolamine) (all three types of phospholipids contain the polyunsaturated docosahexaenoyl chain -dha) and water. experimental data concerning shape of oligomer, conformational changes in metaii, proper interactions and distances among residues have been looked upon. the poster shows results of the molecular dynamic carried in amber force field for ~ ps in the periodic box. conformational changes which took place during simulation caused proper adaptation one another monomers in tetramer and ligands to activated receptors. the human cystatin c (hcc) is a one of known domain swapping proteins. during this process, one of the hcc β-hairpins (β -l -β ) changes its conformation forming long β-strand. this conformational transition destabilizes the monomer structure and leads to domain-swapped dimer. the causative force for changing the βhairpin conformation is assumed to be the alleviation of distortions of the l -loop val amino acid residue's backbone. following the above assumption and our previous conformational studies of the hcc β-hairpin peptide we investigated the influence of the point mutations, v d, v p and v n of the val residue, on the β-hairpin peptide structure. the conformational studies by means of cd spectroscopy and molecular dynamics studies were performed. the study revealed that the hcc peptide with the wild-type sequence has the strongest tendency from all studied peptides to form a β-hairpin structure. on the basis of these results we conclude that the presence of distortions in the val residue of l -loop is unlikely to cause the d domain swapping of the human cystatin c. acknowledgments: this work was financially supported by the ministry of scientific research and information technology of poland under grant t a . temporin a (ta) (flpligrvlsgil-nh ) and temporin l (tl) (fvqwfskflgril-nh ) are small, basic, hydrophobic, linear antimicrobial peptides amide found in the skin of the european red frog, rana temporaria. these peptides have variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and resistant staphylococcus aureus as well as vancomycin-resistant enterococcus faecium strains. to gain further insight into the mechanism of action of these small antimicrobial peptides, we have investigated their conformational behaviour in different environmental conditions. more specifically, we deeply investigated by solution nmr spectroscopy in water and water/dmso ( : ) solutions as isotropic solutions and mm aqueous solution of dpc (dodecylphosphocholine) was used as membrane mimetic environment. understanding the basis of the interactions of temporins with membranes could be crucial for the design and synthesis of potent antimicrobial agents. cripto is the founding member of a family of soluble and cell bound growth factors known as egf-cfc [ ] distinguished by the presence of an n-terminal signal peptide, two distinct cysteine-rich domains (crd) and a c-terminal hydrophobic region involved in cell surface attachment by a post-translational gpi modification. the characteristic crds, known as egf-like and cfc domains (from the first members cripto, frl and cryptic), both span about residues with disulfide bridges [ ] each, which, presumably, beside a possible functional modularity, confer them also a structural independence. in this work we have focused our attention on the cfc domain of mouse cripto. the domain has been produced by ssps, along with variants bearing mutation on h and w , that have been described as crucial for alk receptor recognition. the two variants have been purified and refolded, achieving the correct disulfide bridges, and then comparatively analyzed by cd spectroscopy under different ph conditions; thus obtaining experimental insights on the structural arrangements of this new class of protein domains. furthermore, the binding properties of wild type and mutants cfc domains to alk receptor have been determined by using an elisa-based assay. our results demonstrated that the cfc domain alone can directly bind alk in the absence of additional ligands and, furthermore, confirmed a role of h /w in cripto/alk interaction. there is considerable interest in the pharmacology of the two cholecystokinin (cck) receptors ccka-r (or cck- ) and cckb-r (or cck- ) that mediate the biological action of the cck hormone. they are membrane receptors belonging to the superfamily of g-protein coupled receptors (gcpr) and are predominantly located in the gastrointestinal tract and in the central nervous system, respectively. a library of cyclic peptide analogues derived from the octapeptide c-terminus sequence of the human cholecystokinin hormone [cck( - ), or cck ] has been designed, synthesised and characterised. the peptide analogues have been rationally designed to specifically interact with the cck type b receptor (cckb-r) on the basis of the structure [ ] of the bimolecular complex between cck and the third extracellular loop of cckb-r [namely, cckb-r( - )]. the new ligands showed binding affinities generally lower than that of parent cck . anyway, structure activity relationship data underline that preservation of the trp -met motif is essential, and that the phe side chain and a carboxylic group close to the c-terminal end must both be present. the nmr conformational study in dpc micelles of the compound endowed with maximal binding affinity (cyclo-b , ic = m) shows that this compound presents the turn-like conformation, centred at the trp -met segment, as planned by rational design, and that such conformation is stabilised both by the cyclic constrain and interaction with the micelle. cripto is the founding member of a family of extracellular growth factors called egf-cfc found in mouse, human, chicken, xenopous and zebrafish [ ] . these proteins are characterized by the presence of an n-terminal signal peptide, a c-terminal hydrophobic region and two highly conserved cysteine-rich domains, the egf-like (epidermal growth factor) and the cfc (cripto/frl /cryptic). cripto is strictly required in the early embryonic development and contributes to deregulated growth of cancer cells in adults, since it is highly over-expressed in many solid carcinomas. it has been proposed that each single domain of cripto could bind different protein partners, playing different functional roles [ ] . on this grounds, investigation of the single domains d-structures can have also strong functional implications. we present here an extensive conformational analysis of the mouse cfc domain ( - sequence) and of the w a mutant based on nmr data. sequences have been synthesized by spps and refolded reconstituting the correct disulfide bridges [ ] . the molecular models have been built by computational methods using the nmr data collected under both acidic (ph ) and nearly physiological (ph ) conditions. both domains show a globally extended folding with three strands linked by the three disulfide bridges and two connecting loops, in which h and w , key residues in receptor binding, are exposed to the solvent urantide, a selective antagonist. thus, we carried out a study aiming at the characterization of conformational arrangement and affinity properties of ut extracellular segments.we measured by surface plasmon resonance (spr) technology the binding affinities of the three ligands, u-ii, urp and urantide towards the three extracellular loops of ut. furthermore, the secondary structures of the synthetic receptor fragments in presence of dodecylphosphocholine micelles and interaction with ut ligands were analysed using nmr spectroscopy. spr data showed that the ec loop ii was able to recognize the ligands u-ii, urp and urantide with similar affinities while none of these two ligands were able to interact with the extracellular loop i. furthermore, the absence of binding of urantide, a peptide antagonist, suggested strongly that loop iii would be involved in the signal transduction process and implies that u-ii and urp, but not urantide, would bind to ut according to a common pattern. moreover, the results indicate that potent ut antagonists could be designed by producing highaffinity ligand targeting the extracellular loop ii. also, the spr and nmr studies revealed that the synthetic structural ut domains contained some of the conformational and chemical features essential for the binding of hu-ii, urp and urantide to hut. synthetic cysteine-rich replicates of naturally occurring peptides such as hormones, neurotransmitters, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure. the presence of identical cysteine patterns in the sequence were found to lead to identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. therefore, it is generally accepted to attribute the disulfide connectivities based on the homology of their cysteine pattern. minicollagen- from the nematocysts of hydra is a trimeric protein containing n-and c-terminal cysteine-rich domains involved in the assembly of an intermolecular disulfide network. examination of three-dimensional structures of peptides corresponding to these folded domains by nmr spectroscopy revealed a remarkable exception from the general admitted rule [ ] . despite an identical cysteine pattern, they form different disulfide bridges and exhibit distinctly different folds. additionally, comparative analysis of the oxidative folding revealed for the c-terminal domain a fast and highly cooperative formation of a single disulfide isomer, the n-terminal domain proceeding mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. to our knowledge, this is the first case where two short peptides with identical cysteine pattern fold uniquely and with high yields into defined, but differing, structures. therefore, these cysteine-rich domains may well represent ideal targets for structure calculations to learn more about the elementary information encoded in such primordial molecules. the conformational change of the cellular prion protein, prpc, to its virulent "scrapie" form, prpsc, is believed to be responsible for prion infectivity. and several studies suggest that the prion disulfide bond is important for the stability, structure, and propagation of prion oligomers. to test this hypothesis, we selected two conserved peptides flanking the disulfide bond in the sheep prion protein, and measured the secondary structure of these peptides with circular dichroism, hydrogen/deuterium exchange, and molecular dynamics simulations. our preliminary data suggests that the two peptides do not adopt stable secondary structure, native or otherwise. thus, the folding intermediate of a prion protein seems unlikely to comprise local structure around the disulfide bond. the conformationally labile cα-tetrasubstituted α-amino acid residue bip possesses non isolable (r) and (s) atropoisomers. we have previously reported that in the linear dipeptides boc-bip-α-xaa*-ome with α-xaa* = ala, val, leu, phe, (αme)val and (αme)leu residues at the c-terminal position of bip, the onset of an equilibrium between diastereomeric conformers with unequal populations could be observed by cd and h nmr. the phenomenon of induced circular dichroism (icd) represents the basis for the "bip method", an easy and fast configurational assignment for chiral α-amino acids. in search for an extension of the bip method, we investigated the boc-bip-β-xaa*-ome dipeptide series with β-xaa* = β -hala, β -hval, β -hleu, β -hpro, β -hphe, or the cyclic β , -amino acids ( s, s)/( r, r)-achc and ( s, s)/( r, r)-acpc. low-temperature ( k) h nmr spectra in cd od revealed the presence of two conformers. significant d.r. (diastereomeric ratio) values were observed for all combinations of bip with both β -and cyclic β , -amino acids. cd analysis in meoh solution of the boc-bip-β-xaa*-ome dipeptides allowed us to conclude that the cd resulting from the induced axial chirality in the biphenyl core of the bip residue gives clear information on the β-xaa* configuration for both β -and cyclic β , -amino acids (except the aromatic β -hphe), with a p torsion of the biphenyl axial bond of bip being preferentially induced by (l)-β -xaa* as well as cyclic ( s, s)-β , -xaa* c-terminal residues. we have recently reported that the induced circular dichroism (icd) of the biphenyl core of boc-bip-xaa*-ome dipeptides based on the conformationally labile cαtetrasubstituted α-amino acid residue bip could allow an easy and fast configurational assignment for both α-and β-xaa* amino acid residues. in search for other biphenyl/xaa* architectures in which a transfer of central to axial chirality could result in a potentially useful icd, we considered n-substituted , -dihydro- hdibenz[c,e]azepine (daz) derivatives from α-and β-amino acids as interesting candidates. in the present communication, we report the syntheses, and the h nmr and cd analyses of a series of (daz)xaa*-ome amino esters derived from α-, β -, and cyclic β , -xaa* residues, namely d- β-peptide molecules possess interesting conformational characteristics and biological properties. they may represent a new class of rigid foldamers potentially useful as templates or spacers. d-structures of β-peptides have been experimentally investigated using x-ray diffraction and various spectroscopic techniques, but they have never been doubly spin labelled and studied by epr. a terminally protected β-hexapeptide, based on trans-( r, s)-β-toac and trans-( s, s)-achc, synthesized using classical solution methods, was found by ft-ir absorption and cd techniques to adopt the - -helical conformation. a set of four, terminally blocked, hexapeptide sequences, each characterized by four strongly helicogenic aib residues and all combinations of the two isomeric ile/allo-ile residues at positions and was synthesized by solution methods and fully characterized. a detailed solution (by ft-ir absorption, nmr, and cd) and solid (crystalline)-state (by cd and x-ray diffraction) conformational investigation allowed us to validate our assumption that all four peptides are folded in well developed - -helical structures. however, the most relevant conformational conclusion extracted from this d-analysis is that the handedness of the - -helical structures formed does not seem to be sensitive to the configurational change at the β-carbon atom of the constituent ile versus the diastereomeric allo-ile residues (in other words, the dominant control on this important structural parameter appears to be exerted by the chirality of the amino acid α-carbon atom). these results complement published findings on the diverging relative stabilities of the intermolecularly h-bonded β-sheet structures generated by ile versus allo-ile homo-oligopeptides. taken together, these data represent an experimental proof for the intuitive view that potentially different conformational properties are magnified in a strongly self-aggregated homo-peptide system (as compared to weakly self-aggregated, helical, host-guest peptides such as those investigated in this work). in a first approach to β-sandwich proteins the hydrophobic core between two symmetrical sheets each with four antiparallel β-strands was computationally designed by packing of amino acid side chain conformations (rotamers) in an initially given backbone structure. the proteins were synthesized by coupling four peptides with β-hairpin structure to a cyclic decapeptide template (tasp). an aggregation observed by equilibrium ultracentrifugation with the first designed proteins was decreased to a dimer by increasing the surface charge in two further variants of this protein from - to + and + . replacement of l-pro by d-pro in the loops and the template proved to stabilize the β-structure. these results led us to an improved design of an asymmetric core with algorithms for selection of proteins with a minimal number of atom clashes and cavities in the core, and a maximum number of hydrogen bonds after energy minimization. this protein termed beta-mop (modular organized protein) was synthesized in amounts to allow a characterization by cd, ftir, tryptophan fluorescence during reversible unfolding, and by high resolution nmr. nmr measurements of diffusion indicate a dimeric structure. the β-structure is stable up to °c ( k) as determined by d h nmr showing sharp resonance lines. the d h, h dqf-cosy spectrum at mhz shows a typical βsheet distribution extending well into the characteristic regions > . ppm (for amide protons) and > . ppm (for hα signals). all data indicate a well folded protein with β-structure. a.s. galanis , z. spyranti , n. tsami , g.a. spyroulias , e. manessi-zoupa , g. pairas , i.p. gerothanassis , p. cordopatis angiotensin-i converting enzyme (ace) belongs to the m family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, or as an endopeptidase. among the ace peptide substrates, the most distinguished are angiotensin i (angi) and bradykinin (bk) due to their role in blood pressure regulation. despite the fact that biological data strongly suggest that the two active sites exhibit different selectivity and activity towards physiological and exogeneous substrates none experimental evidence for the interaction of angi and bk with ace catalytic sites, is available so far. a dual approach for studying the structure and physicochemical determinants of ace-angi/bk interaction has been performed. the first involves the application of molecular dynamics simulations (presented elsewhere in this book) and the second is making use of the solid-phase synthesized - aa ace catalytic site maquettes (csm) bearing the native sequence and the application of the nmr spectroscopy, and presented herein. therefore, high-resolution multinuclear nmr spectroscopy was applied to analyze the conformational features of ace substrates angi and bk in dmso or aqueous mixtures. then titration experiments were conducted and ace csms were titrated by angi/bk peptides, while monitored by nmr. d h- h tocsy and noesy experiments were used in order to map the interaction site of both substrates and csm through chemical shift perturbation and comparison of noe signal differentiation. competitive binding studies were also carried out through titration studies of csm-angi/bk and known ace inhibitors. a. carotenuto , p. grieco , l. auriemma , e. novellino , v.j. hruby the melanocortine receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. understanding the conformational basis of the receptor-ligand interactions is crucial for the design of potent and selective ligands for these receptors. the conformational preferences of the cyclic melanocortin agonists and antagonists mtii, shu , [pro ]mtii, and pg (table ) when two chromophores are chirally oriented and close enough to one another in space, their excited states couple and become non-degenerate. this phenomenon, termed exciton coupling, produces a typical bisignate cd curve. the intensity of the cd couplet is dependent on the molar extinction coefficient and the distance between the interacting chromophoric moieties, while the sign is governed by the angle between the effective electron transition moments. in particular, exciton coupling over a long distance can be observed only with strongly absorbing chromophores, e. g. porphyrin derivatives, characterized by their extremely intense and sharp soret band near nm. in this work we examined by the exciton coupled cd method the combined distance and angular dependencies, generated by the seven conformationally restricted β-turn and - -helical spacer peptides -l-ala-[l-(αme)val]n-(n = - ) on a system formed by two intramolecularly interacting -carbamido- , , , -tetraphenylporphyrin chromophores. these porphyrin derivatives are confirmed to be excellent reporter groups. we find that not only the centerto-center separation (from to Å) between the two chromophores, but the orientation (roughly parallel or perpendicular) between the directions of their effective transition moments as well, are responsible for the onset or even for the modulation of the intensity of the exciton coupling phenomenon. in particular, the porphyrin…porphyrin interaction is still clearly detectable over the long distance of ca. Å when the two chromophores are about perpendicularly oriented. a. hetényi , g.k. tóth , c. somlai , t.a. martinek , f. fülöp β-peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the field of β-peptides towards the construction of possible new secondary structures, the replacement of the cα and cβ atoms of the β-amino acid with heteroatoms could be an attractive modification, for example cβ-atom of β-peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies [ ] [ ] [ ] about hydrazine peptides, and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of -amino-pyrrolidine- s-carboxylic acid homo-oligomers (figure ), their potential energy hypersurface were probed at the ab initio b lyp/ - g** level. the calculations predicted the -strand to be the most stable structure. the hydrazino-peptides in question were synthetized on solid support, and their structures were characterized by nmr and cd methods. the results were found to be in good accordance with the -strand structure. cathepsin c [ec . . . ]( ), which belongs to family of cysteine proteases, catalyzes hydrolysis of n-terminal peptide, preferential glyphe. this enzyme may play a part in chronic airway diseases ( ) . also increaser level of enzyme was found in case of cancer, rheumatism and muscle's distrophy ( ) ( ) ( ) . for this reason we have undertook investigations of peptides containing two dehydroamino acid residues, which could act as alkylating inhibitors of this enzyme. to define structure and conformation of investigated peptides we were used different methods of nmr spectroscopy, including standard d experiments, protonproton correlations, proton-carbon correlations, and d noe experiments. to complete structural research computational chemistry methods had been used. in order to predict the biological activity of investigated peptides, the simulation of docking process of these peptides to enzyme active site had been made and after that correlated with results of enzymatic test.. the obtained results suggest, that investigated peptides containing two ∆phe residues (z and e isomers respectively) in solution have bent conformation, which is stabilized by intermolecular hydrogen bonds. these results are confirmed by the results of theoretical calculations. also simulation of docking process have showed two possible peptide's orientation in active site of cathepsin c and allowed the rational interpretation of biological test's results. turns are important elements of secondary structure in peptides and proteins. different types of turns are distinguished according to the number of residues involved. the most abundant is the β-turn, which involves four consecutive amino acids with the co at position i hydrogen-bonded to the i+ nh. the γ-turn is centred at a single residue and is generally stabilized by a hydrogen bond between the i co and the i+ nh. model dipeptides rco-l-pro-xaa-nhr' are the smallest systems able to adopt the β-turn conformation, which is favoured by the presence of proline at i+ . a peptide of this series, incorporating a cyclopropane amino acid (xaa), has been shown to accommodate two consecutive γ-turns in the solid state [ ] , instead of the expected β-turn conformation. the double γ-turn encountered is unique among crystalline short linear peptides. in fact, the γ-turn is observed almost exclusively in low-polarity solvents, and only a few oligopeptides of cyclic structure exhibit a γ-turn in the crystal. this is the first time that the strong tendency of pro-xaa dipeptides to adopt a β-turn in the solid state has been switched to the γ-turn. theoretical calculations [ ] also show the high preference of this cyclopropane amino acid for the γ-turn conformation. [ oxidative stress plays an important part in the development of cardiovascular disease (cvd). haptoglobin is a hemoglobin-binding protein that has a major role in providing protection against heme-driven oxidative stress. there are two common alleles for haptoglobin ( and ), and the three phenotypes, haptoglobin - , haptoglobin - , and haptoglobin - , differ in their ability to function as antioxidants. we determined whether there was a relation between the haptoglobin phenotype and the development of coronary artery diseases. haptoglobin (hp) phenotypes were determined in iranian patients with coronary artery diseases. we performed haptoglobin (hp) genotyping by polymerase chain reaction (pcr) using allele-specific primer-pairs. in multivariate analyses controlling for conventional cvd risk factors, haptoglobin phenotype was a highly statistically significant, independent predictor of cvd. the odds ratio of having cvd in patients with the haptoglobin - phenotype was . times greater than in patients with the haptoglobin - phenotype. an intermediate risk of cvd was associated with the haptoglobin - phenotype. these results suggest that haptoglobin phenotype is an important risk factor in determining susceptibility to cardiovascular disease which may be mediated by the decreased antioxidant and antiinflammatory actions of the haptoglobin allelic protein product. the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different stable conformations. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of different environmental conditions, or the action of chaperones. elucidation of the molecular interactions that occur during the transformation from α-helix to β-sheet and the consecutive formation of amyloids on a molecular level is still a challenge. therefore, the development of small peptide models that can serve as tools for such studies is of paramount importance. we succeeded in generating model peptides that, without changes in their primary structure, predictably react on changes of diverse environmental parameters by adopting different defined secondary structures. these de novo designed peptides strictly follow the characteristic heptad repeat of the α-helical coiled coil structural motif. furthermore, domains that favour β-sheet formation and aggregation can be generated. alternatively, those peptides can be equipped with functionalities that allow either the binding of metal ions or the interaction with membranes. as proof of our concept we showed that the resulting secondary structure of such peptides will strongly depend on environmental parameters. thus, this system allows to systematically study the interplay between peptide / protein primary structure and environmental factors for peptide and protein folding on a molecular level. the pathogenesis of alzheimer's disease (ad) is strongly linked to neurotoxic assemblies of the amyloid β protein (aβ). aβ is a soluble component of human plasma which by an unknown mechanism becomes aggregated and neurotoxic. some genetic mutations within the aβ sequence cause very early onset of ad-like diseases, probably by facilitation of aβ assembly into neurotoxic species. recently, it was found that not amyloid fibrils, but smaller aβ assemblies initiate a pathogenic cascade resulting in ad. therefore, preventing the folding of nascent aβ monomers would have therapeutic benefit. to uncover details of structural changes accompanying the aggregation process, especially its initial stage, we have decided to study the aβ( - ) fragment and its mutation-related variants. our recent studies on this aβ fragment using the cd method and the aggregation test have proved it a good model for structural studies. the obtained results confirmed that the aggregation process follows the scheme with an α-helical intermediate and pointed out differences in the behaviour of aβ variants. to further confirm the scheme of the structural changes accompanying aggregation we have applied ftir spectroscopy and analysed aggregation-induced changes of the amide i band which is directly related to peptide backbone conformations. the ftir spectra analysis indicate that water addition provoked conformational changes are strongly dependent on the aβ( - ) variant and in some cases the formation of α-helical intermediate seems to be preceded by helix formation. to verify this hypothesis the temperature dependent atr ftir spectra will be analysed. supported by ug bw grant. amino acid octarepeats present in the prion protein bind to cu + and are considered as a potential periplasmic copper ion transporters. this octarepeat is located in the unstructured region of the prion protein, which is supposedly not intricately involved in prion aggregation. our group is involved in exploring the function of octarepeats with a special emphasis on their possible role in amyloid fibril formation and aggregation. in this context, we have prepared truncated peptide constructs derived from the prion protein octarepeat phgggwgq and have reported their fibrillation activity. we will present aggregative behavior of a truncated bis-pentapeptide, containing gggwg segment, when tethered with a flexible linker diaminobutane. fibrillar architectures were observed by this bis conjugate after incubation in water which was probed by different microscopic and steady state fluorescence techniques. further investigations with ki revealed a homogeneous environment of the two tryptophan moieties in the conjugate. in the absence of other side-chains, it is likely that fibril formation involves hydrophobic interaction between tryptophan indole moieties and main chain backbone interactions. interestingly, a facilitator role for aromatic-glycine motifs for amyloid aggregation has been proposed based on bioinformatics search of the swiss-prot and trembl databases. collagens are known to fold into a highly ordered rode-shaped triple helix with stretches of lower and higher suprastructural stability and even disruptions to modulate recognition by other proteins that interact with the extracellular matrix [ ] . to increase understanding of folding and stability of the collagen triple helix, we have adressed the design of photocontrolled collagenous peptides. our aim was to crosslink two side chains of the repetitive (xaa-yaa-gly) sequence motifs of collagen model compounds via an azobenzene chromophore in analogy to our previous studies on photomodulation of the conformational preferences of cyclic peptides and more recently of hairpin-peptide model systems [ ] . molecular modeling experiments suggested appropriate sequence positions that could result in triple-helical peptides with conformational stabilities that can be modulated by cis/trans isomerization of the azobenzene moieties. as light switchable crosslinker azobenzene- , '-n-( -iodo- -butynenyl)carboxyamide was synthesized for reaction with two ( s)-mercaptoproline residues placed in suitable xaa and yaa positions, respectively. by this approach a fully folded triple helix was obtained upon thermal relaxation, and unfolding was induced by irradiation at nm. the favorable optical properties of the azobenzene derivative together with the regular suprastructure resulted in a valuable model system that allows for ultrafast time-resolved studies of collagen folding and unfolding. amyloid formation is connected with alzheimer's disease, parkinson's disease, finnish familial amyloidosis. after protein misfolding short peptide sequences act as "hot spots" providing the driving force for protein aggregation in amyloid fibrils. we have identified one of these sequence stretches in the abl-sh domain of drosophila (dlsfmkge) whereas the human homologous region (dlsfkkge) is predicted to be less amyloidogenic. the possible reason for the difference of amyloid formation propensities of the two peptides was investigated by molecular dynamics (md) of β-sheet structures. the antiparallel alanine β-sheets consisting of two and ten strands were constructed, minimized, and mutated to the sequences dlsfmkge and dlsfkkge. all four systems: ) dlsfmkge -two strands, ) dlsfkkge -two strands, ) dlsfmkge -ten strands, ) dlsfkkge -ten strands, were surrounded by Å layer of water molecules over the solute and subjected to md, amber . force field, ntp protocol. the md runs were started at the temperature of k and the temperature was elevated stepwise by degrees till k. the results show considerably higher hydrogen bond percentage for dlsfmkge than that one for dlsfkkge during the course of the simulation, thus suggesting that dlsfmkge is a potential fibril-maker, but dlsfkkge is not. two strand β-sheet systems were stable until k. the ten strand β-sheets are more stable. angiotensin-i converting enzyme (ace) has a critical role in cardiovascular function, which consists of cleaving the carboxy terminal his-leu dipeptide from angiotensin-i producing a potent vasopressor octapeptide, angiotensin-ii. there are two isoforms of ace. the somatic isoform is present in all human cells except the testis cells, where the testicular isoform is produced. the major difference between these two types is that, the somatic form has two active sites, at the n-and c-end respectively while the testicular has only one, which is almost identical to the somatic c-terminal active site. here we report the structural study of a aa peptide (previously expressed in bacteria), which corresponds to an extended domain of the human somatic n-terminal active site of ace (ala -gly ) by circular dichroism experiments, and the overexpression in bacteria, purification and structural study, using circular dichroism techniques, of a aa peptide which corresponds to an extended domain of the human somatic c-terminal active site of ace (ala -gly ). following the subclonning into an appropriate expression vector and the expression, the peptide was isolated from the inclusion bodies using chromatography techniques. the recombinant protein fragment had a molecular weight, measured by esi-ms, of kda which was in consistence with the theoretical calculation based on the dna sequence. the recombinant peptides acquired their theoretically calculated secondary structure only when , , -trifluoroethanol is present at a concentration of ~ %. in order to elucidate their structures, solutions of these peptides, labeled with n and/or c, will be studied by nmr spectroscopy. aggregation of peptides is believed to trigger various degenerative diseases but it also plays an important role for the preparation of peptide fibres and peptide-based biomaterials. it is therefore extremely important to understand the mechanisms involved in peptide aggregation and be able to control them. studies were performed on a library of amphiphilic peptides, designed around the sequence of a model antimicrobial peptide rich in leucine and lysine. the library also included peptide hybrids in which natural amino acids were replaced by non-proteinogenic omega-amino acids, such as -aminohexanoic acid and -aminononanoic acid. the aim was to estimate the aggregation and its correlation with the biological activity by using a fluorescence technique commonly employed to calculate the cmc (critical micelle concentration) of surfactants. peptides and peptide hybrids were synthesized on solid support using the fmoc polyamide protocol. they were purified by semi-preparative rp-hplc and characterized by esi-ms and analytical rp-hplc. the aggregation behaviour of the synthesized molecules was investigated in water by steady-state fluorescence measurements using pyrene as fluorescent probe. peptides were dissolved in water/pyrene or water/pyrene/ . fluorescence spectroscopy has become an extremely valuable technique for conformational studies of biopolymers, the development of peptide-based chemosensors, and biochemical research in general. in this connection, synthetic amino acids as fluorescent probes to be incorporated into a peptide chain may exhibit significant advantages over the related protein (trp and tyr) residues in terms of potentially different and ameliorated properties. we recently designed and prepared a new fluorescent amino acid, antaib, based on a planar anthracene core and belonging to the class of achiral, ciα↔ciα cyclized, cα-tetrasubstituted α-amino acids (strong β-turn and helix inducers in peptides). peptides based on antaib combined with (l)-ala residues were synthesized and subjected to a conformational analysis. more specifically, the protected derivatives boc-antaib-oet (oh) and fmoc-antaib-otbu (oh) were prepared in seven steps from , , conformational transitions in peptides and proteins emerge to play the major role in the genesis and evolution of prion related diseases and alzheimer's disease (ad). in this context, conditions influencing this transition and the following aggregation process are of paramount interest. peptides and proteins that are involved in aggregation processes contain potential metal binding sites. the concentration of metal ions in the brain tissue is naturally high and zn in the mm range has been found in ad amyloid plaques. thus, it is widely accepted that metal complexation is one of the key incidents that lead to conformational transitions and aggregation. we present here a coiled coil based model peptide system with an intrinsic amyloid forming tendency which can be used to study the impact of different metal ions on secondary structure and aggregation. metal complexing histidine residues were incorporated to create potential binding sites which, depending on their position and the nature of the metal ion, dictate folding and aggregation. the time dependent conformational transition was monitored by cd-spectroscopy. aggregates were characterized by cryo tem. high resolution fticr-ms experiments revealed information on the stoichiometry of the peptide-metal complexes. in the absence of metal ions the presented peptides formed amyloids in a time range of weeks. depending on the his positions and milieu conditions, the nature of the metal ion determines folding and aggregate morphology. furthermore, metal binding was shown to inhibit the amyloid formation. a challenge to our understanding of protein folding is the design of a protein from first principle, i.e. starting from geometric restraints and applying properties of amino acids expected to be essential for folding to a defined structure. we developed a program to calculate the backbone coordinates of antiparallel strands to match the surface of an elliptical cylinder. various parameters like the number and shearing of the strands and the ellipticity of the structure can be varied. the relative orientation of the β-strands and the geometrical features of the hydrogen-bonds were derived from statistical analyzes of natural β-sheet structures. iterative cycles of core-packing with amino acid rotamers, molecular dynamics simulation and energy minimization with the charmm forcefield are used to include backbone movements and to minimize the risk of trapping an energetically unfavorable structure. the quality of residue packing is assessed with the help of criteria which proved to be successful in our design of β-sandwiches. at the protein surface, a network of salt-bridges with an excess of positive charges has been designed to increase the stability and the solubility of the protein. the final sequence is synthesized by standard solid phase fmoc-chemistry. insights gained from the analysis of the synthesized structure with ftir and cd spectrometry should help us to refine the parameters for subsequent designs. with this strategy, we hope to contribute to a better understanding of protein folding. the immunoglobulin binding protein g ( igd) from streptococcus species consists of amino acids residues, which form two antiparalell-packed beta-hairpins and an alpha-helix in the middle of the sequence packed to the beta-sheet. the second hairpin was found to be stable in isolation. this fragment is therefore likely to be the first folding initiation site of the protein which could provide an adequate nucleation center on which the rest of the polypeptide chain would find a favorable environment to fold. thus, among the two beta-hairpins, the - fragment of igd corresponding to the c-terminal beta-hairpin was synthesized. in our studies, we investigated different environmental and temperature conditions for formation of the - beta-hairpin structure. its structure was examined by means of cd spectroscopy in water, buffer solutions (ph = to ) and in aqueous solutions of trifluoroethanol. additionally, its structure was investigated in the solid state by ftir spectroscopy. the cd studies revealed that the - fragment of igd in water forms mainly a statistical-coil structure, whereas the ftir technique shows formation of a regular beta-sheet structure. nmr spectroscopy and calorimetric measurements were carried out at various temperatures. our studies show that the - fragment at low temperature exists in an equilibrium between two conformations -a regular beta-hairpin and a statistical-coil. although increasing temperature resulted in shifting the equilibrium in the direction of the statistical-coil structure, the overall beta-hairpin shape of the - fragment was maintained. prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (prpc) into an insoluble, protease-resistant abnormal scrapie form (prpsc) with an highly beta-sheet content [ ] . the analyses of intrinsic structural propensities of the prp c-terminal domain showed an high conformational flexibility for the αhelix fragment which indicates that this region may be particularly important in the prpc→prpsc transition [ ] . therefore conformation-based approaches focalized on helix region appear to be the most promising for the study of prion protein misfolding. recent studies on tetracycline properties showed that this molecule binds and disrupts prp peptide aggregates and inactivates the pathogenic forms of prp [ , ] . a fluorometric titration of the fluoresceinated peptide corresponding to prion protein helix- with tetracycline has been carried out to determine the value of the apparent dissociation constant of this interaction (estimated to be ± nm). remarkably, the fluoresceinated peptide exhibits in water a canonical α-helical cd spectrum, that is maintained even in presence of tetracycline. accordingly, docking calculations and molecular dynamics simulations suggest that tetracycline interacts preferentially with the c-terminal end (residues - ) of helix- with a significant involvement of the treonine rich region. in the last decades, a series of discoveries have shed light on the role played by the carbohydrate moiety in glycoproteins. it has been shown that covalently linked sugar moieties influence peptide/protein properties such as hydrophobicity, conformation, biostability and bioactivity. the design of carbohydrate-peptide analogs with increased, retained or modified biological activity requires an understanding of their conformational preferences both in solution and in the receptor-bound state. in our recent work we have created two classes of well-structured linear and cyclic carbohydrate-modified analogs of opioid peptides, leu-enkephalin and leuenkephalin amide. the first class represents a group of compounds in which the linear peptide is alkylated at the n-terminal position by -deoxy-d-fructose unit, while its cyclic analog possesses an ester bond between the c- hydroxy group of the sugar moiety and the c-terminal carboxy group of peptide, -deoxy-dfructofuranose acting as a bridge between the leu-enkephalin terminal parts. the rigid -membered imidazolidinone ring is characteristic for the second class of compounds. in these adducts an imidazolidinone moiety connects the acyclic sugar residue with the linear peptide chain. in the corresponding bicyclic imidazolidinone analogs -membered ring is formed through an ester bond between the primary hydroxyl group of the d-gluco-pentitolyl residue and the cterminus of peptide. this work reports the comparative cd and ftir spectroscopic properties of the prepared glycopeptides in comparison with data on the non-modified flexible parent peptides performed in different solvents in order to expose the structural and conformational differences caused by a keto-sugar, rigid membered imidazolidinone ring and/or cyclization. the α-helical coiled coil structural motif consists of two to five α-helices which are wrapped around each other with a slight superhelical twist. the simplicity and regularity of this motif have made it an attractive system to study the role of complementary interactions for protein folding. here we present a systematic study showing that intermolecular electrostatic interactions between positions e and g of the helices are in competition with the intramolecular interactions between positions e/b and g/c. those competitive interactions affect folding and stability of the motif which were monitored by temperature dependent cd-spectroscopy. incorporation of oppositely charged amino acids in positions e/b and g/c reduced considerably electrostatic repulsion between equally charged amino acids in positions e and g. in addition coiled coil stability can be increased by the alkyl part of the amino acid side chains in positions e and g. studies with natural and unnatural amino acids showed that the longer this alkyl part the better is the hydrophobic core protected from solvent. therefore the repulsion of equally charged amino acids in positions e and g can be overruled by involving them either into attractive intrahelical electrostatic interactions or into hydrophobic core formation. human cystatin c (hcc) is a amino acid residues protein that reversibly inhibits papain-like cysteine proteases. this inhibitor belongs to the amyloidogenic proteins shown to oligomerize through d domain swapping mechanism. the crystal structure of hcc reveals the way the protein refolds to produce symmetric dimer while retaining the secondary structure of the monomer. the monomeric form of hcc consists of a core with a five-stranded antiparallel β-sheet wrapped around a central α-helix. the hcc dimerization is preceded by an opening movement of l loop from β -l -β hairpin and separation of the β -helix-β fragment from the remaining part of the molecule. the amino acid sequence of β -helix region suggests additional possible partial unfolding in the n-terminal part of helix. in order to investigate the structural stability of β -helix region the peptide corresponding to the helix and its n-terminal truncated analogs was synthesized along with the peptide analogs of helix containing point mutations that could stabilize helical structure of the n-terminus. the peptides were synthesized by the solid-phase method using fmoc/tbu tactics. purified products were identified by maldi-tof. the secondary structure content was calculated from the cd spectra using selcon . the random coil was the predominant structure of the peptide corresponding to the α-helical fragment of hcc and its n-truncated analogs. however, an increase of αhelix content was observed in some of the peptides corresponding to the helix containing point mutations. we expect that these mutations could stabilize the hcc monomer and suppress dimerization. the tumor suppressor protein p is a trarnscription factor that triggers cell-cycle arrest and apoptosis in response to genotoxic stress signals. the tetramereric structure of the p , which is essential for its activity as a transcription factor, is formed as a dimer of dimers. while the primary dimer is constructed from inter molecular formation of a two-stranded anti-parallel b-sheet and a two anti-parallel a-helix bundle, the secondary dimer is stabilized through interactions between residues on the surface of the primary dimer. from various substitution experiments on p , it has been shown that hydrophobicity of phe is critical for the tetramer formation of p . also we have substituted three phenyl groups of p with cyclohexylalanine (cha) and showed that phe cha is dramatically stabilized against temperature, chemical denaturant, and organic solvent by cd measurements. here, to clarify the mechanism of the stabilization of phe cha, we analyzed its three dimensional structure using x-ray crystallography. we obtained two kinds of crystals, one is a hexagonal bipyramid crystal in the space group of p<i> <sub> </sub> </i> with <i>a</i>= . Å, <i>b</i>= . Å, <i>c</i>= . Å diffracted to about . Å, and another is tabular crystal in the space group c<i> </i> with <i>a</i>= . Å, <i>b</i>= . Å, <i>c</i>= . Å diffracted to about . Å. in these crystals, the peptides formed tetramers which are very similar to those observed in the wild-type. the structure of the pocket where the side chain of cha is incorporated was defined to elucidate the hydrophobic interaction to determine the stability. helices shown in proteins, as a secondary structure, almost always form right-handed screw sense. this right-handedness is believed to result from the chiral center at the αposition of proteinogenic l-α-amino acids. among proteinogenic amino acids, l-isoleucine and l-threonine possess an additional chiral center at the side-chain β-carbon besides the α-carbon. however, no attention has be paid how the side-chain chiral centers affect the secondary structures of their peptides. recently, we have reported that sidechain chiral centers of chiral cyclic α,α-disubstituted amino acid (s,s)-ac( )c(dom) affected the helical secondary structure of its peptides, and the helical-screw direction could be controlled without a chiral center at the α-carbon atom. herein, we synthesized a chiral bicyclic α,α-disubstituted amino acid (r,r)-ab( , )c and its homopeptides, and studied the relationship between the chiral centers and the helical-screw handedness of peptides. contrary to the left-handed helices of (s,s)-ac ( ) four threefinger-toxins (tfs) have been purified from the pooled venom of golden krait (bungarus fasciatus, i.e. bf) from thailand and studied previously. these peptide toxins contain - residues and or pairs of disulfide bonds, and are rich in β-structure. we herein analyzed the tf-isoforms in bf venoms from kolkata (eastern india), hunan province (eastern china) and indonesia to study the geographic variations and structure-function relationships of the venom polypeptide family. a total of five or six tfs of low lethality were purified from each of the geographic venom samples, the n-terminal sequences and accurate masses of the peptides were determined. the cdnas encoding some of these tfs were also cloned and sequenced. full peptide sequences were deduced and match with those of the tfs purified from crude venom. intra-species variations of the venom tfs were found to be surprisingly high since sequence-identities between the majorities of orthologous toxins in different geographic samples are only - %. most of the bf proteins were not neurotoxic by electrophysiological assays using chick biventer-cervicis and mouse diaphragm neuromuscular tissues. the toxins appear to be associated with weak toxins or non-conventional snake venom tfs as analyzed by a phylogenetic tree. the reason behind their lack of neurotoxicity would be discussed. v. moussis, e. panou-pomonis, c. sakarellos, v. tsikaris peptides involved in neurodegenerative diseases can adopt at least two different stable secondary structures. amyloid-forming proteins can experience a conformational transition from the native, mostly α-helical structure, into a ß-sheet rich isoform. the latter conformation is probably present in intermediates for the formation of amyloids. the conformational change can be triggered by protein concentration or environmental changes. therefore, our aim was to generate a de novo designed peptide that contains structural elements for both, stable α-helical as well as ß-sheet formation. this model peptide can be used to elucidate the conformational changes dependent on concentration and ph. the design is based on the well studied α-helical coiled coil folding motif. the conformation and structure of the resulting aggregates were characterized by cd-spectroscopy and cryo transmission electron microscopy. as a result, three distinct secondary structures can be induced at will by adjustment of ph or peptide concentration. low concentrations at ph . yield globular particles of the unfolded peptide whereas, at the same ph but higher concentration, defined ß-sheet ribbons are formed. in contrast, at high concentrations and ph . , the peptide prefers highly ordered α-helical fibers. in conclusion, we successfully generated a model peptide that, without changes in its primary structure, predictably reacts on environmental changes by adopting different defined secondary structures. thus, this system allows to systematically study now the consequences of the interplay between peptide primary structure and environmental factors for conformation on a molecular level. cells respond simultaneously to a multitude of different signals. inside a cell signals from activated receptors are integrated by networks of enzymatic reactions and molecular interactions, leading to a spectrum of cellular responses. in order to understand the relationship between a specific cellular stimulus and a cellular response, methods are required to detect in parallel the pattern of molecular interactions. a large number of molecular interactions is mediated by protein domains, binding to linear peptide motifs. lysates of activated and resting cells were incubated on peptide microarrays carrying peptides corresponding to such binding motifs of signalling domains. binding of proteins to a spot of the array was probed by immunofluorescence. jurkat t cells were stimulated either with the phosphatase inhibitor pervanadate or with antibodies directed against cell surface receptors. upon activation of t cells, numerous changes in the pattern of molecular interactions were detected for a total of proteins and peptides. these changes were caused either (i) by masking or unmasking of a binding domain which resulted in a reduced or increased binding of a protein to the microarray or (ii) by recruitment of a protein into a complex that in turn bound to the microarray. the changes were dependent on the nature of the stimulus. the human melanocortin receptor (hmc r) was constructed to contain a flag epitope and a hexahistidine tag at the amino-terminus as well as at the carboxyl terminus to facilitate purification. stably transfected hmc r in human embryonic kidney (hek ) cell lines that expressed the receptor resulted in a kd value of . and . nm respectively in each case when the super potent agonist mtii was competed with [ i]ndp-α-msh. treatment of the tagged receptors in the hek cells with agonist resulted in down-regulation which indicates that these tagged receptors retain their biological functions. the hmc r was solubilized from cell membranes with n-dodecyl-β-d-maltoside and purified at a nickel chelating resin and a newly constructed affinity column. the purified hmc r was a glycoprotein that migrated on sds/page with a molecular mass of kda. the results from matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry was used to identify and characterize peptides derived from the hmc r following in-gel digestion with chymotrypsin. the phosphorylation sites were identified on the purified human melanocortin receptor with agonists (peptide vs small molecule) treatment. the discovery of antibiotics in the s has been one of the most revolutionary events in the history of medicine. however, during last decades, the increase of antibiotic resistance has significantly hampered the application of antibiotics. therefore, further scientific effort to find new antibiotics with novel mechanisms of action is of high importance. insects are the largest and the most diverse group of living animals on earth. they have potentially been confronting high variety of microorganisms. as a result, they have evolved powerful defense system, thus representing vast source of novel potential therapeutics. we chose larvae of fleshfly neobellieria bullata for identification and characterization of new promising molecules, peptides or proteins, which participate in immunity response against microbial infections. the hemolymph of the third-instar larvae of neobellieria bullata was used for isolation. the larvae were injected with bacterial suspension of escherichia coli or staphylococcus aureus to induce antimicrobial response. the hemolymph was separated into crude fractions, which were subdivided by rp-hplc. isolated fractions were characterized by uv-vis spectroscopy, amino-acid analysis, mass spectroscopy, -d and -d sds electrophoresis, capillary zone electrophoresis, ion-exchange hplc, tryptic digests and n-terminal sequencing. we found out significant antimicrobial activities against escherichia coli, staphylococus aureus or pseudomonas aeruginosa in several fractions. using real-time pcr, we followed and compared levels of mrna of different proteins and peptides in induced and non-induced larvae. despite the fact that many technological advances are currently involved in proteome analysis, like two-dimensional gel electrophoresis and mass spectrometry, there is still a great need for the development of novel engineered chemical probes for proteomics and interactomics. here, we describe our approach concerning the study of proteome and interactome of proteins involved in cell-matrix interactions. it relies on the use of a small synthetic inhibitor chemically modified to allow for its immobilisation to magnetic beads or affinity chromatography materials. proteins will be detected together with their native interaction partners because of nondenaturing conditions. this general procedure is applied for the enrichment of metalloproteinases, especially matrix metalloproteinases, which are potential target in tumour therapy. hydroxamic acids are known to be potent inhibitors of metalloproteinases. marimastat is a reversible inhibitor with a good potency and shows activity towards a wide range of metalloproteinases. the synthesis of new marimastat derivatives will be reported. the parent compound is modified with a linker to allow immobilisation on a solid surface. binding studies were performed using surface plasmon resonance. this approach is not only appropriate for the generation of metalloproteinase proteome subsets by affinity column or using magnetic beads, but also to enrich and isolate interaction partners of the target proteins. the present report summarizes the latest data devoted to theoretical and experimental investigation of the high temperature solid state catalytic isotope exchange reaction (hscie) that takes place in peptides and proteins by the action of deuterium and tritium [ ] . the available ms-procedures, designed to estimate the amount of protein, are aimed at derivatization at different stages of sample preparation, and as the best result, it is only possible to achieve quality comparison of the objects involved. the hscie reaction allows the production of evenly deuterium labelled proteins and peptides, and their application makes it possible to create a qualitative mass spectrometry method for protein analysis. introduction of definite amounts of these deuterium-labeled proteins into biological objects, prior to isolation, separation and trypsinolysis, will generate quantitative information concerning the composition of the proteins under study. tritium labelled proteins produced at a temperature of - oc carry the isotopic label in all the peptide fragments and completely retain their enzymatic activity. the proteins' reactivity is dependent on their three-dimensional structure. the hscie reaction has been shown to be used both in the production of tritium labelled proteins and in the investigation of spatial interactions in protein complexes. in addition, evenly deuterium and tritium labelled peptides can be used in studies of the kinetics and transformation paths of peptides in the organism's tissues. immunization of mice with type ii collagen (cii) from rat leads to development of collagen-induced arthritis (cia). susceptibility to cia is associated with the major histacompatibility class ii protein h- aq that binds the glycopeptide epitope cii - ( ) and presents it to helper t cells. [ ] to explore the interactions in the system and to stabilize towards in vivo degradation, amide bond isosteres have been introduced in its backbone.glycopeptide was virtually docked into the binding site of a comparative model of h- aq. based on the hydrogen bonding network between the peptide backbone and h- aq, the amide bond between ala -gly was chosen for isosteric replacement. to vary the geometric and hydrogen bonding properties, mimetics of the dipeptide were synthesized with the amide bond replaced by ψ[ch nh], ψ[coch ] and ψ[(e)-ch=ch], respectively. these were introduced in using solid-phase synthesis to give glycopeptidomimetics that were biologically tested for their ability to bind to the h- aq protein and for recognition by t cells. shown to be a potent neuroprotective factor in various pathophysiological models. despite its therapeutic potential in diverse neurodegenerative diseases, its short in vivo half-life limits its utility as a useful clinical agent. moreover, the development of a peptidomimetic that reproduces the pharmacological activity of pacap is unlikely since the pharmacophores are spreaded throughout the entire peptide chain. therefore, the development of pacap analogues with lower susceptibility to proteolysis represents a first step toward clinical applications. in the present study, derivatives of both pacap and pacap with particular chemical modifications were developed targeting specific peptidase sites of action. results indicate that the incorporation of an acetyl or a hexanoyl group at the n-terminus and modifications at the ser residue contributed to increase stability against dipeptidyl peptidase iv, the major enzyme involved in pacap degradation. moreover, after determination of pacap metabolites in human plasma, the amide bond between residues and was substituted by a ch nh surrogate and this derivative showed increased plasma stability. all modified peptides were tested for their ability to induce pc differentiation. the effects of pacap analogs on pc cells are mediated through the pac receptor which is the major receptor involved in the neuroprotective effects of pacap. this study exposes interesting data concerning pacap metabolism in isolated human plasma and demonstrates the possibility of increasing the metabolic stability of pacap without significantly reducing its biological activity. species of the fungal genus trichoderma are commercially used as bioprotective agents against fungal plant diseases. more than strains were collected from their natural habitats and evaluated for biocontrol properties. seven of the most active isolates exhibiting strong biological activity towards eutypa dieback and esca diesease of grapevine were classified as trichoderma brevicompactum, or shown to be closely related to that species. these strains were screened for production of peptaibiotics. the formation and synergistic action of hydrolytic enzymes and peptaibiotics were to play an important role in mycoparasitism. after background and aims: conotoxins are short, disulfide-rich neurotoxins that target various ion channels and receptors. these peptides have desirable pharmacological properties to become therapeutics for neurological disorders; several conotoxins have already reached clinical development stage. our long-term goal is to improve bioavailability, metabolic stability and pharmacokinetics of conotoxins using a variety of chemical modifications. methods: we designed and chemically synthesized conotoxin analogs containing two distinct types of backbone modifications: ( ) peptide-peptoid chimeras (conopeptoids) of alpha-conotoxin imi and ( ) peptide chimeras of mu-conotoxin kiiia containing non-peptidic "backbone spacers". results: conopeptoid-imi, containing ala replaced by n-methyl glycine potently blocked activity of nicotinic acetylcholine receptors. in mu-conotoxin kiiia, aminohexanoic acid or amino- -oxapemtanoic acid were inserted to be a part of the peptide backbone. the two oxidized analogs containing "backbone prosthesis" differed in their hydrophibicity profile, but they both potently inhibited neuronal sodium channels. conclusion: our results suggest that backbone engineering may become an effective method of producing conotoxin analogs with modified bioavailability. to increase the stability and the therapeutic efficacy of peptide sequences from myelin oligodendrocyte protein (mog) that act as multiple sclerosis (ms) antigens, we grafted them onto a framework of a particularly stable class of peptides, the cyclotides. they are a recently discovered family of cyclic plant peptides with superb intrinsic stability. the limitations of linear peptides as drugs due to their instability and poor bio-availability can be overcome by using the cyclotide scaffold as a framework for novel drug design. peptide epitopes from mog protein were incorporated onto the framework of the model cyclotide kalata b by means of boc-spps approach. after successful backbone cyclisation and oxidation of the cysteine residues, the peptides were purified to high purity with rp-hplc. nmr chemical shift analysis was used to assess whether the grafted analogues have a stable scaffold, similar to that of kalata b . a structure of a representative peptide was determined and it shows remarkable resemblance to the native scaffold of kalata b . the activity of the bioengineered peptides has been tested in vivo. a group of mice injected with one of the peptides have shown a depression in the clinical score and have not fallen ill. this is an exciting result that shows the first active bioengineered cyclotide in an animal model of disease. the structural information from nmr studies will be used in conjunction with the results from the activity studies in a feedback loop to design second-generation lead molecules. a.d. de araujo, p.f. alewood conotoxins are small, disulfide-rich peptide neurotoxins produced in the venom of marine cone snails that enable these molluscs to capture their prey. these compounds exhibit a high degree of selectivity and potency for different ion channels and their respective subtypes, making them useful tools in the investigation of the nervous system. due to the key role of ion channels in many physiological processes, conotoxins are also excellent drug lead candidates in drug discovery, with some examples currently undergoing clinical trials and one recently approved drug (prialt®). like other peptide drugs, the use of conotoxins in drug development is limited by the low oral bioavailability of these compounds due to pre-systemic enzymatic degradation and poor penetration through the intestinal mucosa. peptide analogs that mimic the native structure and incorporate rationally designed non-natural modifications in the disulfide framework or peptide backbone may exhibit increased resistance to proteolytic degradation. in biological environments, disulfide linkages are susceptible to attack by enzymes and reducing agents (such as glutathione). therefore we carried out the synthesis of redox-stable conotoxin analogs in which intrinsic disulfide bonds were replaced by thioether linkages. in this work, wehave explored two solid-phase synthetic routes in the preparation of thioether conotoxin mimetics: the first route based on peptide assembly using a tetra-orthogonally protected lanthionine building block, and the second route based on intramolecular on-bead cyclisation between cysteine and betabromoalanine residues. thyrotropin releasing hormone (trh, l-pglu-l-his-l-pro-nh ), a tripeptide synthesized in the hypothalamus, operates in the anterior pituitary to control levels of tsh (thyroid-stimulating hormone) and prolactin. the thyrotropin-releasing hormone (trh) receptor (trh-r) belongs to the rhodopsin/β-adrenergic receptor subfamily of seven transmembrane (tm)-spanning, g protein-coupled receptors (gpcrs). the two g-protein-coupled receptors for trh, trh receptor type (trh-r ) and trh receptor type (trh-r ), have been cloned from mammals and are distributed differently in the brain and peripheral tissues. the trh receptor subtype- appears to mediate the hormonal and visceral effects, whereas trh receptor subtype- has been implicated in its central stimulatory actions. identification of critical features of the trh, separation of its multiple activities through design of selective analogues and affinity labels have been elusive and unfulfilled goals for more then years. this presentation will highlight our studies on effect of the biological activity of trh with the introduction of alkyl groups of varying sizes at the n- and c- position of the centrally placed histidine residue of trh peptide. the requisite n--boc-dialkyl-l-histidines as building scaffolds have been synthesized in six steps from l-histidine methyl ester dihydrochloride and used for the synthesis of various trh analogues. ghrelin, the natural ligand for the growth hormone secretagogue receptor (ghsr- a), has received a great deal of attention due to its ability to stimulate growth hormone secretion and to control food intake. during these last years, ghrelin analogues or mimetics gained interest for their implication in food intake regulation. in the course of our studies concerning ghrelin analogs, we developed new ligands of the ghsr- a based on heterocyclic structures. interestingly, one heterocyclic family presented high affinity for the ghrelin receptor. a structure activity study was performed within this family and led to potent ghsr- a agonists and antagonists. the binding affinities were determined by displacement of radiolabelled ghrelin and the agonist or antagonist character was measured by intracellular calcium mobilisation. the first in vivo results revealed that in vitro activities and in vivo responses were not correlated. particularly, binding to ghsr- a and in vivo gh release and food intake control were not fully correlated. these results strongly suggest the presence of receptor subtypes that modulate ghrelin actions. some examples will be reported. further investigations are going on in the laboratory. background and aims: alzheimer's disease (ad) related beta-amyloid (aβ) peptides possess high propensity towards aggregation. nowadays one of the major directions of the drug-design against ad is the synthesis of putative amyloid aggregation inhibitor molecules (aai) which are able to hinder the formation of these toxic amyloid aggregates. in the present work we report the design, synthesis and investigation of putative aais derived form the peptide sequence aiigl identical to aβ( - ). methods: aβ( - ) peptide and putative aggregation inhibitors were synthesized manually using fmoc-chemistry and dcc/hobt activation. studies on both the aβ aggregation and the effect of the aais were performed with several instrumental techniques. the total amount of the aggregates was determined by thioflavine-t binding assay, while their size distribution was characterized with dynamic light scattering (dls). aggregated aβ forms were visualized with transmission electron microscopy (tem). the binding affinity of the aais to aβ fibrils was studied in saturation transfer nmr experiments, while in vitro viability assays were performed on cultured human sh-sy y neuroblastoma cells to monitor the neuroprotective effect of the amyloid aggregation inhibitors. results and conclusions: derivatives of aβ( - ) were synthesized and tested concerning their neuroprotective effect against aβ-mediated toxicity. the most promising candidates were examined with physico-chemical methods to characterize their aggregation altering ability. the pentapeptide riigl-amide proved to be the most powerful neuroprotective compound, and it was also able to alter considerably the aggregation kinetics of aβ( - ) . this molecule might serve as a lead compound in the drug-design against ad in the future. m. mattiuzzo, a. bandiera, m. benincasa, i. samborska, m. scocchi, r. gennaro antimicrobial peptides (amps) are ancient molecules of the innate immune defence system. most of them kill bacteria by lysing their membranes. the proline-rich group of amps represents an exception, as they act via a permeabilization-independent mechanism that is likely based on recognition of molecular targets/transporters. however, specific internal targets have not yet been identified for most of these amps. bac is a pro-rich amp isolated from bovine neutrophils that is capable to translocate across membranes to target hypothetical intracellular proteins. in this study, we used a molecular approach to identify putative targets for bac by selection of peptide-resistant mutants followed by identification of the genes responsible of this resistance. to this aim, an e. coli strain susceptible to the fully active fragment bac ( - ) was subjected to chemical mutagenesis and a number of peptide-resistant mutants was obtained. a pool of genomic dna from these mutants was used to construct a plasmid library that was used to transform a susceptible strain. this approach allowed the identification of different clones that provided a high level of resistance to bac . sequence analysis revealed the presence of genes originating from five different regions of the e. coli chromosome. among them, one clone contained ptrb, a gene coding for a serine peptidase broadly distributed among gram -bacteria, which could be involved in resistance by degrading the peptide. other resistanceconferring clones, which encode for membrane proteins that may be involved in peptide translocation across the memmbrane, are currently under investigation. lycotoxins are peptides from the venom of the wolf spider that were predicted to have an amphipatic alpha-helical structure and confirmed to possess significant antimicrobial and pore-forming activities. [ ] we became interested in these peptides as potential leishmanicidal agents against leishmania donovani promastigotes and leishmania pifanoi amastigotes. thus, lycotoxin i (lyi) and lycotoxin ii (lyii), [ ] and shortened analogues lyi - , lyi - , lyii - and lyii - , were synthesized as c-terminal carboxamides. short-and long-term parasite proliferation measurements showed all peptides except lyi - to be active against both promastigotes and amastigotes at the micromolar range, and suggest peptide effects on parasites to be irreversible. lyii, that showed the lower activity, became more leishmanicidal upon residue trimming, whereas the most active lyi displayed the opposite behaviour. a set of complementary techniques showed that lycotoxin peptides are membrane-disruptive to promastigotes. electron microscopy showed that two populations of promastigotes, one with intact parasites and the other extensively damaged, are formed upon addition of the peptides at their ec . all peptides were non-hemolytic for sheep erythrocytes below micromolar. tissue transglutaminase (tg ) is an enzyme that plays a key role in the pathogenesis of the celiac disease. tg is the main autoantigen recognized by the antiendomysium antibodies and, furthermore, catalyzes the deamidation of strategic glutamine to glutamic acid within the sequence of immunodominant gliadin epitopes. recently, another unexpected role for surface tg , in the innate immune response in the celiac disease, has been suggested. it follows that tg inhibitors might represent a potential attractive pharmacological alternative to the gluten-free diet that, nowadays, is the only possible therapy for celiac patients. starting from the sequence of the heptapeptide pqpqlpy, known to be a high affinity substrate of human tg , we have synthesized new analogs replacing pro with different constrained amino acids (d-pro, pip, chg, ind, deg, inp, hyp, thz)) with the aim to develop specific inhibitors of tg . actually, proline residues present in the gluten epitopes are important in determining the immunogenicity of the epitopes and the specificity of tg . herein, we describe the preliminary conformational studies of the synthesized analogs by cd and nmr spectroscopy and molecular modeling methods. the structural features of the peptides have analyzed in different environment. given the role of the domain pqpqlpy in the gliadin proteins, structural analysis on its analogs are of considerable interest. the results of our studies might be useful to clarify the role of the proline residues in the interaction of the gluten epitopes with tg and, consequently, to gain new insight in the molecular mechanism of celiac disease. carnosine and related histidine-containing dipeptides are known to react with high efficiency with the products of lipid peroxidation, namely -hydroxy-trans- , nonenal (hne) and other alpha, beta-unsaturated aldehydes, preventing their reaction with nucleophilic residues in proteins and nucleic acids. histidyl hydrazide alone or conjugated with aminoacids, long chain fatty acids, cholesterol and alpha-tocopherol synthesized in our laboratories demonstrated higher aldehydesequestering efficiency than carnosine, and were also efficient in protecting sh-sy y neuroblastoma cells and rat hippocampal neurons from hne-mediated death. the cytoprotective efficacy of these compounds suggests their potential as therapeutic agents for disorders that involve excessive membrane lipid peroxidation. lantibiotics are antibiotic natural products that are synthesised ribosomally and undergo extensive post-translational modification, resulting in multiple thioether bridges and dehydro amino acids in the mature peptide. one such lantibiotic is mersacidin, which is produced by bacillus sp. and exhibits extremely promising in vitro and in vivo efficacy versus a number of clinically relevant pathogens, most notably methicillin-resistant staphylococcus aureus (mrsa). mersacidin is believed to function by binding to the bacterial cell wall precursor lipid ii, thereby preventing its incorporation into the growing peptidoglycan network. in an attempt to better understand this mode of action and develop more active analogues, we have embarked upon its chemical derivatisation. some of these modifications resulted in an altered antibacterial spectrum, permitting some insight into tentative structure-activity relationships. the characterisation of these structurally complex compounds via a combination of multidimensional nmr and tandem mass spectrometry will also be presented. conjugation of peptides with different moieties, such as peg, lipids, carrier proteins and toll-like receptor ligands is an established approach to improve their pharmacokinetic profile for drug use and/or to enhance their immunogenicity as subunit vaccines. the development of suitable conjugation strategies for peptides of any complexity is therefore fundamental for their effective use in human therapeutic applications. here we describe our strategy to engineer a trimeric coiled coil to obtain a covalently linked, structurally stable construct endowed of extra functionality for further derivatization. we previously showed that covalent stabilization of the designed trimeric coiled coil izn , by interchain disulfide bonds, yielded an extremely potent and broad inhibitor of viral infection, (ccizn ) , [ ] . this potent inhibitory activity makes (ccizn ) not only attractive as an antiviral compound, but also as an immunogen to elicit a neutralizing antibody response [ ] . we have now developed an alternative synthetic strategy to obtain the covalently-linked izn trimer, which allows the presence in the molecule of a free thiol for subsequent chemoselective reactions. first, we showed that stable interchain thioether bonds can be effectively substituted for the disulfides. second, we devised an orthogonal cysteine protection scheme which allows formation of the thioether bonds, while leaving an extra free cysteine on demand. this thiol group can be used for conjugation of the trimeric coiled coil to adjuvant/carrier proteins or, as reported more specifically here, to a peg kda moiety. human igg is a most abundant type of immunoglobulin in serum and most of antibody drugs applied for therapy of cancer and autoimmune diseases also belong to this group of human immunoglobulin. specifically binding peptide to human igg is very useful for detection and purification as an affinity ligand of human igg. although some previous reports described such peptides, we tried here to isolate high-specific and high affinity ligands by employing random peptide-displayed t phage library. our t random peptide phage library possesses a total diversity of powered th consisting of different sequence length constrained by disulfide bond. by biopanning against human igg, we isolated several igg specific clones from our library. the peptides displayed on these phages shared some common sequences in the limited region surrounded by cys residues, which suggests they are essential for binding. these clones bound only to fc region of igg and did neither other types of human immunoglobulin nor igg of other animals. a synthetic peptide derived from a phage clone showed a sub-nanomolar of kd value in binding to human igg fc on spr analysis. these results indicate that the peptide motifs obtained here are a strong candidate of human igg-specific affinity ligand for detection and purification of igg. therefore, we are now going on constructing detection and purification system using modified and improved peptide motifs. synthesis of glp- analogues as potential agents for blood glucose control p. kanda, r.p. sharma, c.p. hodgkinson in this study, we synthesised a panel of glp- analogues stabilised against dpp-iv degradation through either selective amino acid substitutions for ala , or introduction of amide bond surrogates into the peptide backbone between ala and glu . each was made by standard fmoc or boc chemistry, purified by hplc, and characterized by electrospray mass spectroscopy. all derivatives except one bearing a hydrazine modification were stable to dpp-iv proteolysis for up to hours at ph . , o. each was tested for its ability to augment insulin release from a glucose-sensitive, murine insulinoma-derived tc- cell line in culture. it was found that each compound acted as a glp- agonist to varying degrees, with some exhibiting higher activity than native glp- toward promoting insulin release. the most active analogues have been chosen as candidates for stabilisation against renal clearance in efforts to develop new glp- analogues with therapeutic potential. the high propensity of the glucose regulatory hormone human islet amyloid polypeptide (iapp) to misfold and aggregate into cytotoxic beta-sheets and fibrils is strongly associated with beta-cell degeneration in type ii diabetes (t d) and precludes its pharmacological use for the treatment of diabetes. iapp analogs that combine solubility, lack of cytotoxicity, and bioactivity with the ability to block iapp aggregation and cytotoxicity could thus be of high biomedical interest. here we apply a minimalistic conformational restriction strategy to redesign the extremely insoluble and amyloidogenic -residue iapp sequence into a soluble, nonamyloidogenic, noncytotoxic, and bioactive iapp mimic (yan et al., pnas ( ) ). the designed mimic has nearly the same sequence as iapp but is highly soluble, nonamyloidogenic, noncytotoxic and a full iapp receptor agonist. in addition, the mimic binds with high affinity iapp and blocks and reverses with nanomolar activity its cytotoxic self-assembly which makes it to the most potent known iapp cytotoxic self-assembly inhibitor. due to its bifunctional nature, the mimic might find therapeutic application for the treatment of diabetes both as an inhibitor of amyloid cytotoxicity and as a soluble iapp receptor agonist. our findings offer a proof-of-principle of a chemical design approach for generating a novel class of highly potent inhibitors of polypeptide cytotoxic selfassembly which are nonamyloidogenic mimics of the native amyloidogenic sequence as well. such reengineered biomolecules -the design of novel mimics is in progress-are of high biomedical significance for understanding the mechanism of protein aggregation diseases and for the development of prospective therapeutic treatments. peptides that are capable of targeting abnormal changes of living tissue can be very useful in early detection or diagnosis of, e.g., cancer. conjugating a functional agent, an effector unit, to such a peptide may provide the agent with improved pharmacodynamic properties.the specificity of a thiol group for reactive groups offer a unique way to attach effector units to cysteine-free linear or cyclic peptides. tumor targeting peptides were synthesized by fmoc-type solid phase methods. peptides cyclic by cystine were modified by lactam bridges. fluorescein, metal (e.g. lanthanide) chelates and cytotoxins were coupled to tumor targeting peptides via e.g. a peg-type spacer, or the conjugates were immobilized on plates for adhesion assays. the method was uncomplicated and gave stable conjugates with good solubility. the approach is useful in making stable peptide-effector conjugates and sets of them for e.g. detection assays such as delfia method and has prospective use in development of diagnosis and therapy. antibacterial proline-rich peptides -synthesis and antibacterial activity d. knappe, r. hoffmann the antibacterial proline-rich peptide family, originally isolated from insects, shows remarkable activity against diverse bacterial and fungal pathogens. while more and more bacterial pathogens become resistant to common drugs, this part of insect innate immunity provides a new promising approach to develop future peptide-drugs. proline-rich peptides possess a significant sequence homology and share a common mechanism of action. in addition oglycosylated threonine residues of drosocin and formaecin appear to be necessary for full antimicrobial activity, although the significance of the carbohydrate moiety in interaction with intracellular targets is still unknown. we synthesized analogues of different antibacterial peptides on solid phase by the fmoc-tbustrategy. the combination or insertion of sequence regions from different native antibacterial peptide sequences offers several advantageous effects, including further reduction of toxicity and broadening of the antimicrobial activity. furthermore, mimicking the o-glycosylation site and changing the carbohydrate moieties, may yield new synthetic approaches to increase both the activity and the selectivity of these oligopeptides. new immunotherapeutic approaches have been developed for treatment of neurodegenerative diseases of the alzheimer's dementia (ad) type. the identification of a ß-amyloid-plaque specific epitope, aß( - ) (frhdsgy) [ ] , recognised by therapeutically active antibodies from transgenic ad mice, provides the basis for development of new ad vaccines and for molecular ad diagnostics. in order to produce immunogenic conjugates, the aß - epitope was attached via thioether linkage to synthetic carriers of well-defined structures, such as tetratuftsin derivative (ac-[tkpk(clac)g] -nh ) and its elongated version by a helper t-cell epitope (ac-fflltriltipqsld-[tkpk(clac)g] -nh ); sequential oligopeptide carrier (ac-[lys(clac)-aib-gly] -oh) and multiple antigenic peptide (clac-lys(clac)-lys(clac-lys(clac))-arg-arg-ßala-nh ). the epitope conjugates containing a cysteine residue either at the c-or n-terminus, and the chloroacetylated carriers were prepared by spps according to boc/bzl strategy. conjugation reactions were performed in solution under slightly alkaline conditions, and monitored by hplc and high resolution-ms. structures and molecular homogeneities of all epitope peptides, carriers and conjugates were ascertained by hplc, maldi and esi-fticr-ms. conformational preferences of the synthesized compounds in water and in tfe were examined by cd spectroscopy. comparative binding studies of the conjugates with a mouse anti-amyloid protein beta-( - ) monoclonal antibody were performed by indirect elisa. experimental data showed that the chemical nature of the carrier, the epitope topology and the presence of a pentaglycine spacer between the epitope peptide and the carrier, had significant effects on the antibody recognition and on the secondary structures of the conjugates. the new cla analogue , containing ethylene bridge between phe nitrogen atoms, was found to exhibit unexpected stimulatory effect in the model of the in vitro humoral immune response in mice. to disclose the structure-activity relationship the nmr solution conformational analysis was carried out. the solid-state and solution conformational analysis of native cla indicated the existence of this cyclic system as a complex mixture of conformations [ ] . the nmr spectra recorded at mhz in chloroform at k showed the different conformational behaviour of both cyclopeptides: cla exists as one isomer [ ] , peptide is in an equilibrium among at least of three conformers. the picornaviruses are small nonenveloped rna viruses with a single positive strand rna. the virus replication cycle starts after the penetration of the virus in the cytoplasm of the host cell. there are several stages of the virus life cycle used for attack. one of the most useful strategies for attacking of the virus includes inhibition of important for the virus lifecycle enzymes. the key enzymes in the replication of the picornaviruses are c and a proteases. changes in the active center of these enzymes make them incapable to produce polyprotein in vitro, therefore the inhibition by low molecular weight molecules could stop the viral replication in vivo. c protease plays a major roll in the enzymatic proteolysis of the initial viral polyprotein. the target compounds were based on structural modifications in the known as crucial for the cp inhibition activity dipeptides phe-gln by incorporation of additional amino acid and pyrrole moiety. the synthesis was cared out as multiple-peptide synthesis in parallel using stepwise spps, fmoc-strategy. the obtained compounds were tested for antiviral activity by agar-diffusion plaque inhibition test against coxsackievirus b replication in fl cell and on this base some structure-activity interpretations were made. histone deacetylases (hdac) play important roles in various aspects of regulation such as proliferation, differentiation, and aging by counteracting with histone acetyl transferases. the hdacs categorized in class-i and ii have a metalloprotease-related mechanism in its catalytic activity. these enzymes could be inhibited by small molecules bearing various zinc ligands such as hydroxamic acid and mercaptan. based on the structure of chlamydocin, which has a cyclic tetrapeptide framework, cyclo(-l-aoe-aib-l-phe-d-pro-), where aoe is ( s, s)- -amino- -oxo- , -epoxydecanoyl, we have developed potent hdac inhibitors as shown in the figure. in the present study, we examined the effects of the chlamydocin hydroxamic acid ( ) and ss-hybrid ( ) on the diabetes model mice, kk-ay. the peptide ( ) exhibited satisfactory activity to reduce both the blood glucose and blood insulin levels comparable to or even superior to those by pioglitazone, a pparγ agonist. the ss-hybrid ( ) , which is expected to be reduced inside of cells to generate the corresponding thiol-containing cyclic tetrapeptide, also showed a significant effect but less than ( ). the effect was dose-dependent from mg/kg to mg/kg. the effects of hdac inhibitors were also confirmed by the observation of in vivo histone hyperacetylation induced in the lymphocyte cells. synthetic peptides have a number of advantages over current vaccines. however, exploitation of synthetic peptides as vaccines has been limited by the small size; copy number, inefficient delivery, poor peptide immunogenicity and mhc restriction. we have developed chemical methodologies that overcome these limitations by synthesising and polymerising vinyl-peptides. protective b-cell and t-cell peptide epitopes from the oral pathogen porphyromonas gingivalis were identified for three different mhc restricted mouse strains using pepscan techniques. fmoc chemistry for spps was used to synthesize these peptides and a vinyl moiety was incorporated at the n-terminus using acryloyl chloride. after rp-hplc purification free radical polymerisation using ammonium persulphate and temed was used to polymerise the vinyl-peptides in the presence of either acrylamide or other vinyl-monomers to produce single peptide or multi-peptide polymers. size exclusion chromatography indicated that the peptide polymers were > million da. the peptide polymers were used to immunise each mouse strain (balb/c, cba and c bl ) and the t-cell response induced was evaluated using proliferation and cytokine (elispot) assays. the peptide polymers were found to be highly immunogenic, the single peptide polymers were found to only induce a response in their respective mouse strain, however, the multi-peptide polymer containing all of the t-cell epitopes was found to induce a response in all three mouse strains. in conclusion, our data shows that the polymerisation method overcomes all of the limitations in developing a peptide vaccine and most importantly that of mhc restriction. cytotoxic substances are auspicious weapons in tumour therapy. this compounds inhibit cell division and proliferation, hence, they affect all cells that are able to divide. however, all these compounds act intracellularly, i.e. at first they have to enter the tumour cell efficiently. this is a serious obstacle when using highly effective cytostatica and the cause of severe adverse effects by using higher doses. our aim is to overcome this problem by using synthetic hybride molecules composed of the cytostatic agent, in our case derivatives of arglabin, covalently linked to shuttle peptides. in order to identify the most effective possibility we tested two different strategies. by using the peptide hormone npy, whose specific y receptors are often overexpressed by tumour tissues, we intended to address the chemotherapeutic selectively to y receptor expressing tumour cells via receptor mediated endocytosis. on the other hand, the cytostatic agent was covalently bound to a cell penetrating peptide derived from human calcitonin (hct). recently, a c-terminal fragment of human calcitonin was found to internalize into excised nasal epithelium while the receptor activating n-terminal part is lacking. for the class of hct derived carrier peptides previous studies suggested a receptor independent "lipid raft" mediated endocytotic mechanism of uptake. here we present comparative data investigating both strategies, the highly selective receptor mediated delivery and the highly efficient receptor independent delivery. we investigated the peptide uptake by various cell lines and examined the release of the cytostatic agents inside the cells and its toxic effects. background and aims: synthetic peptides provide a straight-forward access to rationally designed inhibitors of molecular interactions based on structural information of proteins. poor membrane permeability may be overcome via cell-penetrating peptides, but low stability remains a major drawback. an increase of stability and bioactivity of peptides by coupling to polymers is intended. methods: for the development of peptide-functionalized cell-penetrating polymer conjugates, peptides were coupled chemoselectivly to hpma (n-( hydroxypropyl)methacrylamide, average mw . kda), as an inert backbone polymer by native chemical ligation. hpma is water soluble and its capacity in drug delivery has been demonstrated. peptide-functionalized polymers and free peptides were incubated with proteases or cell lysates and proteolytic break-down was determined by fluorescence correlation spectroscopy (fcs) deriving information on the number and size of fluorescent particles based on temporal fluctuations of a fluorescence signal caused by diffusion of particles through a femtoliter-size confocal detection volume. the diffusion time depends on molecular size. therefore this technique is suited for the detection of proteolytic fragments. results: efficient chemoselective conjugation of unprotected fluorescein-labelled peptides was accomplished by means of native chemical ligation. our fcs investigations revealed that conjugation of peptides to hpma increased their biostability. this data also indicate that this effect is peptide-dependent. a proapoptotic peptide coupled to hpma and introduced into mammalian cells by electroporation retained its bioactivity. cofunctionalization of hpma with this peptide and nonaargine yielded an efficient cellular import. macrolides, a rather large group of natural or semi-synthetic antibiotics, are widely known translation inhibitors whose structure is based on - -member lactones with carbohydrate substitutes attached. macrolides bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft. carbohydrate residues of the macrolides are spread along the walls of the rt. hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. the goal of this study was to design and obtain peptide derivatives of macrolides interesting both as antibacterial agents and potential probes for investigation of nascent peptide chain topography in the rt. tylosin ( in order to reach target sites inside the cns, neurotherapeutic candidates must overcome the blood-brain barrier (bbb). while several transport mechanisms occur at the bbb, this work has focused on the passive diffusion mechanism. the prediction of a peptide's ability to cross the bbb is not a simple task; hence there exists the need for the rational study of the relevant factors that affect the movement across this physiological barrier. here we present two new approaches based on in vitro non cellular assays for this type of studies. firstly, the evaluation of compound mixtures on parallel artificial membrane permeability assays (pampa). this approach increases the throughput of the study and structure-activity relationships can be easily establish. secondly, the transport and biological activity evaluation in a single assay. this second approach has been applied to the search of inhibitors for cns proteases involved in different neurological diseases (such as prolyl oligopeptidase for schizophrenia) able to cross the bbb. these two new approaches allow assaying compound's permeability in the early stages of a drug development project, and then designing novel analogues with improved bbb transport properties or using blood-brain-barrier shuttles for their delivery. two newly synthesized compounds are derivatives of l-valin and are positional isomers of nicotinic (m- ) and isonicotinic acid (p- ). these functional groups, as well as established good lipid solubility suggest that the main target for their biological action probably will be central nervous system. the presence of aminoacid l -valin, supposes their low toxicity, confirmed by our earlier experiments. the aim of the present work is to study their pharmacological activity as putative drugs. methods: male albino mice ( - g, in groups) were used. next neuropharmacological parameters were studied: analgesic effect (acetic acid test), neuromuscular coordination (rota-rod test), orientation ("hole board" test) and learning and memory (passive avoidance-step down test). results: significant analgesic effect of both compounds was established (more pronounced by dose mg/kg i.p.). slight depressing effect on the orientation reactions in animals was registered, but the neuromuscular coordination and locomotor activity of treated animals were not changed. good dose-dependent effect on learning and memory was established and М- had stronger effect than Р- . the compounds modified the effects of some model substances with central nervous activity. hexobarbital sleeping time was significantly prolonged by p- , but was antagonized by m- . pentileneterazole threshold was increased significantly and suggests some anticonvulsant activity of both compounds. conclusion: as positional isomers m- and p- demonstrated some variations in their pharmacological activity probably due to the differences in their kinetics, metabolism and excretion. registered significant neuropharmacological activity, accompanied by low toxicity motivates the new synthesis and future experimental studies. the glyproline family of regulatory peptides includes pro-gly-pro, gly-pro, pro-gly and also the simplest peptides with hyp substituted for pro. a distinctive feature of these peptides is that they exhibit a broad spectrum of biological activities: the antiulcer activity, the inhibition of blood clotting and thrombosis, the reduction of degranulation activity of mast cells, and the normalization of stressogenic behavioral disorders. alzheimer's (ad) and parkinson's (pd) disease are progressive neurodegenerative disorders which are characterized by amyloid plaques. the main components of the plaques are β-amyloid peptides (aβ - and aβ - ) and α-synuclein. we have previously shown that small peptides structurally related to the sequence of aβ( - ) protect against the neurotoxicity of aβ peptides. recent studies by other groups have shown that β-synuclein can counteract the aggregation of α-synuclein in the neurodegenerative process of pd, hereby might protect the central nervous system from the neurotoxic effects of alpha-synuclein. it was found that a tetrapeptide (kegv) protected against the neurotoxicity of aβ peptides in vivo. based on the previous findings, the following sequences of β-synuclein have been synthesized: kegv-nh and kegv-oh. after comparing the sequences of α-and β-synuclein, we found common sequences which are keqv, regv, kqgv, keqa. we have synthesized these tetrapeptides in amide forms at their c-termini. the peptides have been synthesized on mbha resin using a manual solid phase peptide synthesis equipment and boc-chemistry. the neuroprotective effects of peptides have been investigated in vitro in mtt test in differentiated neuroblastoma cell culture (sh-sy y) and in electrophysiological test on rats using multibarrel electrodes in vivo. the neuroprotective peptides might stop neuronal death and can influence ad and pd progression. the present study was carried out to determine antinociceptive effect in vivo of plant peptide hormone phytosulfokine-alpha (h-tyr( -so h)-ile-tyr( -so h)-thr-gln-oh ) (i) and its selected analogues, such as h-phe( -no )-ile-tyr( -so h)-thr-gln-oh (ii), h-d-phe( -so h)-ile-tyr( -so h)-thr-gln-oh(iii), h-tyr-ile-tyr( -so h)-thr-gln-oh(iv), h-tyr( -so h)-ile-tyr-thr-gln-oh(v) and h-tyr-ile-tyr-thr-gln-oh (vi) in rats. peptides were injected into the lateral brain ventricle at the dose of nmol. in the preliminary investigation we found the psk-as well analogues ii and iii induced a significant antinociceptive effect determined in the test of hot plate. the probable mechanism of this effect was discussed. a.b. bozhilova-pastirova , b.a. landzhov , p.v. yotovski , e.b. dzambazova , a.i. bocheva the tyr-mif- family of peptides (tyr-mif- `s) includes mif- , tyr-mif- , tyr-w-mif- and tyr-k-mif- , which have been isolated from bovine hypothalamus and human brain cortex. all these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides. data in the literature suggest that tyr-mif- `s have antiopioid and opioid -like effects. we used wistar rats to study distribution and density of the tyrozine hydroxylase (th) imunoreactive fibres and nadph-d reactive neurons in the rat ventral and dorsal striatum. our results showed that neuropeptides mif- and tyr-mif- may affect them. opioid peptides have been recognized as modulators of reactive oxygen species (ros) in mouse macrophages and human neutrophils. data in the literature suggest that peptides of the tyr-mif- family -mif- and tyr-mif- have antiopioid and opioid -like effects. these neuropeptides are isolated from bovine hypothalamus and human brain cortex. so far no data about direct scavenger properties of tyr-mifs peptides were available. in this study we tested the hypothesis that they may scavenge ros in vitro. the antioxidant activity of these two substances was studied in the concentration range of - - - mol/l. we investigated the luminol-dependent chemiluminescence to test their ability to scavenge the biologically relevant oxygen-derived species: hydroxyl radical, superoxide radical, hypochlorous acid in vitro. we found that tyr-mif- was a powerful scavenger in all tested systems. the effects were higher for hypochlorous anion and weaker for superoxide radical. mif- had no scavenge activity against the hydroxyl and superoxide radicals and showed a moderate scavenger effect on hypochlorous anion. we have compared different strategies to increase the immunogenicity of an antigenic hiv peptide as a vaccine candidate. our selected b-cell epitope comprises amino acids ( - ) of the v region of hiv- , jy isolate (subtype d), and is in tandem with a t-helper epitope corresponding to the - region of tetanus toxoid. several presentations, including oligomerization, map dendrimer, conjugation to dextran beads or to other macromolecular carriers, have been synthesized and evaluated. murine sera from the different presentations of the v epitope have been compared with regard to antibody titers and cross-reactivity with heterologous hiv subtypes. the map dendrimer version of the peptide, conjugated to recombinant hepatitis b surface antigen protein, was a better immunogen than the dendrimer alone, and showed higher immunogenicity than other multimeric presentations, or than the peptide alone conjugated to dextran beads. the map dendrimer version, either alone or conjugated to hbsag, enhanced cross-reactivity towards heterologous v sequences relative to monomeric peptide. group a streptococcus (gas) responsible for critical diseases (eg. acute rheumatic fever and rheumatic heart disease) are classified over serotypes according to their surface virulence m proteins. development of vaccine to prevent infection with gas is hampered by the widespread diversity of circulating gas strains and m protein serotypes, and multivalent vaccine strategy would contribute to prevention against various gas infections and provide better protective immunity. we have studied the efficacy of incorporating four different epitopes derived from gas m protein into a single synthetic lipid core peptide (lcp) construct, in inducing broadly protective immune responses against gas following parenteral delivery to mice. peptide vaccine was synthesized on mbha resin by manual spps in situ neutralization/hbtu activation in boc-chemistry. immunisation with the mono-or multi-epitopic lcp vaccine led to high titers of antigen-specific systemic igg responses, and the production of broad protective immune responses as demonstrated by the ability of immune sera to opsonise multiple gas strains. systemic challenge of mice after primary vaccination showed that mice were significantly protected against gas infection in comparison with control mice demonstrating that vaccination stimulated long-lasting protective immunity. these data support to the usefulness of lcp system in the development of synthetic multiepitope vaccine to prevent gas infection. glycoprotein d represents a major immunogenic component of the virion envelope of herpes simplex virus and able to induce high titres of neutralizing antibodies. one of its optimal epitopes is the - region ( lkmadpnrfrgkdl ). several cyclic peptides possessing thioether bond and different ring size have been already prepared and some of them were conjugated with tetratuftsin derivative (ac-[tkpkg] -nh ) by thioether bond formation using selectively removable cys protecting groups. antibody recognition results suggested that the size of the cycle has considerable influence on antibody recognition, however, the replacement of met in position by nle is permitted. conjugation of cyclic peptide might increase the antibody recognition, but the binding depends on the structure and/or conjugation site of the cyclic peptide. conjugate with the best binding capacity ( . pmol/ ul) as well as the conjugate containing the linear ( - c) epitope ( . pmol/ ul) were selected for immunization. in order to increase the production of antibodies a new group of conjugates was prepared. in these constructs promiscouos t cell epitope peptide derived from tetanus toxoid (ysyfpsv) was attached to both amino groups of lysine residue coupled to the n-terminus of the carrier (ac-ysyfpsv-k(ac-ysyfpsv)-[tkpk(clac)g] -nh ). the cys containing linear and cyclic epitope peptides were conjugated to the carrier in solution ( . m tris buffer, ph ). this work was supported by grants of the spanish-hungarian intergovernmental program and cost chemistry action. background. primary hyperparathyroidism (pht) is characterized with increased parathyroid hormone (pth) secretion and in % of pht patients with hypertension. it was previously shown that pro-analogue of pth with a reversed sequence (which include strong alkali sequence -arg-lys-lys-) induced significant hypertensive response at dose - m/kg b.w. one of the hypothesis attributed hypertension in pht patients to the presence of fragments of degraded pth possessing -arg-lys-lys-sequence. aim. to compare influence on mean arterial blood pressure (map) of analogue of - pth fragment (amide) and - fragment of pth, with -arg-lys-lys-sequence and also responsible for binding to pth receptor. methods. chosen peptides were synthesized manually by a solid phase peptide synthesis method. the purity of the products was tested by reversed-phase high performance liquid chromatography. the synthesized peptides showed the right molecular mass. the influence on map of synthesized peptides was tested in wistar rats. sequential increasing boluses of each peptide: - , - and - m/kg.b.w. in the same animal were given i.v. blood pressure was measured continuously in carotid artery. results. injection of synthesized analogue of - fragment of pth does not show influence on map vs. control group. synthesized - fragment of pth increased map in min. of experiment for mmhg ± mmhg vs. time of administration of first dose and for mmhg vs. control group. conclusion. it seems to be possible, that in case of alternate degradation of pth, accumulation of - fragment of pth may partially play role in the mechanism inducing hypertension in pht patients. biologically active domains of a high affinity receptor for ig Е (fcεri) were determined, the fragments - and - of the receptor. the program of research of biological properties of synthesized tetrapeptide rnwd and heptapeptide rnwdvyk included the study of their binding with ige, which was contained in standard solutions and in patients' blood serum. the binding of peptides with ige was explored by the ifa method using ige antibodies labeled with horse-radish peroxidase (hrpo). peptides in the concentration of mkg/ml were used for sorption on immunological plotting boards. higher correlation between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and substrate chromogenic mixture (r= . ) was found in the diagnostic system with sorption rnwdvyk-peptide than in the diagnostic system with sorption rnwd-peptide (r= . ). similar investigations were conducted with the diagnostic systems with sorption rnwd and rnwdvyk peptides, but rwnd peptides conjugated with hrpo were used as antibodies against immunoglobulin e, instead of hrpo-labeled monoclonal antibodies. almost equal correlation was found between the concentration of ige in standard serum and serum of allergy patients with the known concentration of ige and the optical density of the solutions after introducing the rnwd peptide, conjugated with hrpo. after introducing allergy patients' blood serum in the holes on the plotting board, the heptapeptide bound . % more ige than the tetrapeptide. our experiments demonstrated a high ige binding activity of synthetic rnwd-and rnwdvyk-peptides. in this study, the information spectrum method (ism) of the ha subunit of the h hemagglutinin protein of the influenza virus, h n , of different reference isolates was performed in order to identify possible antigenic determinants resistant to virus mutations. results of this analysis demonstrated that the primary structures of ha subunit of h hemagglutinins encode a common information corresponding to one characteristic frequency in their iss, which is probably important for the biological function of these proteins, including their possible recognition by the immune proteins targeting this molecule. besides, comparison of the iss of ha proteins of h "spanish flu" and h n isolates demonstrated an informational similarity between them. based on these results, a segment of the n-terminus of ha h n was identified to play a crucial role in the inhibitory and immunological properties of all possible h n variants. the identified core segment, being highly conserved in all h strains, was selected as an antigenic determinant and coupled to the sequential oligopeptide carrier (socn), (lys-aib-gly)n, to the lys-nεh groups, in order to develop a diagnostic immunoassay and formulate a vaccine candidate for the highly pathogenic h n influenza virus. background and aims: thrombin plays a key role in various disorders such as arterial thrombosis, atherosclerosis, restenosis, inflammation and myocardial infarction. insights into the way in which thrombin interacts with its many substrates and cofactors have been clarified by crystal structure and site-directed mutagenesis analyses, but until recently there has been little consideration of how its non-proteolytic functions are performed. we investigated cardiovascular effects of seven modified proline-and rgd-containing peptides designed from three surface-exposed sites of prothrombin, corresponding to residues - , - , and - . methods: cardioprotective effects of synthetic peptides were tested on the two rat models of heart failure produced by coronary artery occlusion ( -or -min) and reperfusion ( -or -min). arterial blood pressures from left carotid artery, heart rate and ecg ii standard lead were measured throughout experiment. at the end of second experiment hearts were morphologically investigated by light microscopy and electron microscopy methods. results: on animal model with short-term ischemia investigated peptides did not protected from myocardium ischemia during occlusion, however, tp-l , bk-mc and tp-h protected rat hearts from ventricular fibrillation, contributed more significant functional recovery during reperfusion and raising survival rate. on the model with prolong ischemia, acceptable cardioprotective effect revealed tp-h and bk-mc. these peptides significantly diminished necrotic zone of left ventricle, protected hearts from ischemia-reperfusion induced functional and morphological changes. conclusions: investigated proline-containing peptides revealed activity on cardiovascular system -decreasing of blood pressure, cardioprotective properties and improved recovery from ischemia. r. mansi, d. tesauro, c. pedone, e. benedetti, g. morelli the widespread use of compounds containing the gamma-emitting radionuclide mtc in nuclear medicine for the scintigraphic imaging, as well as the recent introduction of the beta-emitting radionuclides re and re in radiotherapy, have led to a rapid development of their chemistries, in order to produce novel radiopharmaceuticals. we have developed new peptide based radiopharmaceuticals based on a scaffold in which the radioactive metal ion is complexed by two different peptides that are able to bind two target receptors (see figure) . the + mixed-ligand approach has been used for the preparation of neutral oxotechnetium(v) and oxorhenium(v) peptide complexes. the complex preparation requires the simultaneous action of a dianionic tridentate ligand and a monoanionic monodentate thiolato on a suitable metal precursor. the dianionic tridentate ligand is based on the snn donor set able to stabilize the metal complex. the chelating agent (hsc(ch ) conhch ch(co-r)nh ) was coupled step by step to a bioactive peptide synthesized on solid phase. the second ligand, based on monodentate thiolato moiety, was coupled on n-terminus of the second peptide. labelling procedures and biological tests on tumour cells overexpressing receptors are described for mtc(o) complexed by cck and octreotide peptide derivatives. background: endostatin inhibits the proliferation of endothelial cells and induces their apoptosis. the measurement of serum endostatin can predict tumor vascularity. tumor angiogenesis is a strong prognostic factor in patients with hepatocellular carcinoma(hcc). significantly high levels of endostatin were noted in patients with trabecular-type tumors , and with hepatitis infection. methods : patients with hcc, patients with git malignancies, patients with liver metastasis and without metastasis, and normal persons . all subjects were tested for alfa-feto protein (afp) , ca . , carcinoemberyonic antigen (cea), and endostatin by elisa results : endostatin in normal control persons was . ± . ng/ml with a significant elevation (p< . ) between the hcc group and all the other tested groups .afp was . ± . ng/ml in normal persons with a significant elevation between hcc and all the other tested groups ( p< . ). ca . was . ± . u/ml in normal persons with a significance elevation ( p< . ) relative to hcc., and a significance of ( p< . ) relative to git cancers with metastases. cea was tested to be . ± . µg/l in normal persons , and had a significance of ( p< . ) relative to git metastases. endostatin was elevated in of patients with git cancers not proved to have metastasis. conclusion : endostatin can be used to denote metaplasia and can also detect possibilities of metastasis or liver cell affection even before the frank development of metaplasia affibody® molecules are a novel class of affinity proteins which are generated by combinatorial engineering of the aa three-helix bundle scaffold, originating from the b domain of staphylococcal protein a. we have used fmoc/tbu chemistry for total chemical synthesis of the affibody zher : , binding with picomolar affinity to the cell surface receptor her . the synthetic protein was investigated for molecular imaging of her -overexpressing tumours. in vivo detection of her in malignant tumours provides important diagnostic information which may influence patient management. to enable gamma camera imaging of the tumours, a panel of potential mtc-chelating sequences was designed and introduced into the affibody. the well-studied tc-chelating sequence mercaptoacetyltriglycyl (mag ) was compared to serine-containing sequences with increased hydrophilicity, such as mercaptoacetyltriserinyl (mas ). the total synthetic yield was - % and the her -binding affinity of the affibody conjugates were all in the range - pm. binding specificity of tc-labelled affibody molecules was determined on her -expressing skov- ovarian carcinoma cells. all variants showed receptor-specific binding. the tumour-targeting properties were studied in skov- tumour-bearing nude mice. all conjugates demonstrated high tumour uptake, quick blood clearance and low uptake in most other organs. the biodistribution results further showed that the more hydrophilic, serine-containing chelators resulted in a reduced hepatobiliary excretion, which significantly decrease the background in the abdomen area and provide for more sensitive detection. gamma camera images of mice with grafted tumours showed clear visualization of her -expressing tumours using the mtclabelled mas -affibody conjugate, suggesting a potential future application of this agent for diagnostic imaging. antimicrobial peptides are molecules with a unique mechanism of action. they are widespread in nature and play the role of an effective weapon of innate immune system against bacteria, fungi and viruses. the purpose of this study was to investigate the in vitro activity of natural antimicrobial peptides: citropin, piscidin, protegrin, temporin, uperin and the analogues of antimicrobial peptides: iseganan, pexiganan and omiganan. the peptides were synthesized using the solid-phase method and purified by high-performance liquid chromatography. the peptides were subjected to microbiological tests [mic (minimal inhibitory concentration) and mbc (minimal bactericidal concentration)] on reference strains of bacteria, according to the procedures outlined by the national committee for clinical laboratory standards (nccls). for comparison, conventional antibiotics (vancomycin, rifampycin, piperacillin, chloramphenicol) were included in this research. both the natural antimicrobial peptides and the analogues inhibited the growth of bacteria, but at higher concentrations than did conventional antibiotics. nevertheless, both natural origin of antimicrobial peptides and their low toxicity constitute a considerable advantage and this is an argument for considering the antimicrobial peptides as good candidates for medicines. the linear hexapeptide cypate-grdspk (compound ; the cypate moiety is a near-infrared fluorescent label) whose rgd sequence was rearranged to grd showed high uptake in the αvβ integrin-positive tumor tissues in vitro and in vivo. despite low affinity of to the integrin in the binding assays, the uptake was inhibited by equimolar amounts of the cyclic peptide c(rgdfv), which possesses high affinity to αvβ integrin. these observations led to hypothesis that cell internalization of compound may be mediated mostly by only one of the integrin subunits, as the β one. indeed, blocking of αv integrin by the specific antibody did not inhibit the internalization of in tumor cells, which was in the contrast with successful blocking the cell internalization by the anti-β integrin antibody. similar results were obtained in immunocytochemical assays employing the anti-αv and anti-β integrin antibodies. also, studies utilizing the β -knockout and wild-type mouse cell lines demonstrated that deletion of the β subunit markedly decreased internalization of compound in the β knockout cells. the preferential interaction of compound with the β subunit of integrins relative to the αv subunit was supported also by molecular modeling studies. summarizing, the bulk of our experimental and modeling data emphasizes interaction with the β integrin as the primary mechanism of the uptake of cypate-grdspk by tumors. since this compound showed the superior biodistribution profile in vivo, our results may provide a strategy to image and monitor the functional status of the β integrin in cells and live animals. background and aim: a growing tumor is accompanied by tumor intoxication development. intoxication independs on tumor size and intensity of its break-up. tumor intoxication is one of variant of endogenous intoxication. concentration of tyrosine-contained peptides (tcp) in blood plasma have been proposed as biochemical marker of endogenous intoxication at different organs cancers. our aim was to determine the tcp concentration in blood plasma patients with ovary tumor and its association with the severity of tumor. materials and methods: patients with ovary tumor, mean age is years, were studied. the control group consisted of healthy women without tumor. patients were divided into groups: people with non-malignant and people with malignant ovary tumor. tcp content in blood plasma was estimated by our technique. results: tcp concentration in the control group were , ± , mmol. the tested marker was present in increased concentration in blood plasma of the patients with ovary tumor. the mean concentration tcp in patients with non-malignant tumor was , ± , mmol. the content of this marker in blood plasma of patients from second group was increased , ± , mmol compared with healthy control group. after treatment a significant decrease in tcp content was observed. conclusions: the result indicate that content tcp in blood plasma depends of the type of tumor. it could be suggested that determination of tcp concentration in blood plasma could be useful for improve the diagnostic of ovary tumor and monitoring of its progression. c. strongylis , ch. papadopoulos , k. naka , l. michalis , k. soteriadou , v. tsikaris troponin is a structural protein complex, which is responsible for the regulation of skeletal and cardiac muscle contraction. it consists of three components: troponin i ( kda), troponin c ( kda), and troponin t ( kda), each of which carries out different functions in the striated muscles. cardiac troponins are released into the bloodstream of patients after the onset of a cardiovascular damage. even minimal elevations over the normal values, of serum troponin t and i are being used to diagnose acute myocardial infarction and also to rule out the patients' condition. the development and commercialization of highly specific biological assays for the detection of cardiac troponins is based on the production of specific antibodies against the whole complex or individual subunits. however, the specificity and sensitivity of these assays vary due to problems mainly originated from the fact that cardiac troponins have a high homology with the skeletal isoforms. the aim of this work is to select and synthesize appropriate regions of the cardiac isoforms of troponin i, c and t, suitable for the production of more sensitive and specific cardiac troponin detecting reagents. in order to construct the immunogenic complexes, the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions. using the carrier conjugated troponin sequences, anti cardiac troponin complex specific antibodies in high titers were produced. the increasing problems with the reproductive systems of man and animals are recently linked to the presence of polluting chemicals with endocrine activity, the so called endocrine disrupting chemicals or edc's . the family of edc's is a heterogeneous one and consists of natural and synthetic hormones (like estradiol, ethynylestradiol and diethylstilbesterol), phyto-estrogens (like genistein and coumestrol) and industrial chemicals (like bisfenol-a, ftalates and various pesticides). because of the complexity of the environmental matrices and the low physiologically active concentrations of the edc's there is still a need for an efficient routine analysis protocol. we want to develop a solid-phase bound receptor that possesses a high selectivity for edc's and thus can be used in a simple solid-phase extraction protocol. this receptor must have the right functional groups that bind the edc's with a strong affinity and must be able to create a cavity in which the edc's can fit. by looking at nature's own estrogenic receptor for humans we have found the different amino acids responsible for the specific interactions . in order to create the cavity which mimics the behaviour of the hormone-binding domain of the human estrogenic receptor we have made a tripodal scaffold. this tripodal scaffold has three orthogonal protected amino groups that will allow the generation of three independent peptide chains. milk proteins are a source of opioid peptides. these peptides are liberated from milk proteins during enzymatic hydrolysis. some of these peptides are characterized with agonistic (β-casomorphins) and some with antagonistic (casoxins) properties. the aim of the investigations was to determine the presence of opioid peptides with antagonistic properties in milk products. the experimental material included cheeses, yogurts and kefirs. peptides were extracted with a methanol-chloroform mixture ( : v/v). the peptide extracts were purified by spe method on c or stratax columns and characterized by sds-page electrophoresis. the agonist opioid peptides (casoxins) were identified by hplc using standard agonist peptides. the opioid activity was measured by examining the effects of peptide extracts on the motor activity of isolated rabbit intestine. the results of sds-page electrophoresis indicated the presence of to fractions in peptide extract derived from cheeses and yogurts and to ones from kefirs. the presence of casoxin a ( . - . µg/mg of extract) was proved in all examined the milk products. lactoferroxin a ( . - . µg/mg of extract) was identified only in kefirs and yogurts. those products were also found to contain trace amounts of casoxin c. all peptide extracts showed the antagonistic activity in the relation to motor activity of isolated rabbit intestine. the highest antagonistic activity was reported of peptide extract from kefirs ( . - . %) and gouda cheese ( . - . %), as compared to morphine. the physiological and nutritional function of these antagonist peptides requires elucidation. a. péter , r. berkecz , f. fülöp the past decade has seen a growing interest in β-amino acids, which are important intermediates for the synthesis of compounds of pharmaceutical interest and can be used as building blocks for peptidomimetics. oligomers of β-amino acids (β-peptides) fold into compact helices in solution. recently, a novel class of β-peptide analogues adopting predictable and reproducible folding patterns (foldamers) was evaluated as a potential source of new drugs and catalysts. studies on synthetic β-amino acids can be facilitated by versatile and robust methods for determining the enantiomeric purity of starting materials and products. highperformance liquid chromatography (hplc) is one of the most useful techniques for the recognition and/or separation of stereoisomers including enantiomers. the aim of the present work was to evaluate hplc methods for the separation of enantiomers of eighteen -amino- -aryl-substituted propanoic acids (β-amino acids). direct separations were carried out on different macrocyclic glycopeptide based stationary phases, such as ristocetin a containing chirobiotic r, teicoplanin containing chirobiotic t, teicoplanin aglycon containing chirobiotic tag, vancomycin containing chirobiotic v columns and on a chiral crown ether based column. the effects of different parameters on selectivity, such as the nature of the organic modifier, the mobile phase composition, the flow rate and the structure of the analytes are examined and discussed. the separation of the stereoisomers was optimized by variation of the chromatographic parameters. the efficiency of the different methods and the role of molecular structure in the enantioseparation were noted. the elution sequence of the enantiomers was determined in most cases. a rational approach to evaluating peptide purity a. swietlow , r. lax amgen, inc., pharmaceutics, thousand oaks, ca polypeptide laboratories, inc., torrance, ca, usa recent years have seen an enormous increase in interest in peptide therapeutics. new peptide leads are often chosen by screening procedures using microgram to milligram quantities of peptides, frequently provided by specialized manufacturers utilizing automatic synthesizers to maximize output. the purity of the resulting compounds is often not very high. the use of spps synthetic procedures predetermines that most impurities are closely related and difficult to resolve by reversephase purification. these factors, combined with the use of generic analytical methods not specifically optimized for the peptide in question (e.g. the ubiquitous . % tfa/water/acetonitrile system), lead to erroneous results that frequently severely overestimate the purity of the peptide. the use of poorly characterized materials in pharmaceutical development leads to significant risks of obtaining false negative or false positive results that may cause potential leads to be overlooked or misinterpretation of their pharmacological profiles. we describe a rapid, systematic and reliable hplc procedure for evaluation of peptide purity. utilizing the increased separation efficiency by increasing the column temperature and adjusting the gradient in two steps in reverse-phase buffers containing tfa, naclo , or ion-exchange buffers containing kcl, we demonstrate that methods -suitable for preclinical research -can be developed rapidly. the proposed approach will be illustrated with examples of peptides ranging between and amino acids and a model peptide vypnga. it will be demonstrated that peptides showing an hplc purity close to % are often - % less pure. to approach a high-throughput cell assay format using peptides, we attempt to design and construct a peptide microarray for examination of cell activities of peptides including apoptotic cell death. peptides were immobilized onto solid surfaces via a novel multi-functional linker. the linker enable us to examine various types of peptide cell assays in an array format. we also designed and synthesized peptidyl capture agents on the basis of the cell-active sequences suitable for the peptide microarray. the utility of targeted nanoparticles as fluorescent probes for tissue imaging has recently been subject to widespread interest. one exciting prospect is the further development of nanoparticles conjugated to both targeting peptides and cytotoxic cargoes. these nanodevices could preferentially bind to specific cells and/or tissues to provide effective tools for drug delivery. hence, such multifunctional nanoparticles could provide both diagnostic and therapeutic functions by acting as fluorescent probes that offer targeted delivery of therapeutic agents. we have coated the surface of quantum dots (qdots) with cell-penetrating peptides (cpp) to target and label u m cells for fluorescence imaging. qdots were initially coupled to polyethylene glycol linkers via carboxyl functionalities on their surface. a heterogeneous mixture of poly-arginine peptides of varying lengths (arg( )-arg( )) were covalently coupled via amide bonds to the polyethylene glycol linkers, conferring a cell penetrating capacity to the modified qdots. fluorescence imaging of u m cells, after incubation with the conjugated qdots at concentrations of nm, gave clear signals indicating cell binding and internalisation of the modified qdots across the plasma membrane. we aim to further expand this work by employing racemic mixtures of cpp and cytotoxic agents to engineer conjugates that will facilitate both imaging and the therapeutic delivery of cytotoxic moieties. the ability of cell penetrating peptides (cpp) to deliver biologically active cargoes into different cell types has been successfully applied in several experimental systems. despite the progress and growing number of described cpps, reports about the internalization mechanisms and the intracellular routes of cpps still remain controversial. we have characterized the membrane interaction and cellular localization of proteins delivered into hela cells by cell penetrating peptide transportan (tp) and its shorter analogue tp on ultrastructural level. our previous results obtained by transmission electron microscopy showed that complexes of transportans with gold-labeled streptavidin translocated into cells inducing large invagination of plasmamembrane, suggesting the uptake by macropinocytosis. the complexes of transportan with gold-labeled neutravidin, in contrary, were taken into cells mostly via caveosomes and clathrin-coated vesicles. the cell-transduced transportanprotein complexes localized mainly in the vesicular structures of different size and morphology. most of the complexes-containing vesicles in the perinuclear area contained also lamp protein, marker of late endosomes and lysosomes. still, the transportan-protein complexes were not confined in the membrane-surrounded vesicles, but spread in the cytosol suggesting the escape of transportan-protein complexes from endosomes. our findings show the involvement of different endocytic pathways in the transportan-mediated uptake process of proteins. the concentration of a cpp and the properties of cargo protein seem to determine the pathway for the cellular uptake of a particular construct. rgd peptides (r = arginine; g = glycine; d = aspartic acid) have been found to promote cell adhesion upon interaction with alphav-beta receptors, which are strongly overexpressed during neoangiogenesis by solid tumor associated cells compared to healthy cells. in this study we designed new targeting motifs aimed to deliver various antitumoral drugs specifically to cells involved in tumor vascularization. we inserted this short rgd sequence in tetracyclopeptides closed with various means. we expect these new cyclotetrapeptides to be more specific for the targeted receptor. moreover, these new type of cyclic peptides were multimerized on different scaffold to further improve the receptor avidity. our purpose is first to scrutinize and to quantify the efficient cellular uptake of these molecules and second, to address the specific cell targeting of a fluorescent cargo by differents tools such as fluorescence activated cells sorting (facs) analysis or fluorescence microscopy. these new targeting units were evaluated on two different cell lines: human umbilical vein endothelial cells (huvec) with an over expression of the alphav-beta integrin receptor and a cells expressing a much lower level of this receptor. preliminary results about the selectivity and the efficacy of these new targeting units will be presented. we have recently developed new approaches for obtaining highly immunogenic peptide conjugates: synthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. in this study, epitopes of antigenic parts of surface antigen of hepatitis b virus ( - , - , - and - of the s gene.) had been synthesized.the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem). peptide conjugates of synthetic anionic polyelectrolytes (copolymers of acrylic asid and n-vinylpyrrolidone) were synthesized by carbodiimide condensation following the modification procedures described early. composition and structure of bioconjugates were characterized by hplc (shimadzu), nanospr- , zetasizer nano zs, steady state fluorescence spectrometer qm- and viscotek tda size exclusion chromatography. it was obtained that a single immunization of mice with pe-peptide conjugates without classical adjuvant increased the primary and secondary peptide-specific immune response to hbsag. moreover, these conjugates possess own selectivity for recognizing the antibody in blood sera of hepatitis virus injected people. tissue engineering requires delivery of transplanted cells to organ sites needing repair/regeneration. we have demonstrated that several active laminin peptide-conjugated chitosan membranes enhanced the biological activity and promoted cell adhesion in a cell-type specific manner. the most active laminin peptide (ag : rkrlqvqlsirt)-conjugated chitosan membrane could deliver keratinocytes to a wound bed. when human keratinocytes were seeded onto the ag -chitosan membranes under serum-free condition, more than % of the cells attached within hrs. the membranes carrying keratinocytes were stable enough for handling with forceps and were inverted onto the muscle fascia exposed on the trunk of nude mice. keratinocyte sheets were observed after days and colonies appeared after days on the fascia of host mice. these cells were multilayered on day- and expressed various keratinocyte markers, including cytokeratin- , involculin, and laminin ? -chain. these results suggest that the ag -conjugated chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system, such as delivering keratinocytes to the wound bed. the peptide-chitosan approach may be a powerful cell transplantation tool for various tissues and organs. the fluorescent semi-conductive (cdse/zns) nanocristals possess very attractive optical properties that could be used for tracking individually biological receptors in vivo. our aim is to design functionalized water-soluble semi-conductive nanocristals (or quantum dots) that interact selectively with lipidic or biological membranes. to valid our approach, the interaction between the decorated qd and giant vesicles were observed by optical fluorescent and dark field microscopies. in view to solubilize and selectively bind fluorescent nanocristals to a lipid membrane, heterobifunctionalized peptidic ligands (liipe) that presented an adhesion domain for the nanocristal surface, an hydrophilic spacer and a terminal recognition function, were synthetized. the colloïdal stability of the water-soluble nanocristals (nc-lipe) was checked by dynamic light scattering, optical and electron microscopies the interaction of grafted nanocristals (nc-lipe) with positive or neutral giant vesicles was observed by optical fluorescence and dark field microscopies. as shown in figure, negatively charged nanocristals (nc-lipe) selectively adsorbed onto the surface of positively charged giant vesicles without altering the morphology of the vesicle. the nanocristals appeared as fluorescent patches growing on the surface of the vesicle until completely recovering. therefore these ligands (lipe) permitted to chemically functionalize the nanocristals by keeping their colloïdal stability and their fluorescence in water. furthermore it was possible to selectively label vesicle membrane. creatine analogues for treatment of obesity: us patent bioactive peptides containing pairs of basic amino acids are rapidly metabolized as a result of cleavage by trypsin-like enzymes. to increase the metabolic stability of opioid peptides containing arg-arg and arg-lys pairs, the arg residues were replaced by homoarginine (har) kenes international leprince , f. cavelier , p. gandolfo , m. diallo , h. castel , l. desrues m new bradykinin analogues modified with -aminocyclopentane- -carboxylic acid effect on rat blood pressure and rat uterus o. labudda-dawidowska , d. sobolewski , m Śleszyńska , i derdowska synthesis and heparin binding sites identification f. baleux , f. arenzana-seisdedos chemokines are small proteins involved in numerous biological processes (inflammation, immunity, morphogenesis, tissue repair, and tumour development) the general goal of our project is to elucidate the role that hs plays in vivo in the physiologic and pathologic activities of the chemokine cxcl /stromal derived factor- , and to characterise the molecular and structural determinants accounting for the interaction. three sdf isoforms, alpha ( aa), beta ( aa) and gamma ( aa) have been identified. we previously identified the major heparin binding site on sdf alpha and demonstrated the importance of hs/sdf interaction in hiv entry cell inhibition ( , ). sdf gamma amino acids sequence corresponds to the sdf alpha sequence extended by a c-ter amino acids sequence containing putative heparin binding sites. in order to determine sdf gamma heparin binding sites, wild type and mutants proteins were synthesised by stepwise solid-phase peptide synthesis using fmoc chemistry prusiner proc. natl. acad. sci here we describe xenome's drug development process for the chi family, conopeptide -mria[ ] of the predatory marine snail conus marmoreus, leading to a suitable drug candidate (xen ). xen is highly selective for the norepinephrine transporter (net) compared to other transporters, such as dopamine and serotonin, and inhibits net via an allosteric mechanism. xen is currently in a phase i/iia clinical trial for the treatment of severe pain. an intensive synthetic analogue and screening program around -mria, incorporating early stage animal data xen isomers were synthesized via selective disulfide bond formation to identify the active connectivity. data from alanine-scans, single amino acid mutations and probing of backbone interactions combined with the full d nmr structure, led to the development of a pharmacophore for xen . this model is refined from further studies where structure-activity relationships were developed utilising binding and functional assay data for a range of peptides anti-cancer drug design anti-cancer drug design published structural data for hdac-like protein, a bacterial enzyme sharing high homology to the hdacs in its active site, confirmed that this protein contains a zinc in the active site. for the discovery of specific hdac inhibitors, a number of hydroxamic acis and related compounds have been designed based on the ligating function to the zinc atom. the mechanism also involves an appropriate nucleophile in the active site. chlamydocin is a cyclic tetrapeptide on the other hand, we have been focusing the cyclic tetrapeptide to develop the potent and specific inhibitors of hdacs. in the present report, we employed the chlamydocin scaffold and successfully introduced the series of thioether as the functional group to the cyclic tetrapeptide. it is well argued that the strong inhibition of hdac requires the best combination of zinc ligand, capping group, and appropriate spacer between them jerusalem department of biological regulation department of organic chemistry department of biological chemistry, weizmann institute of science, rehovot, israel estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. the use of estrogen and selective estrogen receptor (er) modulators in treatment of osteoporosis is limited due to substantial risks for breast cancer. recently, we developed peptides having estrogen-like activity peptide emp- had no effect on bone growth in normal mice, and did not influence the ovx-induced bone-loss. we then developed a new µct methodology to evaluate uncalcified and calcified growth-plate parameters. in the ovx mice, peptide emp- reduced volume and thickness of the uncalcified growth-plate, a possible cause for the inhibition of bone longitudinal growth. based on a reported enhancement of er-in female mice during protein biosynthesis, after the release of the nascent polypeptide chain, ribosome recycling factor (rrf) disassembles the post-translational complex. rrf has been shown to be essential for bacterial growth. thus, we are attempting to design suitable compounds to inhibit the rrf function as candidates for new-type antibiotics. we have determined the structure of rrf with amino acid residues by nmr and x-ray analyses and shown that rrf has two domains domain i with three stranded helical bundles and domain ii with &beta;/&alpha;/&beta furthermore, we recently determined the structures of the s ribosome-rrf complex by cryo-electron microscopy and the s ribosome-rrf domain i complex by x-ray analysis using the results of these experiments, we elucidated the interaction profiles between rrf and ribosome and found that the cationic center consisting of three arginine residues on the surface of the helical bundle, which we have shown to be essential for the activity of rrf, is bound to helix - of s ribosomal rna. we synthesized the rna and peptide fragments around this interacting site and characterized them by physico-chemical analyses. the results of cd and biacore experiments to investigate the details of the interactions between them showed that a mer of rna fragment is bound to rrf <i>biochemistry</i> <b> </b> causes an increase of pain by inhibiting the opioid response [ ]. recent research has shown further that melancortin receptors, mainly subtype mc r, produce an increase in response to pain stimuli [ ]. based on this previous work, we are developing chimeric ligands which will be of benefit to therapeutic pain treatment with enhanced opioid efficacy by acting as agonists at opioid receptors and antagonists at both cck and melanocortin receptors cck (i) and melanocortin (ii) pharmacophores were overlapped by trp, and different profiles of opioid pharmacophores (iii) were linked to the n-terminal of the melanocortin pharmacophore (figure). the designed ligands showed moderate to high biological activity at all three receptors depending on their respective structures. design considerations and structure-activity relationships will be discussed in detail along with in-vivo assay results china synthetic exendin- is a -amino acid peptide that exhibits potent anti-diabetic and dose-dependent glucose-regulatory activity. exendin- is susceptible to degradation in plasma, so its activity is limited. our aim is to find sites in exendin- that are susceptible to cleavage and provide information for designing new exendin- analogues. in this study the stability of exendin- in human plasma was evaluated in vitro. exendin- was incubated in plasma at ℃, extracted with sep-pak octadecyl columns and subsequently analyzed using high performance liquid chromatography (hplc). the results showed that exendin- was slowly broken down in plasma with a half-life of . h. the degradation products were identified by quadrupole time of flight mass spectrometry (q-tof-ms) with electrospray ionization pharmacology of exenatide(synthetic exendin- ): a potential therapeutic for improved glycemic control of type diabetes one of the proposed solutions for the pharmacotherapy of obesity, a major health problem in the western world, is to regulate the biochemical pathways that control the metabolic balance in the body. the melanocortin pathway regulates energy balance by binding of the catabolic endogenic neuropeptide αmsh to its mc receptor and thus causes a decrease in food intake. we have synthesized a backbone cyclic peptide library, based on the minimal αmsh sequence phe -d-phe-arg-trp [ ], that activates the mc receptor. all the members of the library shared the same sequence analysis using colorimetric liposomes confirmed that bbc- penetrate the intestinal cells by the transcellular mechanism. moreover, bbc- have high metabolic stability to intestinal enzymes ( % recovery after hr). ec analysis showed that bbc- selectively binds and activate the mc and mc receptors (ec . ± . and . ± . respectively). oral administration of bbc- in mice showed reduces food intake melanocortin tetrapeptides modified at the n-terminus, his, phe,arg and trp positions department of experimental and health sciences graduate school of pharmaceutical sciences graduate school of agriculture graduate school of medicine consisting of amino acids, is a product of the metastasis suppressor gene kiss- . this c-terminally amidated peptide was identified as the endogenous ligand of an orphan g-protein coupled receptor metastin-gpr signaling may regulate gonadotropin secretion and negatively regulate cancer metastasis. it is interesting that activation of gpr signaling negatively regulates the function of sdf- -cxcr axis in cho and hela cell transfectants we conducted the structure-activity relationship (sar) study on kisspeptin- using the neuropeptide-derived rw-amide scaffold to identify five-residue peptide amides as novel gpr agonists equipotent to kisspeptin pro-), where aoe is ( s, s)- -amino- -oxo- , -epoxydecanoyl. in continuation of our study to design and synthesize analogues bearing a zinc ligand to develop potent inhibitors of histone deacetylase (hdac) inhibitors, we shifted the aromatic ring of phenylalanine at the aminoisobutric acid (aib) position and also at the imino acid position. the aim is to explore the interaction of cyclic scaffold with the rim of hdac paralogs. we replaced the epoxyketone moiety of aoe with sulfhydryl group, which is protected as disulfide hybrid, as zinc ligand. benzene ring was introduced to aib structure to design amino- -indane carboxylic acid and amino- -indane carboxylic acid. aromatic ring-containing imino acids, such as d-tic were also employed to replace d-pro. the cyclic tetrapeptides were profiled by the inhibition of hdac , hdac , and hdac in adult goats, however, the infection remains unapparent and the virus may cause abortion, vulvovaginitis or balanoposthitis. the use of a vaccine could provide a powerful tool for the control of cphv- infection. synthetic peptide-based vaccines have advantages of being selective, chemically defined and safe. in order to further localize immunogenic epitopes, glycoproteins b, c and d of cphv- were analyzed with several prediction programs. peptide conjugates incorporating t and b cell determinants in multiple copies in branched architecture are better immunogens for the preparation of goat vaccines, we synthesized peptide conjugates bearing t cell epitope on the n-terminal of the core. b cell epitopes were conjugated via a thioether bond on the ne-amino group of four choroacetylated lysine residues of the core. elisas confirm that the b cell epitopes and the conjugates t celltetratuftsin induce epitope-specific and antibody responses two recorded electrodes implanted bilaterally. eeg recorded after the mirror focus was arises. trh applicated intranasal in ultra low doses ( - m and - m) or intravenously in high doses ( mg, mg, mg). for eeg registration and analysis the computer system conan was used and new modification of fractal analyze of quantized eeg. results: the synchronization of epileptic activity between primary and mirror focuses observed on the third day after operation. intranasal application of trh induced reduction of spontaneous focal epileptic activity as in primary cobalt damage focus as in the mirror focus more than h. the inhibition of mirror focus was more expressed. quite the contrary intravenous trh administration provocated the epileptic discharges in both local focus. the intense synchronized generalized activity was record during - min deraos , t.v. tselios , i. mylonas , g.n. deraos it is known that cyclic peptides are more stable in enzymatic degradation and conformationally restricted compared to linear. the cyclization was achieved using o-benzotriazol- -yl-n,n,n',n'-tetramethyluronium tetrafluoroborate (tbtu) and -hydroxy- -azabenzotriazole, , , collidine allowing fast reaction and high yield final product. the purification was achieved using reversed phase high performance liquid chromatography (rp-hplc) and the peptide purity was assessed by analytical hplc and mass spectrometry (esi-ms). the synthesized cyclic plp analogue was found to exhibit lower agonist eae activity compared to linear plp - epitope in sjl/j mice. this implies that the conformation of cyclic analogue does not trigger autoimmune reaction in the central nervous system and therefore encephalomyelitis the netherlands department of experimental and health sciences on the other hand, in both central and peripheral nervous system, cck acts as neurotransmitter. recently, cck is focused at modulation effects on feeding especially. in this study, we tried to establish a sensitive and specific enzyme immunoassay (eia) for detecting cck and to investigate the effect of some dopamine receptor antagonists using this eia, we measured plasma cck-like immunoreactive substance (is) levels in five healthy human subjects after single oral administration of some prokinetics. the minimum amount of cck detectable by our eia system was . pg/ml, and the ic of the calibration curve was pg/ml. we revealed that domperidone and itopride caused significant decreases in plasma cck-is levels but metoclopramide and sulpiride did not. we established a sensitive and specific eia for cck. furthermore pro-pro-phe-phe-), isolated from linseed oil [ ], possesses a strong immunosuppressive activity comparable at low doses with that of cyclosporin a [ ]. it has been postulated that the tetrapeptide sequence pro-pro-phe-phe is important for biological activity of cla on the basis of this information we have synthesized a series of cla analogues in which the alpha-proline residue was replaced by beta -iso-proline and beta -homo-proline residues, respectively (fig. ). the synthesis of titled beta-amino acids has been achieved according to the literature procedure italy synthetic peptides are largely used as antigens in solid-phase immunoenzymatic assays (elisa) for recognition of antibodies (abs) in biomedical research and, most importantly, in the set up of diagnostic methods. it is well known that the method of peptide immobilization on the solid support is very important for a correct ab recognition atherosclerosis in patients infected with helicobacter pylori (hp) we synthesized appropriate oligopeptides immobilized on cellulose via n-or c-termini, using standard -alanine linkers as well as a new linker, developed for this particular studies glc)ghsvflapygwmvk) we found the strongest recognition when the peptide was linked to the cellulose support via the c-terminus. however, in the case of ureb f hp urease smallest epitope (sikedvqf), and epitope ub- ( - hp fragment: chhldksikedvqfadsri) the strongest reactions with sera of atherosclerosis suffering patients were obtained for n-terminally anchored peptides the synthetic dipeptide gamma-d-glutamyl-l-tryptophan (scv- ) has been shown to stimulate t-lymphocyte differentiation and specific immune responses, and enhance il- and inf-gamma production. due to this preferential activation of th cytokine production, scv- may show utility in treatment of infectious diseases cba mice at a dose of , µg/kg. the same animals were used for all three methods of administration with a dosing interval of weeks. blood samples were taken from the right retro-orbital sinus. for determination of the scv- concentration in blood samples, an "eia-scv- " competitive solid-phase immuno-enzyme assay was developed (loq ng/ml) with a mean residence time of minutes. minutes postdose, indicating very rapid absorption. mean concentrations then declined and were measurable through . and hours postdose (mrt and minutes, for i/p and p/o, respectively). the estimated bioavailability of scv- after i/p and p/o administration was almost equivalent gastrin- (g ) is a peptide which promotes gastric acid secretion, cell proliferation, and occasionally gastrointestinal cancer in the gastric antrum. g also promotes the growth of cancerous colonic epithelial cells, but the cck /gastrin receptor, which mediates its activity, is largely not expressed on such cells. instead, our previous studies have shown that some other receptor mediates stimulation of proliferation of dld- and ht human colonic carcinoma cells by ncarboxymethyl gastrin (g gly) at namomolar concentrations and inhibition at micromolar concentrations, indication separate binding sites. we have shown previously that g ( - )-oh stimulates cell proliferation of ht- cells - )-nh , in order to determine their selectivity for and activation of the putative proliferation-stimulatory receptor. the results revealed that g ( - )-oh is not selective for a single receptor, but binds both sites as do g and g gly. g ( - )-nh promotes dose-dependent and non-biphasic proliferation of dld- cells and binds a single receptor with low affinity. m comparative study of ige-binding activity of synthetic peptides rnwd and rnwdvyk v th involvement of l-name in the antinociceptive effects of newly synthesized analogues of tyr-mif- peptide in rats tyr-mif- is able to interact with opioid receptors with a higher potency at m sites as well as to its specific non-opiate receptors in the brain. nitric oxide and tyr-mif- `s are potent modulators of opioid activities. involvement of no in nociceptive effects is well documented in various physiological and pathological processes in the cns. l-name when administrated i.c.v. or systemically exhibit antinociceptive activity in rats as evaluated by the pp test. in the present study, we investigated the involvement of l-name in the antinociceptive action of newly synthesized analogues of tyr-mif- peptide: nα-(me)tyr-mif- , d-tyr-(me)-mif- , tyr(cl )-mif- and tyr(br )-mif- . the experiments were carried out on male wistar rats ( - g). the changes in the mechanical nociceptive greece paclitaxel is one of the most important anticancer drugs used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. however, the development of resistance to paclitaxel, the side effects and low solubility of this drug remain major obstacles for its optimal use in the clinical practice. in this work, we present the synthesis of various analogues in which paclitaxel is covalently bound to peptides or as multiple copies to synthetic carriers. these peptide-paclitaxel derivatives possess greater solubility in water, could be suitable in producing anti-paclitaxel antibodies and inhibit the proliferation of human breast, prostate and cervical cancer cell lines. although, no major differences were found concerning the extent of the antiproliferative effect between various paclitaxel derivatives and paclitaxel, the analogue with four molecules of paclitaxel covalently bound to synthetic carrier [ac-(lys-aib-cys) -nh ] when used at low concentrations inhibited cell proliferation more potently than paclitaxel th involvement of the histaminergic system in the nociceptin-induced pain-related behaviors in the mouse spinal cord the purpose of the present study was to determine whether histamine-containing neurons in the spinal cord are involved in nociceptin-induced behaviors in mice. the i.t. injection procedure was adapted from the method of hylden and wilcox. immediately following the i.t. injection, the time spent for nociceptive behaviors including scratching, biting and licking were measured. the i.t. administration of nociceptin resulted in nociceptive behavioral responses, which were eliminated by the i.t. co-administration of opioid receptor like- (orl- ) receptor antagonists. the nociceptive behaviors were significantly attenuated by the i.t. co-administration of the h receptor antagonists, but not the h receptor antagonists. i.t. co-administration of the h receptor antagonist significantly increased the behavioral responses, whereas the behavioral responses were completely attenuated by the i.t. co-administration of the h receptor agonist. an antiserum against histamine injected i.t. reduced the nociceptin-induced behavioral responses. the same result was observed by i.p. pretreatment with histidine decarboxylase inhibitor. in conclusion, i.t.-administered nociceptin elicits the orl- receptor-mediated nociceptive behavioral responses. the activation of the orl- receptor by nociceptin may induce the release of histamine in previous studies, we demonstrated that highly constraint cyclic (s,s)-cxaac-containing peptides inhibit platelet aggregation and fg binding [ , ]. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. conformational studies revealed that orientation of the charged side chains toward the same side of the molecule increase the anti-aggregatory activity of the inhibitor. in this work we present the synthesis and the inhibitory activity of new cyclic compounds. for the design of the studied compounds we combined the available information from the -cdc-containing inhibitors and the gpiib - (ymesradr) sequence which has been shown to inhibit the adp induced human platelet activation however, its pharmacological effects and physiological functions are still unclear. the present study was designed to characterize the nociceptive behaviours induced by intrathecal (i.t.) administration of hk- in mice. the i.t. administration was made in conscious mice according to the method described by hylden and wilcox. immediately after the i.t. administration, the accumulated time for nociceptive behaviours was measured for min. the i.t. administration of hk- dose-dependently produced characteristic nociceptive behaviours consisting of scratching, biting and licking, which peaked at - min and almost disappeared by min after injection. the subcutaneous pretreatment with morphine dose-dependently attenuated the hk- -induced nociceptive behaviours. the nociceptive behaviours elicited by low-dose of hk- were significantly inhibited by i.t. co-administration with nk receptor antagonist, however, the nociceptive behaviours elicited by high-dose of hk- were not affected by i.t. coadministration with nk receptor antagonist. on the other hand, nmda receptor antagonists significantly suppressed both high-and low-doses of hk- -induced nociceptive behaviours in a dose-dependent manner. these results suggest that the nociceptive behaviours induced by low-dose of hk- may be mediated through both nk and nmda receptors, whereas high-dose of hk- may induce the nociceptive behaviours through nmda receptor the bacterial lp are strong modulators of the innate immune system. until recently, it was generally assumed that triacylated lp, like the synthetic pam cys-sk , are recognized by tlr /tlr heteromers, whereas diacylated lp, like fsl- , induce signalling through tlr /tlr heteromers. contrary to this model, we could show that depending on the peptide moiety, diacylated lp also signal in a tlr -independent and tlr -dependent manner. the aim of this study was to analyse more closely the structural basis of this heteromer usage. the synthesis of lp was carried out by fully automated solid phase peptide synthesis and fmoc/tbu chemistry on tcp or rink amide resin. information on the structural factors determining the tlr /tlr versus tlr /tlr heteromer usage was obtained by testing of ligands with cells obtained from tlr , tlr , and tlr -deficient mice. when stimulating b-lymphocytes of wild-type mice we found that ester-bound long-chain fatty acids are necessary to induce considerable responses. for triacylated lp with long chain length ester-bound acyl residues (like pam c-ssnask ) the response in tlr -deficient cells was only slightly decreased, whereas for lp with short length ester-bound fatty acids (like pamoct c-ssnask ) the response was completely abolished. in summary, a tri-acylation pattern is necessary but not sufficient to render an lp tlr -dependent and a di naples department of organic chemistry "ugo schiff sera from patients suffering from autoimmune disorders often contain multiple types of autoantibodies, some of which can be exclusive of a disease and thus used as biomarkers for diagnosis. identification of these autoantibodies, as disease biomarkers, should be achieved using native antigens in simple biological assays. however, post-translational modifications, such as glycosylation, may play a fundamental role for specific autoantibody recognition. in line with these observations, we previously described synthetic glycopeptides able to detect high autoantibody titers in sera of patients affected by multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system. we also demonstrated that glycopeptides able to reveal high antibody titers in multiple sclerosis sera are characterized by a type i we describe here the result of a conformationally driven rational design exercise, which led to the preparation of new, optimized glycopeptides endowed with enhanced antigenic properties. most importantly, the same approach, based on structure alignment, was used to shed light on the native antigen(s), target of pathogenetic autoantibodies involved in demyelination processes vitro and in vivo evaluation for cholecystokinin-b receptor imaging istituto nazionale per lo studio e la cura dei tumori ome (f )<br> (aib, α-aminoisobutyric acid) to investigate its binding properties to tb(iii) ions. according to our published spectroscopic results f populate a set of ordered conformations involving /α-helical segments and compact structures generated by the formation of a turn around the flexible gly -gly central motif. cd experiments showed that the binding of tb(iii) to f gives rise to a structural transition of the peptide chain from a helical to a folded conformation. peptide binding is also responsible for the dramatic increase in the tb(iii) fluorescence intensity, suggesting that the tb(iii)/f complex may represent an interesting system for imaging applications or bioanalytical sensing the kda protein of mycobacterium tuberculosis provokes specific immune response, therefore related epitope peptides and peptide-conjugates can be considered as potential diagnostics. in our previous study we have determined the functional human t-cell epitope within the - region. based on this we synthesised two groups of peptides: a) nand c-terminal alanine and beta-alanine elongated variants of the - epitope and b) - peptides with alanine substitution at different position according to the hla dr and tcr binding sites. peptides were prepared by solid phase synthesis using boc/bzl or fmoc/tbu strategy. the homogeneity and the primary structure of peptides were checked by analytical rp-hplc, amino acid analysis and esi-ms. the t-cell stimulatory activity of the compounds was investigated using in vitro assays (proliferation and ifn-gamma production) on the - epitope specific human t-cell clones and pbmc (peripherial blood mononuclear cells) from patients and healthy (ppd+, ppd-) subjects. the effective peptides were conjugated to branched polypeptides with polylysine backbone (sak, eak), tetratuftsin derivative (h-[thr-lys-pro-lys-gly] -nh ) and lysine dendrimer (h-lys-lys(h-lys)-arg-arg-beta-ala-nh ) (map) carrier via thioether bond formation. the subtitution degree of the conjugates was determined by amino acid analysis. pbmc and human t-cell clones were stimulated with the free peptide alone or with peptide-conjugates containing an equimolar amount of peptide or with a mixture of free peptide and carrier italy we demonstrated, for the first time, that an aberrant post-translational modification (ptm, n-glucosylation) is possibly triggering autoantibody response in multiple sclerosis. this was possible because of a "reverse approach", which led to csf (glc), a structure based designed glycopeptide, as the first multiple sclerosis antigenic probe accurately measuring high affinity autoantibodies (biomarkers of disease activity) in sera of a statistically significant patients' population universal peptide scaffold" to be modified with a series of glycosyl amino acids (different in sugars and linkages), in the aim of developing personalized diagnostic/prognostic tests. the csf beta-turn structure, exposing at the best the aberrant ptm specific for antibody-mediated forms of other autoimmune diseases, will lead to a family of antigenic probes to be used in diagnostic this information is encoded by the distribution of the electron-ion potential (eiip) of amino acids along the sequence and is represented by the frequency components in is. proteins with the same biological functions or interacting proteins (e.g. antibody/antigen) share the information corresponding to the common frequency components in their iss. investigation of the hiv- envelope glycoprotein gp , as a model system for hypervariable proteins, revealed that this information is strongly conserved and is not significantly affected by natural mutations. the c-terminus of the second conserved region (c ) of gp , encompassing ntm peptide is important for infectivity and neutralization of hiv- , while human natural anti-vasoactive intestinal peptide (vip) antibodies reactive with gp play an important role in control of hiv disease progression. ntm/vip multiple copies were coupled to an artificial sequential oligopeptide carrier for developing an immunoassay (elisa) as a reproducible, reliable and sensitive tool for the detection of anti-ntm/vip derived antibodies these peptides have been utilized in an immunopeptidometric assay for specific measurement of active, noncomplexed psa. however, this assay has not been sensitive enough for the measurement of active psa in clinical samples. therefore, we aimed to develop an improved assay utilizing the same principle as previously, but using a more sensitive detection method based on proximity ligation assay. methods: in the assay, psa is first captured on a solid phase by a psa antibody czech republic rapidly increasing knowledge of new gonadotropin-releasing hormones (gnrh)of different species of the animal kingdom induces the need to prepare new synthetic derivatives and fragments of these peptides with higher potency and metabolic stability and suitable for the formulation of new immunogens. the species related differences in the sequence of the native mammalian gnrh pglu-his-trp-ser-tyr-gly-leu-arg-pro-glynh concern predominantly the positions , and , particularly tyr in position is replaced for his or leu, leu in position by val or trp, and arg in position is substituted by lys, ser, asn or gln. several gnrh derivatives with with the above substitution and gnrh fragments were prepared by solid phase peptide synthesis and purified by rp-hplc. purity of the synthetic peptides was checked by capillary zone electrophoresis (cze); peptides were analysed as cations in acidic backround electrolytes (ph . - .quantitative analyses for determination of their effective electrophoretic mobilities and the estimation of their effective charges.supported by grant of ministry of agriculture of cr-nazv qf by rants of ga cr nos we use peptaibiomics for the structural determination of peptaibiotics from fungi grown on single agar plates thus avoiding time-consuming isolation and purification procedures. the method comprises fast and effective solid-phase extraction followed by on-line rp-hplc coupled to tandem esi-ms. here we present a survey of the peptaibiome of hypocrea species. in extracts of hypocrea semiorbis, h. vinosa, h. dichromospora, h. gelatinosa, h. nigricans, h. muroiana and h. lactea a multitude of short and long-chain peptides containing aib could be characterized. the formation of new and known peptaibiotics could be established by comparison with sequences stored in data bases japan process scale rp hplc purification of peptides and proteins is increasingly important in bio-pharmaceutical production. besides selectivity, other crucial factors are loadability, recovery loadability is believed to depend on the surface area of the packing material. consequently, smaller pores providing larger surface area should lead to increased loadability. this principle is misleading in the case of large molecules, because they cannot penetrate smaller pores. therefore the chromatographically accessible surface area has to be taken into account. recovery problems like irreversible adsorption or aggregation are frequently caused by hydrophobic surface properties of ods phases. the less hydrophobic c is a substituent to avoid considerably these problems. however c is less durable than ods under extreme acidic conditions. our new proprietary c modification technology combined with a perfect end-capping minimizes the presence of residual silanol groups and protects the silica surface sp- -c -bio demonstrates high mechanical stability by no obvious alteration of back pressure and particle size after repeated packing cycles in dac columns. by overcoming the common weaknesses of the conventional c rp silica phases, daisogel sp- -c -bio opens new avenues for process scale separation of peptides and proteins. m determination of peptide: protein molecular ratio in conjugates by seldi-ms method synthetic peptides are widely used as antigens in various research and practical areas of biology and medicine. peptides with molecular masses < kda should be conjugated with carrier proteins in order to ensure their immunogenicity and protect from proteolysis. in these cases the comparison of peptide immunogenicities and immunotest system development should be performed having in mind exact peptide-to-protein ratios. conjugates of peptide fragments of hepatitis c virus envelope protein e with ovalbumin, bovine serum albumin, and myoglobin were prepared using glutaraldehyde (ga), m-maleimidobenzoyl n-hydroxysuccinimide ester, dimethyl suberimidate (dms), -ethyl- -( -dimethylaminopropyl)-carbodiimide as conjugating reagents. the rough evidence of the peptide-protein conjugate formation was obtained by page. the exact peptide:protein molar ratio was estimated in all conjugates by seldi-ms. almost all conjugates had oligomeric structures due to the formation of intermolecular linkages between proteins. the peptide : protein molar ratio in conjugates varied from : to , : . conjugates obtained with the ga were more diversified in the number of peptide molecules linked to carrier proteins (peptide:protein ratios ranged from : to : ) than other conjugation reagents mazur-marzec poland posttranslational modifications (ptms) like phosphorylation, acetylation, or methylation have been shown to play a significant role in directing the function of various proteins [ ]. in eukaryotes, most of proteins have been shown to be posttranslationally regulated by a variety of different modifications. many effects of ptms include a change of enzymatic activity capillary electrophoresis (ce) has been used to study electrophoretic behavior of ptm-peptides gal-nh , gal( - )-nh by capillary electrophoresis. using a phosphate buffer most of ptm-peptides were poorly separated at acidic or neutral ph. the best results were obtained using trifluoroethanol containing separation buffers. optimization of ce separation of maps of peptides containing ptms should allow to detect ptm-proteins and characterize their role in the living cell. comparison of modification events occurring in diseased and healthy cell may iran the purpose of this study was to use the application of multiplex reverse transcription polymerase chain reaction(rt-pcr) assay for detecting the two most common leukemia translocations t( ; ) and t( ; ) in childhood acute lymphoblastic leukemia in iranian children. cases of leukemia patients were screened with the rt-pcr assay. this assay will identify the all type bcr-abl transcripts encoded by the t( ; ) and all described variants of the e a-pbx transcript encoded by the t( ; ). rna was isolated from leukocyte cells of patient's samples. through the construction and optimization of specific primers for each translocation,we have been able to set up multiplex rt-pcr reactions.then pcr products was electrophoresed on agaros gel and were compared with size markers and expected fragtments key words: acute lymphoblastic leukemia -multiplex rt-pcr tu study on the syntheses and lc/esi-ms analyses of the glutathione conjugates of bile acids t. wakamiya , m. sogabe a carboxylic acid-containing drug, is metabolized to a glutathione (gsh) conjugate in vivo, and the conjugate is excreted in human urine [ ]. although bile acids, compounds with carboxylic acid in molecules, are also expected to form gsh conjugates in liver, no evidence is so far obtained to confirm such metabolism, since there are no suitable standard samples for the research. in the present paper we report the syntheses of the gsh conjugates of main bile acids in human, i.e., cholic acid (ca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), ursodeoxycholic acid (udca) and lithocholic acid (lca) as shown below, and the detailed analyses of these synthetic conjugates by means of linear ion trap lc/esi-ms. furthermore, the evidence for conversion of cholyl adenylate [ ] and ca-coa thioester into ca-gsh conjugate will be presented these peptides do no exhibit such strong side effects as csa, but their practical application is hindered because of their poor solubility in water. the - fragment of tat protein and its analogs, including oligoarginine sequences, are known for their unusual ability to cross cell membranes, skin, and blood-brain barriers. moreover, these peptides are able to transport other substances into cells. this strategy was successfully applied in cases of csa, taxol, and other drugs to improve their bioavailability. now we have synthesized a series of analogues of cyclolinopeptide a, clx, and the immunosuppressive fragment of ubiquitin, covalently bound to the cell-penetrating fragment of tat and its analogues. the ability to cross the biological membranes and the immunosuppressive activities of these conjugates were tested. the conformation of the peptides was determined by circular dichroism methods: we used fluorescein-labelled cationic cell-penetrating peptides and analyzed the uptake efficiency (flow cytometry) as well as the intracellular distribution (confocal laser scanning microscopy). the bioactivity of a proapoptotic cargo-peptide, delivered into the cells either via electroporation or via cpps was quantified using a caspase- activity assay and cellular assays. to address the integrity of cpps during their trafficking, a fluorescent double-labelled antp peptide was designed and used as an intracellular fret-sensor. results: endocytosis-mediated uptake of the cpp-cargo conjugate led to a significant reduction of cargo bioactivity compared to its direct transfer via transient membrane permeation. this finding was related to the sequestration of peptides within endocytic vesicles but also, in the case of the tnf response, to the induction of receptor internalization during cell entry. moreover, during endolysosomal passage peptides undergo significant proteolytical degradation. conclusions: the endocytosis-dependent uptake mechanism of cholesterol pullulan (cp), in which maltose moieties are partially modified by cholesterol, is unique in forming self-assembled nanoparticles ( - nm) in water. combination of these characteristics is considered to be promising for development of effective non-viral vectors without toxicity. a conjugate of hiv-tat and cp was synthesized and its gene expression efficiency was evaluated. fully protected hiv-tat-( - )-cys(snm)-gly-nh-r was obtained by conversion of the corresponding cys(acm) peptide which was synthesized by the solid-phase method [snm: (n-methyl-n-phenylcarbamoyl)-sulfenyl] [ ]. the sulfhydryl function was introduced to the hydroxyl groups of cp by acylation with trt- -mercaptopropionic acid followed by acid treatment. resulting -mercaptopropionyl-cp was coupled with cys(snm) peptide to form disulfide bridge and the protecting groups of the peptide were removed to give the cp-tat conjugate. cp-tat and pcmv-luc complex was transfected into cos cells and luciferase activity was analyzed after h. cp-tat elicited remarkable cytoplasmic luciferase activity and low toxicity this finding provides a possibility to use gnrh-iii as a targeting moiety for intracellular drug delivery. therefore we have prepared methotrexate and doxorubicine conjugates of gnrh-iii. the drug molecules were attached to the lys side chain in position of gnrh-iii by thioether bond formation through the gflg spacer elongated with cys either at the c-or n-terminus. since we found earlier that the dimer derivative of gnrh-iii was more effective, new dimer derivatives with a combination of antitumour agents were also prepared. branched gnrh-iii derivative (pyrhwshdwk(clac-glfgc(acm))pg-nh ]) was synthesised by spps. the drug molecules were attached to this compound by thioether bond and finally disulfide bridge was formed between two peptide chains. the cytotoxicity of new derivatives was characterised by mtt test th oligopeptide antifungals are exceptionally active against multidrug-resistant yeast previous studies have demonstrated that longer sirnas that are processed by dicer can result in more potent knockdown than the corresponding standard -mer sirnas. dicer-substrate - -mer sirnas were conjugated with different structural classes of peptides and their cell uptake properties evaluated. peptides were conjugated to the ' end of the sirna sense or antisense strand via a thioether bond under denaturing conditions to prevent aggregation and precipitation. the ability of conjugates to translocate fluorescently-labeled sirna across the plasma membrane was evaluated by flow cytometry. the results indicate that some peptides can mediate higher efficiency uptake of sirna into cells compared with lipofectamine or cholesterol-conjugated sirna. the peptide-sirna formulation with -mer sirna conjugates showed higher knockdown of tnf-alpha mrna and protein levels in activated human monocytes in vitro compared to the conjugated -mer sirna species. the products resulting from in vitro digestion of peptide-conjugated rna duplexes with recombinant human dicer were identified using esi-ms and consisted predominantly of the desired -mer sirna several peptides containing the sequence arg-gly-asp (rgd) were studied and developed for their nanomolar affinity to the membrane receptor alfa v beta and alfa v beta integrins, which are over-expressed by endothelial cells during proliferation and by tumor cells. to improve the pharmacological profile of some camptothecin derivatives (cpts), five conjugates were designed, where the cytotoxic drugs were covalently attached to the rgd peptide analogues for preferential uptake into tumor cells. the peptides to be used have been selected among a series of new pentacyclic peptides bearing at -position a trifunctional pseudoamino acid with a carboxy-terminal side-chain. peptide analogues showing the highest affinity to alfa v beta and alfa v beta integrins were coupled with cpts at different positions. the conjugates have been optimized for binding to the receptors, proteolytic stability and an overall improvement in tumor selectivity. the nature of the linkage between rgds and cpts has a major impact on stability and biological activity of the conjugates. the conjugates with amide bond, but not those with ester bond, are sufficiently stable and show in vitro antitumor activity against a and a cell lines combination of amaranth protein with other plant proteins (cereals) enables to formulate the composite protein (near to milk or beef protein, but exclusively on vegetal basis). it is shown on graphs. the aim of the projects' proposals is a development and realization of the technology for fractionation of amaranths defatted flour product is a top protein obtained by removing starches and next polysaccharides decomposed on soluble monosaccharide by specific enzymes. there are shown the chromatograph measuring. we can see complete disintegration of starch and the unchanged proteins. the separate solution monosaccharide is usable for others fermentative processes or as a nutrient solution for yeasts there are description methods for isolation amaranth protein -extraction processes, enzymatic removal starch. the product is a isolate protein rich in essential amino acids. the waste monosaccharide solution was used to production yeasts biomass rich in proteins vitamins amaranth protein isolate have high nutritional value and can be used as food ingredients, for functional, probiotic formulation to begin with, we made epitope mapping with the highly sensitive spot array method ( ) in order to study antigenic regions of parvovirus b vp and vp capsid proteins. epitope mapping identified highly reactive, immunodominant early epitope on parvovirus surface that centered to kyvtgin residues of vp . in the subsequent phases we developed the kyvtgin epitope type specific (ets) igg serodiagnostics. a correlation between enhancing igg avidity to b capsid and a transient reactivity with the point-of-care kyvtgin peptide was clear. together the two assays enhanced the value of early diagnosis of b infections ( ) acute phase-specific heptapeptide epitope for parvovirus b diagnosis synthetic peptide arrays on membrane supports-principles and applications human parvovirus b infection during pregnancy--value of modern molecular and serological diagnostics laboratory of peptide & protein chemistry & biology department of organic chemistry "ugo schiff" and cnr-iccom department of chemistry department of pharmaceutical sciences glc) able to recognize specific autoantibodies in multiple sclerosis (ms) patients' sera has been developed by the laboratory of peptide & protein chemistry and biology [ and bio-plex suspension array system, biorad. the biosensor technology and bio-plex suspension array system will offer advantages such as rapid analysis, and high sensitivity for a high throughput screening. immobilisation will be based on different strategies that are anchoring the synthetic antigen on different solid supports such as polystyrene well plates (elisa) optimisation of the different techniques was performed with anti-csf (glc) autoantibodies isolated using affinity chromatography from ms patients' sera. the analytical parameters such as specificity, sensitivity, and matrix effect were evaluated. the different technologies have been used for a high throughput screening of ms sera which control specific cell adhesion. here we discuss the route for preparation of amphiphilic block copolymers composed of hydrophobic polylactide and hydrophilic polyethylene oxide (peo) blocks, carrying various cell-adhesion oligopeptide sequences at the end of peo block. fully protected peptide fragments were prepared by solid-phase peptide synthesis by using fmoc strategy and chlortrityl resin. the side-chain protected peptides were cleaved from resin by % hfip solution in dcm. the copolymers peptide-polytehyleneoxide were prepared by coupling of the activated peptide fragments with α-amino-ω-hydroxy-peo in dmf using pypop as an activation reagent. subsequently, the polylactide block was grafted to the ω-hydroxy end group of the peptide-peo copolymer via a controlled rop polymerisation of lactide r)- -aminocyclopentanecarboxylic acid (acpc) and beta-methylphenylalanine (beta-mephe) were designed and synthesized to obtain more potent and selective mu-opioid receptor ligands with higher stability against proteolytic enzymes. we have prepared the peptides by spps methods using racemic amino acids. the diastereomeric peptides were separated by hplc. the configuration of the unnatural amino acids in the peptides was determined by chiral tlc using enantiomeric standards. radioligand binding assays and in vitro gpi and mvd assays indicated that several analogues showed high, subnanomol affinity and high selectivity for mu-opioid receptors having agonist or antagonist properties. the incorporation of alicyclic amino acids into the endomorphins resulted in enzyme resistant peptides. the most promishing analogues (dmt-pro-phe-phe-nh and tyr-( s, r)acpc-phe-phe-nh ) were labeled with tritium using precursor peptides containing dehydroproline or dehydro-( s, r)acpc amino acids and tritium gas and pd/baso catalyst. the novel peptides and their radiolabelled analogues with high specific radioactivity ( . - . tbq/mmmol) have become useful pharmacological and biochemical tools for the opioid research iran background and aims: injectable drug delivery based on polymer solution platforms has gained in resent years, particulary for protein-based therapies. the influence of polymer molecular weight (rg h, rg h) on the morphology, erosion of matrices and also on their in-vitro drug release behavior over a period of days was assessed for leuprolide acetate in this study. methods: each formulation was composed of % (w/w) polymer and % (w/w) leuprolide acetate dissolved in nmp. release studies were performed in a home-made diffusion cell at °c. the polymer erosion was studied using two different methods as follows. (a): l-lactic acid detection (b): ph change study. the morphology of the matrices was then analyzed by scanning electron microscope as is shown, the lower molecular weight polymer formulation shows higher porosity and pore diameter due to a rapid phase inversion phase i) can be divided into three more phases with different release rates. results showed that burst effect for rg h, %, was significantly (p< . ) higher than rg h ( %) italy fabrication of photocurrent-generating systems based on bioinspired organic-inorganic hybrid materials is currently of great interest. more specifically, the photoelectronic properties of nanometric films formed by peptide self-assembled monolayers have been actively investigated. in this work interdigitated gold microelectrodes were modified by covalently linking a hexapeptide ester functionalized by a lipoic acid (lipo) at the n-terminus. the peptide chain [lipo-(aib) -trp-aib-otbu] comprises five α-aminoisobutyric acid (aib) residues and one trp, a fluorescent amino acid with strong absorptions in the uv region. due to the very high percentage of conformationally constrained aib residues in the chain, the peptide adopts a rigid - -helical structure. cyclic voltammetry measurements indicate that the peptide forms a homogeneously and densely packed monolayer on the gold surface, while current/voltage curves exhibit interesting rectifying properties of the peptide sam. photocurrent generation experiments, performed on the peptide-layered microelectrode, show peculiar modifications of the spectrum. at nm a notably higher photocurrent/voltage response was observed for the peptide-modified electrode, suggesting that a photoinduced electron transfer process from trp to gold does take place with high efficiency this may lead to randomly orientated enzymes and subsequently limited activity. the aim of this work is to selectively activate enzymes at their c-terminal position in order to allow specific immobilisation. we chose akr a , an enzyme of the aldo/keto reductase superfamily, for the synthesis of an artificially monolabeled redoxprotein. akr a is a monomeric enzyme and catalyzes the nadph dependent reduction of aliphatic/aromatic ketones and aldehydes. to produce monofunctionalized enzymes we applied the strategy of expressed protein ligation (epl). accordingly, we used the impact®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. through intein mediated splicing we could produce c-terminal thioester of the akr a . in the next step, the thioester was coupled to a biotin containing peptide by native chemical ligation. this specifically modified enzyme was immobilised on avidin coated surfaces. the attachment on the surface was tested by tryptic digestion, followed by maldi-tof-ms analysis since safe organic solvent waste disposal is an important environmental problem, we aimed to perform peptide synthesis in water. we have reported solid phase peptide synthesis in water using water-soluble n-protected amino acids, such as -[phenyl(methyl)sulfonio]ethoxycarbonyl and -( -sulfophenylsulfonyl)-ethoxycarbonyl amino acids. following to study on water-soluble n-protected amino acids, we developed a new technology based on nanochemistry for solid phase peptide synthesis in water. the new technology is based on coupling reaction of suspended nanoparticle reactants in water. fmoc-amino acids are used widely in peptide synthesis, but most of them show poor water-solubility. we prepared well-dispersible fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol) (peg). the size of fmoc-amino acid particles was - nm. to evaluate the utility of this technique, leu-enkephalin was prepared using the nanoparticulate fmoc-amino acids on a peg-grafted rink amide resin in water supramolecular structures formed from n-lipidated oligopeptides immobilized in the regular pattern on the cellulose surface are able to bind ligand molecule, thus acting like artificial receptors. due to the conformational flexibility of lipidated oligopeptide chains, the supramolecular structure is highly flexible, forming the cavities with the shape and prosperities adjusted most effectively to requirements of the guest molecule. structural requirements for a peptide providing the most efficient fit the guest molecules are not known, therefore an array of the artificial receptors have been synthesized and used in the studies. thus, even in the case, when the single receptor in an array does not necessarily have selectivity for a particular analyte, the combined fingerprint response can be extracted as a diagnostic pattern visually, or using chemometric tools in order to improve the sensitivity of the competitive binding and to study the mechanism of molecular recognition, experiments involving fluoresceine and fluoresceine marked acp fragment were performed. we found that λmax and intensivity of fluorescence depends on the structure of the peptide motif and lipidic fragment of receptor this mts was linked with dhhp- by disulfide bond, and the new molecular was named mts~dhhp- . the peroxidase activity of mts~dhhp- ( . x u•µmol- ) was tested and similar to that of mp- ( . x u•µmol- ). mts~dhhp- coated with quantum dots (qds) [ ] were observed to accumulate into neonatal rat cardiomyocytes (nrcms) of wistar rats and co-localized with mitotracker red in mt. these results suggest that mts~dhhp- is an excellent apx mimics and may have potential proceedings of the twenty-eighth european peptide symposium, kenes international, israel, , . references: elastin-based polypeptide, poly(val-pro-gly-val-gly), undergoes self-assembly called coacervation, in which microcoacervate droplets with approximately nm diameters are formed [ ]. nanoparticles cross-linked by cobalt- γ-irradiation of these microcoacervate droplets are useful as drug release devices. to investigate the size optimization of nanoparticles, the stability of nanoparticles in the treatment of enzyme, and the drug release profiles from nanoparticles, the three copolymers; poly[ (val-pro-gly-val-gly), (val-ala-pro-gly-val-gly)], poly[ (val-pro-gly-val-gly) application of polyelectrolytes and theoretical models the synthetic heptapeptide rnwdvyk is a fragment of a high affinity receptor (fcεri) for immunoglobulin e (fragment - ). it is the active domain for binding with ige. the program of studies of biological properties of the heptapeptide included the investigation of its binding to ige contained in standard solutions and in patients' blood serum. the binding of rnwdvyk with ige was investigated by the ifa method using the ige antibodies labelled with horse-radish peroxidase (hrpo). we determined the optimum sorption concentration of the peptide in this experimental immunoenzyme system to be mkg/ml. the ability of synthetic rnwdvyk peptides to bind with ige was studied as a function of ige concentration in standard serum ( . to ng/ml ige). a high correlation was found between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and the substrate chromogenic mixture (r= . ). similar investigations were conducted using the allergy patients' blood serum. the serum with a known concentration of ige was added to immunological plotting boards with sorbed synthetic rnwdvyk peptides. a high correlation was also found between the concentration of ige in the patients' blood serum and the optical density of the solution after introducing monoclonal antibodies labelled with hrpo and substrate chromogenic mixture (r= . ). our experiments showed the high ige binding activity of synthetic rnwdvyk peptides. we demonstrated the possibility of construction of diagnostic systems for the quantitative determination of ige and ige-antibodies. the fusion protein nucleocapsid-dutpase is present in virions of mason-pfizer monkey betaretrovirus and in virus-infected cells where it potentially contributes to rna/dna folding and reverse transcription (barabas, et al., ; bergman et al., ; berkowitz, et al., ) . in addition to trimeric dutpase core, the protein possesses flexible n-and c-termini consisting of the nucleocapsid segment, and a peptide motif conserved in dutpases. to analyze the function of the flexible cterminal peptide segment, reconstitution experiments were designed with truncated enzyme lacking the c-terminal mer oligopeptide and the synthetic oligopeptide prepared on rink-amid resin by solid-phase peptide synthesis, using fmoc strategy. the truncated enzyme proved to be practically inactive. addition of the synthetic mer (pyrgqgsfgssdiy) at fold molar excess resulted in partial complamentation of the catalytic activity (to % of original). a mixture of the truncated enzyme and the mer oligopeptide (this latter at fold excess) was put to crystallization trials. we conclude that the c-terminal mer is essential for catalytic activity. antifolate drugs are inhibitors directed to interfere with folate metabolic pathway. methotrexate (mtx) and pemetrexed (alimta®) are known folic acid analogues used in cancer treatment. different peptide conjugates of mtx have been prepared for intracellular delivery. ( ) in octaarginine conjugates one of the carboxylic groups of mtx was attached to the n-terminal of the peptides. ( ) however, as results showed, that both carboxylic groups are required to the biological effect of mtx. therefore we decided to synthesize peptide conjugates of folic acid analogues in which the carboxylic groups are untouched. octaarginine, penetratin and a cyclic peptide cgnkrtrgc, which can deliver a cargo molecule in the lymphoid system, were used as delivery peptides. we introduced squaric acid or aminoxy acetic acid as linker moiety between the peptides and cargo molecules. the conjugation was monitored by rp-hplc, the crude products were purified by rp-hplc and were identified by mass spectrometry. the biological activity of the conjugates was evaluated in vitro on sensitive and resistant human leukemia (hl- ) cell lines. besides its endocrine activity, trh (the tripeptide pglu-his-pro-nh<sub> </sub>) has also been long recognized as a modulatory neuropeptide with broad range of physiological and pharmacological activities in the central nervous system (cns). although numerous centrally active and metabolically stable analogues and peptidomimetics have been synthesized using trh as a template, selectivity of their cns action has remained an issue to be addressed. we aimed at discovering novel analogues with enhanced cns-selectivity by incorporating pyridinium building blocks. the design also allowed for enhancing transport across the blood-brain-barrier and increasing residence time in the cns through prodrug strategy. solid-phase chemistry was used to prepare the analogues and novel methods previously not used to incorporate pyridinium moieties into resin-bound peptides such as the zincke reaction were also introduced. comprehensive evaluation included measurement of affinity to trh-receptor, acetylcholine-releasing, analeptic and antidepressant-like activity in animal models, as well as prediction of membrane affinity, determination of in vitro metabolic stability, and pharmacokinetics and brain uptake/retention studies that employed in vivo microdialysis sampling. a strong connection between acetylcholine-releasing potency and analeptic effect in animals was obtained for close analogues of trh, while pyridinium compounds designed from the structurally related pglu-glu-pro-nh<sub> </sub> maintained the antidepressant-like effect of the parent peptide, while showing significant decrease in analeptic action.in conclusion, an increase in the selectivity of cns-activity profile was obtained by the incorporation of pyridinium moieties. we have also demonstrated the benefits of the prodrug approach on the pharmacokinetics, brain uptake and retention of the analogues upon systemic administration. the use of small radiolabelled compounds such as peptides is a very attractive tool for the diagnosis of several different pathologies, specially cancer, through the use of nuclear medicine techniques. among the various membrane receptors, the two cholecystokinin receptors ccka-r and cckb-r are very promising biological targets for radiolabelled compounds due to their overexpression in many tumours . in order to develop radiolabelled peptide derivatives able to target these receptors, the binding mode of the c-terminal cholecystokinin octapeptide (cck ), toward the two cholecystokinin receptors ccka-r and cckb-r has been, recently, structurally characterized. the structural data suggest that modifications on the n-terminal end of cck obtained by introducing chelating agents and their metal complexes should not affect the interaction of the derivatized cck peptides with both ccka-r and cckb-r. here we report the labelling procedures and the in vitro and in vivo characterization of new mtc cck derivatives. a stable mtc-nitrido complex is obtained by using the coordinative set formed by: ) the n-terminal amino group and the sh cystein of the cck derivative cys-gly-cck peptide; and ) a pnp aminodiphosphine used as coligand. several phosphines are used in order to define the best labelling procedures and to optimize the in vivo biodistribution properties of the mtc labelled peptides.references various combinations of pore size and chemistry of silica-based materials were investigated for high performance liquid chromatography (hplc) of peptide separation. incorrect pore size and ligands have been suggested to cause peak broadening, poor resolution and poor recovery. our study suggests that an appropriate combination of pore sizes and ligands is necessary to obtain the most efficient usage of reversed-phase hplc columns according to the molecular weights of peptides and proteins. we will also show the possibilities of an improved method development for the separation of complex peptide mixture by ph or additives.the development of new biopolymer materials as drug delivery systems is of enormous interest on biomedicine. dendrimers are polymers with particular properties; they are highly branched polymers with well-defined chemical composition, and show compact globular shape, monodisperse size and controllable surface functionalities. peptide dendrimers incorporates amino acids in their structures and have additional features such as biocompatibility and biodegradability.in previous studies we described the solid-phase synthesis of a new class of polyproline-based dendrimers. these biopolymers have the capacity to cross the mammalian cell membrane and moderate toxicity. these promising results open up the possibility to explore these dendrimers as delivery systems.to design more versatile polyproline dendrimers, we have developed a methodology that involves the combination of solid-phase and solution strategies. diverse multivalent peg-and proline-based cores were synthesized to attain dendrimers with distinct topologies. dendrimers were synthesized by iterative building block addition [(glypro ) imdoh], around an inner core, using a peptide solution convergent approach. a variety of coupling methodologies and protecting groups for the n-terminal function were explored. the novel high throughput protein detection system using designed peptide arrays has been successfully indicated on their capabilities as the "protein-chip" [ ] [ ] [ ] [ ] . our concept has many advantages especially for high-quality industrial production and practical applications compared to arrays with antibodies or recombinant proteins. the deposited peptide solution can be dried without covalent immobilization, although, when the resulted arrays are exposed in protein-solution they showed planned conformation [ ] . based on these basic results, several hundreds of α-helical, β-loop and β-sheet peptides, which involved a cysteinyl residue for covalent immobilization and tamra as a fluorescent label, were successfully synthesized, characterized and used as capture molecules. a novel material for chips made from amorphous carbon suitable for our concept has been developed, which has significant advantages over conventional glass or polymer plates, such as no selffluorescence, mechanically more stable, easy manufacturing in the aspects of precised and high throughput processing. thus, chip-plate with nano-l wells could also be easily manufactured. peptides were deposited on these chips surface covalently as well as non-covalently in picol/spot (diameter: ca µm). the resulted chips were used for protein detection. a part of this work was funded by the okinawa-bio-project and nedo-grant. key: cord- - quigar authors: nan title: posters date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: quigar no abstract is available for this article. laboratory of molecular biology and immunology, department of pharmacy, university of patras, patras, greece antimicrobial peptides (amps) are an important component of innate immune system of most living organisms. they have recently gained much attention as new anti-infective drugs with new modes of actions and few or no side effects. their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses . fish have proven to be a rich source of antimicrobial peptides. three chrysophsin peptides (chrysophsin- , - , - ) have been identified in the gills of the red sea bream, chrysophrys major, which are all bactericidal to pathogenic bacteria at low concentrations . they are cationic α-helical peptides, rich in histidine residues and all end in an unusual rrrh motif. however, in addition to its high antimicrobial potency, chrysophsins have considerable hemolytic activity. the development of new analogues which would preserve high antimicrobial potency, but would lack the undesired hemolytic activity, could be a useful tool with possible commercial and clinical applications. in the present study, we synthesized a series of analogues of chrysophsin- with different ratios of lys and leu residues, utilizing the fmoc/but solid phase methodology . the synthesized analogues were purified and isolated by rp-hplc. the antimicrobial properties of the above peptide analogues are currently testing in gram positive (s. aureus, s. epidermidis, e. faecium) and gram negative (e. coli, p. aeruginosa) bacteria. the goal is to identify the minimum bacteriostatic and bactericidal concentrations of the analogues, under conditions that simulate the best possible that of the human organism. hemolytic or cytotoxic activity of the peptides will also be determined. the rise of antibiotic resistance demands the development of new antimicrobial agents. these should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. such criteria are difficult to meet. however, cationic host defence peptides (hdps) have emerged as promising candidates. hdps target and disrupt bacterial membranes. in order to evade such a threat a bacterium would need to make substantial changes to its membrane composition disfavouring the development of resistance ( ) . however, exact role and mechanism of hpds in the regulation and monitoring of microbial invasions remain to be established. herein we will present new potential mechanisms of antimicrobial regulation by helical hdps using de novo ( ) and native systems ( ) . biophysical and microbiology aspects of the experimental designs will be discussed. the low number of the newly discovered antibiotics, emergence of multiple-drug resistance, and the alarming death rate due to the infection disease led to development the alternative means to combat the infections. the researchers accumulate information about antimicrobial drugs that could be result of the innate immunity mechanisms. armed only with the innate immunity, the insect has developed into the most widespread class in living kingdom. they produce several antimicrobial peptides with complementary and rapid mode of action. so far there are hundreds of antimicrobial peptides isolated from insect and lot of them are waiting to be discovered. the fleshly neobellieria bullata was chosen for isolation of these active compounds. its larvae in the third instar were squeezed to collect the haemolymph, which was gradually centrifuged and precipitated by acidified methanol. supernatant was subsequently separated by chromatographic methods (spe column, rp-hplc) to obtain fractions of short peptides. identification and characterization of these fractions were performed by tricine electrophoresis, mass spectrometry maldi-tof analysis and n-terminal sequencing. several fractions showed antimicrobial activity against institute of chemical kinetics and combustion, novosibirsk, russian federation in this work, we extracted d-structural information on newly synthesized, medium-length, double spin-labeled peptaibiotics using peldor spectroscopy. we investigated the magnetic dipole-dipole interactions between spin labels and the orientation selectivity effects. in particular, the medium-length peptaibiotics tylopeptin b , and heptaibin , double spin-labeled with the nitroxyl probe toac ( -amino- -oxyl- , , , -tetramethylpiperidine- -carboxylic acid), were studied by means of x-band peldor spectroscopy. this study was conducted on tylopeptin labeled at positions and (t ) and heptaibin labeled at positions and (h ) in frozen glassy methanol solutions at Κ. peldor data analysis was carried out using the theory developed for short interspin distances. the distance distribution functions between spin labels for Τ (maximum at . nm, halfwidth of . nm) and Η (maximum at . nm, half-width of . nm) were determined. the intramolecular distances observed between the labels allowed us to assign an essentially α-helical conformation to Τ and a largely prevailing -helical structure to Η under the aforementioned experimental conditions. are amidated at the c-terminus, as a result of a posttranslational enzymatic reaction. temporins are particularly active against gram-positive bacteria and are not toxic to eukaryotic cells. in this study we designed a series of analogues of tb with the aim to improve the peptide antimicrobial activity against both gram negative and gram positive strains and then to structurally elucidate the mechanism of interaction of active peptides with lps. the peptides have been synthesized substituting one or two amino acids with an alanine and lengthening the sequence with positively charged amino acids. among the designed peptides, one of the analogues, tb_kkg a, showed highly increased activity against gram negative bacteria and also a slightly increased activity against gram positive bacteria with a total lack of hemolytic activity. to develop ll- -derived short amps with prokaryotic selectivity and lipolysaccharide (lps)neutralizing activity, a series of amino acid-substituted analogs based on ig- (residues - of ll- ) were synthesized. analog a showed the highest prokaryotic selectivity, but much lower lps-neutralizing activity compared to ll- . the analogs, a , a , a and a with higher hydrophobicity displayed lps-neutralizing activity comparable to that of ll- , but much lesser prokaryotic selectivity. these results indicated that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of lps-neutralizing activity and prokaryotic selectivity. to increase lps-neutralizing activity of the analog a , we synthesized trp-substituted analogs (a -w and a -w ), in which phe or phe of a is replaced by trp. despite their same prokaryotic selectivity, a -w displayed much higher lps-neutralizing activity compared to a -w . this result suggested that the effective site for trp-substitution when designing novel amps with higher lps-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. furthermore, d-enantiomeric peptides (a -w -e and a -w -e) of a -w and a -w possessed not only more improved prokaryotic selectivity and retained lpsneutralizing activity compared to a -w but also protease stability. taken together, a -w -e and a -w -e can serve as promising templates for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. department of zoology, faculty of science, charles university, prague, czech republic antimicrobial peptides (amps) are among the most promising lead compounds for developing medicines in the fight against resistant pathogenic bacteria. we have already shown that the venom of wild bee is a rich source of pharmacologically interesting antimicrobial peptides [ ] [ ] [ ] [ ] . from the venom of solitary bee macropis fulvipes, we isolated and characterized the novel antimicrobial peptide named macropin (mac- ). by edman degradation and mass spectrometry, its primary sequence was established as gfgmalkllkkvl-nh . mac- possesses potent antimicrobial activity against both gram-positive andnegative bacteria and moderate hemolytic activity against human red blood cells. cd spectra confirmed that mac- can form an amphipathic α-helical secondary structure in the presence of membrane-mimicking substances as sodium dodecyl sulfate or organic solvents like trifluoroethanol. we prepared a series of mac- analogs to study the effect of incorporating d-amino acid residues into the sequence in various positions on antimicrobial and hemolytic activity, α-helicity and serum stability. the substitution of l-amino acid residues at n-terminal part of sequence by d-amino acid residues led to the improving hemolytic activity with maintaining or increasing antimicrobial activity. these modifications increased peptide stability in human serum. effect of the incorporation of d-amino acid residues into the mac- sequence on its α-helical structure will be discussed. the neutralization of endotoxins (lipopolysaccharide, lps) by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of gram-negative bacteria. the active endotoxic center of lps is lipid a, its lipophilic part. an effective antimicrobial peptide against gram-negative bacteria is magainin , which was originally found in the skin of an african frog. here, we studied the interaction of hexa-acyl bisphosphoryl lipid a prepared from erwinia carotovora lps with magainin with some minor substitutions in the amino acid pattern. by using fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid a, the conformation of their phosphate groups due to peptide binding, and the profile of the secondary structure of the peptides was investigated. the zeta potential of lipid a aggregates in the presence of the peptides was determined by measuring the electrophoretic mobility. small-angle x-ray scattering was performed for the elucidation of the aggregate structures in the absence and presence of the peptides, and isothermal titration calorimetry was applied for evaluating the thermodynamics of binding between peptides and lipid a. the data show that asp-or glusubstituted peptides improved the binding activity to lipid a correlated with characteristic changes in the physical parameters, which were stronger expressed for the aspsubstituted peptide. the new hydrogen bond connection between glu and asp by carboxylic acids apparently leads to a more pronounced -structure of the peptide. the conformation change of the peptide enhanced the activity of incorporation into the lipid a aggregates, along with changes in biochemical and biophysical parameters. royal college of surgeons, dublin, ireland cationic antimicrobial peptides (caps) have been reported to exhibit anticancer activity . one such peptide, p , has been shown to inhibit the growth of several cancer cell lines, with inhibiting concentration (ic ) in the range of to μm . however the concentration at which p and other caps act is too high to be clinically relevant. the enhancement of their activity can be achieved through the modification of their amino acid composition or the addition of other molecules. conjugation of naturally produced hydroxylated fatty acids to p showed a -fold improvement in its anticancer activity on a variety of human-derived cell lines. in addition to the enhancement of activity we wished to understand the mechanism of action of the peptide and conjugates. we investigated the uptake of conjugated and unconjugated peptides into hela (cervical) and miapaca (pancreatic) human cancer cells and the localisation of the peptide in the cell once taken up. we investigated the effect of altering the carbon number of the hydroxylated fatty acids ranging from hydroxyhexanoic acid (r ) to hydroxydodecanoic acid (r ) conjugated to p peptide and tested on hela and miapaca cell lines. circular dichroism studies were performed to investigate the effect on α-helical content due to amino acid composition alteration and hydroxyalkanoic acid conjugation. the effect of the position of the hydroxyl moiety on enhancement of activity was also investigated. in the current study p and its derivatives also lacked haemolytic activity with concentrations up to fold higher than ic values needed to observe any haemolysis. when current antibiotics become less efficient, there is a promise that some antibiotics can be replaced by other nature's substances, e.g. peptides. halictines are novel antimicrobial peptides isolated from the venom of the eusocial bee halictus sexcinctus. we obtained four analogues of the native peptide hal from iocb av cr. they already characterized structural properties of these peptides and their antimicrobial activity against selected bacteria . the analogues were prepared by point mutations of native peptide, which could increase antimicrobial activity and decrease undesirable hemolytic activity. our aim was to characterized membrane permeation activity of halictines through the use of a basic model of biological cells -large unilamellar phospholipid vesicles luvs. we prepared two basic types of leakage assays based on luvs with free dyes entrapped inside and one assay with laurdan content. we used classical steady state fluorescence spectroscopy and advanced fluorescence methods for study of dyes escape from luvs and we also used laurdan generalized polarization technique gp for better understanding peptide insertion into membrane. in this way we received complementary information and we can conclude that the most active peptides are the native hal and analogue hal / . however hal / requires presence of negatively charged phospholipids in membrane which may explain its higher selectivity against bacteria. furthermore, fcs results have shown that the leakage happens via pore formation. results from gp revealed that peptide insertion in the membrane do not lead directly to formation of pores. against a wide range of microorganisms, mainly by perturbing the permeability of bacterial membranes through the formation of pores. however, amps effects on membrane properties probably extend beyond poreformation. we performed a systematic spectroscopic analysis of the effects on membrane structure and dynamics of two very different amps: the cationic pmap- , which creates pores according to the "carpet" model , and alamethicin, which forms "barrel-stave" channels . by using fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we measured peptide effects on membrane fluidity and order. laurdan spectral shifts provided indications about water penetration in the bilayer. in the case of pmap- , it was possible to focus specifically on the lipids surrounding the peptide by following the membrane-probe fluorescence due to fret from the peptide trp residues. finally, peptide-induced perturbation of lateral mobility and domain formation were determined by several methods. all experiments were compared with liposome-leakage measurements: while for pmap- all membrane-perturbing effects are correlated with the vesicle leakage process, alamethicin does not significantly influence membrane dynamics at the concentrations in which it forms pores. surprisingly, in all cases the most significant peptide-induced effect is a reduction in membrane fluidity. we have reinvestigated -residue peptaibols named metanicins from an ascomycetous fungus originally described as metarhizium anisopliae strain cbs . (cbs = centraalbureau voor schimmelcultures, utrecht, the netherlands). however, due to unusually shaped conidia and based on rna-sequencing of its internal transcribed spacer (its) region, the identification of cbs . as metarhizium has been withdrawn and this particular strain is currently under taxonomic reinvestigation . sequencing of four isolated peptides by fab-ms, esi-ms and edman degradation of partial hydrolysates revealed structural relationship to -residue peptaibol antibiotics paracelsins from trichoderma reesei (=hypocrea jecorina). sequences determined are: ac-u-a-u-a-u-a(u)-q-u-v-u-g-l-u-p-v-u-u(j)-q-q-fol (exchange positions in parenthesis; ac, acetyl; u, aib, α-aminoisobutyric acid; j, d-isovaline; fol, l-upmc univ paris laboratoire des biomolécules; cnrs umr ; ens lbm; address: laboratoire des biomolécules, ens dpt de chimie, , rue lhomond f- , paris, france current data suggest that the cellular uptake of cellpenetrating peptides (cpps) occur by two processes: direct translocation across the plasma membrane and endocytosis . the large diversity of cpp sequences described in the literature (derived either from fragments of proteins, structurally constrained synthetic peptides, peptide libraries or dendrimers) has hampered the identification of general rules for their efficacy of internalisation. we have used a reductionist approach, restricting the cpp functional groups (amide and guanidinium) and tailoring cpp amphiphilic properties. two families of cpps have been designed: ) primary amphiphilic cpps corresponding to tetra-arginines functionalised with fatty acid chains of different lengths and ) secondary amphiphilic cpps containing arginine and alanine or tryptophan residues . these cpps were linked by a disulfide bridge to a peptide inhibitor of protein kinase c (pkci). the efficiencies of internalisation of the conjugates were quantified by a method based on maldi-tof mass spectrometry previously developed in our group . the mechanism of internalisation was studied by comparing the amounts of cell-surface bound and internalized pkci cargo on cho-k cells and glycosaminoglycan-deficient cho cells at o c and o c. conjugates were found to enter by both direct translocation and glycosaminoglycandependent endocytosis. in addition, the primary amphipathic cpps were found to be more efficient than the secondary amphipathic ones. furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to fda approval. therefore, modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures. vancomycin is a glycopeptide commonly used as a front line treatment for infections caused by methicillinresistant staphylococcus aureus (mrsa). the emergence of vancomycin-resistant enterococci (vre), vancomycinintermediate s. aureus (visa) and vancomycin-resistant s. aureus (vrsa) has prompted the development of semisynthetic glycopeptides . we have generated a variety of glycopeptide derivatives that show superior antibacterial activity against mrsa and vre compared to vancomycin and second generation lipoglycopeptides. this was undertaken by employing a combination of solid phase and solution phase chemistry to attach a membraneassociative element that selectively binds to bacterial membranes in preference to eukaryotic membranes, thus increasing the local concentration at the lipid ii d-ala-d-ala peptidoglycan cell wall precursor target site. three novel antimicrobial peptides, named panurgines (png), were isolated from the venom of wild bee panurgus calcaratus. one of them is dodecapeptide with sequence lnwgailkhiik-nh (png- ). the next two peptides are almost identical. these are cyclic peptides containing amino acid residues and two intramolecular disulfide bridges ldvkkiicvackixpnpackkicpk-oh (x=k png-k and x=r png-r). all peptides exhibited antimicrobial activity against gram-positive bacteria and gram-negative bacteria, antifungal activity and low haemolytic activity against human erythrocytes. we prepared analogues of α-helical amphipathic png- with the aim to improve its biological properties and a linear analogue of png-r to elucidate the importance of disulfide bridges for its activity. in the second part of the study, we followed the effect of panurgines on the degree of membrane disruption by observing the leakage of fluorescence dye (calcein) entrapped in artificial phospholipids vesicles [ ] . specifically, we investigated membrane interactions of pngs with the vesicles made from negatively charged : dopc/dppg and : dopc/dopg vesicles as a general model of bacteria membrane and : : dopc/dopg/cl as a possible model for a membrane of bacillus subtilis. the membrane interaction of pngs was also investigated on uncharged dopc vesicles as potential model membrane for erythrocytes. pngs exhibited weak dye-leakage activity for neutral vesicles, while they effectively induced dye leakage in the presence of negatively charged vesicles. these results indicate that pngs have stronger potency to disrupt bacteria-mimicking anionic membranes than those which mimic eukaryotic cell membrane. department of biochemistry and toxicology, university "lucian blaga", sibiu, romania a common tool to bias the conformation of linear peptides is the insertion of side-chain modified amino acids or sidechain/main-chain conformationally restricted building blocks. an alternative approach is a simple backbone modification. in this connection, backbone amide replacements with (almost) isosteric surrogates were extensively used. these modifications may impart resistance to enzymatic degradation and better bioavailability to the peptides, but also influence the secondary structure. a thioamide (ψ[cs-nh]) is perhaps the closest structural mimic of an amide. however, it possesses different and attractive features: (i) its nh group forms stronger hydrogen bonds, being more acidic than that of the amide. (ii) its c-n bond undergoes cis/trans isomerization by irradiation at nm (π→π* transition). (iii) it may act as a "minimalist" fluorescence quencher. for all these reasons, we started a programme aimed at exploring how the endothioamide bond affects peptide folding and bioactivity. in this communication, we describe the synthesis and conformational results of the three analogs of the membrane-active peptaibiotic trichogin ga iv listed below: n-octanoyl-aib-gly-ψ[cs-nh]-leu-aib-gly-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-ψ[cs-nh]-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-gly-leu-aib-gly-ψ[cs-nh]-ile-leu-ome ( / ) the syntheses of the three peptides were accomplished in solution according to a fragment condensation approach. appropriate thioamide-containing tri-or tetrapeptides were prepared by treating the corresponding all-amide precursors with the lawesson reagent. ft-ir absorption, d-nmr and cd conformational investigations on the three analogs were conducted in comparison with the naturally occurring peptaibiotic. all three analogs maintain the capability to interact with the dope/dopg model phospholipid membranes and exhibit a comparable bioactivity against s. aureus. peptide-peptide interaction of lactococcin g class iib two peptide bacteriocin h. etayash, w.soliman and k. kaur* faculty of pharmacy and pharmaceutical sciences, university of alberta, edmonton, alberta, t g e lactococcin g, a class iib two-peptide bacteriocin, consists of two complementary peptides lcng-α and lcng-β that act as one functional unit with optimal antimicrobial activity achieved by the presence of both peptides in approximately equal amounts. in this study we have investigated the mechanism of pairing of the two complementary peptides as well as explored any specific interaction that could take place between the peptides. molecular dynamics (md) simulation was employed to study the interactions at the atomistic level. four different md simulations with the peptides in a lipid bilayer system were conducted. md results from these simulations confirmed and pointed out that (i) the two putative gxxxg motif, g xxxg in lcng-α and g xxxg in lcng-β, were attracted and came closer to each other, showing the role of these motifs in attracting the two peptides to each other. closer views, however, showed no clear interactions between these two motifs. most likely, nonspecific interactions play a role in bringing the two peptides together; (ii) variations and loss in the secondary structure in both the peptide fragments were confirmed among the four simulations. on the contrary, stability of helical regions was identified between residues w -g and d -q in lcng-α and v -e in lcng-β; and (iii) role of tryptophan at the n-terminal regions in positioning and setting the peptide orientations were confirmed which matched the previous reported results. faculty of pharmaceutical sciences, unesp -univ. estadual paulista, araraquara, sa͂ o paulo, brazil antibiotic resistant bacterial strains represent a global health problem. antimicrobial peptides (amps) are promising novel antibiotics because they have displayed little or no resistance effects. it is well known that the charge, amphipathicity, hydrophobicity and helicity of the peptide are fundamental for the biological activity. in addition, covalent dimerization appears as a new parameter to be studied. in this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages like enhanced antimicrobial activity, solubility and proteases resistant. however, the effect of this modification is unclear since dimeric versions of some amps are toxic . to evaluate the effects of dimerization on the structure and biological activity of the amp aurein . , the monomeric version (au) and the c-and n-terminal dimers, (au) k and e(au) , respectively, were synthesized. circular dichroism results indicated that dimeric versions showed more defined structures in aqueous solution. e(au) showed "coiled coil" structure while (au) k an αhelix structure. in contrast, au displayed typical spectra for disordered structures. in tfe and lpc, all the peptides acquired a high amount of α-helix structure. the antimicrobial activity against bacteria and yeast decreased with dimerization. however, dimeric peptides promoted the aggregation of c. albicans. hemolytic and vesicle permeabilization assays showed that au has a concentration dependence activity, an effect that can be assigned to a "carpet" like mechanism peptide, whereas this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action. in conclusion, our studies showed that the effects of amp dimerization are complex and still unclear. , the first antimicrobial peptide generated in vivo and isolated from the gut contents of the cattle tick boophilus microplus . we have shown that these peptides are equally lethal to candida albicans mdm and practically not active on human erythrocytes . to examine the properties and mode of action of hb - a, we synthesized it and its fluorescently labeled analogue (fam-hb - a) by the solid-phase method at o c, purified them by rp-hplc and characterized their purified forms by lc-esims. at low salt concentration, both peptides were found to inhibit the growth of candida albicans atcc , candida parapsilosis atcc and candida krusei atcc , but hb - a was two-fold more active (mics of . ; . and . μm, respectively). at those concentrations, both peptides also kill the fungi. assays with human erythrocytes showed that, likewise hb - a, fam-hb - a present activity lower than % at μm. apparently, hb - a targets the membrane cell because confocal microscopy analysis revealed that, at the half of mic value, fam-hb - accumulates on the fungal cell membrane. in contrast, fluorescence activated cell sorting (facs) analysis revealed that, at the mic, more than % of the fam-hb - a penetrates into the cell. membrane permeability assay using hb - a, c. albicans atcc and the kit live/dead funga light confirmed progressive membrane damage associated with an increase in peptide concentrations. the use dibac ( ) and facs analysis showed that hb - a alters the plasma membrane potential, leading to cell death. supported by fapesp, cnpq and capes. lasso peptides form a growing class of to residue ribosomally-synthesized and post-translationally modified peptides produced by bacteria. they share a rigid and compact interlocked structure consisting of a macrolactam ring at the n-terminus and a c-terminal tail that is looped back and threaded through the ring, forming a typical [ ] rotaxane , . the macrolactam is formed by condensation of an asp or glu side-chain with the free amino group of a gly or cys . the lasso fold is stabilized either by steric hindrance assumed by bulky amino acid side-chains and/or by disulfide bonds between cysteines from the tail and the ring. given this structure, lasso peptides display a high stability against proteolytic and chemical degradation. they are biologically active on various enzymatic targets, which confer them in some cases an interesting antimicrobial activity. given its characteristics, the lasso scaffold thus represents a promising tool for biotechnological application in the development of bioactive peptides. until now, nine peptides had been structurally characterized as lasso peptides. based on a genomics-based approach, we identified a novel lasso peptide from streptomyces sviceus that we termed sviceucin. it was produced in high yield by heterologous expression in s. coelicolor and submitted to structural analysis by mass spectrometry and nmr. sviceucin is residue long and stabilized by two disulphide bonds. their connectivities were identified mainly from the typical noes between the beta-protons of the cysteines. the lasso structure of sviceucin was obtained by nmr-based molecular modelling. sviceucin was shown to exhibit antibacterial activity directed against gram positive bacteria, while gram-negative bacteria and fungi showed resistant. the penicillium chrysogerum antifungal protein (paf) is a cysteine-rich, cationic protein that inhibits the growth of a variety of filamentous fungi without toxic effect on mammalian cells . although paf is used to be produced in p. chrysogerum or a similar microorganism, preparation of analogues of the protein for structural and functional investigations requires an efficient chemical method. the unsuccessful continuous synthesis of the -mer small protein prompted us to use native chemical ligation . the syntheses of the fragments were performed by solid-phase method applying tboc chemistry. using the acid-labile tboc protecting group, the thioester end of the n-terminal fragment remains intact during the course of the synthesis. the first attempt was the synthesis of peptides with pmethylbenzyl groups on the side chains of all of the six cysteine residues. under no circumstances oxidative folding provided the natural disulphide bridge pattern. the failed attempts led us to orthogonal protection of the sulphydryl groups. different sets of protecting groups were tried and evaluated. our experiments showed that basic treatment triggered rearrangement of the previously formed disulphide pattern. thus, base-labile protecting groups (such as -fluorenylmethyl, fm) have to be avoided in the synthesis of paf. the alarming increase and spread of antibiotic resistance among bacterial pathogens has stimulated the development of new antibacterial agents with innovative mode of action. antimicrobial peptides with broad spectrum activity are widely distributed in nature and play an important role in innate immunity in several species, including humans. tigerinins are a unique family of -to -residue antimicrobial peptides found in skin secretion of the indian frog rana tigerina , . characterized by a disulfide-bridged loop composed of nine amino acids, tigerinins do not show primary structural homology to any known antimicrobial peptides from amphibians. tigerinins could provide novel lead compounds for the design of effective antimicrobial peptides with a new mode of action. the peptide murdp has been identified after the screening of phage display libraries against pseudomonas aeruginosa cell wall biosynthesis murd amide ligase enzyme . murdp is a low micromolar range inhibitor of murd enzyme and showed good antimicrobial activity. composed of nine amino acids, it is also characterized by a nine residues disulfide-bridged loop containing two prolines. this great similarity with tigerinins, led us to investigate if murd enzyme could be a potential target for these peptides. in silico analyses using modelling, molecular dynamics and docking with p. aeruginosa murd showed that murdp and tigerinin- and - make similar interactions in the binding site. these results suggest that murd may be an intracellular target for tigerinin- and tigerinin- . synthesis, murd enzymatic inhibition assay, antibacterial activity evaluation and structure-activity relationships of murdp and tigerinins analogs will be presented. h. etayash, l. norman, t. thundat*, k. kaur* faculty of pharmacy and pharmaceutical sciences, department of chemical and materials engineering, university of alberta, edmonton, alberta t g e , canada listeria monocytogenes is a gram positive bacterium that accounts for about % of the deaths resulting from food borne illnesses in north america. moreover, l. monocytogenes is considered one of the most difficult bacteria to detect in contaminated food products. while standard microbiological and biochemical assays currently used are accurate and sensitive, they are time consuming and often require specialized instruments operated by a trained user making on-site testing difficult. to this end, we propose the development of an antimicrobial peptide (amp) or peptide fragment sensor for the on-site detection of l. monocytogenes. leucocin a, which is a naturally occurring amp consisting of a amino acid sequence, is known to exhibit specific activity against l. monocytogenes at pico to nanomolar concentrations. for this reason, we have synthesized a shorter peptide fragment of leucocin a consisting of amino acids using solid phase peptide synthesis. the peptide was purified by reversed phase hplc and maldi-tof mass spectrometry indicates the desired biological entity was achieved. by including an n-terminal cysteine group, the tailored amp was readily immobilized at a gold interface. the resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicate that the helical peptides were adsorbed on the interface with a preferred orientation parallel to the surface. the bacterial specificity of the anchored leucocin a fragment was tested against three gram positive bacteria and results reveal that the adsorbed amp exhibits a limit of detection of approximately one bacterium/μl which is a clinically useful detection range. faculty of science, university of south bohemia, České budějovice, czech republic during the last few years we have identified three novel defensins from arthropods. two of them, lucifensin and lucifensin ii were purified from various tissues of lucilia sericata and l. cuprina larvae, respectively. larvae of these flies are routinely used in the hospitals around the world for the treatment of non-healing infected wounds in the procedure known as maggot therapy. these amino acid residues and three disulfide bridges peptides differ from each other only in one amino acid residue in position (val-ile). linear precursor of lucifensin was prepared by fmoc-spps chemistry which was then subjected to the oxidative folding yielding a peptide with a pattern of disulfide bridges identical to that of native lucifensin and other insect defensins. this was examined by the identification of the fragments resulting from the thermolysin digestion of lucifensin by means of mass spectrometry. however, this cyclization reaction proceeded via an intermediate having incorrect pairing of disulfide bridges. from the hemolymph of blood sucking tick dermacentor marginatus (d.m.) we purified defensin containing amino acids and three disulfide bridges. its sequence determined by edman degradation and mass spectrometry was identical to that previously determined by molecular biology methods . sequence of d.m.defensin shows no homology to insect defensins. by spps prepared linear precursor of d.m.-defensin was subjected to oxidative folding under the open air. the linear peptide was straightforwardly folded into cyclic one which was identical to the native peptide. in antimicrobial assay using a set of different bacteria all three studied defensins show activity preferentially against gram-positive bacteria including staphylococcus aureus but are inactive against gram-negative ones. the importance of disulfide bridges on tertiary structure of defensins and their antimicrobial activity will be presented. recently, the chemical structure and conformation of pseudodesmin a has been determined through x-ray diffraction and nmr spectroscopic analysis . in this way pseudodesmin a was identified as a new member of the viscosin group of antimicrobial peptides (amps). in addition, it was demonstrated that individual molecules self-assembly in apolar environment into a supramolecular pore-like structure, providing structural support for its biological activity , . to further explore the structure-function relationship, a viable synthesis strategy for pseudodesmin a analogues was developed, based on side-chain attachment of the first amino acid to the solid support, followed by stepwise fmoc solid-phase synthesis of the linear peptide precursor and on-resin head-to-tail cyclization. nmr study confirmed the molecular structure and thus the development of an efficient and successful synthesis of this type of amp's. these results and the synthesis route will be presented. trichogin ga iv, isolated from the fungus trichoderma longibrachiatum , is the prototype of lipopeptaibols, a subclass of short-length peptaibiotics exhibiting membranemodifying properties. its primary structure is as follows: n-oct-aib -gly-leu-aib-gly-gly-leu-aib-gly-ile -lol, where n-oct is n-octanoyl, aib is α-aminoisobutyric acid, and lol is the , -amino alcohol leucinol. this peptaibol is predominantly folded in a mixed -/αhelical conformation with a clear, albeit modest, amphiphilic character . in this work, we synthesized by solution and solid-phase methodologies a set of trichogin ga iv analogs in which the four gly residues, lying on the poorly hydrophilic face of the helical structure, are substituted by one (or more) strongly hydrophilic lys residues. moreover, we synthesized another set of analogs where one (or more) aib residues are replaced by leu. the conformational preferences of these analogs were assessed by x-ray diffraction, cd, and d-nmr techniques . we tested the role played by the substitutions on the peptide bioactivity, e.g. protease resistance, cytotoxicity, and hemolysis. cytotoxicity was tested using three in vitro cell-based assays: (i) human red-blood cells lysis; (ii) cell mortality in total human blood leukocytes and in separate subpopulations; (iii) cell mortality in three tumor-derived stable cell lines (hela, a , and a ). our data show that some of our trichogin analogs are active against tumor cells, leaving the leukocytes unaffected. a convenient post-screening ring opening approach for the decoding of one-bead one-compound cyclic peptide libraries a. girard, e. biron* faculty of pharmacy, université laval and chuq research center, quebec, canada combinatorial chemistry has been widely used as an effective method for the generation and screening of synthetic peptide libraries. amongst the different combinatorial methodologies to discover new bioactive peptide-based compounds, we were particularly interested in the one-bead one-compound (oboc) approach . this powerful approach fully exploit the great molecular diversity accessible with peptides and has been used to identify a great number of ligands and modulators for a wide variety of biological targets. however, its use with cyclic peptides is limited by difficulties in sequencing hit compounds by edman degradation or tandem mass spectroscopy due to the lack of free n-terminal amine and complicated fragmentation patterns, respectively. this problem has been overcome by pei and coworkers by using a bead segregation strategy in which the outer layer exposes the cyclic peptides and the inner layer the linear counterpart for sequencing . more recently, lim et al. reported an elegant method to prepare and sequence oboc cyclic peptoid libraries without encoding by using a ring opening approach with triazine-based cyclic derivatives . unfortunately this method is incompatible with amino acids bearing some functionalized side chains. based on this strategy, we have developed an efficient method to prepare oboc cyclic peptide libraries that does not require encoding by using a simultaneous ring opening/cleavage approach. the procedure is compatible with commonly used amino acids and allows rapid and efficient sequencing of selected hits after on-bead screening. the synthesis of an oboc cyclic peptide library, ring opening methodology and sequencing by mass spectrometry will be presented. cyclotides are a very abundant class of plant peptides that display immense sequence variability around a conserved cystine knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability . their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals . however, laborious isolation and purification prior de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development . here we extend the knowledge about their sequence diversity by analyzing the cyclotide content of a violet species native to western asia and the caucasus region . using an experimental approach, which we named 'sequence fragment assembly' by maldi-tof/tof-based peptidomics, we were able to characterize novel cyclotides from viola ignobilis. amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analyzing mixtures containing multiple peptides. using in-source decay and high energy collision induced dissociation of digested cyclotides allowed to distinguish isobaric residues ile and leu. overall this work underlines the immense structural diversity and plasticity of the unique cyclotide framework. the presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides. glycation is a nonenzymatic reaction occurring between reducing sugars and reactive amino groups of biomolecules. the reaction leads to a formation of a heterogeneous mixture of compounds which are classified as early, intermediate or advanced glycation end products (age). these compounds, especially advanced glycation end products, are involved in many pathological processes, mainly diabetic complications, and could be markers of certain diseases. detection of early products of glycation (amadori products) is a relatively easy task and can be performed by various methods including e.g. ms/ms techniques, isotopic labeling and affinity chromatography on immobilized boronic acid , . however, the diverse structures of ages make detection of these compounds more challenging. the aim of the study was testing a new method of ages identification based on isotopic c labeling. a model protein (hen egg lysozyme) was modified with an equimolar mixture of [ c ]glc and [ c ]glc. then the glycated protein was subjected to reduction of the disulfide bridges followed by enzymatic hydrolysis. the obtained digest was analyzed by lc-ms methods. the glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. this method allowed for identification of early maillard reaction products and different structures of the glycation end products. isotopic labeling technique combined with lc-ms is a new and very sensitive method for identification of the advanced glycation end products even if their structures are unknown. this method could be also used as an alternative method of detection of amadori products. in the course of a project aimed to assess the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus hypocrea phellinicola growing on its natural host phellinus ferruginosus . using a peptaibiomics approach , , we detected -and -residue peptide sequences by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of an agar plate culture of h. phellinicola cbs (ex-type) grown under laboratory conditions. notably, h. phellinicola could be identified as a potent producer of -, -, (culture) and -residue (specimen) peptaibiotics of the suzukacillin-type . minor components of the -residue peptaibols, herein named suzukacillins c, are assumed to carry a c-terminal residue tentatively assigned as tyrosinol (tyrol). in addition, the previously isolated suzukacillin b was sequenced and shown to be a microheterogeneous mixture of -residue peptaibols. in order to further investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the fungicolous fungus hypocrea pulvinata growing on its natural hosts piptoporus betulinus and fomitopsis pinicola . using a peptaibiomics approach , we detected -, -, -(major sequences), and -residue peptide sequences in the five specimens analyzed by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens . major, -residue peptaibols were assigned as deletion sequences of the trichosporins b lacking the ala/aib residue in position . our results corroborate that: i) peptaibiotics are, indeed, biosynthesized in the natural habitat, thus, ii) their membrane-perturbing formation of ion channels may support the parasitic life style of a fungicolous fungus. based on methodology that we have developed in our lab , we identified specific and selective substrates for these serine proteases. we used a , membered pnaencoded peptide library to screen , possible peptide substrates in a single experiment. the library was incubated with the protease of interest and then hybridized on a custom designed dna microarray. microarray scanning and data analysis allowed the measurement of the changes in fam/tamra ratios resulting from the protease activity and the determination of the protease specificity. to verify the predicted activity and specificity, fret peptides were synthesized, incubated with the enzymes and the hydrolysis reaction was followed by monitoring fluorescence emission. specificity constants kcat/km were calculated and the cleavage sites of the peptides were identified. dubs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets . several directions have been pursued in the search for lead anti-dub compounds. however, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay . in this study, we report on the design and preparation of a fret-based assay for dubs based on the application of our recent chemical method for the synthesis of ub bioconjugates . in the assay, the ubiquitinated peptide was specifically labeled with a pair of fret labels and used to screen a library comprising compounds against uch-l . such analysis identified a novel and potent inhibitor able to inhibit this dub in time-dependent manner with kinact = . μm and ki = . μm. our assay, which was also found suitable for the uch-l enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of dubs in health and disease. . more recent work based on rna interference experiments on a mouse model suggested that isoform-specific inhibitors against nmt might be effective anti-cancer agents as a knockdown of nmt inhibits the tumour growth, whereas knockdown of nmt has no effect . if residual nmt activity can compensate for loss of nmt function in healthy cells, potential toxicity may also be minimised. we developed a method to identify peptide or protein substrates of nmt and/or nmt . peptides/ proteins are exposed to nmt and/or nmt and an alkyne-tagged analogue of myristoyl coa. subsequent azide-alkyne "click" cycloaddition allows visualisation of the myristoylated substrates in fluorescence or chemiluminescence, using a fluorescent or a biotin moiety on the capture reagent. this labelling technology was applied to peptide libraries prepared on microarrays to investigate nmt / isozyme substrate specificity using recombinant nmt and nmt . peptides made of the first or amino acids at the nterminus of known myristoylated proteins were functionalised with a biotin moiety at the c-terminus and immobilised on an avidin-functionalised glass plate before being screened for activity. selective peptide substrates will be developed as isozyme-specific inhibitors and applied in cancer cell lines. using chemical proteomics and the labelling technology, a selective nmt or nmt inhibitor could also be used to identify protein substrates of one isozyme. for this purpose computer programs are created which can generate fragments of one compared structure and to reveal homology by their scanning along the amino acid sequence of another. our analysis was performed by comparing the primary structures of all possible protein fragments with the amino acid sequences of all presently known natural regulatory oligopeptides. the oligopeptides were extracted from the erop-moscow database which at the time of analysis contained data on the structures and functions of more than , natural oligopeptide regulators. the structure-function analysis was performed using a specialized software package. the input data were the complete amino acid sequences of the proteins used as a source of fragments with a specified length. then the initial sequence was fragmented in a stepwise manner. for example, in the case of dipeptide fragments, this procedure produced fragments with the following numbers of amino acids from the n-terminus - - , - , and so on until the fragment that started at the second residue from the c-terminus. the cases when the amino acid sequence of a fragment coincided with part of the primary structure of a natural oligopeptide were recorded in the total protein chemical synthesis requires a case by case design and optimization which is governed by factors such as the solubility of the individual peptide segments, their primary sequence and in particular the presence of "difficult" amino acid residues at ligation junctions such as proline or the location of cysteines. usually, a subset of chemical tools are selected among a vast array of methodologies to match the specificities of the target protein. in this context, methods enabling the assembly of three peptide segments in the n-to-c and c-to-n direction play a central role and must be considered as complementary as they can be selected for building subdomains of the target protein. to date, most of the proteins were assembled in the c-to-n direction. only few methods are available for the n-to-c sequential assembly of proteins, whose design is highly challenging. we have recently reported that sea ligation, that is the reaction of a bis( -sulfanylethyl)amido group (called sea) with a cysteinyl peptide, allows the formation of a native peptide bond in water and at neutral ph . in this communication we will show that native chemical ligation and the unique chemical properties of sea group , can be combined in order to design a highly efficient one-pot three segments protein assembly procedure, working in the n-to-c direction amylin is one of the most amyloidogenic peptides, its fibrils are responsible for causing type ii diabetes. amyloid formation mechanism is investigated both to find amyloid inhibitors as potential medical drugs, and to use amyloids as potential self-assembling biomaterials [ ] . amyloid formation of amylin - , its reverse and designed analogue beta-sheets and beta-sheet stacks was studied by molecular dynamics (md), amber . , f force field. md revealed that for amylin - and its reverse analogue both the parallel and antiparallel beta-sheet and beta-sheet stack structures are stable suggesting that this could explain the high tendency of amylin to form amyloid fibrils. parallel amylin - beta-sheet stacks are kept together by two hydrophobic cores, while for the antiparallel system the dominating is the backbone hydrogen bonding between neighbor strands. also the bent form of the amylin - beta-sheet is stable. this is in concordance with transmission electron microscopy (tem) experiments stating that all three peptides, amylin - , its reverse and designed analogues, exhibited significant fibrillar polymorphism [ ] university of gdansk, poland molecular dynamics (md) of two peptides dlsfmkge (mk) and dlsfkkge (kk) not related to any known disease was run to investigate the mechanism of the amyloid formation. the parallel and antiparallel [ ] betasheets of mk and kk peptides were simulated by molecular dynamics (md), amber . , f force field, ntp protocol. it was found that antiparallel beta-sheets both of mk-and kk-peptides show much higher stability than the corresponding parallel beta-sheets. this md result was supported by atr-ftir spectroscopy [ ] . the betasheet stacks built from six ten stranded antiparallel beta-sheets of mk-and kk-peptides: x xmk and x xkk, were subjected to md. it was found that the mk-system, x xmk, is strongly kept together due to hydrophobic core built from two metionines, two phenylalanines and two leucines, but the kk-system, x xkk, which differs only by one mutation m k dissolves already at ns of md run, because the separate beta-sheets don't hold togather in the betasheet stack due to lost hydrophobic core. the hydrophobic core of the mk-system consists of hydrophobic units centered on the two phenylalaninetwo metionine hydrophobic interactions, and two leucines from the both sides stabilize the unit. this mechanism could be used in amyloid based biomaterials. urokinase plasminogen activator (upa) is a serine protease involved in the metastasis of several tumor types. upa is therefore an interesting target in cancer therapy. upain- is a new analogue of a highly specific peptidic inhibitor (upain- ) of upa. the peptide contains twelve amino acids and is cyclized through the cysteines at its termini (s -s cyclo-ac-cswrglenhaac-nh ). upain- inhibits upa with a ki of approximately μm. one method to improve binding affinity is multivalent exposure of the inhibitor, where the local concentration at the binding site is increased. fusion of upain- to the trimeric tetranectin showed improved binding affinity compared to the single peptide. here, we report efforts towards novel chemically linked upain- peptides to allow multivalent display. the ki value of an upain- dimer, linked by a short peg chain through the n-termini, was almost halved compared to that of the single peptide ( μm). this motivated us to explore the role of the site (n-or cterminal) and the size of the linking segment on the binding affinity. additionally, the influence of the number of upain- peptides in the molecule (two vs. four) was investigated by synthesizing a carboprotein that displayed four upain- peptides. we present two novel nmr spectroscopic approaches to study reversible self-assemblies in solution. both methods were applied on the self-assembling pseudodesmin a, a pseudomonas produced cyclic lipodepsipeptide that has the capacity to form pores in cellular membranes. , the first method is based on the dependence of the c α relaxation rate constants on the anisotropy of the assembly. when the monomer conformation is known and the multiple ch bonds in the monomer sufficiently sample all orientations, the rotational diffusion coefficients can be assessed, revealing assembly shape information. in addition, the orientation of the monomer within the assembly is obtained. the second method is based on fitting translational diffusion coefficient data as a function of concentration in a model-free way, i.e. without assuming an oligomer shape beforehand. here, it is assumed that the diffusion coefficient's dependence on the oligomer size behaves as a power law, which dramatically simplifies the expression for the average diffusion coefficient (measured by pfg-nmr) as a function of concentration. the fitted value of the exponent of the power law fully embeds all shape information of the assembly, and may be related to the socalled fractal dimension of the oligomer. moreover, this approach reveals mechanistic information concerning the assembly formation. both methods thus allow structural information of the assembly to be obtained, even when there is little or no prior knowledge available on the mechanism of the selfassembly. nucleotides and α-amino acids are crucial building blocks for living organisms. these chiral molecules are the biosynthetically precursors of two of the most important classes of biopolymers, dna and proteins, respectively. the d-structures of biomolecules are currently studied using a variety of techniques, while helical handedness is routinely detected by means of light pulses of opposite circular polarization. the difference in the uv absorption of these two circularly polarized pulses is called electronic circular dichroism (ecd). in nature, biomolecules explore a wide range of conformations with intrinsically strong ecd signals in the - nm region, but these signals are essentially absent in the visible. nanomaterials such as metallic nanoparticles (depending on their sizes) display absorptions in the visible region but are achiral. as a result, when biomolecules are co-assembled with nanomaterials their chirality is transferred to create a plasmon-induced ecd signal in the visible region. in this work, we present our results which underscore the occurrence of moderately strong ecd bands in the range - nm resulting from a series of appropriately thiolfunctionalized peptide oligomers (based on alternating l-ala and aib residues) covalently anchored to - . nm sized gold nanoparticles. we related the (positive or negative) signs of the ecd plasmonic signal with the oligopeptide length, that in turn is strictly associated with their secondary structure. this latter property was simultaneously monitored via ecd in the - nm range. we believe that in our systems a peptide-tometallic surface chirality transfer would take place. light can be controlled with high temporal and spatial precision. if a specific molecule is made light-sensitive, then a precise spatiotemporal control of some of its properties can be achieved. azobenzene is the most widely used photochromic group due to its propensity to pass reversibly from the cis to the trans state under irradiation with light of the appropriate wavelength. the cisand transazobenzene isomers exhibit different spatial arrangement of the aromatic moieties that give rise to significantly distinct physical and chemical properties. the design of novel azobenzene-based molecules with precisely placed photochromic groups able to induce photomodulation of macroscopic properties is currently attracting much interest. in this work, we explored the behaviour of the conjugate formed by linking each of the four hydroxyl groups of pentaerythritol to the carboxylic function of bis[p-(phenylazo)benzyl]glycine. this c α -tetrasubstituted α-amino acid bears two side-chain azobenzene groups. the resulting system exhibits tetragonal symmetry, with a total of eight azobenzene moieties, and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. up to eleven (out of the possible fifteen) discrete states produced by sequential trans-to-cis isomerization of the individual azobenzene units have been observed depending on the time of exposure to uv-light. this process is fully reversible (cis-to-trans) under vis-light irradiation for several cycles. in addition, this compound has been shown to exhibit photomodulated physical properties, such as polarity and hydrodynamic volume. moreover, it shows a high propensity to self-assemble in aqueous solution, giving rise to supramolecular vesicles. light-scattering and electron microscopy experiments confirmed that a conformational reorganization of the vesicles can be triggered under exposure to uv or vis light. the total chemical synthesis of native or modified proteins is gaining increase importance in the study of protein function, but also in the development of protein therapeutics. it is usually achieved by assembling in water unprotected peptide blocks using so-called native peptide ligation methods. recently, our group has developed a novel native peptide ligation method based on a peptide featuring a bis( sulfanylethyl)amido (sea) group on its c-terminus in reaction with a cysteinyl peptide in water at ph . we will discuss in this communication the scope and limitations of sea native peptide ligation. for this, model sea peptides featuring all the possible proteinogenic amino acids were synthesized. their rate of sea native peptide ligation with a model cys peptide were determined in the absence of presence of guanidinium hydrochloride or other additives frequently used in ncl. we will present also experiments intended to clarify the mechanism of sea ligation such as the effect of ph on the rate of ligation, or the ability of the transient thioester sea form produced by in situ n,s-acyl shift to participate in thiol-thioester exchange , . overall, the data show that sea ligation is an interesting method for native peptide ligation at various x-cys junctions, and thus an interesting alternative to ncl. plga copolymers were used as the support for inducing controlled biomarkers releasing system. visualization of the penetration in the hippocampus of mice with confocal microscope was carried out by testing both peptide-free and peptide-bearing nanoparticles, previously labeled with the phthalocyanine fluorescent probe. the encapsulation degree of the larger ( - ) segment was less effective than the others thus stressing the importance of the peptide length to this internalization process. the results showed that all peptide-containing nanoparticles were able to cross the blood-brain-barrier thus indicating improved bioavailability and uptake for peptide delivery into the brain. in regard to the radiolabeling approach, the m tc radioisotope was used to label the peptide sequences at his residues, as previously described . stable metal-peptide complexes were obtained in - - - m peptide concentration range. noteworthy, higher metal labeling yield was achieved with peptide segments bearing his residues at peptide c-terminal position, thus pointing to a positiondependent effect for the m tc coupling reaction. in conclusion, the findings indicate potentials for the proposed encapsulation and radiolabeling strategies applicable for in vitro and in vivo diagnostic assays with these peptides for the study of amyloid plaques. we have used bifunctional short peptides (ac-cg n c-nh , n= , , ) to selectively link gold nanorods in an end-to-end manner. additionally, we have manipulated the gap distance between the rods by changing the length of the peptide linker. the presence of the peptide in the gaps was shown by incorporating a propargylglycine residue in the sequence, which was detected with surface-enhanced raman spectroscopy (sers). the acetylene moiety will allow further chemical modification of the linker in the gaps, opening a wealth of interesting molecular systems to be placed and studies inside self-assembled nanogaps. in this work, the fragmentation pathways of alitame, neotame and andvantame in comparison to those of aspartame and aspartame-d , were studied by negative ion electrospray ionization (esi) high resolution mass spectrometry (thermo orbitrap mass analyzer). accurate mass spectra of the dipeptides allowed proposing specific fragment ions. neotame and advantame, which are the n-( , dimethylbutyl) and n-[ -( -hydroxy- -methoxyphenyl)propyl] derivatives of aspartame, presented similar fragmentation to that of aspartame. for neotame and advantame, the "diketopiperazine'' pathway seemed to be the major one, while a pathway resulting to the formation of a pyrrolidine- , -dione derivative, through the involvement of the side chain carboxyl group of aspartate, was also observed. for alitame, the "pyrrolidine- , -dione" pathway was recorded. similarities in the fragmentation using either orbitrap or triple-quadrupole mass spectrometry have been observed. elucidation of the fragmentation is very useful for the trace-level determination of the artificial dipeptide sweeteners in complex matrices. generation of silver nanoparticles in the presence of oligoproline derivatives p. feinäugle, h. wennemers* eth zürich, switzerland in the last years, the generation of silver nanoparticles (agnps) attracts, due to its unusual physical and chemical properties, more and more attention. agnps offer great opportunities for applications in molecular electronics, catalysis, imaging and for antimicrobial coatings. the characteristics depend on their shape and size. many efforts have been made to optimise the generation process by, for example, varying the reducing agents, which usually are used for the synthesis or using manifold additives which should guide the nucleation and also stabilize the resulting particles. nevertheless, the generation of agnps in defined sizes and shapes still remains a challenge. we address this goal by utilizing functionalized oligoprolines that form a conformationally well-defined and rigid helical secondary structure (ppii) as additives. recently, we showed that by decorating this template with aldehydes which allow for in situ reduction of the silver, they act as scaffolds in the generation process and allow the formation of defined nanoparticles. we will report the results of the generation of agnps with various oligoprolines as additives, which differ in the attached functional groups as well as in the length of the peptides. laser desorption/ionization mass spectrometry (ldi-ms) using specific inert surfaces to promote ion formation has been widely investigated the last decade [ ] . in addition to porous silicon through the original dios technique, different materials were tested as potent ldi-promoting agents. we explored a variety of inert silicon-based uvabsorbing materials that were presenting different physico-chemical properties for the analysis of peptides [ ] [ ] [ ] . both material architecture (amorphous powders, structured particles, structured surfaces) and material hydrophilic/hydrophobic character tuned by specific chemical derivatization (oxidation, silanization) were probed as crucial parameters for achieving efficient and robust detection of an home-made array of model peptides covering a wide structural and mass diversity. through this set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking into account peptide detection sensitivity (down to femtomolar concentrations) and reproducibility/repeatability (intra-spot/inter-spot signal variations) as well as the method robustness using conventional maldi-tof/tof instrument. having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in the same run, high-throughput identification of protein tryptic digests by a rapid ms profiling and subsequent ms/ms analyses was achieved. comparison of the ms and ms/ms data with those obtained with sample conditioned in organic matrix [ , ] showed a great behavior for low mass responses demonstrating the capability of ldi on nanostructured silicon supports to be a complementary method to maldi in proteomic workflow. the dipeptides aspartame, alitame, neotame and advantame are low caloric artificial sweeteners. advantame , which is the n-[ -( -hydroxy- -methoxyphenyl)propyl] derivative of aspartame, is the most recent among them. an application for its approval has been applied in usa, australia and new zealand. such sweeteners are used in food products and beverages and they can help in managing body weight and disorders like obesity and diabetes. in this work, the simultaneous determination of aspartame, alitame, neotame and advantame by negative and positive electrospray ionization (esi), under hydrophilic interaction chromatography (hilic), is presented. advantame, neotame and intermediates were synthesized in our laboratories for the present application. the key-step for the synthesis of advantame and neotame was the reductive amination of h-asp(obu t )-phe-ome with -( -hydroxy- methoxyphenyl)propanal and , -dimethylbutanal, respectively. the chromatographic behavior of the artificial sweetener dipeptides was studied on two hilic columns: kinetex hilic (a fused core silica column) and zic-hilic column (a sulfoalkylbetaine column). the separation of dipeptides was achieved on kinetex hilic using mm ammonium formate buffer ph . / methanol / acetonitrile ( / / ), with a flow rate of μl/min at o c column oven temperature. at this ph, silica is neutral and the dipeptides are in positively charged form. the retention mechanism of all analytes seems to be partition to the water layer as well as hydrogen bonding. département de pharmacologie, université de sherbrooke, sherbrooke, qc, canada plasma and in vivo stability are essential requirements for the successful development of potential drug candidates or diagnostic imaging probes. rapid degradation of compounds in plasma may result in insufficient concentration to produce the desired pharmacological activity or to be used as a diagnostic agent. there are several strategies to improve plasma half life of peptides including pegylation, modification of nand cterminal fragments of peptide, replacement of labile amino acids, and cyclization . we have previously reported on probes which specifically detect matrix metalloproteinase- (mmp- ) activity with magnetic resonance and optical imaging , . mmps are zincdependent endopeptidases degrading the extracellular matrix (ecm) and involved in cancer progression. the main goal of this work was to find more stable probes without sacrificing enzyme specificity. we have selected specific mmp- substrates and their stability was evaluated in three different conditions: in plasma, in plasma with a mmp inhibitor and in a mmp- solution. the samples were analyzed by hplc to detect the degradation pattern of our compounds and by lc-ms to determine the molecular mass of peptide fragments. based on these studies, the most stable peptide was selected and incorporated in a solubility switchable probe with radiolabelled ( )ga-dota. its in vivo stability was estimated up to minutes, making it a suitable candidate for further investigations. cancer of thyroid gland is the most common malignancy of the endocrine system. the treatment improvement could be achieved by early diagnosis. the aim of the study was to identify cancer specific markers using the libraries of artificial receptors immobilized on the cellulose. an array of supramolecular structures formed from n-lipidated peptides attached to cellulose via aminophenylamino- , , -triazine was prone to formation of monolayer of "holes" and "pockets" in dynamic equilibrium. this selforganized structures were found capable of binding small guest molecules very efficiently recognizing the shape, size, and polarity of ligands, thus resembling arti cial receptors . recognition and binding properties of guest molecules by artificial receptors depends mainly on the character of peptidic pockets and structure of the fatty acid. proper construction of the binding pocket allows selective binding components of mixtures of compounds from a living organism . the preliminary data indicates that it is possible to construct an array of artificial receptors with diversified structures of peptidic pockets which are able to distinguish between components of homogenates from tumor and normal tissue. century. most of opioid alkaloids and their derivatives have μ-opioid affinity, while endogenous enkephalins are rather δ-than μ-selective. morphine is still the drug of choice for treating severe pain caused by cancer or surgical operation, but its side effects are the reason for the searching and development of new, selective mor agonists. the aim of our study is to choose within recently published crystallographic structures templates for homology modeling of the human μ-opioid receptor. we generated several models using different templates and all of them were evaluated by docking procedure (gold . ) ligands used in this investigation were synthesized and evaluated for their biological activity in our previous studies. they are enekphalin analogues with substitutions in second position. the best model of the human mu-opioid receptor was chosen according to data obtained from docking and in vitro biological activity of analogues and endogenous enkephalins. acknowledgments: this work was supported by nfsr of bulgaria project dvu / and cost action cm project do - / . . . pneumoniae, h. pylori, proteus sp. are considered as important factor contributory to development of rheumatoid arthritis (ra). the aim of this study was to investigate the level and specificity of antibodies binding to the synthetic peptides corresponding to the bacterial ureases "flap" region sequences in the rheumatoid arthritis patient's sera. for these investigations, peptides with amino acid sequences derived from "flap" regions of different ureases were synthesized using -( , -dimethoxy- , , -triazin- -yl)- -methylmorpholinium tetrafluoroborate (dmt/nmm/bf ) as coupling reagent. peptides were immobilized on a cellulose membrane. the level of antibody binding as well as specificity of them was analyzed by quantitative dot blot method using sera sera from rheumatoid arthritis patients (rap) and sera from volunteer blood donors (vbd). the results of studies suggest that "flap" region may be involved in arising antibodies participating in autoimmunological processes but not to fight infection. this effect indicates that the peptides analyzed by us could be useful for investigation of ra pathogenesis. this suggestion was confirmed by the antibodies absorption experiment which indicates that specificity of antibodies present in rap serum is slightly lower in comparison with vbd serum. it has been found that antibodies present in rap serum recognize not only a specific peptide but also peptides containing fragments with different amino acid sequences. it means that immune system of rap is unstable and may produce a wide spectrum of antibodies recognizing not only a specific epitope but also a set of similar structures. autoantigen-specific t-cells also play a crucial role in the initiation and perpetuation of dsg /dsg -specific t-cell responses. t-cells recognize epitopes from dsg protein and produce different cytokines, e.g. interferon-γ (ifnγ). functional t-cell epitopes of dsg protein have outstanding importance in immunopathological research, development and the design of novel diagnostic tools. our previous studies have shown that certain t-cell epitope peptides are able to stimulate the peripheral blood monomorphonuclear cells (pbmc) of pv patients more effectively than those of healthy donors. our aim was to select a set of t-cell epitope peptides as potential synthetic antigens which are reliably able to distinguish between donors based on the in vitro t-cell stimulating activity. we have prepared synthetic dsg oligopeptides by fmoc/tbu solid phase methodology. after cleaving from the resin with tfa the peptides were purified by rp-hplc, and they were characterized by rp-hplc, mass spectrometry and amino acid analysis. pbmc of pv patients and healthy donors were isolated; and the cultures were stimulated by dsg peptides in a concentration of . mm for hours, and the rate of ifnγ production was determined from the supernatants in sandwich elisa. synthetic dsg oligopeptides induced different in vitro ifnγ production rate on pbmc obtained from pv patients and healthy controls determined by elisa. our approach identified a synthetic antigen set as a promising biomarker for pemphigus vulgaris. [ ] . in particular, cap b (pelyafprvamide) has been shown to elicit antidiuretic activity in the green stink bug acrosternum hilare [ ] , an important pest of cotton and soybean in the southern united states. analogs of cap b containing either an (e)-alkene, cispro or a transpro isosteric component [ ] were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretions of malpighian tubules isolated from a. hilare [ ] . at a concentration of μm, the conformationally constrained transpro analog demonstrated significant antidiuretic activity, whereas the cispro analog failed to elicit any activity. the results provide strong evidence for adoption of a trans orientation for the pro in cap b neuropeptides during interaction with the receptor associated with the antidiuretic process in the stink bug. the work further identifies a scaffold with which to design biostable mimetic cap b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting cap bregulated diuretic systems. the enkephalins are pentapeptides (tyr-gly-gly-phe-met/leu) with a proven antinociceptive action. it is believed that the interaction between them and the lipids composing the membranes is important for converting the peptides into a "bioactive" conformation , . using langmuir's monolayer technique the interaction of a synthetic methionine-enkephalin (met-enk) and its amidated derivative (met-enk-nh ) with mixed lipid monolayers composed of palmitoleoylphosphatidylcholine (popc), sphingomyelin and cholesterol was studied. the surface pressure-area (π-a) isotherms with regard to πmin, π max and the hysteresis curve shape of the pure lipid monolayers and after the addition of the respective enkephalins were detected. in addition, by using brewster angle microscopy (bam), the surface morphology of the mixed lipids-enkephalins monolayers were determined. our results suggest that there is a strong penetration effect of the enkephalins studied into the mixed monolayers. moreover, our results demonstrate the potential of lipid monolayers formed in langmuir's through in combination with bam to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface. acknowledgments: this work was supported by bulgarian ministry of education, youth and science, projects n do - / , drg / and my-fs- / . dept pharmacolgy, temple univ, philadelpha, pa , usa bioinformatic algorithms has predicted the existence of several potential hormone-like peptides transcribed from the ecrg gene . previous publications indicated a highly level of gene expression of ecrg products has been found in the pancreas , choroids plexus, epithelial cells, leukocytes, and macrophages . however, the presence in the hypothalamus and the major form of derived peptides in each tissue haves not been clearly identified. knowingledge of the precise peptide generatesd within a given tissue is essential to understanding its functions. we have generated the peptide specific antibodies to against ecrg -derived pprepro-augurin( - ) and developed a specific ria kit for the quantification of the such peptide in question. a method for the purification of endogenous ecrg -derived peptides from bovine hypothalamus also has been established. using ria to monitor the immunoreactive fractions and maldi-tof to identify the endogenous peptides, we foundhave detected the presence of ecrg derived molecular of bovine preproaugurin( - ) from the homogenates of bovine hypothalamus. immunohistochemistrycal staining by antibody aalso confirmed the presence of thee peptide in some of the hypothalamic cells. of hypothalamus. the amount of prepro-augurin( - ) in the hypothalamus although not soas high as pancreas, but is one third of the augurin level of the pituitary. conclusions: the native peptide derived from augurin preproteinecrg has been discovered.identified. we have confirmed the property of purified peptide,s prepro-augurin( - ), along with the synthetic peptide standards. the present study provides the necessary procedures such as the elution from ( ) c column, ( ) p sizing column, and ( ) a further purification conditions for hplc in order to enhance the immunoreactivity from tissue fractions and yield enough amount for identification. this dsip-related peptide (kn-dsip or knd) differs from dsip by only amino acid residues in positions and . we do not consider the homology between dsip and knd as accidental, bearing in mind functional significance of histone demethylases of the jmjc-group. methylation-demethylation of histones is known as an important mechanism of posttranslational modification playing a prominent role in epigenetic regulation of chromatin structure and gene transcription. dsip is also known as an effective "normalizer" and protector from homeostatic disorders induced by stress related disturbances. we suggest that histone demethylase of the jmjc-group containing dsip-related region can be considered as a possible protein precursor of endogenous peptides with dsip-like activity. in order to test our hypothesis we synthesized knd and studied its biological effects. in a preliminary assay cited below [ ] knd showed similar and probably more pronounced effects than dsip as an agent that stimulates endurance and stress-resistance of animals in the forced swimming test. also knd provided a more active detoxifying action after administration of a semi-lethal dose of the cytostatic agent. in the present work we assessed neuroprotective and antioxidative potency of both peptides in vivo and confirmed the higher efficiency of knd. this study is supported by the moscow government. is a tridecapeptide (pglu -leu -tyr -glu -asn -lys -pro -arg -arg -pro -tyr -ile -leu ) highly expressed in the central nervous system. this peptide elicits an analgesic response following peripheral or central administration. importantly, nt exerts a more potent analgesia than morphine at an equimolar dose, without having the associated side effects of opioid drugs. structure-activity studies have identified the c-terminal fragment nt( - ) as the biologically active minimal sequence. however, nor the full or truncated peptides cross the blood-brain barrier (bbb), thus hampering its clinical development. the substitution of pro by an unnatural amino acid silaproline (sip) increased bioavailability and plasma stability. structural properties conferred by the pro were also retained as determined by nmr and ir. aiming at delineating the mode of action of cl, three new cl derivatives bearing suitable labeling moieties, i.e the fluorescent molecule fitc, the streptavidin-counterpart biotinyl-group and the m tc-radiometal chelating unit dimethylgly-ser-cys, were designed, synthesized, purified, and characterized to be applied in in vitro and in vivo evaluation studies. the structure of the cl derivatives in aqueous solutions was studied with nmr, in parallel and in comparison with the parent molecule cl, in order to examine whether the presence of the labeling moieties has induced changes to the structure of the biologically active part of cl. cell survival assays with cl and the cl derivative bearing the fitc moiety were conducted in the pc cell line in order to explore their rescue effect. in parallel, the cl derivative bearing the dimethylgly-ser-cys moiety was successfully radiolabeled with m tc and its stability was assessed over time in its synthesis reaction mixture and in plasma. this m tc-radiolabeled derivative was subsequently administered to swiss albino mice in order to determine the biodistribution of cl in the living organism and its route of excretion, a study that has not been carried out so far for any peptide of the humanin family. furthermore, the potential interaction of cl with β-amyloid peptide, the hallmark of ad pathogenesis, was explored with circular dischroism. the results of this multifaceted approach to the biological action of cl will be presented. institute of biochemistry and biotechnology, martin-luter university, halle-wittenberg, germany kinins, such as the nonapeptide bradykinin, are important mediators of various physiological and pathophysiological responses including inflammatory disease, asthma, rhinitis, cell division, pain, vascular permeability, allergic reactions, pathogenesis of septic and endotoxic shock. there are two types of receptors for kinins, known as b and b . b receptors are constitutively expressed in wide variety of cells and required entire bk sequence for recognition, while b receptors have normally very limited expression and respond to [desarg ]bk. b receptors gene is turned on following either tissue damage or inflammation. accumulated evidence indicates that most of the clinically relevant effects of bk are functions of b receptors this being the reason why research on their antagonists is a topic of great interest. in our previous study we described the synthesis and some pharmacological properties of four new analogues of bradykinin (bk), designed by substitution of position or of the known [d-arg ,hyp ,thi , ,d-phe ]bk antagonist with l-pipecolic acid (l-pip) (both analogues were also prepared in n-acylated form with -adamantaneacetic acid (aaa)). our results showed that presence of l-pip in position slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. replacement of thi by l-pip in position also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. in the present study we continue our previous investigations to find structural requirements which in the case of bk analogues result in high b antagonistic activity. several new bradykinin analogues modified in their cterminus with d-pipecolic acid were synthesized using spps method. the biological properties of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous bk in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by bk. acknowledgements this work was supported by the university of gdansk (ds/ - - - ). peptides with beta-turn structure in peptide/mhc complexes a. stavrakoudis department of economics, university of ioannina, greece major histocombatibility complex (mhc) molecules interact with small peptides and form complexes. in most of the cases, peptide's structure in these complexes is found in extended conformation. however, notable exceptions exist where the peptide forms a beta-turn structure. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the c-terminal of class ii complexes [ ] . several peptide/mhc complexes were, derived with xray studies, were extensively subjected to molecular dynamics simulations [ ] in order to investigate the stability of this turn-like structural feature and to explore the factors that possibly contribute to this stability. it was found that both intra-peptide and peptide/mhc interactions might be responsible for peptide's conformation. the peptides were found to undergo several structural transitions indicating conformational plasticity and not a completely rigid structure inside the mhc groove. the results might be of special importance in designing defective peptide vaccines and beta-turn pharmaceuticals. the heptapeptide met-enkephalin-arg -phe (merf) with the sequence of yggfmrf is a potent endogenous opioid located at the c-terminus of proenkephalin-a (penk), the common polypeptide precursor of met-and leuenkephalin. our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different penk prepropeptides among vertebrate animals. four orthologous heptapeptides with single or double amino acid replacements were identi ed among animals, such as yggfmgy (zebra sh), yggfmry (newt), yggfmkf (hedgehog tenrek) and yggfmri (mudpuppy). each novel hepta-peptide, together with the mammalian consensus merf and metenkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and g-protein activation assays in rat brain membranes [ ] . equilibrium binding af nities changed from good to modest as measured by receptor type selective [ h]opioid radioligands. the relative af nities of the heptapeptides reveal slight mu-receptor (mop) preference over the delta-receptors (dop). [ s]gtpγs assay, which measures the agonist-mediated g-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory g-proteins. all peptides were effective in promoting the agonist induced internalization of the green uorescence protein-tagged human mu-opioid receptor (hmop-egfp) stably expressed in hek cells. thus, the c-terminally processed penk heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution. corticotropin releasing factor (crf) exerts most of its physiological and pathophysiological actions by interacting with its type receptor (crf ) and activating different intracellular signalling pathways. the crf is a plasmamembrane protein, which belongs to the family b of g-protein coupled receptors (gpcrs) and like the other gpcrs consists of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, mostly hydrophobic, membrane-spanning segments (tm -tm ), connected by alternating intracellular (il) and extracellular loops (el). binding of crf and its related peptides, such as sauvagine, to the extracellular regions of crf is associated with receptor activation and subsequent activation of different g-proteins and regulation of diverse signalling pathways. using a mutagenesis approach in combination with a radioligand binding study we found that trp and phe in the second extracellular loop of crf interacted with the amino-terminal portion of crf and sauvagine. interestingly only the interaction of sauvagine with trp and phe is important for crf -mediated stimulation of camp accumulation. in marked contrast the interaction between crf and the residues trp and phe was unimportant for the activation of adenylate cyclase. thus it is possible for trp and phe of crf to regulate distinct signalling pathways, or different sets of them, after their interaction with different peptides. we are now performing experiments to fully elucidate the signalling pathways that are regulated by the interaction of crf and sauvagine with trp and phe . these studies will advance the development of crf -selective selective signalling-specific peptides that would be extremely useful for the elucidation of the role of crf in many physiological and pathophysiological situations, and possibly for the treatment of several crf -related diseases. thymus humoral factor gamma- (thf-γ ), an octapeptide, purified from crude thf, retains essentially all the biological properties of thf [ ] [ ] . it regulates clonal expansion, differentiation and maturation of t-cell precursors, stimulates the production of lymphokine, maitains the normalization of impaired ratios between helper(cd +) and suppressor / cytotoxic (cd +) subsets and augments il- production in spleen cells. thf-γ has a calculated molecular weight of and has the following amino acid sequence: leu-glu-asp-gly-pro-lys-phe-leu. its poor stability towards protein enzyme limits its extensive application. with the inte ntion to promote its bioavailability, bioactivity and develop ideal immunoregulatory drug candidat, four series of derivatives of thf were designed and synthesized: . n-and cterminal acylation. .restitution the flexible segment gly-pro by unnatural amino acids -aminohexanoic acid (aca) in order to shorten the synthetic steps and simultaneity improve the bioavailability and biostability of peptide; . reserve protected group of some amino acid residus as spot mutation. . mannich-based cyclization was carried out on resin [ ] , phe was replaced by tyr serving as the active hydrogen component, a proline was introduced at the n terminal as the amine component and formaldehyde was used as the only component in solution. the bioactivity of synthesized products were detected. the leukocytopenia model in mice was induced by cyclophosphamide intraperitioneal injection. white blood cell count, thymus index and spleen index were detected to evaluate the immune function of compounds in mice. the results show that those compounds play a significant role in improving immune function in mice. the activity of compound lhl and lhl are also better than authentic compound tp- and tα . marine organisms have been recognized as a promising source for the development of new pharmaceuticals. in the course of screening for antitumor substances from marine organisms, we found cyclic peptides containing many nonribosomal amino acids such as hydroxyasparagine, hydroxyleucine, or other supporting a hydrophobic side chain that were shown to be a key element for their biological activity. the laxaphycine b, a cyclic lipopeptide isolated from marine cyanobacteria anabaena torulosa harvested in french polynesia constitutes an example of this peptide class. this compound has attracted our attention because of its micromolar cytotoxic activities on different cancer cell lines as well as its antiangiogenic properties which seems to be due to an interaction with the vegf receptor- - . the synthesis of the non-natural amino acids - and of laxaphycine b analogues will be presented along with their preliminary biological activities. immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases including dermatology. cyclolinopeptide a (cla) c(pro -pro -phe -phe -leu -ile -ile -leu -val -), a cyclic, hydrophobic nonapeptide isolated from linseed, possesses strong immunosuppressive and antimalarial activity. it has been suggested that both the pro-pro cis-amide bond and an 'edge-to-face' interaction between the two aromatic rings of adjacent phe residues in tetrapeptide unit are important for biological activity. this edge-to-face interaction can be influenced when phenyl rings are replaced by naphtyl substituent. in this communicate new analogues of cla modified by naphtylalanine ( -nal) in positions or or both and ( - linear analogues, - cyclic analogues) will be presented. the synthetic strategy and biological activity as well as conformational analysis will be evaluated. the onset of type ii diabetes mellitus (t dm) coincides with the deposition of fibrillar material in the islet of langerhans in the pancreas that is a clinical hallmark of more than % of patients suffering this disease. the main component of the pancreatic amyloid deposits is a -residues polypeptide hormone called islet amyloid polypeptide (iapp) or amylin. in this work we have examined, by means of cd spectroscopy and tht-fluorescence, the conformational polymorphism of both full-length - hiapp, and the related fragment hiapp - , and compared the results with the respective rat counterparts. moreover, the cytotoxic activity was determined toward different pancreatic β-cells lines in the attempt to correlate iapp's fibrillogenic properties with the ability to mediate cell death. together the results suggest that β-sheet conformational transition, that generally preludes to fibril formation, is not a prerequisite for eliciting toxicity toward β-cells cultures. interestingly, confocal microscopy indicated that both hiapp - and hiapp - can enter the cell and might exert their toxic action at intracellular level. acknowledgments: this work was supported by miur, firb-merit project rbne hwlz. due to its physiological functions, s proteasome is considered the target molecule in overcoming several diseases [ ] . its core particle s has three types of active sites: chymotrypsin-, trypsin-and caspase-like. many natural and synthetic compounds were tested for their ability to inhibit proteasome. a recent report describing the inhibition of s by the serine proteases inhibitor -bovine pancreatic trypsin inhibitor -was considered by us with great attention [ ] . our scientific interest is focused on peptide inhibitors and their interaction with serine proteases. sunflower trypsin inhibitor (sfti- ) is the smallest and the most potent peptide inhibitor in the bowman-birk family. owing to its size and the rigid structure (disulfide bridge and "head to tail" cyclisation) sfti- is willingly chosen as the lead structure in the search for new inhibitors [ ] . its sequence is shown below (lys the p residue responsible for specificity): & gly-arg-cys(& )-thr-lys -ser-ile -pro -pro-ile-cys(& )-phe-pro-asp& since native sfti- is not able to inhibit s [ ] , we have designed its monocyclic analogues (with disulfide bridge only) with lys or arg in position (p ) and at least one basic amino acid (lys or arg) in positions (p ') and/or (p '). all analogues inhibit chymotrypsin -(ic at the range of ÷ μm) and caspase-like (ic at the range of . ÷ μm) activities in vitro, whereas their activity towards trypsin-like specificity is much weaker. in several rat tissues, our view on ras has changed. metabolism of the ang-( - ) may represent alternative pathway of ang ii formation, importantly, independent on renin and ace activity , . ahmad et al. have described metabolism of ang-( - ) by human atrial tissues and showed that ang ii is formed mainly by chymase. this renin-inependent ang ii production could explain the "resistance" regarding use of ace inhibitors in patients with hypertension or diabetic nephropathy. noteworthy, the role of ang-( - ) in circulation is still unclear and there are no information about possible pharmacological modulation of its metabolism. in our study, we compared the ex vivo metabolism of angiotensinogen (fragment - ) in hypertensive (shr) and normotensive (wky) rats in organ bath of aorta and heart using lc-ms method . surprisingly, we identified ang-( - ) formed via reninindependent pathway to be a main product of angiotensinogen metabolism in rat aortic tissue and heart. in this setting, ang-( - ) appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of ang i and ang ii. as compared to wky rats, formation of ang ii, from ang-( - ), was much higher in shr aortas but not in the heart. the functional consequences of these findings require further investigation. this study was supported by the grant n n polish ministry of science and higher education. the lysosomal cysteine protease cathepsin c (cat c), also known as dipeptidyl peptidase i (dppi), activates a number of granule-associated serine proteases with proinflammatory and immune functions by removal of their inhibitory n-terminal dipeptides. activity of this protease is associated with several pathologies in human body [ ] . in this work the characterization of cat c specificity using combinatorial chemistry methods will be described. the main goal of this work was to determine of substrate specificity of the prime region of this enzyme. the chemical synthesis and deconvolution of two libraries will be described. the hemostatic mechanism has the crucial role to prevent loss of blood from injured blood-vessels. this loss is prevented by the integrity of the vessel walls, by platelets aggregation or by blood coagulation, which in normal conditions is limited onto the local trauma of the vessel wall. in generally, the blood coagulation mechanism is important for maintaining vascular integrity and thus for the precaution of an organism from bleeding, which may also occur by blood coagulation caused by thrombin production. the diversion rate of this production leads to an expansion of thrombin to the general blood circulation. thus, when thrombin generation is not controlled by the mechanisms of inhibition, a widespread undesirable intravascular thrombosis is occurred. the whole process of platelets adhesion requires the presence of clotting factor viii (fviii), a necessary for the blood coagulation cascade glycoprotein, which takes part in the intrinsic pathway and acts as a coenzyme for the activation of factor ix, a serine protease depended on the thrombin production. the target of the present research is the synthesis of biologically active cyclic, head to tail, peptides, analogs of the sequence - of a subunit of fviii, which are potentially capable to block fviiia-fixa complex, reducing the thrombin production and thus the blood coagulation. the synthesized peptides are investigated for their inhibitory activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. the blood coagulation is part of an important host defence mechanism, which under pathological conditions results in inappropriate intravascular coagulation when thrombin is produced. clotting sequence is the result of a cascade of two biochemical pathways, intrinsic pathway, so called because all components are present in blood, and extrinsic pathway, in which tissue factor is required in addition to circulating components. the activated form of factor viii (fviiia) is a key component of the fluid phase of the blood coagulation and plays an important role formatting a trimolecular complex with factor ixa, ca + and negatively charged phospholipids of the cells membrane. this complex is called tenase and participates in activation of prothrombin, which acts on fibrinogen to generate fibrin monomer, polymerized rapidly to form fibrin clot. the fviii is comprised of a heavy (a -a -b) and a light (a -c -c ) peptide chain, both cleaved by proteases at three sites, resulting in alteration of its covalent structure and conformation. its deficiency is known as haemophilia a. our research effort is focused on the synthesis, identification and biological evaluation of peptide analogs, expected to inhibit selectively the increasing of thrombin production. their sequence is based on the regions in which the fviii interacts with fix, specifically on the sequence - of the a subunit. the synthesized peptides are examined for their activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. inhibitor with the following structure: ac-llllrvkr-nh , which has potent effects on the proliferation of prostate cancer cells. the potency and stability of this compound was subsequently enhanced by substitution of arg residue in position p with its conformationally restricted mimetic - -amidinobenzylamine (amba). nevertheless, the specificity toward pace was significantly reduced by this modification. thus, in order to improve its selectivity without sacrificing inhibitory potency we decided to use positional scanning approach. in this study we present synthesis of two series of peptide libraries, which were designed by substitution of leu in the p , p position of our control peptide (ac-llllrvkr-amba) with each of nineteen amino acid residues in order to verifying its influence on activity and selectivity of the resulting analogues. all peptides were synthesized by a combination of solid phase peptide synthesis and solution synthesis and tested for their inhibitory potency against furin and pace . the p -p fragments were synthesized by fmoc/tbu spps strategy on hydrazinobenzoyl or acid labile chlorotrityl chloride resin. then coupling of the -amidinobenzylamine · hcl was performed. the best modifications were combined to give as several multipoint substituted inhibitors. we believed that our work, will provide new important information about structure-activity relationship of these class of analogs in order to obtain potent and highly specific pace inhibitor. institute for research in biomedicine, parc cientific de barcelona, barcelona -spain a bacterial toxin-antitoxin (ta) system is composed of two genes organized in an operon encoding a toxin and an antitoxin that regulate the growth and death bacterial cell under various stress conditions. the operon parde encode a ta system formed by pare toxin and its antitoxin pard. pare is a kda protein that inhibits dna gyrase activity and thereby blocks dna replication. however the pare-gyrase interactions and the gyrase activity inhibition mechanism have not been explored. as an approach for understanding of this mechanism and to elucidate the pare region responsible for protein-protein interactions we have designed and synthesized a series of linear analogues of pare and investigated the ability of peptides to inhibit dna topoisomerases activity. so, based on structural data inferred from pare three-dimensional model , peptides were synthesized by solid-phase method. four peptides (parelc , parelc , parelc and parelc ), showed complete inhibition of dna gyrase supercoiling activity, by gel electrophoresis assay , with an ic of to μmol.l - . in addition, intrinsic fluorescence and fluorescence anisotropy assays showed that inhibition process must occur by interaction with the gyra subunit. differently of wild type pare, the peptide analogues were able to inhibit the dna relaxation of topoisomerase iv with lower ic values. interesting was that only parelc displayed inhibition of the relaxation activity of human topoisomerase ii. our results suggest a new class of molecules with simultaneous inhibitory activity in dna gyrase and topoisomerase iv. furthermore, we have obtained the first example of a synthetic peptide from a bacterial toxin with inhibitory activity on human topoisomerase ii. institute of experimental endocrinology, slovak academy of sciences, bratislava, slovakia the renin-angiotensin system (ras) has long been recognized as an important regulator of systemic blood pressure and electrolyte homeostasis. our understanding of ras has experienced remarkable change over the past two decades. besides, angiotensin ii, the new biologically active peptides [e.g. ang-( - ), ang-( - ), ang iv, ang-( - )] and pathways [e.g. angiotensin converting enzyme -ace ] have been described ; some of them, like ang-( - ) may oppose many actions of ang ii. importantly, despite all components of classical ras are found in adipose tissue , the data about fat formation of various angiotensins remain scarce. in our study, we compared the ex vivo metabolism of angiotensinogen, ang-( - ) and ang i in hypertensive (shr) and normotensive (wky) rats in organ bath of retroperitoneal and periaortic fat tissue using lc-esi-ms method. additionally, qpcr measurements of mrna expression of main enzymes involved in ang i metabolism were performed. both in the periaortic and epidydymal fat, the formation of ang-( - ) was higher than production of ang ii. fat tissue formation of two main ang i conversion products, ang ii and ang-( - ), differed significantly between shr and wky rats. compared to wky rats, the formation of ang-( - ) in periaortic fat tissue was decreased in shr. in opposite, in epidydymal fat tissue formation of ang-( - ) and ang ii was higher in shr. interestingly, there were no differences in aorta formation of ang ii and ang-( - ) between shr and wky rats. our results suggest that in hypertension visceral fat production of angiotensin peptides is increased, while generation of "beneficial" ang-( - ) in periaortic fat is decreased. however, the functional importance of such finding require further investigation. department of chemistry and biochemistry, university of washington, usa phospholipases a (pla ) are a superfamily of enzymes involved in various inflammatory diseases. in particular, human secreted giia spla is an attractive target for the development of novel medicines. we have shown that oxoamides based on γ-or δ-amino acids are potent inhibitors of cytosolic giva pla . very recently, we have demonstrated that a long chain -oxoamide based on (s)leucine displays inhibition of human and mouse giia spla s (ic nm and nm, respectively). a combined experimental/computational study was undertaken to further understand the role of the α-amino acid of -oxoamides for the inhibitor-enzyme binding. the crystal structure of giia spla s reveals a highly conserved ca + -binding loop and a catalytic dyad consisting of his /asp . -oxoamides based on hydrophobic αamino acids showed better binding score prediction compared to polar α-amino acid derivatives. a number of new -oxoamides based on α-amino acids were synthesised and tested for their inhibitory activity against giia, gv and gx pla . the -oxoamide based on (s)-valine displayed potent inhibition of giia spla (ic nm) in accordance with the predicted docking score. docking results reveal that (s)-valine-based inhibitor forms key interactions with the active site of the enzyme. the carboxylic group participates in a hydrogen bonding with gly and lys , and -carbonyl group with gly . furthermore, both carbonyl groups are in the proximity with ca + . the side chain of (s)-valine adopts a suitable orientation to interact with tyr and lys . the long aliphatic -oxoacyl chain is accommodated in the hydrophobic region of the active site and creates proximal contacts with leu , ile , his and phe . the search for novel classes of pharmaceutical molecules with enhanced therapeutic power has been the subject of numerous research groups all over the world. moreover, systems of immobilization and controlled release which are adapted to these new classes of molecules, has proven to be an area of extreme importance to provide the same therapeutic efficacy. using solid-phase chemistry a series of ccdb toxin analogous peptides were synthesized and were synthesized and tested against the capacity of inhibition of bacterial enzymes dna gyrase and topoisomerase iv (topo iv). subsequently those peptides were detained in drug delivery systems (dds) to be tested against the inhibition of growth of different bacterial species. in this data we could observed that the analogue ccdbsg could inhibit only dna gyrase and not the topoisomerase iv. in the other hand the analogue ccdbsg presents a hard inhibition potential against topo iv specially because of their structural difference. is possible conclude that topoisomerase iv presents the tertiary structure very similar to dna gyrase, but those mechanisms of action must be clearly distinct . in the in vitro studies, as expected, results revealed that the drug delivery systems are the key to the power efficiency of peptide analogues against the bacterial growth inhibition which cannot be observed when the peptides are free in solution. some of the different lipid compositions of the dds are demonstrating to be more efficient in the membrane cell transverse and this data previously assumes that it is possible to apply different types of dds to promote the peptide molecules transport across the cellular membranes according to several specific therapies. with this studies we have obtained more knowledge about the interaction system of enzyme-toxin and hopes which helps in future studies to development a new antimicrobial molecules class. it is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. we propose to target an ancestral form of the proteasome, the hslvu protease, which is present in the parasite's single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. originally discovered in eubacteria, this complex is constituted by two central hexameric hslv protease rings sandwiched between two hexameric hslu atp-ase rings. as hslv shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. according to studies on bacterial hslvu, , the c-terminal segment of hslu is essential in hslv activation and in complex assembly, therefore representing a privileged target. we produced recombinant hslv, which is inactive alone, and showed that a synthetic c-terminal hslu peptide was able to induce the digestion by hslv of a fluorogenic substrate that we developed. with this enzymatic test in hands, we started the characterization of the interaction of the c-terminal portion of hslu with hslv. we will present the results obtained with various series of analogues of the original c-terminal hslu peptide, including truncated forms, ala scan, constrained analogues and multivalent constructions. helped by molecular modelling studies, the aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the hslu and hslv rings, obligatory for the degradation of proteins by the hslvu complex. finally, we checked that hslv was inhibited by classical active-site directed proteasome inhibitors like bortezomib. f. babos a,d , e. szarka b , gy. nagy c , z. majer d , g. saŕmay b , a. magyar a , f. hudecz a,d citrullinated filaggrin peptide (ccp) were detected in ra sera and anti-ccp positivity is widely used for diagnostic purposes. identification of new epitopes of filaggrin would be useful in the diagnosis of anti-ccp seronegative patients. in order to achieve optimal immune recognition of biotinylated epitope peptides it is important to analyse the effect of the labelling moiety on antibody binding. for these studies -as well as -mer peptides with nor c-terminal biotin were synthesised manually by spps, using fmoc/ t bu strategy. biotinylation was performed by using biotin, biotinyl- -aminohexanoic acid or , , -trioxa- , -tridecanediamino succinic acid linker modified biotin. labelled peptides were used in an indirect elisa, on neutravidin pre-coated plates and the binding was detected by anti igg-hrp. to examine the role of the presence/position of biotin in the secondary structure of the peptides, electronic circular dichroism (cd) method was used. we found that the ccp + serum samples specifically recognized the c-terminally biotinylated -mer filaggrin peptides, while showed no binding with the n-terminally biotinylated compounds. in case of the -mer epitope peptides there was no difference between the recognition of nand c-terminal biotinylated analogues. data presented suggest that the position of the biotin in case of the short filaggrin epitope peptides markedly influence the serum antibody binding. upon activation process, they are released from the granules and then involved in immunoresponse of the organism. when out of the cell those enzymes remain in free form or become associate with the cell membrane. the physiological role of this proteases is manifestated in several processes such cytokine and chemokine processing, platelet activation, and degradation of extracellular matrix's proteins [ ] . in this work results of the specificity of two members of nsps pr and hne evaluated using the combinatorial chemistry methods will be presented . both enzymes share primary specificity and to obtain the selective substrate that will be recognized only by one enzyme, the prime sites should be investigated. the general formula of the designed library is as follows: where in positions x ', x ' and x ', the set of proteinogenic amino acids (except cys) was introduced. abz is -amino benzoic acid served as donor of fluorescence and -nitro-l-tyrosine as acceptor. eukaryotic proteasome is a highly organized protease complex comprising a catalytic s core particle (cp) and two s regulatory particles (rp), which together form the s structure. the main function of this large intracellular protease is to degraded ubiquitine labeled proteins. the catalytic particle of the proteasome displays three distinct enzymatic activities: trypsin-like, chymotrypsin-like and glutamyl-like. the increase activity of the proteasome is associated with several disease including cancer [ ] . the main aim of this work is to synthesized the cell permeable fret displaying peptides that will selective cleaved by single proteasome activity. additionally each peptide when independently cleaved by the proteasome subunit, should emit the fluorescence energy in a different spectral region. our intention was designing substrates which would allow to monitor simultaneously (in a single experiment) and independently of three proteasome activities in this report, we will describe the chemical synthesis of several peptides modified at on cand n-termini by synthetic fluorescent amino acids the general formula of these peptides is as follows: where x is a non proteinogenic amino acid that serve as a donor of fluorescence, y amino acid that is a acceptor of fluorescence. the obtained fluorescent peptides were examined for their ability to cross the cell membrane. also kinetic parameters (k cat, km, kcat/km) with proteasome will be presented. approximately dubs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. recently, several groups have introduced various methods for linking ubiquitin to different substrates via nonhydrolyzable isopeptide bonds, which resist the action of dubs. using these methods, one could explore the function and the mechanism of dubs and apply them in activity based profiling. here we present a new and convenient strategy for preparing nonhydrolyzable ubiquitinated peptides and proteins by nmethylating the isopeptide bond. using this method we prepared several nonhydrolyzable ubiquitinated peptides with different lengths derived from ubiquitinated h b and examined their affinity to different dubs. f.i. nollmann, c. dauth, d. reimer, h.b. bode* goethe universität, frankfurt, germany bacteria of the genus xenorhabdus and photorhabdus are gram negative gamma proteobacteria that live in symbiosis with nematodes of the genus steinernema. undergoing their partly entomopathogenic life cycle these bacteria not only produce antibiotics , and insecticides but also several different small molecular compounds and peptides. for the most part the biological benefits of these secondary metabolites have not fully been understood yet. with the help of inverse feeding experiments, hr-ms and nmr as well as molecular engineering we were able to characterize and/or isolate some of these peptides. since they are mostly produced in trace amounts, we synthesized them in order to make them accessible to continuative testing. given that not only linear but also highly methylated or cyclic peptides are produced, the synthesis was quite challenging. nevertheless, we were able to establish in our laboratory a general synthesis route for cyclic peptides and depsipeptides , as well as highly methylated hydrophobic linear sequences. testing several of these peptides has revealed activity against insect cells and against the causative organisms of neglected tropical diseases. cyclotides are a large class of plant peptides defined by a head-to-tail cyclized backbone and three conserved disulfide bonds in a knotted arrangement. these unique structural features confer them with remarkable stability and due to a range of bioactivities they are extensively investigated as templates in drug discovery . based on the use of oldenlandia affinis in traditional african medicine for its uterotonic principle we investigated crude plant extracts and semi-pure cyclotide fractions for the ability to induce uterine contractions using a collagen-gel contractility model . pharmacological analysis of the effects led to the identification of the oxytocin receptor, a representative of the g-protein coupled receptor (gpcr) family, as a molecular target for cyclotides. mass spectrometry-based sequence analysis of 'active' fractions revealed cyclotides with high similarity to the human oxytocin (h-ot) peptide that exhibited weak binding to the human oxytocin receptor. we further analyzed synthetic cyclotide-derived small ot-like peptides and grafted the h-ot sequence into the stable cyclotide frame. these peptides showed increased binding and activation as compared to native cyclotides. these findings may open new avenues for the discovery of gpcr ligands from natural peptide sources. gpcrs are promising drug targets and ~ % of currently used drugs act via binding to these receptors. natural combinatorial peptide libraries are likely to play an important role in identifying novel gpcr ligands . particularly plant cyclotides cover a large chemical space based on their high sequence diversity. together with their range of bioactivities and unique stable structure suggests that cyclotides are of current and future interest for drug discovery and development. acknowledgements: this work is funded by the austrian science fund fwf (p ). drosha and dicer are two key endonucleases for biogenesis of micrornas (mirnas) that regulate target mrna. drosha converts pri-mirna to ~ nucleotide (nt) pre-mirna in nucleus and dicer converts pre-mirna to linear ~ nt single-stranded mirnas in cytosol. even though dicer is potentially important to control availability of mature trans-acting rnas in cytosol, the enzyme itself does not seem to be the suitable target controlling mirna processing due to the lack of its substrate specificity. nature, however, might be intelligent enough to differentiate a variety of pre-mirna, so that a certain specific pre-mirna is converted to mature mirna in case it needs. therefore, other component(s) in the enzyme complex could be involved in recognition of auxiliary proteins from out sources to give extra specificity. we have synthesized trp-containing amphiphilic peptides against several pre-mirna. peptide b showed a picomolar binding affinity and a large specificity against pre-let a- . in vitro mirna processing, dicer activity was also selectively enhanced in the presence of this peptide. on treatment with this peptide on hct colon cancer and p ec cell lines, let a- mirna was more processed than reference mirnas. the toxicity of furan is known to rely on its selective oxidation in the liver by cyt p enzymes transforming it into the very reactive butenedial, which quickly reacts with proximate nucleophiles. this principle was used in our laboratory to develop a high yielding dna interstrand crosslinking methodology. in view of the demonstrated site-selectivity, the method further holds promise for sitespecific crosslinking dna to its binding proteins, which is highly relevant in the study of transient protein-dna interactions. furthermore irreversible dna binding can be achieved through such a covalent linkage, which is potentially useful for new generation therapeutics. the reactive furan moiety can in principle be incorporated either in the dna or in the protein. in the former case, a furan modified nucleotide was built into an oligonucleotide positioning the furan moiety at the periphery of the dna, to avoid interstrand crosslinking. for the latter approach, we initially chose to synthetically access a furan modified dna binding protein mimic. next to a previously described non-covalent gcn mimicking dimer, we have also investigated a new type of steroid-based dipodal dna binders. synthesis of the latter constructs has proven challenging in view of the immobilization of two peptide chains with helix forming tendency at close distance on the template. results, showing the power of microwave assistance will be discussed. in an alternative approach, a full length protein was modified with furan by amber suppression based on the structural similarity between a furan modified amino acid and pyrrolysine. pharmaceutical institute, university of bonn, an der immenburg , bonn, germany human matriptase- is a kda protein with trypsin like specificity. this protein exhibits a domain organization similar to family of membrane-bound serine proteinases known as type ii transmembrane serine proteinases. among many ascribed function in human body, this enzyme is a potent negative regulator of hepcidin, the peptide involved in iron homeostasis [ ] . matriptase- has a similar fold as other tmsp members, however their detailed specificity still remain unclear. the aim of this study was to determine the substrate specificity of this physiological important enzyme using combinatorial chemistry approach. in order to characterize the matriptase- specificity, the tetrapeptide library with c-terminal amide of aminocoumarin (acc-nh ) that serve as a fluorophore, was synthesized. its general formula is given below: x -x -x -x -acc-nh , where in position x -x the set of proteinogenic amino acid residues are present, whereas in position x lys or arg was introduced. deconvolution of such library was performed using iterative approach in solution. the results obtained indicate that matriptase- display diverse p -p specificity as compare to matriptase- . the most efficient hydrolyzed amino acid residue in position p appear to be ile, that is followed by arg in p and ser in p . the arg in position p is % faster hydrolyzed then lys. for selected substrates, the kinetic parameters (kcat, k m ) were determined. amyotrophic lateral sclerosis (als) is a chronic progressive disease. it is characterized by degeneration of upper or lower motor neurons, but its pathogenesis is still unknown and no effective treatment currently exists. it is known that antibodies to gangliosides have been found in some als patients, and these antibodies are also well known to be present in the patients affected by a variety of autoimmune diseases including multiple sclerosis. up to now anti-gangliosides antibodies are detected in clinical immunology laboratories using isolated non consistent antigen mixtures. therefore, we are interested in developing reliable and univocally characterized synthetic antigens for efficient antibody detection. csf (glc) is a family of structure-based designed glycopeptides that we previously developed as multiple sclerosis (ms) synthetic probes. these n-glucosylated peptides are able to detect specific autoantibodies in the sera of an antibody-mediated form of ms. autoantibody recognition was favored because of the exposition of the sugar amino acid on the tip of type ' β turn structures. aim of this study is the introduction, in the type ' β turn peptide structure, of the sugar moiety specific for anti-gangliosides antibody recognition by synthesizing specific building blocks. these building blocks are amino acids carrying glycans mimicking the biological activity of complex oligosaccharides. we selected sialic acids (in particular the n-acetylneuraminic acid -neu ac) because they are involved in a significant number of biological events. neuraminic acid and its derivates are widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. therefore, we synthesized fmoc-l-asn(neu ac)-oh and fmoc-l-ser(neu ac)-oh. these building blocks will be introduced in the type ' β turn structure for the detection of anti-gangliosides antibodies in als. as a distinct pattern of ms could involve an antibodymediated demyelination, identification of autoantibodies as specific biomarkers is a relevant target. even if interesting data focused on the diagnostic and prognostic role of the detection of antibodies to myelin oligodendrocyte glycoprotein (mog) in adults' serum, its value remains dubious due to many other contrasting results. our research group identified csf (glc), an nglucosylated peptide, able to detect disease-specific autoantibodies in the sera of a statistically significant number of ms patients. , since this synthetic antigen may be considered as a mimic of aberrant post-translational modification (i.e. n-glucosylation) of myelin protein(s) triggering autoimmunity in ms, our goal is to obtain the extracellular domain of mog properly glucosylated thanks to a simplified native chemical ligation approach. for this purpose, the n-glucosylation will be introduced in a synthetic peptide fragment following the building-block approach by spps. the other protein fragment bearing an n-terminal cysteine will be expressed in e. coli after introduction of a selective point mutation into mog. finally, our aim is to test the semi-synthetic protein by sp-elisa to study the ability to detect autoantibodies in ms patients' sera and to find a potential cross-reactivity with csf (glc). this peptide is an endogenous ligand of the opioid receptorlike (orl ), previously referred to as "orphan" receptor, structurally and functionally related to the classical opioid receptors. also the hexapeptide ac-ryyrwk-nh is shown to be a selective ligand for the nop receptor with marked analgesic effect. with a view to developing ligands for the nop receptor with more potent analgesic activity, new series of the ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β tryptophan analogues were synthesized . the aim of the present study was to examine the effects of naloxone (nal) and jtc- (nop receptor antagonist) in the analgesic activity of newly synthesized hexapeptide analogues. all peptides ( μg/kg), nal ( mg/kg) and jtc- ( , mg/kg) were injected intraperitoneally (i.p.) in male wistar rats. antinociceptive effects were evaluated by two nociceptive tests -paw-pressure (pp) and hot-plate (hp) and statistically accessed by anova. the results will be discussed compared to the referent compound in both tests used and mechano-and thermo-receptors are involved. [ ] . socs and socs have many similarities as well as some intriguing differences. both can block signalling by direct inhibition of jak enzymatic activity yet apparently require different anchoring points within the receptor complex. while the primary socs interaction is with a critical py residue within the jak catalytic loop [ ] it interacts also with py residues in the ifnαr and ifn r subunits in a jak -independent manner; the socs -sh domain also interact with y in jak , albeit with slightly lower affinity, but subsequent studies demonstrated a high affinity interaction with py residues located within receptor subunits [ ] . mutagenesis studies identified small regions at the n-termini of the socs and socs -sh domains, and at the c-terminus of the socs -sh domain, which were critical for phosphotyrosine binding. in order to gain insights in molecular discriminants for the interaction of both socs and socs toward jak and tyk we designed and synthesized peptides encompassing regions involved in proteins recognition. we set up a spr assay to evaluate the affinities of complexes formation. then through an alascanning approach we have designed new peptide sequences containing un-natural amino acids that are able to better recognize wild sequences and whole proteins. cellular experiments on stat activation signaling suggest their potential application as modulators of disorders involving socss overexpression. targeting proapoptotic death receptors (drs) to trigger apoptosis in cancer cells is a promising anticancer therapeutic approach. trail (tnf-related apoptosis inducing ligand) is a transmembrane homotrimeric protein belonging to the tnf family that triggers selective tumour cell apoptosis upon binding to its cognate receptors dr and dr . several strategies are being developed to exploit the unique cancer selectivity of the trail-dr pathway in therapy, including the use of recombinant trail targeting dr or dr . [ ] recently, a disulfide-bridged macrocyclic -mer peptide (derived from phage display) that binds selectively to dr has been identified. [ ] oligomeric versions of this macrocyclic peptide display increased binding avidity to the receptor and exhibit the capacity to activate the trail apoptotic pathway both in vitro and in vivo. [ ] however, disulfide bonds are susceptible to reduction and scrambling in vivo potentially resulting in the loss of the desired biological activity. among alternative linkages with increased redox stabilities, lanthionine thioethers, in which one of the sulfur atoms of the disulfide bond is removed have previously been introduced into biologically active peptides with some success. [ ] disulfide bridges can undergo a -elimination in alkaline conditions, followed by a michael addition to give a thioether bridge. optimization of this reaction led to the desulfurized analogue of the dr -binding peptide. the native dr -binding peptide and its desulfurized analog have been compared for their structural (nmr conformational analysis) and biological properties (affinity to dr and signaling pathways). the apelin/apj complex has been detected in many tissues and is emerging as a promising target for a number of pathophysiological conditions. in the central nervous system, apelin/apj was detected in brain regions involved in spinal and supraspinal control of pain, such as the amygdala, hypothalamus, dorsal raphe nucleus and spinal cord. we propose the hypothesis that apelinergic agonists represent a potential new approach to pain modulation and that the synthesis of stable analogues would lead to compounds with antinociceptive properties. there is currently little information on the structure/activity relationship (sar) of the apelin hormone. in an effort to better delineate sar, we synthesized analogs of apelin- modified at selected positions with unnatural amino acids, with a particular emphasis on the c-terminal portion. analogs were then tested in binding and functional assays by evaluating gi/o mediated reduction in camp levels and by assessing β-arrestin recruitment to the receptor. the plasma stability of new analogs was also assessed. several were found to possess increased binding and higher stability compared to the parent peptide. there is compelling evidence that the neuropeptide rfa and its cognate receptor gpr , are involved in the control of food intake and bone mineralization. among the gpcrs whose structures have been solved, gpr exhibits the highest sequence homology with the beta adrenergic receptor. the aim of this work was to experimentally characterize predicted ligand-receptor interactions by site-directed mutagenesis of gpr and design of point-substituted rfa analogs. starting from the x-ray structure of the beta -adrenergic receptor, a d molecular model of gpr has been built. the bioactive c-terminal octapeptide rfa( - ), kggfsfrf-nh , was subsequently docked in this gpr model and the ligandreceptor complex was submitted to energy minimization. in the most stable complex, the phe-arg-phe-nh part was oriented inside the receptor cavity whereas the n-terminal lysine remained outside. a strong intermolecular interaction was predicted between the arg residue of rfa and the gln residue located in the third transmembrane helix of gpr . in order to study this interaction, we have investigated the ability of rfa and arg-modified rfa analogs to activate the wild-type (wt) and the q amutant receptors transiently expressed in cho cells. the platelet receptor αiibβ plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affinity for fibrinogen. the αiibβ activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, the most important is that of talin with the β cytoplasmic tail. it has been recently suggested that talin-mediated αiibβ activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) β regions with talin f domain and that the -n ply -motif of β , which can be phosphorylated at y , plays a critical role in this process. to evaluate the interaction of talin with the β tail of integrin we designed and synthesized three peptides corresponding to the md and mp parts of β in their carboxyfluoresceinlabeled form (md: cf-r akwdtannplyke -nh , cf-n nplykea -nh and mp: cf-k llitihdrke -nh ). emission and anisotropy fluorescence spectroscopy was used to quantitatively assess the affinity of these peptides for talin. furthermore, to challenge the role of the y phosphorylation in talin-α iib β interaction we also studied the binding of talin to the modified analogues of md, cf-r akwdtannpl(ptyr)ke -nh and cf-n npl(ptyr)kea -nh . our experiments revealed that the md and mp parts of β bind tightly to talin and that y phosphorylation has an inhibitory effect on this binding. functionalized oligoprolines as multivalent scaffolds in tumor targeting p. wilhelm, h. wennemers* eth zurich, zurich, switzerland oligoprolines are known to be structurally well-defined molecular scaffolds. in aqueous media, even short chain lengths of six proline residues adopt a polyproline ii helix (ppii). this secondary structure is a highly symmetric helix where every third residue is on top of each other in a distance of about . Å. [ ] the incorporation of azidoproline (azp) allows facile and versatile functionalization either via copper-catalysed azide-alkyne cycloaddition (cuaac) or an acylation that followed a staudinger reduction. [ ] based on the structural integrity of the oligoproline scaffold, targeting vectors can be conjugated via coppercatalysed azide-alkyne cycloaddition in defined distances. recent studies on radiolabeled oligoproline-bombesin conjugates, to target the gastrin-releasing peptide receptor (grp-r), showed in vitro and in vivo superior internalization in prostate cancer cells compared to the established monovalent ligands. [ ] a facile route to synthesize alkynylated ligands has been developed successfully. we are currently expanding this concept to the integrinligand c(rgdyk) as well as to [tyr ]-octreotide, which binds to somatostatin-receptors. the monovalent analogue of the latter, dota-[tyr ]-octreotide (dotatoc), is well established for diagnosis [ ] and therapy [ ] of somatostatinpositive tumors such as neuroendocrine tumors. the cu i -catalyzed azide-alkyne addition (cuaaa), the useful variant of "click chemistry," has emerged as a powerful technique for specific addition. that chemistry is also commonly used for conjugation, and cyclization of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhance potency or selectivity. another useful application of the cuaaa, which we are reporting, is the n-terminal crosslink of two synergic peptides to gain their potency. cuaaa reaction is performed on solid phase (merrifield resin) where one of the peptide components with azido group on the linker ( azido-hexanoic acid) is "clicked" with second peptide component in solution, made by fmoc strategy in partially protected form containing at n-terminal side alkyne group (fmoc-l-propargylglicine). cuaaa coupling is performed in dmf/t-buoh/h o with presence of cui and sodium ascorbate when reacting mixture was degassed. linked peptides are cleaved finally from resin and purified. as an application example we picked two endothelin active peptide analogues: bq derivative (a highly potent and selective eta antagonist) and irl- derivative angiogenesis is a key step in the transition of tumors from a dormant state to a malignant state. the vascular endothelial growth factor (vegf) is a major contributor to tumor angiogenesis. its pro-angiogenic activity is mainly mediated through binding to two tyrosine kinase receptors located predominantly on the surface of endothelial cells: vegfr- and vegfr- . vegf binding to these receptors triggers the activation of different signal transduction pathways responsible for the proliferation, survival and migration of endothelial cells . vegf/vegfr system constitutes a target to stop tumour growth. an attractive approach is the development of peptides, or small-molecules, with a high affinity for the extracellular domain of the receptors to prevent vegf binding. based on the x-ray structure of vegf and the d domain of vegfr- , cyclic peptides had been developed in our group . such peptides, mimicking simultaneously the - loop and helix · of vegf, can bind to d domain of vegfr- and inhibit receptors phosphorylation and thus map kinase pathway . we describe here our strategies to optimize peptidic antagonists of vegfr- . chemical modifications are made in order to better mimic peptide conformations and to increase their receptor binding affinities. we introduce a hydrophobic functional group at the c-terminal of the original cyclic peptide , some of such modified peptides reveal improved vegfr- binding affinity. otherwise, as the helix · presents most of the important residues in vegfr binding according to alanine-scan in the literature , we try to stabilize the helical conformation by insertion of aib residues or by peptide cyclisation. the peptides affinities are evaluated by an elisa test developed previously . institute of chemistry and biochemistry, freie universität berlin, thielallee , d- berlin, germany new polypeptide was isolated from the azemiops feae viper venom by combination of gel filtration and reversephase hplc and called azemiopsin. its amino-acid sequence (dnwwpkpphqgprpprprpkp) was determined by means of edman degradation and mass spectrometry. it consists of residues and does not contain cysteine residues. according to circular dichroism measurements, this peptide adopts a β-structure. peptide synthesis was used to verify the accuracy of the determined sequence and to prepare sufficient peptide amount for biological activity studies. azemiopsin efficiently competed with α-bungarotoxin for binding to torpedo nicotinic acetylcholine receptor (nachr) (ic . ± . m) and with lower efficiency to human α nachr (ic ± μm). ala scanning showed that amino-acid residues at positions - , - and - are essential for binding to torpedo nachr. in biological activity azemiopsin resembles waglerin, a specific blocker of muscle-type nachr from tropidechis wagleri venom. however the sequences of these peptides are markedly different, and azemiopsin is the first natural toxin to block nachrs that does not possess disulfide bridges. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, shiga - , japan while neutrophils infiltrate into damaged sites immediately after tissue injury, endogenous factors which induce their acute transmigration and activation have not been thoroughly elucidated. for the candidates, we recently identified two novel neutrophil-activating cryptides, mitocryptide- (mct- ) and - (mct- ), which were hidden in mitochondrial cytochrome c oxidase and cytochrome b, respectively [ ] [ ] [ ] . in addition, the presence of many neutrophil-activating peptides other than mct- and - was observed during their purification. these findings suggest that neutrophils are regulated by many unidentified peptides. here, we purified a novel neutrophil-activating octadecapeptide whose primary structure was identical to mitochondrial cytochrome c ( - ) from porcine hearts. we named this functional peptide as mitocryptide-cyc (mct-cyc). the structure-activity relationships of cytochrome c on β-hexosaminidase release from neutrophilic differentiated hl- cells demonstrated that cytochrome c ( - ) was the most potent cryptide among cytochrome c-derived peptides. since cytochrome c is known to be involved in the apoptotic process, our present results suggest that cryptides produced from cytochrome c play an important role in scavenging toxic debris from apoptotic cells by neutrophils. anthracis spores are very resistant and can remain dormant in soil for decades. therefore, an effective detection system for b. anthracis is urgently needed. recently, it was found that one of the components of the b. anthracis exosporuim is a collagen like protein whose carbohydrate portion is composed of the tetrasaccharide with the highly specific monosaccharide upstream terminal, named anthrose. since anthrose was not found on other bacterial spores, including those closely related to b. anthracis, this monosaccharide is an attractive target for the development of new b. anthracis detection and identification methods. peptide cyclization represents particularly interesting approach for the design of artificial receptors for anthrose, because cyclic peptides provide the possibility of having a spherical lipophilic binding site of appropriate size and shape for a particular carbohydrate substrate. the presence of hydrogen donor/acceptor groups within a three-dimensional structure permits carbohydrate substrates to be encapsulated, thereby allowing their binding in water. in order to determine whether the cyclic peptide receptor can selectively detect the anthrose, we have successfully prepared cyclic peptide combinatorial library (total peptides) by the process of divide, couple and recombine ("tea-bag" technology) using standard fmoc solid-phase peptide synthesis. prepared combinatorial library is screened for anthrose binding in fluorescence-based assay, and individual cyclic peptides with enhanced affinity toward anthrose are identified by the positional scanning deconvolution process. cyclization of linear sequences is a well-known approach used to restrict the flexibility of peptides. cyclization often increases selectivity of peptides towards one specific receptor type, increases metabolic stability and generally increases lipophilicity, which often improves the bloodbrain barrier permeability of peptides. in our previous study [ ] we have reported on the synthesis of a cyclic endomorphin- (em- ) analog, tyr-c(d-lys-phe-phe-asp)-nh , which elicited analgesia after peripheral administration. encouraged by the fact that this analog was able to cross the blood-brain barrier we designed and aliskiren is the first orally active, direct renin inhibitor to be approved for the treatment of hypertension. its structure and conformational analysis were explored using molecular dynamics (md) simulations. for the first time, md calculations have also been performed for aliskiren at the receptor site, in order to reveal its molecular basis of action. it is suggested that aliskiren binds in an extended conformation and is involved in several stabilizing hydrogen bonding interactions with binding cavity (asp / , gly ) and other binding-cavity (arg , ser , tyr ) residues. of paramount importance is the finding of a loop consisting of residues around ser that determines the entrapping of aliskiren into the active site of renin. the details of this mechanism will be the subject of a subsequent study. additionally molecular mechanics poisson-boltzmann surface area (mm-pbsa) free energy calculations for the aliskiren-renin complex provided insight into the binding mode of aliskiren by identifying van der waals and nonpolar contribution to solvation as the main components of favorable binding interactions. adamantyltripeptides and phospholipids in liposomal bilayers . now, we were primarily interested to study incorporation profile of mannosylated adamantyltripeptides. we have demonstrated that the adamantyl moiety, due to its liphophilic properties, penetrates into the lipid core of the bilayer while the hydrophilic part with the mannosyl moiety is exposed on the liposome surface. after concanavalin a (con a), a lectin, which specifically binds α-d-mannosyl residues, was added to the liposome preparation, increase in liposome size and appearance of aggregates has been observed. the enlargement of liposomes was ascribed to the specific binding of the con a to the mannose present on the surface of the prepared vesicles. the afm analysis revealed that the adamantyltripeptide molecules grouped into small domains that raise above the bilayer surface. the molecule size and molecular geometry, as well as the hydrophilic and hydrophobic surfaces in the structure of mannosylated adamantyltripeptides, are responsible for arrangement of molecules in the lipid bilayer. this approach might be a useful model for investigation of specific protein interactions with membrane receptors. also, the adamantyl moiety may be considered as a potential membrane anchor for different carbohydrate or other molecules of interest, which could be bound on it and thus exposed on liposome surfaces and as such used in targeted drug delivery. the assay is carried out in a well format p and images are captured throughout the course of the assay, thus we can not only determine a ligand's propensity to induce internalization, but also its efficacy and internalization rate. addition of test compound, followed by the standard agonist at a later interval, enables differentiation between agonist or antagonist activities. in the positional scanning format [ ] , while the possibility of agonists and antagonists working against each other within a mixture exists, the effects are minimized in screening the whole library as there are as many arrangements of the sub-libraries as there are defined positions. therefore while an agonist and antagonist might be present in a particular mixture in one sub-library they will be in different mixtures in all other sub-libraries. we have used this assay format to simultaneously screen for novel agonists and antagonists against the orexin receptor. assay development and library screening will be presented. [ ] . since the pro residue in position of em is very important in the proper conformational alignment of the two aromatic residues tyr and phe in em molecule at the receptor site, it is possible that structural modification around the pro residue yields compounds with unique biological properties and improved metabolic stability. in the present study, we synthesized seven em analogues containing isopro or constrained residues with oxopyrrolidine or oxopiperadine ring, instead of pro residue in position . all peptide analogues were synthesized solid phase method. incorporation of oxopyrrolidine and oxopiperadine rings were carried out on a solid support by the methods of gellerman, et al. [ ] and mohamed, et al. [ ] , respectively. opioid receptor binding activity for μ and δ-receptors using the development of resistance to mainstay cancer therapies has become a major limitation for the treatment of many cancers. there is an urgent need to develop new antineoplasic agents with innovative anticancer approaches. to overcome resistance to cancer therapies, our attention has turned to proteins that regulate multiple signalling pathways essential for tumour survival. among the few known nodal proteins upregulated in cancer cells and involved in many hallmarks of cancer, we are interested in survivin. an essential regulator of cell proliferation and apoptosis, survivin is sharply overexpressed in cancer cells and plays a major role in resistance. being a small protein, its bioactivity is relies mainly on protein-protein interactions (ppi) with different partners. a critical point for its multiple functions in cancer is its association with hsp , which is required for its stability. a nonapeptide from survivin called shepherdin has been shown to modulate the interaction of survivin with hsp by binding to hsp and to induce death of tumour cells. unfortunately, shepherdin is not cell permeable, has low proteolytic stability and shows poor bioavailability, limiting its use as anticancer therapeutic agent. to improve pharmacological properties of shepherdin, cyclic and peptidomimetic analogs of shepherdin have been synthesized followed by structure-activity relationship studies. in hsp binding studies, some cyclic hexa-and heptapeptidic analogs showed increased affinity compared to shepherdin. the synthesis of cyclic and peptidomimetic analogs and the results from the binding assays and the conformational analyses will be presented. the hexapeptides with formula ac-ryyr/kw/ir/k-nh have been identified as shortest peptide sequence with high nop receptor affinity, selectivity and marked analgesic effect. it was found that the following peptides act as partial or full agonists or antagonists of nop receptor in different in vivo and in vitro systems. these hexapeptides were used as chemical templates in sar studies , . the aim of the present study was the synthesis and the biological screening of new analogs of ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β -tryptophan analogues . these non natural amino acids were prepared using reaction of asymmetric friedel-crafts alkylation of various indoles with a chiral nitroacrylate to provide optically active β-tryptophan derivatives. the four newly synthesized ligands for the nociceptin/orphanin fq (n/ofq) receptor (nop) have been prepared by solidphase peptide synthesis-fmoc-strategy. these compounds will be tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of wistar rats. bacterial infections are a common problem associated with dermal wounds. these infections can prolong or impair wound healing. hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. in this work, we describe the design and utilization of injectable gels prepared from self-assembling β-hairpin peptides having a high content of arginine. these gels were found to be extremely effective at killing both gram-positive and gramnegative bacteria, including multi-drug resistant p. aeruginosa. importantly, no added antibacterial agents are necessary since the nanostructure of the gel, itself, is the active agent. using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. structure-activity relationships studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. these studies culminated in an optimized gel, composed of the peptide pep r. pep r gels prepared at . wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible towards mammalian mesenchymal stem cells. the general mechanism by which pep r exerts its action was explored and it is suggested that involves membrane disruption that occurs when cells come in contact with the gel's surface. atomic force microscopy (afm) was used to study the effect of the gel on the cell envelope morphology of e. coli. rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. they are intimately involved in the molecular process leading to the delicate nano-patterned silica shells of diatoms. deciphering the mechanisms of silica-biogenesis in diatoms will inspire the development of novel routes for the biomimetic synthesis of silicon-based materials under mild conditions and expand the scope of biotechnological applications, e.g. for immobilization of enzymes in silica matrices. we synthesized silaffin peptides derived from c. fusiformis that carry posttranslational modifications such as phosphorylation or polyamines linked to lysine side chains. a distinct alteration of silica precipitation activity depending on the particular modifications of the silaffins emerged. these modified silaffin peptides were covalently linked to recombinant proteins by expressed protein ligation leading to stable protein-silaffin conjugates. using egfp as model protein, we could show that egfp-silaffin conjugates can induce biomineralization of silica and ensure an efficient and homogeneous immobilization of egfp into silica particles, superior to simple co-biomineralization approaches. moreover, a significant stabilization of immobilized egfp against denaturing agents was observed. we established a method for controlled immobilization of biomolecules based on covalent attachment of silaffin peptides with well-defined silica precipitation properties. currently this method is applied to the immobilization of biotechnological relevant enzymes in order to test their activity and the stabilization effect. herein, we present the covalent functionalization of multiwalled cnts (mwcnts) with organocatalysts based on proline or proline derivatives carrying either a dipeptide or a sulfonamide moiety. two different approaches were followed, namely, covalent grafting of the organocatalysts either at the tips or at the sidewalls of the cnts. for the former approach, mwcnts were oxidized in order to introduce carboxylic units at their tips and make them easily dispersed in aqueous solutions. then, oxidized mwcnts readily reacted with proline-based derivatives carrying a free amino unit yielding the corresponding hybrid materials. for the latter approach, the functionalization methodology based on in-situ generated aryl diazonium salts was followed. in this context, mwcnts were modified with aryl units carrying free amino terminal groups, which were subsequently conjugated with proline-based derivatives carrying a free carboxylic unit. all newly formed hybrid materials were fully characterized with complementary spectroscopic (atr-ir, raman), thermal (tga) and microscopy (tem) techniques. the catalytic evaluation of the activity of the cnt-based organocatalysts in aldol reactions is in progress. financial support from gsrt/ΕΣΠΑ - ΣΥΝΕΡΓΑΣΙΑ through ΣΥΝ- - -ΝΑΝΟΚΑΤΑΛΥΣΗ project is acknowledged. novel organogels based on self assembly of rationally designed pseudopeptides c. pappas, n. sayyad, a.g. tzakos, i. plakatouras section of organic chemistry and biochemistry, department of chemistry, university of ioannina, ioannina, gr- , greece self-assembly is becoming a rather intriguing way to build an array of nano-and micro-structured materials. low molecular weight organogelators can self-assemble into various architectural types in organic solvents through weak intermolecular interactions. such organogelators have potential applications in the generation of novel materials for nanobiotechnology . herein, we report the synthesis of rationally designed pseudopeptides and the conditions to form organogels. the obtained gels are responsive to temperature, and the sol-gel process is thermoreversible. the architecture of the constructed organogels was characterized via tem and spectroscopic techniques. diffusion ordered nmr spectroscopy (dosy) was further utilized to determine differences in the molecular shape of the different pseudopeptides. applications of the resulted compounds in nanotechnology will be reported. since , organocatalysis has met such a great rate of expansion that is nowadays considered the third major branch of modern asymmetric catalysis along with the transition metal catalysis and biocatalysis. after the seminal work of list, lerner and barbas on the enantioselective aldol reaction between acetone and -nitrobenzaldehyde catalyzed by proline, it became clear that amino acids and peptides could serve as an abundant pool full of potential to develop novel organocatalytic motives. following our recent report that the combination of a prolinamide with a thiourea group having as a spacer a chiral diphenylethylenediamine leads to an efficient organocatalyst for the aldol reaction, we recently considered the possibility to couple the prolinamide unit with an urea moiety. one of our main interests was the substitution of the diphenylethylenediamine spacer by a gem diamine derived from an α-amino acid. the gem diamine is easily synthesized via a curtius rearrengement of the corresponding acyl azide. after synthesis and evaluation of a number of potential catalysts, the prolinamide derivative bearing a gem diamine derived from (s)-phenylalanine and an aryl urea moiety proved to provide the best results in the reaction between cyclic ketones and aldehydes. utilizing mol% of our organocatalyst, the aldol products were obtained in high to quantitative yields (up to %), high to excellent diastereoselectivities(up to > : ) and high to excellent enantioselectivities (up to % ee). peptide self-assembled monolayers are of current interest to study physicochemical properties of modified metal (e.g. au) surfaces. rigid peptide scaffolds could enhance the interaction between gold surfaces and labels by reducing and precisely monitoring the distance between the supported monolayers and gold. the c α -tetrasubstituted αamino acid -amino- , -dithiolane- -carboxylic acid (adt) , which contains a cyclic disulfide system, is interesting in this respect because it may allow the parallel binding of the peptide helical chain to the metal surface. adt occurs in nature and has been utilized in medicinal chemistry and in a model compound of [fefe] hydrogenase. we synthesised a series of constrained helical peptides, based on the ala-ala or the ala-aib sequence, containing one or two adt residues. these peptides were functionalised with spectroscopic or opto-electronic labels. among the large number of reactions involving the formation of carbon-carbon bond, the addition of ketones to nitroolefins is a powerful tool for the synthesis of γ-nitrocarbonyl compounds, useful intermediates for pharmaceutical industry. our recently reported primary amine-thioureas based on tert-butyl esters of natural amino acids exhibit excellent performance for the michael reaction of ketones with nitroolefins providing the products quantitatively and almost stereospecifically (> % ee). , using this methodology, enantiopure baclofen and phenibut (analogs of gaba) have been synthesized. polymersupported organocatalysts constitute a great challenge for the michael reaction. in the current study, we report the immobilization of amine-thiourea catalysts containing ( s, s)or ( r, r)-diphenylethylenediamine and tert-butyl aspartate, on various polymer supports, either directly or through spacer units. the solid-supported catalysts evaluated in the reaction between acetone and βnitrostyrene and highlighted the importance of the choice of the polymer as well as the presence of the spacer or not. the direct attachment of the primary amine-thioureaaspartate to a crosslinked polystyrene-divinyl benzene resin containing a uniform distribution of aminomethyl groups provides a supported catalyst that affords the product of the reaction between acetone and β-nitrostyrene quantitatively and in high enantioselectivity ( % ee a. theodorou, g.n. papadopoulos, c.g. kokotos* laboratory of organic chemistry, department of chemistry, university of athens, athens, greece after the pioneering report that proline can catalyze efficiently the intermolecular aldol reaction between acetone and a variety of aromatic aldehydes, it became evident that amino acids and peptides can afford a plethora of different structural scaffolds for novel catalysts. along the first decade of its life, organocatalysis has grown to such an extend that now it is considered the third major branch of asymmetric catalysis. recently, researchers have paid special attention to other amino acids rather than proline. some primary amino acids have already been applied to a number of transformations with success. usually improved catalytic properties are observed when derivatives of primary amino acids are utilised. we have undertaken a study on the application of simple and cheap primary amino acids and amino acid derivatives, either commercially available or easily obtained, as organocatalysts for the asymmetric α-amination of aldehydes. in the present work, we report that the use of simple derivatives of primary amino acids like phenylalanine and aspartic acid can efficiently catalyze this transformation leading to products in high to quantitative yields and enantioselectivities up to % ee. the majority of the organocatalysts developed up to now for asymmetric organic transformations employ more than one functionalities in the catalytic mechanism that act through either covalent or non-covalent interactions. for example, proline employs the pyrrolidine nitrogen and the carboxylic acid group, while chiral thioureas combine the thiourea functionality with a tertiary or a primary amino group. we have recently shown that an amide of proline with a diamine carrying a thiourea group is a very good catalyst for the enantioselective aldol reaction. trying to improve the activity, we have found that a tripeptide-like thiourea having as building blocks (s)-proline, ( s, s)diphenylethylenediamine and (s)-di-tert-butyl aspartate provides the products of the reaction between ketones and aromatic aldehydes in high to quantitative yields and high stereoselectivities (up to : dr and % ee). a number of structural modifications of the catalyst were undertaken in order to understand the role of the hydrogen bond donors of the catalyst, i.e. the prolinamide hydrogen and the two hydrogen atoms of the thiourea group. we have come to the conclusion that the importance of the hydrogen bond donors of the catalyst follows the order: thiourea hydrogen originated from aspartate › amide hydrogen › thiourea hydrogen originated from diphenylethylenediamine. g eldrug s.a., patras , greece a convenient and facile synthesis and in vitro biological evaluation of n-substituted -butylimidazole derivatives as potent angiotensin ii (ang ii) receptor type (at ) antagonists have been reported in the present study. a series of imidazole based compounds bearing the biphenyl moiety at the n- position, a halogen atom at the c- and polar substituents such as hydroxymethyl at the c- position were synthesized. , these compounds were evaluated for binding to human at receptor and for ang ii antagonism in vitro on isolated rat uterus. in particular, butyl- -[[ ΄-( h-tetrazol- -yl)biphenyl- -yl]methyl]imidazole derivatives complexed with the at receptor and showed high binding affinity. these analogues were also found to be active in the rat uterotonic test. importantly, their binding affinities and potencies were comparable to those of losartan. these results indicate that the hydroxymethyl at the c- position of the imidazole ring is favorable for high affinity binding and antihypertensive activity and in line with the activities of the losartan counterparts. experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/at receptor interactions. z-leu-glu-his-asp-aluc, suc-leu-leu-val-tyr-aluc) are good substrates for bioluminescence assays, for example in the detection of caspase activity during apoptosis . these substrates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. luciferase-based assays are typically -to -fold more sensitive than the comparable fluorescent assays (rhodamine , -amino- -methylcoumarin (amc) and -amino- trifluoromethylcoumarin (afc)). the synthesis of different type peptide-amino-luciferin conjugates and their precursors have been published and some of them are commercially available. however, because of their high price the in vivo application of these conjugates is limited. to solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from -chloro-benzothiazole. moreover this products have excellent purity (> %) and adequate yield ( - %). major health problems arising from bacterial resistance towards existing antibiotics make discovery of antibacterial drugs with new mechanisms of action pertinent. although proof of concept for a novel antimicrobial approach using peptide nucleic acid (pna) antisense targeting of essential bacterial genes was obtained a decade ago, this technology is still limited by the lack of carriers that facilitate effective bacterial delivery and confer optimal pharmacokinetic properties to the prospective drugs. [ , ] in the past two decades, parallel efforts of exploiting naturally occurring antimicrobial peptides (amps) as drugs have been made. the cationic amp subclass appears to be directly involved in the innate immune response towards microbial infections. [ ] so far only few cell-penetrating peptides, with activity on mammalian cells, and other membrane-active peptides, have been investigated as potential vehicles for bacterial delivery. for instance, cationic amps with an internal target appear not to have been investigated for bacterial delivery of antibiotics. the aim of this project is to develop highly potent genetic antibiotics by exploiting naturally occurring antimicrobial peptides as potential delivery vehicles for antisense peptide nucleic acid oligomers. the amps are chosen from amps reported to act via intracellular targets, and thus must possess an inherent ability to permeate bacterial cell membranes without direct killing of the bacteria. faculty of chemistry university of gdansk, gdansk, poland azt ( '-azido- ' '-dideoksythymidine), a modified nucleoside used in antiretroviral therapy and peptide plant hormone -systemin were used as substrates of , -dipolar cycloaddition (click chemistry). systemin is -aa peptide defense hormone released in response to plant (tomato, tobacco) damage or pathogen attack. we examinated whether systemin's fast movement through plant tissues could be used for cargo (azt) transport. the huisgen cycloaddition also known as , -dipolar cycloaddition is a chemical reaction belonging to the larger class of cycloadditions. reaction between organic azide and alkyne appended substrates allows the synthesis of the desired conjugate in high purity and yields irrespective of the sequences and functional groups on either of the two substrates [ , ] . conjugate of azt-systemin has been synthesized by click chemistry, using systemin modified at n-terminus with propiolic group and azt. the conjugation was catalyzed by cu(i). the reaction was fast, efficient and regioselective. its progress was easily monitored by capillary electrophoresis (ce). ce was also applied for characterization of systemin and azt-systemin stability and movement throughout tomato leaf and stem. despite the fact that systemin moves rapidly through tomato tissues, our calorimetric (itc) studies showed that the peptide does not interact with liposomes-cell membrane model. universitätsklinik für nuklearmedizin, inselspital, bern, switzerland regulatory peptides (e.g., somatostatin, bombesin) have been shown to be suitable vectors for the specific delivery of radioactivity to tumors for diagnostic and therapeutic applications in nuclear oncology. a potential drawback of such vectors is their inherent instability in vivo. thus, new strategies are needed for the stabilisation of radiopeptides in order to improve their bioavailability and, consequently, increase their accumulation in the targeted tissue. it has been suggested that , , -triazoles, readily obtained by cuaac, are suitable amide bond surrogates which are resistant to proteases. in the present study, we report the synthesis and pharmacological evaluation of radiolabelled, triazole-containing analogues of the gastrin releasing peptide receptor (grpr) targeting peptide bombesin (bbn). to study the effect of backbone modifications in the minimal grp-binding sequence, we synthesized a series of analogues of [nle ]bbn ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , in which each amide bond is individually replaced by a , -disubstituted , , triazole. after radiolabelling of the peptidomimetics, their binding affinity and internalization kinetics were determined using pc- cells. metabolic stability was evaluated in blood serum. a number of the novel tumor-targeting peptide analogues presented exhibit both a retained high affinity (nm) towards the grpr and an improved serum stability. first preclinical data on the in vivo evaluation of the most promising candidate will be presented. to the best of our knowledge, this is the first report of the systematic replacement of amide bonds with , , triazoles within the binding sequence of linear, high affinity peptides. the methodology can be applied to a variety of peptide vectors and thus, holds great potential for the development of novel, stabilized peptide-based radiopharmaceuticals. dna is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a nonspecific target of cytotoxic agents. although this is true for chemotherapeutics, other agents that were discovered more recently have shown enhanced specificity. the development of new site-specific dna binders, which are associated with the recognition of the dna major groove, are based on the design of transcription factor mimics that bind the dna as a dimer , and prevent specific genes from being transcribed. these could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. in order to approach this biological constructs, we choose the bzip leucine zipper transcription factor as a model to mimic. as the entire structure cannot be synthesized without expensive, complicated and time-consuming biotechnological methods, the substitution of the dimerization domain by a less complex scaffold is the first step in the design. thus, we consider a steroid based scaffold as a candidate. the specific choice of the steroid scaffold as substituent is inspired by its known ability to enhance proteolytic stability of attached peptides, by its conformational properties ensuring correct positioning of the two appended chains and by its potential to increase bioavailability. this transcription factor binds specific dna sequences by dimerization and inserting short α-helices into the dna major groove. in order to attach the peptides to the scaffold, different strategies were studied. firstly, applying the well-known click chemistry, functionalizing the scaffold with an alkyne moiety, the peptide with an azide and viceversa. secondly, via the unknown resin to resin transfer reaction (rrtr), which has not been applied on peptide chemistry so far. this unprecedent methodology consists on the reaction of a peptide, which is attached on a safety-catch resin, with a second resin bearing a nucleophilic amino terminus resulting in amide bond formation. during the process, the peptide on solid support undergoes cleavage. an hexapeptide was synthesized on a preloaded safetycatch resin. deoxycholic acid derived scaffold with orthogonally protected amines was attached to tentagel resin that acts as acceptor resin. rrtr experiments were performed at both c and c positions of the deoxycholic acid derivative. in addition, this convergent strategy can be applied to other different peptide conjugated systems. we recently described a new kind of cyclized peptide in which the cyclization is performed between the side-chains of two diaminoacyl residues via a diversely substituted guanidine bridge. we showed that the degree of bridge substitution could impact on the orientation of the bridge inside the cycle and therefore the peptide conformation. we prepared two series ( and atoms cycle size) of cyclic enkephalin analogues to assess the potential effect of this kind of bridge on the biological activity. the compounds were synthesized on the solid support via the formation of a thiourea bridge and with the variable substituent being introduced at the last step before cleavage. it is noteworthy that the synthesis afforded at least two stable and separable conformers for each analogue of the shortest cycle series. generally, one major and one minor species were recovered. but in the case of di-substituted compounds with a cyclic moiety (pyrrolidine or piperidine substituents), three significant species were obtained. analogues were submitted to various biological assays (binding to μ and δ opioid receptors and functional assays). we observed a significant variation in affinity and selectivity for the receptors as a function of the degree of bridge substitution. a structural analysis by d nmr has been undertaken and correlated the variation in activity with a variation in conformation. the origin of the multiple conformers observed for the analogue with a pyrrolidine susbtituent was also investigated. this kind of cyclization could represent a useful tool to easily modulate the conformation and biological activity of a unique peptide sequence. the t-cell response is triggered by the formation of the trimolecular complex between the major histocompatibility complex (mhc), the immunodominant myelin protein epitopes and the t cell receptor (tcr). herein, we report the design and synthesis of non-peptide analogues with the ability to mimic the immunodominant epitope - of mbp , . the mimetics were designed to block the formation of the trimolecular complex and therefore the t-cell activation , . more specifically, indole analogues were synthesized with substitution at positions and or . these molecules contain a carboxyl or an ethyl ester group in position and a benzylamino or phenylamino group in position or . the synthesis of the indole ring was achieved by fischer reaction followed by catalytic hydrogenation, reductive amination or arylation and ester hydrolysis. the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, japan amyloid β peptide (aβ), the main component of senile plaques in the brain of alzheimer's disease (ad) patients, is formed by proteolysis of amyloid precursor protein (app). as β-secretase (bace : β-site app cleaving enzyme ) triggers aβ formation by cleavage at the aβ domain nterminus, it is a molecular target for ad therapeutic intervention. previously, we reported potent pentapeptidic and non-peptidic bace inhibitors containing a substrate transition-state mimic. although these inhibitors exhibited potent inhibitory activities, their molecular-sizes appeared a little too big (mw> ) for developing practical drugs. in this study, we designed a series of small molecular peptides, with bace inhibitory activity, lacking the p -p ' region on the basis of the conformational structure bound in bace . design and synthesis of new '-peptidyl-trna analogues, in particular "hydrolysable" analogues, which represent covalent conjugates of peptide-nucleic acid (pna) with "stop-peptides," were carried out. such compounds are of interest as tools to study the ribosome functioning and as inhibitors of protein biosynthesis. ( aminoethyl)glycine pna models '-end trna sequence cca in designed structures. computer simulations showed the formation of watson-crick pairing of the pna cytosine residues with s rrna nucleotides g and g involved in interactions with peptidyl-trna during its specific binding in p site of the ribosomal peptidyl transferase center (ptc). short "stop-peptides" were planned for conjugation with pna. these peptides form stable complexes with the ribosomal tunnel (rt) that leads to ribosome stalling and translational arrest. structures of "hydrolysable" 'peptidyl-trna analogues that could form peptide bond with amino acid residue of aminoacyl-trna in a-site of ptc included '-deoxyriboadenosine instead of the pna adenine containing residue. such conjugates would permit to identify the chemical nature of specific sites localized in rt and responsible for interactions with amino acid residues of the nascent polypeptide chain. pna and "stop-peptide" as well as pna-"stop-peptide" conjugates were prepared by solid phase synthesis on sasrin polymer using fmoc/bhoc(boc) strategy. synthesis of "hydrolysable" conjugates included modification of the 'hydroxyl of '-protected '-deoxyadenosine by n-blocked "stop-peptide", deprotection of the '-hydroxyl, its conjugation with n-protected pna and removal of protecting groups from the resulted conjugate. the binding of the new '-peptidyl-trna analogues with ribosome will be tested by chemical probing and in the cell free translation system. this study was supported by the russian foundation for basic researches (grant - - -a). a close structural similarity of endomorphin- and another atypical opioid peptide, morphiceptin, which both have a phe residue in the third position, encouraged us to study antinociceptive activity of these two peptides and their analogues. in order to improve the affinity and chemical stability of these opioid peptides, we have designed, synthesized, and analyzed novel analogues. the first modification included endomorphin- and morphiceptin analogues, where halogenated phenylalanines in position or were incorporated as surrogates of the native phenylalanine. another important modifying element is non-protein amino acid canavanine (cav) and its analogue (sarg). it is well documented that cav and sarg exhibit strong analgesic activity. two new morphiceptin analogues were synthesized by introducing cav and sarg in position . we further characterized their antinociceptive activities by the paw pressure (pp) test. the experiments were carried out on male wistar rats ( - g), treated with i.p. doses of mg/kg. e eldrug s.a., patras , greece the renin angiotensin system (ras) has been a prime target for the therapy of cardiovascular diseases. angiotensin ii type (at ) receptor mediates vast majority of biologically detrimental actions. non-peptide at receptor blockers are presently the most specific means to block the ras enzymatic cascade. the dupont group was the first to develop losartan (dup ), an orally effective angiotensin ii receptor blocker, which is metabolized in vivo to the more potent antagonist exp . herein, we report on the preparation of e-urocanic acid based analogs, focusing our attention on the introduction and structural modifications of the substituents on the imidazole ring as well as the modifications on the acrylic side chain. in particular, we have designed and synthesized a series of urocanic acid analogs bearing the biphenylmethyl tetrazole moiety at the n- of the imidazole ring. additionally, the rigid acrylic chain was lengthened by esterification resulting in the ethyl ester and on the other hand the latter was readily converted to the corresponding acrylic alcohol or aldehyde which may proved to be effective structural elements for enhancing biological activity. finally, a lipophilic alkyl chain such as the n-butyl group was introduced at the -position of the ring which may possibly enhance the antihypertensive activity. docking studies and biological evaluation of the synthesized analogs are being undertaken. university of athens, department of chemistry, laboratory of organic chemistry, , panepistimiopolis zografou, athens, greece the backbone modification of bioactive peptides with replacement of a scissile peptide bond in enzymatic hydrolysis is a well-established strategy for developing protease inhibitors. in particular, for zinc metalloproteases, which contain a zinc atom in their active site, several successful modifications have been reported over the past years. phosphinic pseudopeptides are among the best candidates when addressing the challenge to potent and selectively inhibit zinc proteases. a thorough search in the literature revealed the absence of any reference regarding thiophosphinic pseudopeptides. we thought that this class of compounds would add a valuable tool in the field of zincbinding groups. in the present study, we describe in detail the first synthesis of a new class of phosphorous compounds, thiophosphinyl dipeptide isosters (tdis). we prepared several fully protected thiophosphinate pseudodipeptides of the general formula pg-phe-Ψ[p(s)(ox)ch ]-gly-pg' starting from the corresponding phosphinate pseudodipeptide using lawesson's reagent. selective deprotection of these compounds was also studied and the results are disclosed. these compounds can be used as building blocks for the synthesis of longer thiophosphinic pseudopeptides after suitable deprotection and elongation as well as transition transition state-mimicking inhibitors for several zinc metalloproteases. in the last decade, trypsin inhibitor sfti- isolated from sunflower seeds [ ] has become one of the most studied peptidic inhibitors of serine proteases. owing to its small size and a strong trypsin inhibitory activity (ka = . × m - ), sfti- is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [ ] . it could also serve as an affinity probe for the isolation of trypsin like (sfti- ) or chymotrypsin like ([phe ]sfti- ) proteinases. following this idea, we decided to synthesize a set of cell-permeable monocyclic sfti- analogues with a fluorophore moiety attached at their n-termini. the presence of the fluorophore in the molecule enabled us to show that the analogues can cross the cell membrane. the cell penetration assay was performed using multiple cell lines (hela cells and human fibroblasts cell line ( br. n) was obtained from european collection of cell cultures (ecacc)). for all the obtained peptidomimetics, we determined the association constants with cognate proteinases. selected peptides were also used as a probes for the detection of inhibitor -proteinase complex, which was achieved by the means of gel filtration chromatography equipped with fluorescence detector and acrylamide native gel electrophoresis. the functional reconstruction of folded protein surfaces with peptide-based mimics is an enormous scientific challenge. the majority of proteins show activity through a small area of their folded surface: "the binding site". however, linear peptides are too flexible and seldomly adopt the correct d-structure of the binding site spontaneously. therefore, they show limited or no activity at all . crucial for activity is to control the secondary (αhelix, β-sheet and/or β-turn) and tertiary structure (relative orientation of subdomain structures). we present the development of a new type of watersoluble scaffolds that have the potential to control both secondary and tertiary structure of discontinuous (i.e. double-loop) protein mimics. the new scaffolds contain a first pair of reactive functionalities to constrain the linear peptide conformation via a 'clips' reaction , stabilizing the secondary structure. next to this, a second functionality allows for ligation of two dissimilar constrained peptides to form a discontinuous binding site mimic via oxime-ligation or click-reaction. these ligations offer the ability to position different peptide loops in d, thus mimicking the tertiary structure of the native protein. most unique to our approach is the fact that all chemical conversions are performed in aqueous media, using side-chain unprotected peptides . growth hormone-releasing peptide (ghrp- ) is a synthetic hexapeptide (his-d-trp-ala-trp-d-phe-lys-nh ), which interacts with two kinds of receptors: growth hormone secretagogue receptor a (ghs-r a) and cluster of differentiation (cd ). the latter is a membrane glycoprotein member of the class b scavenger family, and decreases the internalization of oxidized lipids into macrophages, as well as causes inhibitory effects on angiogenesis associated with binding to thrombospondin. to increase activity and selectivity for the cd receptor, different analogues of ghrp- were synthesized. in particular, substitution of trp in ghrp- by aza-amino acids has given selective analogs, due likely to induction of a β-turn secondary structure. for aza-peptide synthesis, a submonomer solid-phase approach has proven effective to introduce side chains onto the semicarbazide residue. studying influences of benzylidene, benzhydrylidene and fluorenylidene residues during the alkylation of the semicarbazide, superior conversion was observed with fluorenone derivative, and mild alkylation conditions employing et noh as base have improved yields and minimized racemisation. our presentation will focus on the improved submonomer synthesis method for optimization of selective and potent cd- ligands with antiatherosclorotic and anti-angiogenic effects. for instance, the integrin αvβ , vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. integrin αvβ also play a significant role in tumor growth, invasion and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (rgd) tripeptide sequence. rgd has been shown to be potent antagonist of the integrin αvβ , and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. in this context, drug design based on the rgd structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer. we designed and synthesized series of short rgdmimetics containing the sequence xaa-gd, where xaa is arg-mimetic. as promising candidates we have chosen canavanine (cav) and canaline (can) instead of the basic residue arg. in order to improve antitumor activity of the parent molecule, c-terminal modifications were also applied. their cellular uptake was determined on human breast (mcf ) cancer cell lines. furthermore, the in vitro cytostatic effect was evaluated by mtt assay on human liver hepatocellular carcinoma (hepg ) and human breast (mcf ) cancer cell lines after , and hours of treatment. in the case with the human tumor cell lines (hepg , mcf ) and c-modified analogues, statistically reliable results were achieved for the most of concentrations used. acknowledgements: this work was supported by bulgarian ministry of education and science, project my-fs- / . microwave assisted solid phase synthesis of urea and urea/amide based foldamers k. pulka, c. douat-casassus, g. guichard* european institute of chemistry and biology, university of bordeaux -cnrs umr , pessac, france foldamers are fully arti cial molecules that structurally and functionally mimic variety of biopolymers. among them, aliphatic n,n'-linked oligoureas with proteinaceous side chains can adopt extremely robust helical folds stabilized by intramolecular three-centred h-bonds. owing to their resistance to enzymatic degradation, diversity of side chains and structural predictability urea-based foldamers represent unique scaffolds to elaborate functional mimetics of α-polypeptides. of note, heterogenous oligo(urea/γamides) backbones obtained by substituting nh groups by ch display very similar folding propensities. in our laboratory we are investigating the solid phase synthesis of urea and urea/γ-amide oligomers. urea bonds are incorporated into the growing chain by reaction of active succinimidyl carbamates. previously we have applied two different strategies involving fmoc-or bocchemistry, but both methodologies suffer some limitations. therefore a new strategy (compatible with the use of tfa sensitive linkers and side chain protecting groups) featuring azide as a masked amine group has been developed. the synthesis of new azido protected succinimidyl carbamate building blocks is reported. they were obtained in steps from α-amino acids ( - % overall yield). the staudinger reduction with pme was successfully applied to restore the amine group after urea formation on solid support. in addition, microwave irradiation has been found to dramatically accelerate the synthesis. overall, this azide-strategy combined with microwave irradiation was found to be very effective for the solid phase synthesis of oligoureas and related hybrids, surpassing previously developed approach utilizing fmoc chemistry. these antibiotics should have a mechanism different from currently used antibiotics to circumvent existing resistance mechanisms . previous results have shown that "genetic" antibiotics operating by gene silencing in bacteria via rna interference may be successful new candidates. efficient silencing requires efficient crossing of cell membrane. this step can be alleviated using cell penetrating peptides (cpp) as carrier of drug candidates, such as peptide nucleic acids (pnas) which inherently have poor internalization properties . the aim of this study is to elucidate mechanisms of uptake in bacteria using pna-cpp conjugates, which previously have shown promising antibacterial effects . the fate of the pna and cpp parts of the conjugates, once inside the cell, is investigated regarding localization and possible degradation within the cell. furthermore, a method for toxicity testing of pna-cpps is being developed using histamine release in rbl- h cells as a quantitative measure of allergenicity of pna-cpps. the prospect of this information is to define boundaries within which cpps can be found, thereby rationally designing novel efficient antibacterial biomolecular drug delivery systems. oxytocin and its fragments have the potential to influence behavioral and cognitive functions, including their disturbances in some brain disorders. therefore, there is an interest to synthesize new peptide-steroids chimeras for potential therapeutic use. oxytocin analogue was synthesized in solution by coupling azido-phenylalanyl residue or p-azidopegylated handle to the n-terminal end of oxytocin molecule. its c-terminal fragment pro-leu-gly-nh (mif- ) was elongated at proline residue by the same type of azido handles as well. both peptides were marked for fluorescent detection of their possible binding on brain slices. peptide chimeras with the suitable steroids were prepared via azide click to the triple bond on the modified steroid counterpart like ( α)- -hydroxypregn- -en- -yn- -one, -norchol- -en- -yn- β-ol. steroidyl-peptides were then used in the trials using rat-brain slices. the sites of the peptide-steroids chimeras bound to the brain tissue were identified with the aid of fluorescent microscopy. the suitable chimeras will be tested for their penetration through blood brain barrier for the pharmacological effects. indicating that the orientation of the n-butyl group is of primary importance. docking studies revealed that the highly active analog affords an additional hydrophobic binding feature compared to losartan which fits to an extra hydrophobic cavity. these results may contribute to the discovery of new biologically active molecules by a convenient and cost effective synthetic strategy. the context of pain research, the co-administration of opioid agonists and nk antagonists previously led to an enhanced antinociceptive potency, and recently largent-milnes and co-workers have shown that a hybrid opioid-nk octapeptide was able to attenuate tolerance development, related to sustained opioid treatment. our group has prepared a compact opioid agonist-nk antagonist peptidomimetic chimera dmt-d-arg-aba-gly-nme- ', '-bn(cf ) that served as a lead structure. we report a solid phase method for the synthesis of the amino- , , , -tetrahydro- -benzazepin- -one (aba) structure, which is used as a central unit in the investigated dual ligands. this method allowed the rapid assembly of new bifunctional ligands containing the aba structure. variations of the d-arg , gly and n-benzyl substituents were made. the introduction of d-cit , a gly → β-ala substitution and the removal of the trifluoromethyl substituents in caused considerable shifts in receptor binding. the obtained structure-activity relationships will be presented. hence, a promising approach for the treatment of dmd is the use of drugs to force ptc readthrough. (+)-negamycin is a dipeptidic antibiotic containing a hydrazide structure. although (+)- was not clinically developed due to some toxicity, it was recently reported that (+)- restore dystrophin expression in the muscles of mdx mice, an animal model of dmd. therefore, (+)- is a promising therapeutic candidate for diseases caused by nonsense mutations. based on our own efficient total synthetic method of (+)- , structure-activity relationship (sar) study was perfromed to discover derivatives with a potent readthrough-promoting activity. we found a derivative, ( r)- -hydroxy- -aminohexanoyl-glycine exhibited not antimicrobial activity but a similar readthrough activity to (+)- , suggesting that the ptc readthrough mechanism can be distinguished from the antimicrobial mechanism. moreover, we synthesized -epi-negamycin and found that this analog exhibited a similar activity to (+)- in in vitro readthrough assay. this result hence prompted us to synthesize a -dehydro-derivative, e.g., -dehydro- -epinegamycin , which is a natural product with little antimicrobial activity. surprisingly, we found that showed a higher in vitro readthrough-promoting activity than (+)- . this result suggests that mother nature independently evolved readthrough-promoting products like suppressor trna, in distinction from aminoglycosides, which show both antimicrobial and readthrough-promoting activities. agricultural university of athens, athens, greece high interest has been paid to synthetic structural motifs that promote specific conformations because of their importance for the development of new therapeutic peptidomimetics. in addition, such motifs may show catalytic activity for asymmetric organic transformations. during the last two decades, various synthetic structural motifs that promote reverse turns have been studied. following our interest on chiral prolinamide-thioureas that present interesting organocatalytic activity, we have undertaken a combined experimental/computational study to understand the structural features that may stabilize a reverse turn in short-length peptidomimetics containing a thiourea functionality. compounds with the sequence r-pro-diphenylethylenediamine-thiourea-asp(obut)-obut (r: boc or fmoc, or boc-ala), were synthesized and studied by nmr spectroscopy (tocsy, h- c hsqc, noesy, roesy spectra) for the sequential assignment and the exploration of the dipolar connectivities. sampling of the conformational space was driven by the noe intensities while molecular dynamics simulations were further applied to the consistent with the experimental data conformers in order to monitor the stability of the formed hydrogen bonding interactions in the course of time. energy refined produced conformers were subsequently modified by applying all combinations of d-and l-amino acids at each site in a stepwise manner. the modelled structures were studied in silico aiming to explore the combinations of heterochiral residues which would promote a folded structure and would favour the potential of β and γ turn motif. the most promising combinations were chosen for synthesis and subsequent nmr characterization. in this research project we will deal with chemical strategies to produce suitable surface modifications in order to induce multidirectional cellular migration along gold surfaces. to achieve this objective we want to use and characterize self-assembled monolayers (sams) of thiolated dna chains (dna-sh) adsorbed on gold surfaces through the hybridization with complementary modified single-stranded pnas. pna is a structural dna mimic obtained by polymerization of n-( aminoethyl) glycine monomers that replace the ribose-phosphate backbone characteristic of natural nucleic acids. it is an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability, and it can bind complementary dna strands with higher affinity than the corresponding dna sequences.for all these reasons we have chosen pna as a key molecule to promote and assist the movement of cells. by producing a chemical gradient of dna-sh along a gold surface in the presence of a chemotactic molecule it will be possible to obtain and control a directed cellular migration. the norwegian structural biology centre and the centre for theoretical and computational chemistry, department of chemistry, university of tromso, troms , norway renin is a highly selective aspartic protease which catalyzes the hydrolysis angiotensinogen, a protein secreted from the liver, to the decapeptide angiotensin-i. angiotensin-i is further processed by the relatively nonspecific angiotensin converting enzyme (ace) to give the octapeptide angiotensin-ii, a potent vasoconstrictor and the dominant peptide produced by the reninangiotensin system. renin catalyses the rate determining step in the formation of angiotensin-ii, and has for several decades been an established therapeutic target for drug development in relation to hypertension. in the search for renin inhibitors, substituted piperidine derivatives have been identified as promising, - and piperidines have proven to be efficient scaffolds for the development of novel non-peptide aspartic protease inhibitors, particularly towards renin. [ ] [ ] [ ] we herein describe a series of -triazolyl substituted piperidine derivatives that have been synthesized from n-boc protected trans- -ethynyl- -hydroxy piperidine and tested as novel renin inhibitors. piperidine derivatives containing a -substituted , , -triazol- -yl substituent were found to be most active and molecular docking experiments provides a rank order that is in very good agreement with experimental data. the cxcr /sdf- axis is involved in many biological processes such as hematopoiesis, immune cell migration, as well as in cancer metastasis. cxcr also mediates the infection of t-cells with x -tropic hiv functioning as a coreceptor for the viral envelope protein gp . cxcr , as a pharmaceutical target, is of utmost importance but the lack of synthetic agonists has seriously slowed down drug development. it has been recently described by our research group , that grafting the sdf- n-terminus onto a side-chain of the inverse agonist t . generated high affinity synthetic agonists as well as partial agonists for the chemokine receptor cxcr . to remain stable towards proteases and act as useful pharmaceutical tools, the pk-adme properties need to be improved with a gradual transition to peptidomimetic structures. medicinal chemistry witnessed major advances with the discovery of small synthetic molecules that mimic the natural peptidic substrates. these small molecules do not undergo proteolytic degradation, an advantage they hold over natural counterparts. in order to improve stability against proteases, part of the sdf- chain was replaced with variable lengths of polyethylene glycol and unnatural amino acids at differents positions. here, we have produced a series of compounds, most of which showing nanomolar affinities for cxcr and some are displaying partial agonistic properties. tlrs are the innate immunity receptors that recognize the epitopes found on surfaces of various cells and therefore they initiate and sustain the atherogenic inflammatory response [ , ] . we assume that the use of small stat mrna−binding pna−inhibitors to manipulate the activity and expression of stat could prove an attractive therapeutic strategy in treatment of atherosclerosis. to that end we synthesized a specific stat mrna−binding pna inhibitor as well as a non-specific pna to compare their inhibition of gene expression. in our work we developed effective method of synthesis of pna−peptides conjugates by means of "click chemistry". determination of optimal conditions for conjugation (connection of pna with the peptide) will allow for the design of compounds useful in gene therapy. the specificity of pna hybridization to complementary dna fragment was verified by capillary electrophoresis (ce). as an artificially synthesized somatostatin analogue, tyr octreotate (toca) can specifically bind to somatostatin receptor (sstr), which are usually over-expressed on many tumor cells. carbohydration of n-terminus of toca has resulted in improved pharmacokinetics and tumor targeting ( ) . f is an ideal nuclide for positron emission tomography (pet) imaging; there may be significant uses of f labeled glucitol-toca and its analogues as tumor probes for the diagnosis of sstr-positive tumors. in order to explore a novel pet probe for diagnosis of sstrpositive tumors, we designed a synthetic route to synthesize n-gluc-lys(nota)-toca, which uses , , -triazacyclononane- , , -triacetic acid (nota) as the chelating reagent. n-gluc-lys([al f]nota)-toca is radiosynthesized quickly and efficiently using the chelation reaction of al f complex and n-gluc-lys(nota)-toca. the aim of this study is to develop an efficient method for the synthesis of monomers of triazolic nucleic acid (tna), a new class of artificial nucleic acids. but- -yne- , -diol and nucleobases derivatives will be substrates of the monomers synthesis. tna oligomers could be used as specific inhibitor of tar rna hiv- , the regulatory rna structure crucial for hiv replication. "click chemistry" based on , -dipolar cycloaddition will be used to conjugate an alkyne and azide derivatives of monomers subunits. a ru (ii) complex will be used as a catalyst of internal alkyne (but- -yn-based) cycloaddition. the reaction gives exclusively of , , -trisubstituted derivative of triazole ring . the monomers will be characterized using rp hplc, capillary electrophoresis (ce) and h and c nmr. the resulting monomers containing fmoc-protected amino group and a free carboxyl group will be used for the classical spps method to synthesize tna oligomers. tna sequences will be designed against tar's bulge and an external loop. through the recognition that the repertoire of polypeptide conformations can be greatly expanded by the creation of structures incorporating β-amino acids. moreover, the numerous advantages of hybrid (mixed α-and β-) backbone peptidomimetics with respect to homogeneous ones were quite recently outlined. we describe here various β-amino acid-based β-hphe-β-hphe dipeptide derivatives, also conformationally constrained, and their application to the synthesis and biological evaluation of hybrid analogues of the opioid endogenous peptide endomorphin- (em- ). the opioid system mediates a wide variety of pharmacological and physiological processes, including pain perception and modulation. the amidated tetrapeptide em- has been shown to be μ-opioid receptor (mor) agonist exhibiting a very high μ-receptor affinity and selectivity, and it is an important model in the search towards new analgesics. structural investigation of em- reveals the high conformational freedom of the phe side chains and also the inherent flexibility of the peptide backbone, indicating many probable bioactive conformations, ranging from βturns to extended conformations. with the aim of better clarify the relevant role of the proper spatial orientation of the aromatic rings and in particular of the benzyl side chains at position and , h nmr studies, molecular modelling, and molecular docking to a homology mor model of our hybrid analogues are currently under way. the lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity.the lipid ii -nisin complex is responsible for pore-formation since the c-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. in order to increase the metabolic stability of nisin, the oxidationsensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin ab(c) analogs containing alkane/alkene bridges [ ] . to obtain more insight into the importance of the cross-bridged de-ring structure (i→i+ , i+ →i+ connectivity) on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged de-ring mimic, using ring-closing metathesis. all four diastereoisomers were obtained by hplc and structurally characterized by nmr spectroscopy. an orthogonal protection scheme was used, to enable the independent n-or c-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. via cu(i)-catalyzed cycloaddition chemistries, alkyne-functionalized natural abc-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by hplc purification, have been conjugated to synthetic de-ring mimics to obtain novel nisin derivatives and their affinity toward lipid ii and pore-forming capacity have been studied. herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic de-ring mimics, and their use as synthons in cu(i)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics. università di ferrara, dipartimento di biochimica e biologia molecolare, ferrara, italy mirnas play an important role in regulation of gene expression, being involved in numerous processes such as cell proliferation, cell differentiation, apoptosis and also in the progress of diseases as cancer and cardiovascular disorders. mirnas associated to diseases recently become targets for the development of new drugs based on antisense oligonucleotides or analogues complementary to the chosen mirna, in order inhibit the binding of the mirna to its mrna target. therapeutic silencing of mirna has been also observed in several animal disease model. in this work we propose a new approach to interfere in the mirna function, based on peptide nucleic acid (pna) oligomers designed to be complementary to selected regions of the mirna precursor (pre-mirna). as the pre-mirna bases belonging to the stem are not perfectly complementary, we hypothesized that the mismatched duplex of the pre-mirna could be opened by pnas inhibiting of its maturation into mirna. two pna sequences, targeting respectively the "sense region" and the " ' end region" of the pre-mir were designed. pnas were conjugated to different carrier peptides, hiv-tat, r , k and two nuclear localization signal (nls and binls), in order to increase their cellular uptake. to verify the ability of the designed pnas to give strand invasion on the pre-mirna, we conjugated also pnas to the thiazole orange, a probe which lights-up upon hybridization the development of privileged molecular scaffolds efficiently mimicking reverse turn motifs has attracted remarkable interest when structural constraints are exploited to increase both binding and selectivity of model peptides. one of the successful approaches to restrict peptide conformation is the disubstitution in the α position of an α-amino acid, leading to a conformational constraint and a stereochemically stable quaternary carbon center. in particular, spirocyclic scaffolds are able to provide, upon the attachment of appropriate functional groups, useful high-affinity ligands, relevant to the field of drug discovery. at present, we are interested to spirocyclic tryptophan (trp) analogues, in order to develop new reverse turn nucleating moieties able to be inserted into pharmacologically relevant peptidomimetic compounds. among peptides sharing a tryptophan-containing β-turn motif of which the trp residue is critical for binding, we looked at the hormone peptide somatostatin, acting in various organ systems as a neuromodulator and a neurotransmitter, as well as a potent inhibitor of various secretory processes and cell proliferation. somatostatin and its analogue octreotide (sandostatin® drug, clinically used for the treatment of endocrine tumors and acromegaly) are thought to interact with the sst - receptors mainly by inserting a β-turn substructure, carrying a lysine (lys) and a trp side chain into a pocket of the g protein-coupled somatostatin receptor. we report here the preparation and structural characterization of a new , , , -tetrahydro-β-carboline (thbc)-based spirocyclic lactam as type-ii β-turn model compound and the application of its core structure to the synthesis of a somatostatin mimetic, whose biological evaluation is under way. the analogues of sfti- modified in the p position by, βand γ-amino acids and n-substituted β-alanines r. lukajtis, a m. filipowicz, a a. legowska, a d. debowski, a a. lesner, a k. rolka a a faculty of chemistry, university of gdansk, - gdansk, poland serine proteinases play very important roles in many physiological processes in humans, such as: food digestion, fertilization of the ovum, blood clotting and dissolution of blood clots, immune response. however, their uncontrolled activity can evoke serious pathological conditions. therefore, serine proteinase inhibitors are considered to be a promising class of therapeutic agents. trypsin inhibitor sfti- , on which we focused our attention in the last decade, is an attractive template for the design of such compounds. its primary structure is shown below: & gly-arg-cys(& )-thr-lys -ser -ile-pro-pro-ile-cys(& )-phe-pro-asp& the inherent feature of natural peptides and proteins is their low stability towards proteases, which seriously reduces their bioavailability. there is a growing need for the development of artificial biopolymers with diverse side chains, capable of mimicing peptide function. β-and γpeptides are an interesting class of peptidomimetics with significant chemical and biological properties. the present communication describes the chemical synthesis and inhibitory activity of a series of trypsin inhibitor sfti- monocyclic analogues (with disulfide bridge only) modified in p position by βand γamino acids and n-substituted β-alanine (β-peptoid units). the following mimetics of proteinogenic lys or phe were used: β hlys, β hphe, γ hhlys, γ hhphe, βhnlys, βhnphe. all compounds were synthesized manually on solid support. β-peptoid monomers were introduced into the peptide structure by two steps method [ ] . newly obtained sfti- analogues modified in p position by β-derivatives of lys and phe were able to inhibit bovine β-trypsin and bovine αchymotrypsin, respectively, whereas the remaining ones (except for [βhnphe ]sfti- ) appeared to be inactive. the notion that early soluble aß intermediates are endowed with cytotoxic effects suggests that a major effort should be directed toward the inhibition of amyloid aggregation at very early stages. inhibiting aß self-oligomerization could, therefore, provide a useful approach to treating and controlling the pathogenic pathways underlying alzheimer's disease (ad). likely, agents that target the basic molecular recognition process preceding the formation of early intermediates are the most valuable candidates. we have conjugated a trehalose moiety to the known ß-sheet breakers pentapeptides lpffd. trehalose has received a special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation. the glycosidic moiety was covalently linked to different regions of the peptides' primary sequence, including the n-terminus or c-terminus or the aminoacid side chain. this new class of peptides showed an increased resistance to proteases. in this work, the inherent ability of these peptides to recognize and bind the monomeric form of recently reported a d-amino acid-containing hiv protease inhibitor with a sulfonyl group showed an activity enhancement against drug resistant viruses. x-ray crystallographic study of the derivative revealed existence of four bridging water molecules. we suggest that the additional indirect interactions through water molecules induced the inhibitor's flexibility in binding conformation, keeping the affinity with the mutated proteases. oxalyamide, so-called oxamide, has two carbonyl oxygen atoms as hydrogen bonding acceptor similar to sulfonyl group, which is promising to interact with water molecules. to increase the numbers of bridging water molecules, we built-in two oxamide structures to both terminals of pseudo-symmetric compounds with hydroxymethylcarbonyl-hydrazide isostere. the derivatives were tested for inhibitory activity using wildtype hiv protease and a highly mutated protease with lopinavir resistance. we found that the loss of potency against the mutated protease was relatively small in the oxamide derivatives. the molecular dynamic simulations suggested the ability of bridging water formation of the two oxamide groups. optimization of the pna-synthesis using different bases for fmoc-deprotection s. rawer , k. braun , r. pipkorn life technologies, darmstadt, germany dkfz, heidelberg,germany pna (peptide nucleic acids) are considered as highly sensitive and specific tools for antisense strategies especially conjugated with cell penetrating peptides. individual designed shuttle systems can be applied in cancer diagnostics and possible therapy ( ) . it is, however, undisputed that proper pnas' syntheses prove to be a challenge for coupling and fmoc-deprotection. due to the structure-formation the success of the synthesis depends strongly from parameters, like activator's quality and deproctection kinetics correlating to the length of the pna polymer spps product. using the example of the spps pna synthesis' results of the coding sequence of c-myc human exon ii, different bases, acting as fmoc-deprotection reagents, are compared and analyzed aiming at optimizing the pna synthesis strategy ( ) peptidoglycans are central structural components of the cell wall of bacteria. several plant receptors are known to recognize peptidoglycan fragments. it is believed that these receptors form part of the defense mechanism against bacterial infections in several plant species. peptidoglycans consist of long chains of alternating β( - )linked glcnac and murnac moieties that are crosslinked by short, non-ribosomal peptides. these peptides consist of several d-amino acids and the symmetrical (r,s)diaminopimelic acid (meso-dap). in particular, the latter complicates the synthesis of peptidoglycan fragments due to the requirement for individually addressing the two pairs of functional groups. some chemical syntheses of peptidoglycan fragments have been reported [ ] [ ] [ ] [ ] , hhich involved multi-step formation of an orthogonally protected dap moiety, and elaborate oligosaccharide synthesis. here we present a new and simple approach to peptidoglycan synthesis which is based on the use of commercially available building blocks for the dap and oligosaccharide components. this allows easy access to a range of peptidoglycan fragments for structure-activity studies. the introduction of solid-phase peptide synthesis (spps) and the subsequent refinement of resins, linkers, coupling reagents and amino acid protecting groups allowed access to a wide range of peptides. therapeutic peptides, in particular, have benefitted from the maturation of spps, as complex peptides can be synthesized more efficiently in comparison to conventional solution phase synthesis. however, peptides containing multiple disulfide bonds often still remain difficult to make due to a lack of orthogonal cysteine protecting groups that can be used in routine spps. the cysteine protecting group s-tertbutyl mercapto (s-tbu) is commercial and orthogonal to other cysteine protecting groups. removal of the protecting group is facilitated by reducing agents (e.g. thiols or phosphines) and is stable to tfa and piperidine, hence compatible with fmoc/o-tbu peptide synthesis. however, the protecting group cannot be used in routine spps due to long deprotection times ( - h) . in certain cases it has been shown to be impossible to remove due to proximity of bulky protecting groups and sensitivity to certain sequences. additionally, reports of desulfurization of s-tbu protected cysteine to dehydroalanine, by the use of prolonged exposure to reducing agents, show the limitations of this protecting group. the concept of cysteine protecting groups labile to reducing agents is promising due to orthogonality to other cysteine protecting groups and the limitations of s-tbu initiated an investigation into novel reductive cysteine protecting groups. herein, we introduce s-tmp as a novel cysteine protecting group that is very labile to reducing agents. the increased lability, in comparison to s-tbu, allows utilization of reducing agent labile protecting groups in routine peptide synthesis of disulfide containing peptides. as modern automated spps protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. although different methods have been developed for monitoring spps, we observed that the use of colorimetric test or continuous-flow uv absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis. in this study we demonstrate the usefulness of the combination of a mw-assisted mini-cleavage protocol and the uplc-esi-ms analysis for monitoring the quality of the reaction steps. as a proof of concept, based on this strategy, we monitored the synthesis of pthrp( - )nh (synthesised by fmoc/tbu rt-spps, liberty™, cem), characterised by a cluster of arginine residues in the - region. by the use of mw irradiation during the mini-cleavage protocol, we optimized time for mini-cleavages particularly in case of multi-arginine containing peptides, protected by pbf group. the results obtained by uplc-esi-ms showed that the complete removal of the pbf groups from the arginine sidechain residues required h at rt. on the other hand, the mw-assisted mini-cleavage monitoring let us to obtain final results just in min, confirming that the use of microwave irradiation in mini-cleavages is an efficient strategy to monitor also difficult peptide couplings, such as multiarginine peptides. identification of some deletion sequences was helpful to recognise critical couplings in order to adopt more efficient coupling strategies and therefore to optimise the final yield and purity of the crude peptide. development of green sustainable chemistry is currently regarded as a challenge in science and technology to reduce the use of organic solvents and utilize less toxic solvents instead. water and aqueous-based solvent systems represent an increasingly significant choice for replacing traditional solvents in synthetic chemistry. until recently, peptide synthesis in aqueous solution has remained largely unexplored. this is because the most common building blocks are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. we have developed a method for solid-phase peptide synthesis in water, which utilizes water-insoluble fmoc-amino acids that are converted to water-dispersible nanoparticles. in this way, the solubility problem is overcome. our technology, which uses suspended nanoparticle reactants for the coupling reaction, offers many advantages in terms of reaction efficiency over inwater synthesis using water-soluble or non-disperse reactants. however, there are two main problems with this nanoparticle approach; (i) slow reaction rates compared to general peptide synthesis in ordinary organic solvents (ii) poor yields for the synthesis of long peptides because the protected peptide chains on the resin have a tendency to aggregate in water. mw assisted spps is particularly attractive because of the widespread availability of the new technology, including automated peptide synthesizers. a trial of mw assisted inwater solid-phase synthesis using non-disperse boc-amino acids has been reported by albericio previously. currently, fmoc-amino acids are routinely used as building blocks for solid-phase peptide synthesis. with this in mind, we have developed a mw irradiation procedure aimed at reducing reaction time and increasing reaction yield for in-water solidphase synthesis using water-dispersible fmoc-amino acid nanoparticles. and we demonstrated in-water solid-phase synthesis of difficult sequence peptide with mw irradiation. m. lebl, z. flegelova spyder institute praha, czech republic cotton was shown as a convenient solid phase support earlier - , but did not find wide acceptance by the peptide community. we decided to try its application as (i) a support of choice for the synthesis driven by combination of capillary forces and gravity, (ii) support for synthesis utilizing in situ neutralization boc based protocol, (iii) support for combinatorial synthesis based on easy labeling and physical separability of cotton substrate, and (iv) support for multisupport synthesis. -we have built a simple synthesizer in which the cotton carrier (functionalized thread) is placed inside the capillary tubing and the appropriate reagents are introduced by connecting the inlet with appropriate reagents. the speed of "pumping" the reagents is driven by the difference between the elevation of the inlet and outlet of the capillary tubing. -we have shown that boc solid phase synthesis utilizing in situ neutralization is compatible with cotton substrate and provides high quality products. combining with the fact that cotton by itself acts as the self-association breaking agent, makes cotton a suitable carrier for synthesis of "difficult" sequences. -labeling of individual solid support particles can be easily based on the length of the cotton thread pieces, number and positions of knots, or their attachment to a secondary carrier. in addition, it is possible to synthesize peptides differing by the partial structure (alternative linkers, terminal modifications, etc.) in a mixture of classical resin with labeled cotton carriers, which are easily separable at the end of the synthesis. . use of microwave irradiation provides peptides in a fraction of time compared to conventional methods, and the peptides are also often generated in higher yield and purity. while microwave technology is particularly suited for the synthesis of "difficult" to synthesize peptides, this tool can routinely be used for the synthesis of a wide variety of peptides without the need for extensive method optimization. the focus of this study is to demonstrate how a peptide can be synthesized on a small scale (for example μmol) up through larger scales (> mmol) with ease. as a biologically relevant model peptide the last residues of the human platelet factor protein (hpf - ) was selected due to its significant antimicrobial activity. however, the problem of developing a robust fmoc thioester method is that the deprotection of the fmoc group with base at each cycle is not compatible with an active ester at the c-terminus. many ingenious approaches have been developed to generate the required thioester peptide. , , the most popular has been to use an nacylsulfonamide as a base and acid stable (safety-catch) linker for peptide synthesis. alkylation of the sulfonamide after peptide assembly makes the linker labile to cleavage with nucleophiles. whilst popular, it has been plagued by notoriously low yields which originate from the incomplete acylation of the resin-bound sulfonamide with the c-terminal residue, incomplete alkylation of the sulfonamide and the incomplete thiolysis of the resin-bound protected peptide. in this poster we describe the development of a novel dual linker strategy , involving anchoring of the sulfonamide linker to a standard acid-labile resin. this variation overcomes many of the current limitations of the sulfamylbutyryl linker approach and provides a simpler and scalable method for peptide ligation via fmoc spps. m. ziovas, d. tataraki, p. manousou, n. parveri, f. satoglou, d. gatos and k. barlos department of chemistry, university of patras, patras, greece solid phase peptide synthesis is traditionally performed by the attachment of the c-terminal amino acid through its α-carboxyl function on a suitable solid support and elongating peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. several thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals. in contrary to the attachment of the c-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side-chain on suitable resins and their application in spps is described in a small number of publications and patents. most of these publications describe the attachment of the amino acids through a side-chain carboxyl group of asp and glu. in the present work peptides were synthesized very efficiently in high yield and purity by anchoring of side-chain hydroxyl, amino or thiol groups of amino acids, amino acid amides, amino alcohols or small peptides on resins of the trityl or benzhydryl type. several peptides of pharmaceutical interest, such as exenatide, octreotide, pramlintide, calcitonin, bivalirudin, insulin b-chain and others were produced as examples of this technology, either by the step-by-step procedure or by fragment condensation in solution and on solid phase. step given that some of these diseases are caused by a mutation and/or malfunction of an essential protein, a better understanding of the structure and function of such proteins will allow us to prevent, slow down or even cure these diseases. to increase our knowledge, the synthesis of the target protein, a fragment involved in its activity or interacting peptides that modulate the protein activity is often required. in some cases the preparation of a protein analogue that improves its efficacy is envisage. however, conventional solid-phase peptide synthesis methods have some limitations when attempting to achieve these complex sequences of considerable length. using novel technologies, such as a microwave-assisted solid phase synthesis, commonly found in many peptide synthesis labs, here we performed the step-wise solidphase synthesis of a protein holding more than residues (d-vegf). this synthetic achievement indicates the suitability of this approach for synthesis of long proteins or their analogues. the detailed synthesis of the enatiomeric version of vegf and the selenomethionine substituted analogues of huprp ( - ), proteins involved in angiogenesis and prion protein amyloidoses respectively, are described as study cases, where the use of microwaves allow us to obtain them in a fast and efficient manner. therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. the pharmacological evaluation of a bioactive peptide [des-gly ,tyr (ome),d-leu ,aze-nhet ]gnrh, analogue , is presented herein. in vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatographytandem mass spectrometry. analogue , an agonist of the gnrh receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix. repeated dosing studies in mice demonstrated that analogue was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. analogue also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (lncap) cells. on the basis of pharmacokinetic advantages, we expect that analogue or analogues based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. cortexin is a polypeptide drug isolated from cattle and porcine brain cortex. cortexin is effective in monotherapy and in combination with traditional methods of treatment. cortexin produces tissue-specific, regulatory, and reparative effects on the brain cortex and contains active low-molecular-weight neuropeptides (< kda) penetrating through the blood-brain barrier. cortagen is a tetrapeptide h-ala-glu-asp-pro-oh (geropharm) produces nootropic and neuroprotective effects. oleylcortagen is a lipophylic analog of cortagen c h o-ala-glu-asp-pro-oh was created for increased proteolytic stability and increased penetrating through the blood-brain barrier. the main aim of our investigation is the analysis of psychopharmacological profile of peptide preparations in comparison with piracetam. have been shown oleylcortagen ( mg/kg) and piracetam ( mg/kg) possess activating effect on motor and research components of behavior in «open field» test. two of peptides (oleylcortagen, cortexin) decreased period of immobilisation and demonstrated antidepressant effects on rat behavior in the porsolt's test, on other hand cortagen demonstrated depressant action. therefore, the significant psychoactivating properties are typical for oleyl-cortagen, cortexin. the mechanism of the action of these peptides can be explained from the viewpoint of the regulatory cascade. they produce a direct information impact on cell structures of the brain, and then promote release of the regulatory peptides, which in turn, induce the release of the next group of peptides. neurology, queen square, london wc n bg, uk one of the hypotheses of alzheimer's disease neuropathology involves beta-amyloid (βa) binding with proteins on neuronal cell surface which leads to cell lysis and amyloid plaque formation. according to the latest data α -type of the nicotinic acetylcholine receptor (achr) and the prion protein can be the target for betaamyloid toxicity [ , ] . aggregated βa causes many pathological changes in cultures of mixed neurons and astrocytes such as sporadic cytoplasmic intracellular ca + -signal, activation of reactive oxygen species (ros) production and cell death. in the present work we demonstrated the ability of affinity purified antibodies to synthetic fragment - of α -subunit of the achr (achrabs) or to peptide - of the prion protein (prpabs) to protect cells from βa induced cell death. we also showed that both antibodies did not block βa induced ca + -signal in astrocytes. however, preincubation of cortical co-culture of neurons and astrocytes with achrabs or prpabs significantly reduced the rate of caspase activation and the rate of βainduced ros production via modulating nadph-oxidase. more detailed research of involvement of α -type achr revealed that α-bungarotoxin was also very effective in the inhibition of caspase activation and superoxide production. the observed positive effect of antibodies to α -type achr or to the prion protein gives an additional explanation regarding the involvement of these proteins in ad pathology and provides new approach into an anti-ad vaccine design. capturing and macrophage-aided clearance of amyloid beta by surface modified proteineous particles m. richman, s. rahimipour department of chemistry, bar-ilan university, ramat-gan , israel imbalanced homeostasis and oligomerization of amyloidβ (aβ) peptide in the brain are hallmarks of alzheimer's disease (ad). microglia and macrophages play a critical role in ad progression by clearing aβ from the brain or inducing inflammation. recent evidence suggests that the phagocytic pathway of aβ may be defective in ad microglia/macrophages that contributes to the build-up concentration of aβ in the brain. , therefore, efforts have been directed toward developing treatments that trigger these cells to clear aβ through alternative mechanisms. we have recently demonstrated that protein microspheres modified at their surface with multiple copies of an aβrecognition motif can strongly bind aβ, inhibit its aggregation and directly reduce its toxicity by sequestering it from the medium. here, we describe how the aβ-bound microspheres can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of aβ. the phagocytosis was mostly effective with microspheres having diameter size of about . - mm and introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. moreover, while aggregated aβ induced a significant inflammatory response that was manifested by the release of tnf-α from the microglial cells, the aβ-bound microspheres dramatically reduced the amount of the released cytokine. our data suggest that surface-modified microspheres could be utilized to detoxify other pathogenic or misfolded proteins that their accumulation may lead to genesis of other diseases. vasoactive intestinal peptide (vip) and its derivatives have been thought to be promising drug candidates for airway inflammatory diseases. however, the therapeutic potential of vips is highly limited because of rapid metabolic degradation and systemic side effects following systemic administration. previously, to overcome these drawbacks, our group developed a novel vip derivative, [arg , , , leu ]-vip-grr (ik ), with improved metabolic stability ( ), and respirable powder (rp) formulation of ik (ik -rp) for pulmonary administration ( ) . these attempts successfully led to enhanced pharmacological effects of ik in the airway system and reduced systemic exposure; however, further chemical modification of ik with a focus on metabolic stability might provide better clinical outcome. the present study aimed to design a pegylated vip derivative with improved metabolic stability and to develop its respirable powder (rp) formulation for inhalation therapy. ik was chemically conjugated with peg ( kda, p k), the physicochemical and biochemical properties of which were characterized by cd spectral analysis, binding assay, and metabolic stability. the rp formulation of pegylated ik (ik /p k) was prepared with a jet mill, and in vitro inhalation performance and in vivo pharmacological effects in antigen-sensitized rats were also evaluated. the cd spectral analysis demonstrated that peg conjugation had no impact on the solution structure of ik . although receptor-binding activity of the ik /p k (ic : nm) was estimated at ca. -fold less than that of ik (ic : . nm), metabolic stability for the ik /p k was highly improved. according to the laser diffraction and cascade impactor analyses, ik /p k-rp had fine in vitro inhalation performance. insufflation of ik /p k-rp ( μg of ik /p k) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decrease of inflammatory biomarkers and granulocyte recruitment in pulmonary tissue at h after antigen challenge. from these findings, pegylation of vip derivative, as well as its strategic application to the rp formulation, might be a viable approach to improve its therapeutic potential for treatment of airway inflammatory diseases. the previous studies have shown that trkb (tropomyocin receptor kinase) acts as an oncogenic agent and its binding to bdnf (brain derived neurotrophic factor) activates signaling angiogenesis of tumor proliferation [ ] . for finding the most stable and potentially effective peptides against the trkb, we applied the following protocol. at the first step of this protocol we designed a peptide library by using sequence tolerance method in rosetta . package, then peptide energy optimization performed by backrub protocol for finding the most stable peptides. the five best peptides in energy optimization selected based on backrup scores by using r package [ ] . d-structure prediction of the selected peptides was performed by using molecular dynamic in hyperchem software. docking of peptides with trkb receptor was carried out in haddock software. we used cyclotraxin, a selective trkb inhibitor as positive control for this protocol. cyclotraxin and the peptides were compared by anova or t-test. the peptides are going to be tested against the trkb in an in vitro model. dirucotide (mbp - ) is a synthetic peptide analog of , that consists of amino acids and tested in a phase trial were failed to reach his tolerance level on previous phase ii in rpms patients. one of the major disadvantages of peptide therapy is the activation of proteolytic enzymes, leading to peptide degradation. to address this problem cyclic peptide analogues have been synthesized. thus we synthesise a linear and cyclic analogues of dirucotide. the two analogues were synthesized by changing the amino acid residue at position from to the synthesis of the linear peptide, as well as of the cyclic one, was carried out by the fmoc/tbu methodology, utilizing the -chlorotrityl chloride resin (cltr-cl). the purification was achieved using semi-preparative rp-hplc and the identification was assessed by analytical rp-hplc and by mass spectrometry (esi-ms). the linear and cyclic peptide analogues will be used in human t-cell cultures to test their immunogenicity in patients versus healthy controls. in the first approach a reporter moiety was introduced to diagnose and treat cxcr related diseases. therefore, an anchor point to attach additional molecules to the ligand was elucidated. using sar studies to optimize the linker from the ligand to the detectable moiety the excellent receptor affinity could be retained. in a final step the reporter moiety was introduced to give a ligand for diagnosis via pet imaging and for possible endoradiotherapeutic applications. originating from the dimeric motif present in many active cxcr ligands several dimers were prepared using a monomeric ligand identified in the prior study. comparison of monomers and dimers yielded a possible subsite binding mode explaining why the dimers exhibit enhanced affinity using a model derived from the origins of the monomer. several peptidomimetic modifications were introduced to the ligand to reduce the peptidic structure. in a conformationally guided approach introduction of a peptoid motif could enforce a single active conformer that was enhanced through subsequent modifications. this yielded a compound times better than the original cxcr antagonist which is the most affine cxcr ligand reported so far. our previous studies have demonstrated that pace represents a potential therapeutic target for the treatment of prostate cancer . moreover, we have developed potent and selective pace inhibitor, ( -fold specificity over furin), known as multi-leu or ml peptide, which has a significant inhibitory effect on the proliferation of prostate cancer cell lines. peptide-based drug candidates can be limited by their poor metabolic stability and low bioavailability. thus, we performed structure-activity relationship studies to improve the pharmacokinetic properties of our ml inhibitor. we have designed and synthesized new ml peptide analogs having various chemical modifications. first, based on our previous results, we combined the most effective modifications of position p (d-amino acids) and p the arginine mimetic amidinobenzylamine (amba) to improve overall properties of our leading compound. second, the n-terminus of the resulting analogs was modified with a fatty acid, in order to enhance their cell permeability properties. third, we modified the inhibitors with a peg moiety to increase their stability and bioavailability. we tested the inhibitory activity, stability in plasma, cellular uptake, and cytotoxicity of each inhibitor. the results of this study demonstrate that the presence of the n-terminal extension (c or peg ) does not affect activity of our inhibitors. on the other hand, we show that introduction of the peg moiety does not increase cytotoxicity of ml analogs. it is interesting to note that the pegylated analog ac-peg -d-leu-lllrvkr-amba has better cell-permeability activity than its counterpart without peg unit. this combination of pharmacological properties makes our new ml analogs promising candidates for the development of potential anti-prostate cancer agents. [ ] peripheral coadministration of rf with opioid analgesics led to confirm the involvement of npff receptors as a part of the antiopioid system. indeed, rf was able to reverse the opioid induced hyperalgesia in rat (randall selitto test). then, a complete structure-activity relationships analysis was performed with rf , assessing the involvement of each moiety for affinity and selectivity towards both npff receptors. a first exploration of the n-terminus part of rf (> synthesized derivatives) led to replace the hydrophobic adamantane moiety by a hindered aromatic group, providing a subnanomolar npff ligand with more than two log-units selectivity against npff . then, the removal of the cterminal amide function led to reduce the dipeptide arg-phe-nh into an arginine derivative. in spite of an initial loss of affinity, optimization of the phenethyl moiety at the cterminus part of arginine led to non-selective nanomolar ligands for both npff & receptors. next, we applied efficient methodologies in order to synthesize non-natural analogs of arginine, leading to various compounds exhibiting selectivity for either npff or npff receptors. in particular, compound rf was identified as a selective npff antagonist (npff , ki = nm; npff : ki > μm). lacking of analgesia properties, oral administration of rf ( mg/kg per os) to rats was able to fully reverse fentanylinduced hyperalgesia. rf is the first orally available npff antagonist capable of reversing opioid induced hyperalgesia at low dose. moreover, this result allows identifying for the first time npff receptor as a key-partner of the anti-opioid system. administration of multiple antiplatelet agents has become the mainstay in the treatment of acute coronary syndromes in everyday clinical practice. we have previously reported significant antiplatelet effects of novel synthetic peptides' single administration on experimental carotid artery thrombosis in rabbits . in the present study we sought to investigate the peptides' effects when administered in marginally effective doses (significantly lower than those utilized in the past), in animals that had previously received low doses of aspirin. the peptides when co-administered with aspirin preserved the carotid artery's blood flow, in contrast to the total artery occlusion observed in animals receiving aspirin and placebo. blood flow at min after electrical stimulation was reduced to . ± . % and . ± . % in the ymesradr and psrcdcr-nh groups respectively (p< . vs aspirin and control). thrombus weight was significantly reduced in animals receiving ymesradr and psrcdcr-nh versus aspirin and control ( . ± . mg and . ± . mg, vs . ± . mg and . ± . mg respectively, p< . ). platelet aggregation was significantly inhibited in the ymesradr and psrcdcr-nh groups by . ± . % and . ± . % for adp (p< . vs aspirin and control), and . ± . % and . ± . % for aa (p< . vs aspirin and control), respectively. blood loss did not significantly differ among the various groups. administration of novel synthetic peptides, even at marginally effective doses, in animals previously treated with low doses of aspirin results in enhanced antiplatelet effects in an experimental model of arterial thrombosis. the study of peptide metabolism is particularly important when examining anticancer peptides; it can provide pivotal clues for the evaluation and improvement of stability of a peptide drug leading to enhanced pharmacokinetics and efficacy. gnrh analogues are used for the treatment of prostate cancer. as with other peptides a drawback is their short half-life due to their metabolic degradation. in order to examine the stability of these analogues we have developed several in vitro peptide stability and metabolism assays using specific tissues, isolated membranes, cancer cells that are analyzed subsequently using lc-ms/ms based approaches. such in vitro studies are followed up with pharmacokinetic studies in mice in order to establish the correlation between in vitro and in vivo approaches. the kidney is the main metabolic organ for peptide metabolism and for that reason we employed a peptide stability and metabolism assay previously described by our group using isolated mouse kidney membranes for the evaluation and comparison of different gnrh analogues. we tested gnrh analogues in other tissues such as mouse plasma, which is the "distributing" tissue for these drugs and mouse brain homogenate, a tissue known for its abundance in peptidases and relevance for centrally mediated effects. stability studies were also performed in cancer cells. in all cases lc-ms/ms based assays were developed for measurement of peptide drug and the resulting metabolites. for triptorelin, and a series of gnrh analogues, degradation and metabolite formation was studied by our mouse kidney membranes assay. studies of coadministering the peptide of interest with inhibitors that are presumed to block the metabolism in mice are ongoing. the vulnerability of gnrh analogues was verified after incubation with plasma and brain homogenate and by metabolite identification we obtained clues about the key cleavage sites. the described in vitro/in vivo protocols provide valuable information that could lead to the synthesis of more stable anticancer peptides with improved anticancer efficacy. in this work, we explored the use of a high-throughput synthesis and screening approach with peptide arrays to identify and structurally optimize shortened hiv- fusion inhibitors. the peptide array technology involves miniaturized synthesis of immobilized peptides, followed by affinity testing with a five-helix bundle, as a mimetic of the fusogenic gp protein. this exercise resulted in the identification of a class of truncated peptides which demonstrates a surprisingly high antiviral potency, despite the absence of the pocket-and the lipid-binding domain. the propensity of these peptides to adopt the bioactive α-helical conformation in solution as determined by circular dichroism, could be the key factor for this unexpected potency. these peptides are promising leads for the treatment and prevention of hiv. the pathological role of platelets in cardiovascular disease (cvd) is well established. platelets secrete adp to recruit additional platelets to a thrombotic site. we have previously identified novel cell-permeable peptide modulators of platelet function by using a bioinformatic screen based on patterns of evolutionary conservation in transmembrane proteins . in this study we further explored peptides derived from cadherin cell adhesion molecules. we explored a range of overlapping peptides derived from different cadherins varying from - amino acids long. peptides are synthesized and analyzed in a high-throughput platelet adp secretion assay. peptides ( . - μm) were assessed in the presence and absence of thrombin receptor activating peptide (trap; μm). we identified cadh- and proteins, but not cadh or in human platelets using western blotting and mass spectrometric analysis. peptides derived from paralogous juxta-membrane, cytoplasmic regions of these cadherins are potent modulators of platelet secretion. by systematically deleting amino acids from c or n-terminus of active peptides, we established that the minimal functional sequence for biological activity was a short six-residue motif, which corresponds to the known catenin-binding region of cadherin tails (kepllp) . peptides alone have no biological activity. however, they potentiate the response induced by the platelet agonist trap at low doses. thus we have identified a cadherin-derived peptide that can modulate platelet secretion events. this highlights a previously unknown role of cadherins in the regulation of platelet function. agents that interfere with cadherin signaling in platelets might have a therapeutic role in cvd. ageing of the brain leads to impairments in cognitive and motor skills, and is the main risk factor for several common neurological disorders such as alzheimer's disease (ad) and parkinson's disease (pd). altered protein handling (proteolysis, repair system, chaperones) forms a basis for a large number of protein conformational disorders. extra-and intracellular, as well as intranuclear accumulation of abnormal proteins, in the form of protein inclusions and aggregates, and dysfunction of the quality control mechanisms are common in all these disorders. alterations in protein homeostasis occur with age, causing molecular changes such as protein misfolding and aggregation. many biologically active proteins lack stable secondary and tertiary structure, these are called intrinsically disordered proteins (idps), some of them (e.g. β-amyloid, α-synuclein) are coupled to neurodegenerative disorders. idps exist as assemblies of rapidly fluctuating structures undergoing coupled folding and aggregation process. protein aggregation is characterized by polymorphism, where a mixture of soluble oligomers, amyloid fibrils and amorphous aggregates is the final product. soluble oligomers are inevitable formed during the self-association process and might initiate the neurodegenerative cascades of ad, pd and similar diseases. the emerging consensus that protein misfolding (leading to idps) is the cause of several neurodegenerative disorders now offers the opportunity to develop a general therapy. soluble oligomers with id regions are potential drug targets. recently short peptide fragments and small peptidomimetic molecules have been found also in our laboratory; these molecules bind to the id regions of β-amyloid and are putative drug candidates. precise control of bleeding is ensured by anticoagulant mechanisms, which under normal conditions prevail over coagulants mechanisms. disrupting the balance between procoagulant and anticoagulant systems due to congenital or acquired defects leading to thrombotic disorders. anticoagulants are substances that inhibit blood clot formation. their action consists in inhibiting the synthesis of prothrombin, the substances forming thrombus as well as some coagulation factors. many peptides and proteins with different molecular weight, such as antistasin (ats), ghilantens, hirudin, etc. showing high anticoagulant activity are isolated from salivary glands of ticks and leeches. they inhibit the action of serine proteinases from blood coagulation cascade. this creates an opportunity for targeted synthesis of low molecular weight analogues of some of these proteins, which can be used in the prevention and treatment of thrombotic disorders. ats is competitive, slow-binding inhibitor of factor xa. it is well known that blood coagulation could be blocked at different stages of the coagulation cascade through inhibition of various enzymes. therefore, it is interesting to determine the place of action of newly synthesized antithrombotic agents in the blood coagulation cascade. this can be done by determining the inhibitory constants of newly synthesized peptides on different enzymes of this cascade. herein we report on our kinetic investigation of newly synthesized peptide amides, analogues of isoform of ats . ki, km, vmax and type of inhibition on the factor xa, thrombin and plasmin were determinate. some interesting differences between type of inhibition of ats, free acids and amide analogues of ats were revealed. to evaluate the relative anti-platelet aggregation activities of each peptide, the lebetins were chemically synthesized and fully characterized. here we described the synthesis, the solution structure of lebetin g -g from the venom of vepera lebetina by h bidimensional nmr and the relation structure-activity. this peptide has been demonstrated to be associated with a potent anti-platelet aggregating activity. the g -g three dimensional structure consists in a compact β-bulged hairpain core from which emerges one loop and the cterminus and the n-terminus. we report on an approach whereby ligands are designed to bind and stabilize the - region of aβ in an α-helical conformation. these ligands reduce aβ toxicity to cells in culture and to hippocampal slice preparations. in addition, when these inhibitors are administered to drosophila melanogaster expressing human aβ ( - ) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed . stabilization of the central aβ α-helix appears to counteract polymerization into toxic assemblies and indicates that this approach holds promise for the development of orally available compounds against alzheimer's disease. encouraged by the above results we are currently developing a second generation of designed ligands. this involves synthesis of different new peptoids and unnatural amino acids. additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the aβ central helix as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation . synthesis and methodology for new ligands, which includes synthesis of novel amino acids, will also be presented. triostin a is a well-known natural product with antibiotic, antiviral, and antitumor activities. it inhibits rna synthesis by bifunctional intercalation into dna base pairs through its two quinoxaline units showing cpg selectivity. triostin a must adopt an altered conformation upon dna bisintercalation that is substantially different than its preferred native x-ray or solution conformation. this fact suggests that the destabilizing conformational change in the cyclic octadepsipeptide counteracts much of the gains derived from a second intercalation. nonetheless, the wide range of pharmacological activities exhibited by this compound prompted interests in identifying novel and additionally potent lead compounds with improved pharmacokinetic properties for clinical applications. herein, a library of twelve simplified triostin a analogues has been synthesized by solid-phase peptide synthesis. the introduction of the key quinoxaline units was carried out in solution. the analogues' conformation corresponds to the staple form that bisintercalator cyclic (depsi)peptides adopt when binding to dna and, in addition, some of the synthesized compounds showed improved solubility. our library was evaluated for its antiproliferative activity against four human cancer cell lines and one analogue showed greater cytotoxicity than triostin a and even comparable activity to doxorubicin, a very commonly used drug in cancer chemotherapy nowadays. surprisingly, little is known about its mechanism of degradation in solution or the degradation products. a recent study identified monomeric polysulfides and dimeric degradation products, postulated to derive from β-elimination followed by deamidation and dimerization. we recently reported that degradation of oxytocin and its analogues in aqueous solution at ph . produced monomeric polysulfides with up to sulfur atoms as well as dimeric products. unexpectedly, incubation of ot or of various analogues modified in position resulted in identical dimeric degradants. we concluded that β-elimination via breakage of the c-s bond of cys must be a key step of the process, and that the resulting Δala residue would have to undergo further modifications to yield the same dimeric products independently of the substitution of the n-terminal nitrogen. here we further clarify the degradation mechanism and propose a structure for the dimers. we postulate that hydrolysis of the Δala residue yields an n-pyruvoyl linear peptide, which loses one sulfur atom and subsequently forms dimers, which we found are linked by one disulfide bridge and one non-reducible bond. the putative linear n-pyruvoyl oxytocin intermediate was synthesized and found to degrade to the same dimers as the ones in the incubations of ot. a [u- c]cys ot analogue gave degradation products with c-nmr spectra consistent with a non-stereospecific aldol-type condensation. detailed experimental procedures, structures of the degradants and the postulated mechanism of ot degradation in near neutral solutions will be presented. inserm u paris, france αiibβ is the main platelet integrin and is responsible for platelet aggregation. a lipid-modified peptide corresponding to αiib intracellular sequence - (palmitoyl-k-l eeddeege , pal-k- - ), is platelet permeable and inhibits human platelet aggregation induced by thrombin . ymesradr, a peptide corresponding to the extra-cellular sequence - of αiib, is a platelet activation and aggregation inhibitor . the aim of the present study was to investigate the cooperativeness of the intracellular and extra-cellular peptides on platelet aggregation and their effect on the phosphorylation of fak and erk. pal-k- - together with the extracellular ymesradr peptide, at concentrations lower than their ic values, showed cooperative inhibition of platelet aggregation. the peptide combination inhibited also fibrinogen and pac- binding to activated platelets. fak phosphorylation is a postaggregation event related to outside-in activation of the receptor. the combination of peptides inhibited fak phosphorylation. erk phosphorylation is independent from platelet aggregation, and is enhanced by rgd-peptide inhibitors. the combined peptides inhibited erk phosphorylation. ovarian cancer (oc) is considered a rare disease and represents the fifth most common cause of death from cancer in women. the standard first-line treatment consists of a combination of paclitaxel and carboplatin (ddp) or carboplatin alone. in the case of progressive disease or drug resistance to platinum-based agents, either alone or in combination, investigational compounds should be used ( ) . the mechanisms behind acquired resistance to ddp and its derivatives are not clear yet, although it is evident that the process is multifactorial, including enhanced dna repair processes. some peptides designed from the interface subunit of the human thymidylate synthase (ts) have been identified recently ( ), as effective anticancer agents against sensitive and resistant oc cells. one of them was also able to recover the cellular sensitivity towards cisplatin in resistant oc cells in the μm range. to improve its potency and selectivity structural studies have been performed in combination with cellular assays aimed at understanding the mechanism of action. a label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. structure-activity relationships (sar) have been performed to improve the lead peptide pharmacodynamics. all the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of oc cell growth. hepatitis c is a liver disease provoked by a virus known as hcv. the disease is insidious. hcv causes anorexia, nausea, vomiting, fever, fatigue and jaundice. in about % of sufferers the disease is short, but others become chronic. in the chronic form in about % of cases the final result is cirrhosis of the liver and in the remaining % it leads to liver cancer. hcv is a very serious problem today. about % of people infected with hcv worldwide, i.e. about million are residents of europe. million people carry the disease as a chronic illness with the potential to develop into cancer in their liver. all these people represent a "reservoir" for storage and distribution of hcv. ribavirin, the nucleoside analog -β-d-ribofuranosyl- , , triazole- -carboxamide, known by the trade name virazole (also known as rebetron in combination with interferon-α), exhibits antiviral activity against a variety of rna viruses (paramyxoviruses, flaviviruses, etc.) as well as some dna viruses. in humans ribavirin is currently used in combination with interferon-α to treat hcv infections. this lack of strict specificity and a broad spectrum of activity are due to its multifunctional mechanism of action against viruses. these characteristics have made ribavirin a drug of substantial research interest. unfortunately, ribavirin shows a significant toxicity, causing bleeding in accumulation [ ] . herein, we report a total synthesis of modified in position ' of ribose residue, ribavirin in order to be further linked to cell penetrating peptides (cpps). in addition the synthesis of some cpps as well as bonding between two parts of final hybrid molecules will be reported. in our case the design of new hybrid molecules is done in order to: (a) vectorize ribavirin into liver cells; (b) transport ribavirin molecule trough cell membrane and (c) decrease toxicity of ribavirin. to obtain oligomeric aβ peptide, our laboratory uses a precursor depsipeptide of aβ. this precursor, termed as "iso-aβ" has an ester bond between the side chain of ser and gly . at physiological ph this ester bond becomes an amide bond via an o→n acyl shift. binding partners by which the oligomeric aβ mediates its toxic effect has not been yet investigated in the proteome or subproteome level. we used protein array technology to study the interaction of oligomeric aβ with recombinant human proteins, immobilized on a protein chip. aβ binding proteins were identified with the aid of a monoclonal aβ antibody. altogether proteins were found to interact with our aβ-oligomers. these proteins were grouped according to their function. one of the major groups contained proteins participating in translation. these proteins were found in ribosomes. to prove our proteomic results ribosomes from rat hippocampus were isolated. elisa experiment revealed that aβ binds to ribosomes in a dose-dependent manner. using the sequence of the aβ-binding proteins a homology search was performed to find oligopeptides, that possibly bind to aβ. based on these sequences a peptide chip containing hexapeptides was prepared. aβ interacting peptides were identified with a monoclonal antibody. several peptides were synthesized and tested on mtt assay. two out of four compounds inhibited the toxicity of aβ on rat hippocampal slices. summarizing our results aβ binding proteins and peptides were identified. knowledge about aβ binding proteins can help to understand the pathogenesis of ad, such us the possible involvement of ribosomes. oligopeptides can be lead compounds of future drug development. huge proteolytic complex named proteasome catalyzes protein degradation in every eukaryotic cell. it consists of subunits forming four stacked rings and one or two regulatory caps. two inner rings of the proteolytic part contain three catalytic β-subunits that possess different substrate specificity. higher vertebrates can express γinterferon-inducible immuno-β-subunits. proteasome plays an essential role in continual turnover of intracellular proteins and in antigen processing. autoimmune diseases such as multiple sclerosis and its murine model eae are believed to rise from breakdown of tolerance of the immune system. it assumed that immunoproteasome could play an important role in autoimmune diseases. several classes of chemicals proved to be inhibitors of proteasome and the most active are boronate peptide derivatives. these inhibitors totally inactivate proteasome and result in full stop of intracellular protein turnover and cell death via apoptosis. another class of inhibitors, epoxy ketones, was shown to be more selective for immunoproteasome and could be used not for full stop of proteasome function, but for fine tuning of altered proteasome functioning. we examined properties of several inhibitors of four different classes, namely peptide boronate bortezomib, peptide aldehyde mg , lactam lactacystin, and peptide epoxyketones epoxomicin, mg ek, uk and pr- . for inhibition experiments we used proteasome isolated from eukaryotic cell lines cho, nso and hek, treated and non-treated with γ-interferon, as a model cells contatinig constitutive and immunoproteasome. the upregulation of proteasome immunosubunits was revealed in cho and nso cells treated with γ-interferon. the ic values for all studied inhibitors were obtained, and ki in some cases were calculated. the epoxyketones were shown to selectively inhibit in submicromolar concentrations the proteasome sample which contain high amount of immunosubunits. in order to find an effective antimalarial, this study refers to some angiotensin ii (aii) analogues which were considered the important physicochemical characteristics described by silva et al. to verify the biological activity against plasmodium gallinaceum and to understand the hydrophobic cluster influence, explained by tzakos et al. these analogues were synthesized and characterized as described by silva , as well as the biological assays and comprises, to verify: the hydrophobic cluster activity -a) drvyhipf; b) drvypr; c) ryhipf and d) fphiyvrd; the importance of these residues in aii molecule -e) rypf; the importance of aromatic residues -f) yhpf and the action of these hydrophobic residues, when interacting with the parasite membrane -g) vipf. it was observed that in a ( % of bioactivity), the phenol group of tyr is close to imidazole group of his that could promote a hydrogen bond formation. besides that, could occur van der waals interactions between ile and phe residues due its proximity and non-polar characteristic. these interactions could not be effective in native aii ( %) , because ile residue promote a steric influence on the organization of his and tyr residues that not exist in b ( %). in c ( %) and e ( %) analogues, the influence of the arg residue could promote a cation-π interaction with tyr residue and the cluster may have suffered slight destabilization and its antiplasmodial activity was compromised subtly. in d ( %), the electrostatic change, obtained with the total inversion can have disordered its interaction with parasite membrane, since it is not related to membrane receptors, because d-aii presented % of biological activity. moreover, hydrophobic and aromatic residues importance was confirmed through the results obtained % and % of activity, with g and f, respectively. we conclude that hydrophobic cluster modifications and interactions of amino acid side-chain influences in the biological activity. closed joint-stock company "vertex", st-petersburg, russia creatine (cr), a small molecule synthesized in the kidney, liver and pancreas plays important role in atp synthesis, replenishing its store even in the absence of oxygen. cr is able to protect brain cells against ischemic damage; however it has poor ability to penetrate the blood-brain barrier without specific carrier protein. thus, synthesis of stable hydrophobic derivatives capable of crossing the bbb by alternative pathway is of great importance for the treatment of different neurological diseases including stroke, traumatic brain injury and hereditary crt deficiency. here we describe the synthesis and biological activity of new hybrid compounds -creatinyl amino acids. originally the title compounds were synthesized by guanidinylation of sarcosyl peptides. however, for large scale synthesis better results can be obtained using direct cr conjugation with amino acid or peptide derivatives by isobutyl chloroformate method. addition of equivalent amount of ptoluenesulfonic acid as lipophilic counterion ensures efficient cr dissolution in dmf along with its simultaneous protection towards intramolecular cyclization. it excludes the application of expensive guanidinylating reagents and permits to simplify the synthetic procedure. purification of final product and its conversion into appropriate salt form can be achieved by iec followed by crystallization from organic solvents. synthesized creatinyl amino acids and peptides exhibited significant biological activity in different assays including platelet aggregation test, ischemic stroke and nano -induced hypoxia model. one of the most effective compounds -creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. these data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. the efficient recognition and destruction of tumor cells via specific cellular markers is a major goal in cancer therapy. various growth factor receptors such as egfr, hgfr, vgfr and their downstream signaling networks have been proven to be effective molecular targets, as they are frequently involved in cancer proliferation and metastasis. downregulation of these receptors and/or blocking their signaling pathways have clear anti-tumoral effects. drugs based on monoclonal antibodies (mab) targeting such cell surface receptors have attracted a lot of attention as a new generation of therapeutics. however, their production is costly and identifying new, variable routes to modified molecules with similar properties is currently a major focus. , here we present an approach to chemically synthesize a molecule that combines the mode of action of antibodies with the advantages of smaller, chemically accessible molecules. these "synthetic antibody" (sab) molecules contain a chemoattractant that activates the innate immune response and resembles the fc domain of a typical antibody. specificity is imparted by two binder peptides that assume the function of the variable antibody domains and bind to a cell surface target. the fc and fab domains of the sab molecules are connected via polyethylene glycol linkers. sab molecules are prepared by solid phase synthesis, a flexible technique that allows fast production, full control of their properties and targeting two different cell surface receptors (bispecific tumor targeting). they are currently tested in vitro and in vivo for their effect on the innate immune system, general toxicity and selective binding to cancer cells. the key enzyme in the processing of polyproteins translated by viral rna genome of sars-cov is a kda protease called c-like protease ( cl protease). sars cl protease is a cysteine protease containing a cys-his catalytic dyad, and cleaves precursor poly proteins at as many as conserved site involved a conserved gln at the p position and a small amino acid (ser, ala, or gly) at the p' position. due to its functional importance in the viral life cycle, sars cl protease is considered to be an attractive target for drug design against sars. recently, we found tetrapeptide aldehyde, ac-thr-val-cha-his-h, showed high inhibitory activity with ic value of nm toward cl-r i mutant protease , . to compare the inhibitory activity of small compounds with those containing active functional groups, we synthesized serine-derivatives within the essential functional groups and evaluated its inhibitory activity. the synthetic scheme was started from fmoc-ser(tbu)-oh, following modification of c-terminal carboxyl group with p , n-terminal amine with p and side chain alcohol with p functionalities. steps overall reaction led to obtain novel serine derivatives for the small molecular inhibitors of sars cl protease. the assay with cl r i mutant protease was examined to evaluate the inhibitory activity of the synthetic serine derivatives. then, molecular docking study of complex of cl protease with the ligand was carried out. docking simulation experiment with r i (pdb id: aw ) and the inhibitor, which has the best activity in the serine derivatives, indicated that p fitting s ' pocket. at the result of assay, p , p and p positions of the inhibitor should be modified by benzoyl group, cyclohexyl group and cinnamoyl group, respectively. their bioactivities are underpinned by their distinctive structure with exceptional stability, thus making cyclotides exciting, not only for agricultural and pharmaceutical purposes, but also as a template in drug design. in all of the reported activities, cell membranes seem to be the primary target for cyclotide activity. to unravel the importance of lipid membranes on the reported activities of cyclotides, a set of cyclotides belonging to möbius and bracelet subfamilies were compared in their mode of action. the lipid selectivity and membrane affinity were compared with their efficiency against different target cells (e.g. red blood cells, bacteria, hiv particles). we have found that the bioactivity of cyclotides is dependent on the lipid composition of the target cell membrane and independent of a protein chiral receptor. in particular, all the native cyclotides tested target the cell membrane through specific binding to phospholipids containing phosphatidylethanolamine (pe)headgroups, but the membrane binding affinity is further modulated thorough non-specific peptide-lipid hydrophobic interactions, which are dependent on the specific cyclotide. in addition, the bioefficiency of cyclotides broadly correlate with their ability to target and disrupt the cell membrane. overall, we have shown that even with a common specificity for membranes containing pe-phospholipids, a fine selection was found across the family. in particular, each cyclotide inserts and disturbs the membrane in a distinct way, which explains the diversity of this family but also their distinct activities. the observation that all the tested cyclotides have a preference for a specific lipid makes this family truly intriguing and brings insights to optimize the use of the cyclotide template in drug design. malaria is a disease that affects around million people causing . - million of deaths annually. based on our previous studies, angiotensin ii (aii) presented antiplasmodial activity against plasmodium gallinaceum, but due to pressure activity, it cannot be used as an antimalarial drug. in an attempt to increase antiplasmodial activity and reduce hypertensive activity, we synthesized by solid phase method, cyclic analogues of aii with i-(i+ ) and i-(i+ ) lactam bridge scaffold using asp and lys residues. the bridge was more effective when inserted next to n-terminal extremity , probably this insertion, on another portion of the peptide, provides a change in the conformation of the molecule and its hydrophobic cluster formed by tyr, ile and his , which may have influence in the peptide-membrane interaction. thus, we have focused in the n-terminal extremity, testing new analogues, using glu/asp/orn/lys residues as bridgeheads components in i-(i+ ) lactam bridge scaffolds, which showed that antiplasmodial activity is increased using glu residue and that larger lactam rings are better to biologically active. therefore, new restrict peptides by i-(i+ ) and i-(i+ ) lactam bridge were designed, using glu residue as bridgehead element, but the same effect was not verified, getting a maximum of % of bioactivity. on the other hand, we promoted an increase in the hydrophobic character of the molecule, replacing the asp residue of aii sequence by fmoc-glu and asp(ofm), in order to improve the interaction of these compounds in the sporozoite membrane. the replacement by fmoc-glu provided a decrease of activity, while that asp(ofm) kept the aii activity, because there are changes of charge in the peptide, which may have modified the conformation in physiological medium. this kind of approach may offer the basis for development of new drugs and chemotherapy against malaria. animal venoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors such as ion channels or g-protein coupled receptors. venoms can therefore be seen as large natural libraries of biologically active molecules that are continuously selected and highly refined by the evolution process. the vision associated with the venomics project is to investigate in depth the enormous structural and pharmacological diversity of venom peptides through the development, integration and implementation of a novel research paradigm combining cutting-edge "omics" technologies in a high-throughput workflow. this new paradigm enclosed in venomics aims at replicating in vitro the diversity of venoms to generate original peptide banks to be used in drug discovery programs. herein, we show the different strategies we adopted for efficient solid phase synthesis and folding with an easy purification of peptides rich in cysteine and containing posttranslational modifications (ptm). angiogenesis depends on the adhesive interactions of vascular cells. the adhesion receptor integrin av b was identified as a marker of angiogenic vascular tissue. the αν β integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of a-amino acids by aza-β -amino acid analogs in cyclic rgd-peptides as αν β -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. we synthesized cyclic rgd peptidomimetics that include aza-β -amino acid residues. modifications were added to the rgd skeleton in order to optimize the peptide activity. then, we investigated the pharmacokinetics activity of these pseudopeptides in hek (human embryonic kidney ) and endothelails cells huvec (human umbilical vein endothelial cells) cell by analyzing cell viability and protein involved in the angiogneisis processes. since tenascin c is a factor expressed highly in the tumorassociated matrix, targeting it would be a desirable first step for targeting the tumor-specific microenvironment in fact, a high level of tenascin c expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. therefore, the targeted binding of tenascin c in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. we isolated a peptide that bound to tenascin c by phage display peptide library selection, and the selected peptide specifically recognized tenascin c protein in xenograft mouse tissue. we also observed exclusive staining of tenascin c by the selected peptide in tumor patient tissues. moreover, the peptide reduced tenascin c-induced cell rounding and migration. we propose that the tenascin c targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors. radiolabeled pansomatostatins are expected to enhance hsst - tumor-uptake and to broaden clinical indications as compared to currently established sst -prefering radioligands. previous experience has revealed [ in-dota ,dtrp ]ss- ([ in]at s) as a true pansomatostatin analog, exhibiting however poor in vivo stability. in order to enhance metabolic stability, we introduced a second disulfide bridge to the at s motif by formation of extra / -amino acid (aa) or / -aa ring generating at s and at s, respectively. the orthogonally protected sequences were assembled on the solid support, deprotected and cleaved from the resin with tfa. the first cys -cys (at s) or cys -cys (at s) cyclization was performed in dmso, while the second was completed with iodine oxidation after in situ deprotection of cys (acm) and cys (acm). during hsst - +-autoradiography, at s showed unexpected total loss of sst - affinity, whereas at s showed high affinity (ic in nm) to all hsst - (hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; and hsst = . ± . ). consistent with this finding, only at s stimulated sst internalization during immunofluorescence-based internalization assays, showing agonistic properties for sst . furthermore, [ in]at s internalized rapidly and specifically in sst + ar - j and hek -hsst +-cells. hplc analysis of min ex-vivo mouse blood samples revealed that > % [ in]at s remained intact. after injection in scid mice bearing ar - j and hek -hsst + tumors [ in]at s specifically localized in the rsst a+ ( . ± . %id/g vs. . ± . %id/g + nmol tate at h postinjection (pi)) and in hsst + implants ( . ± . %id/g vs. . ± . %id/g + nmol ke at h pi). this study has shown that introduction of an extra disulphide bridge in at s confers high metabolic stability. however, in a / member ring combination it leads to total loss of affinity. the reasons for this effect are currently investigated by nmr conformational studies. transporter compounds are useful tools to solubilise and increase the delivery of therapeutic molecules in the human body. one system to improve the cellular uptake of such therapeutic molecules are cell-penetrating peptides (cpps). these short peptide chains are either polycationic (containing several arg and lys) or show a more amphiphatic structure. it is known that the multivalency effect -the presentation of several copies of a cpp motif on a single molecule -can increase the cellular uptake. peptide dendrimers represent a group of tree-like, multivalent macromolecules, which are synthesized for different chemical and biological applications in our group. we now combine linear cpps with peptide dendrimers to get a well defined branched molecule made up of only natural amino acids. in our systematic study of peptide dendrimers decorated with different cpps we found that the potency of the single cpp as a transporter for small molecules can be increased and that these peptides show usually low cytotoxicity. additionally we designed new dendritic cell penetrating peptides with similar activities like linear cpps. all compounds are covalently linked to fluorescein for visualization with flow cytometry and confocal analysis. the results show that the peptides can transport efficiently a hydrophobic cargo into the cells. chemical stability of esters of acyclovir with amino acid and cholic acids k. chuchkov, r. chayov, i. g. stankova* south-west university "neofit rilski", blagoevgrad, bulgaria amino acid esters of antiviral drugs are a very good solution for improving oral bioavailability of the actual medicine. one of the most effective and tolerant prodrugs is valine ester of acyclovir -valaciclovir. taken orally exhibits three to four times higher bioavailability of acyclovir. the chemical stability of amino acids ( -fphenylalanine) (r,s) and bile acids (deoxycholic acid and chenodeoxycholic acid) esters of acyclovir was studied in experimental conditions simulating some relevant biological medias (ph . and . , °c).the chemical stability experiments revealed that the examined amino acid ester of acyclovir were relatively unstable in acidic ph, but bile acid ester is stable in the same ph. the examined amino acid and bile acid esters of acyclovir in neutral ph are relatively stable. in ph , all of tested compounds are more stable than valacyclovir (t / = h) -the first effective prodrug of acyclovir. in acidic ph acyclovirdeoxycholat and acyclovirchenodeoxycholat are more stable than valacyclovir. acyclovirchenodeoxycholat is the most promising anti-ebv prodrug candidate with high activity and satisfying chemical stability. cell-penetrating peptides (cpp) have become efficient tools for the cellular internalization of bioactive molecules due to their ability to cross the plasma membrane of diverse cells and cell lines. [ ] we recently reported that the cpp sc , which consists of the residues - of the c-terminal region of the cationic antimicrobial peptide cathelicidin (cap ), is an effective carrier peptide for small organic molecules like fluorophors and toxic peptide sequences into various cell lines [ ] . however, in general linear peptides are more susceptible to proteolytic degradation than their cyclic analogs [ ] . therefore, we investigated the cyclization of cpp derived from sc by means of cui-mediated azidealkyne cycloaddition (cuaac) [ ] . furthermore, we examined their conformation and proteolytic stability as well as their internalization efficiency and toxicity against various cell lines, in comparison to their linear equivalent and to other cpp. looking for the proper prodrug: a peptidomimetic approach to identify and inactivate bacterial mono-adp-ribosyltransferase toxins m. beich-frandsen, r. jørgensen division of microbiology and diagnostics, statens serum institute, copenhagen, denmark mono-adp-ribosylation is an endogenous posttranslational modification in eukaryotic cells, simultaneously utilized as virulence strategy by deadly secreted bacterial toxins. many bacterial toxins have been found to act as mono-adp-ribosylating enzymes, targeting anything from g-proteins to the actin skeleton. the diphtheria toxin from c. diphtheriae and exotoxin a from p. aeruginosa, both target the diphthamide-group of a unique modified histidine in eef , inhibiting protein synthesis by ribosome mimicry , . we aim to inactivate these nad+-utilizing toxin enzymes by nad-conjugated peptidomimetics, in a target-specific prodrug-approach. the adp-ribosylation reaction follows a random third-order s n mechanism. in the proposed model for the transition state of the reaction, the cleavage of the n c -nn bond of nad + releases strain and generates a oxacarbenium ion intermediate with a positively charged nicotinamide (n)ribose, subject to a nucleophilic attack from the substrate , , . adp-ribosylating toxins are commonly characterized by a artt-motif involved in substrate recognition . studies suggests conformational rearrangement of the residues surrounding the substrate binding site to be required for optimal geometry of the initial glycosidic nc -nn bond cleavage within nad + . subtype specific nad-conjugated peptides, designed based on previous structural analysis of the adpribosylation reaction, act as substrate for the enzymatic adp-ribosyl-transfer, and hereby attach covalently to and inactivate the nad + -utilizing toxin. relying on previous structural studies, and established ligand-binding and kinetic data, an initial peptide library, designed by bioinformatics and evaluated for specificity of common targets in-silico identifies initial leads. lead-scaffolds are implemented in rational peptide-design, based on high-resolution structural-and biophysical studies of multiple peptide-enzyme complexes, to identify possible prodrug-strategies for enzyme inactivation. nanoparticles play a crucial role in medicine for their potential application as in vivo carriers of active principles [ ] . liposome display unique pharmacokinetic properties slowly releasing drugs loaded in the inner aqueous cavity. in the last years we have developed supramolecular aggregates labeled by bioactive peptides able to recognize overexpressed receptors on tumour cells membrane delivering doxorubicin chemiotherapeutic drug [ ] . neurotensin(nt), a amino acid peptide, has dual functions of neurotransmitter or neuromodulator. the cterminus short fragment - preserve the activity but the half life of wild type form in vivo is very short. nt receptor type (nts ) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. we have designed new amphiphilic molecules containing in the hydrophobic moiety two aliphatic chains and in the hydrophilic moiety a the bioactive portion able to aggregates with phospholipid molecules achieving liposome. we have synthesized neurotensin wild type sequence, the truncated form and the tetra-branched neurotensin(nt - ) or a truncated form(nt - ) tetrabranched peptides(nt ) adopting an opportune synthetic strategy on solid phase. all liposome were formulated adding the neurotensin amphiphilic monomer in ratio : with dopc in order to evaluate the capability to recognize selectively receptors overexpress on cell membrane surface. the liposomes size was determined by dynamic light scattering measurements, values for the hydrodynamic radius(rh). the selective internalization and cytotoxicity of fully doxorubicin loaded liposomes as compared to pure dopc liposomes, was tested in ht human colon adenocarcinoma and te human rhabdomyosarcoma cells. recently, small interfering rna (sirna), one kind of rna interference (rnai) technology represent the most common and, to date, the most effective method to inhibit target gene expression in human cells. it is also a common recognition that non-toxic delivery of sirna is an urgent problem for the therapeutic application of sirna. for the efficient gene silencing in vivo, prolonged circulation of sirna with take efficient and non-toxic cellular uptake and resistance against enzymatic degradation are indispensably required. ) telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. in this paper, we present the efficient and non-toxic cellular uptake of sirna using novel amphiphilic peptides and the application to silencing of htert in human cancer cell lines. in the present study, we investigated the intracellular delivery of sirna using some amphiphilic peptides and the silencing effect of sirna targeting htert mrna in human cancer cell lines, jurkat, hela and k . the complex of sirna and a specific amphiphilic peptide or its hybrid with an intracellular transport signal peptide could be effectively taken up into cells. the complex also showed a high silencing effect against htert mrna. moreover, the combination of sirna-nes conjugates and the amphiphilic peptides improved silencing effects up to . %. the amphiphilic peptides and their hybrids showed almost no cyto-toxicity and protected sirna against intracellular nuclease digestion in % fbs (half life time was over h). tumor targeting with the decapeptide gonadotropinreleasing hormone (gnrh) or its analogues is based on the discovery that gnrh receptors are overexpressed in many tumor cells, compared with their expression in normal tissues. using these peptides as carriers/targeting moieties in a conjugate with therapeutic agents can increase the selectivity and the stability of the conjugates, or eliminate the toxic side effects of the drug. gnrh-iii (<ehwshdwkpg-nh ) is especially favoured as a targeting moiety because of its antiproliferative effect and weak endocrine activity in mammals. the aim of our work was to synthesize novel short-chain fatty acid (scfa) acylated daunorubicin-gnrh-iii bioconjugates with enhanced enzymatic stability, cellular uptake, in vitro and in vivo antitumor activity. ser in position of gnrh-iii was replaced by lys and its ε-amino group was acylated with various fatty acids. gnrh-iii( lys(x), in conclusion, glu - /dglu - substitution in gi-derived radiopeptides enhanced stability and reduced renal values by ~ . fold. on the other hand, removal of the (glu) sequence in mg suppressed renal uptake but undermined stability. both modifications, however, compromised tumor targeting, demonstrating that more efficacious structural changes in the gi motif are warranted to improve the in vivo profile of resulting radioligands. synthesis and characterization of a mtc-labeled rhodamine-conjugated angiotensin peptide as a potential cardiac function imaging agent s.m. okarvi king faisal specialist hospital & research centre, cyclotron and radiopharmaceuticals department, riyadh , saudi arabia angiotensin ii (ang ii) is an -amino acid peptide that has been known to play a vital role in cardiovascular system. the actions of ang ii have been implicated in many cardiovascular conditions, such as coronary heart disease and heart failure. in an effort to develop a cardiac imaging agent with enhanced targeting capacity, we attached ang ii to rhodamine (rh), a lipophilic cation that specifically accumulates in the myocardium. the rh conjugated ang ii was evaluated for its potential as a heart imaging agent. rh-lys-gly-cys-asp-arg-val-tyr-ile-his-pro-phe-conh was prepared by solid-phase peptide synthesis according to fmoc/hbtu methodology. nhs-rh was attached to the ang ii peptide via the free amino group of lys residue by manual synthesis. rh-ang ii conjugate was labeled with tc- m by the stannous-tartrate exchange method. in vitro stability was determined in human plasma and in excess of cysteine. in vivo biodistribution and biokinetics were performed in balb/c mice and sprague dawley rats. the rh-ang ii conjugate radiolabeled efficiently with tc- m (> % labeling efficiency) as determined by hplc analysis. tc- m-rh-ang ii exhibited good chemical stability against cysteine transchelation and sufficient metabolic stability in human plasma. in mice, the bioconjugate displayed efficient clearance from the blood and excreted mainly through the renal route with some excretion by the hepatobiliary pathway. the uptake in the heart was . ± . % id/g as early as min post-injection; whereas, the uptake in the lungs, liver, stomach and kidneys varied between - % id/g. in rats, the bioconjugate displayed relatively better pharmacokinetic characteristics, with low uptake in the major organs (< % id/g). the uptake in the heart ( . ± . % id/g) was found to be higher than the uptake in the blood and muscle, resulting in good heart-to-blood and heart-to-muscle uptake ratios. this initial study towards the development of an effective cardiac imaging agent advocates that the use of hybrid conjugates appears to hold a great promise as a new and attractive approach for rapid and efficient imaging of heart. in humans two isoforms of gnrh are exist, gnrh-i (<ehwsyglrpg-nh , where <e is pyroglutamic acid) and gnrh-ii (<ehwshgwypg-nh ). in contrast to gnrh-i, exact function of gnrh-ii is not well identified. however, it was indicated that gnrh-ii derivatives have more potent antiproliferative activity on human endometrial and ovarian cancer cells in vitro than gnrh-i analogs. some kinds of tumor cells (e.g. breast, prostate, colon) produce gnrh-i and gnrh-ii, and express their receptors. however, the presence of functionally active gnrh-ii receptors on cancer cells is still not clear - . on the other hand, gnrh-ii analogs, both agonists and antagonists, induce apoptosis in many different cell types. in contrast to gnrh-i agonists, gnrh-ii agonist [d-lys ]gnrh-ii does not activate nfκb, therefore does not protect the cancer cells from apoptosis [ ] [ ] [ ] . in the regulation of apoptosis, not only the pro-and antiapoptotic proteins play a role , but other molecules also attend (e.g. ck , casein kinase ii enzyme) . our aim was to synthesize new proapoptotic peptide or enzyme inhibitor containing [d-lys ]gnrh-ii based derivatives and investigate their in vitro cytotoxic, cytostatic and apoptotic effects. the sythesis of [d-lys ]gnrh-ii, which is one of the most potent gnrh-ii agonist, was carried out on solid phase using fmoc/tbu strategy. different proapoptotic peptides or enzyme inhibitors were attached directly to the gnrh-ii derivative. in vitro cytostatic and cytotoxic effects of gnrh-ii conjugates were investigated by mtt-assay using different human breast (mcf- and t -d) and colon carcinoma (ht- ) cell cultures. early apoptotic effect of gnrh-ii conjugates was studied by flow cytometry on mcf- , t- d and ht- cell lines. according to our results, these conjugates might be potential candidates for cancer treatment, because the combination of a targeting moiety (gnrh derivative) with proapoptotic peptides or enzyme inhibitors and drug molecules might have synergistic activity resulting in highly potent multifunctional therapeutic agents. department of pharmaceutical and pharmacological sciences, university of padua, padua, italy melanoma is the most deadly skin cancer with an increasing incidence. its therapy continues to be a challenge since, regardless of the treatment used, longterm survival is quite uncommon. melanoma cells are characterized by high levels of glutathione s-transferase p - (gstp - ) isozyme. this enzyme is also highly expressed in solid tumours and drugresistant cells, where a role for this protein in the regulation of cell proliferation has been described. in the past, lyttle et al. synthesised glutathione (gsh)linked anticancer prodrugs activated by physiological concentration of gsts. structure and mechanistic studies showed that in the gst-gsh complex, the gst tyr residue placed near the s atom of the cys in gsh, is in the phenoxide form to facilitate the deprotonation of the sulfhydryl group in gsh, making it able to react with electrophilic species. by taking advantage of this active-site geometry, we synthesised two tyrosinase activated melanoma prodrugs ( -methoxyphenol and n-acetyl- -s-cysteaminylphenol) linked to gsh. in these compounds an ethoxycarbonyl group was placed between the gsh sulphur, oxidate to sulfone, and the tyrosinase-activated prodrugs. in this way the tyr phenoxide (tyr in gstp and tyr in gstm isozyme) would be able to abstract one of the acidic methylene protons linked to the sulfone moiety. such a deprotonation should trigger a beta-elimination, resulting in the release of melanoma prodrugs. the stability of the obtained putative prodrugs has been evaluated in different ph buffers and in rat liver and kidney cytosolic fractions. synthetic peptides represent first choice biomolecules, with respect to proteins and mab, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. it has been demonstrated that the surface molecule integrin alfa-v beta- could be an ideal target for the interaction with melanoma directed radiolabeled peptides. this integrin is indeed overexpressed both in tumor cells and in tumor vessels endothelium, while it is not expressed in the majority of normal tissues. cyclic peptides with the sequence arg-gly-asp (rgd), are known to bind the integrin alpha-v beta- with high affinity and selectivity and could conveniently target a radiotherapeutic to melanoma cells. we previously demonstrated that the cyclic rgdfk peptide interact with high affinity to a human melanoma cells. the aim of this work was to conjugate the mtc to the peptide by using the [ m tc(n)(pnp)] + system (pnp=aminodiphosphines) to obtain a radiolabeled peptide useful in melanoma imaging. , spectrum. a nh(i)→βch(i- ) cross peak more intense than the nh(i)→βch(i) cross peak is observed when the peptide adopts the . -helixl conformation. by contrast, an opposite trend of intensities of the same noe cross peaks indicates the occurrence of a -helical conformation. by using this method, we were also able to determine that polar solvents, such as mecn or meoh, may induce a helical structure in deg homo-peptides which otherwise adopt the . -helix conformation in cdcl . this finding allows us to rapidly switch these peptide series from "short" ( -helix) to "long" ( . -helix) by changing the solvent only, thus making them extremely attractive tools. school of biological sciences, university of auckland, auckland , new zealand naturally occurring anti-freeze proteins (afps) enable organisms like polar fish to survive the freezing temperatures of their natural habitat. as well as being cryoprotective, afps exhibit properties of ice recrystallization inhibition (ri). the ability of afps to influence the size, morphology and aggregation of ice crystals can be used in food technology, where the growth of ice crystals in frozen foods is of primary concern. chemical synthesis of afps enables access to pure material for structural and functional studies. our new research programme in the 'food and health' area, prompted us to develop tailor made anti-freeze peptides for potential applications in the frozen food industry. 'multiple salt-bridge analogues' of the naturally occurring winter flounder afp were synthesized and evaluated for their antifreeze activity in comparison to the natural sequence. details on the molecular modelling, structural characterization and anti-freeze activity analysis on the synthetic afps will be presented. several disorders are now believed to be conformational diseases caused by misfolding in the structure of specific proteins leading to the gradual aggregation and deposition of these abnormal proteins. attempts to develop effective therapies for these proteopathies (such as alzheimer's, parkinson's and huntington's disease) have been directed toward reducing the production of the proteins, inhibiting their aggregation, or promoting their removal. conformation-dependent antibodies that specifically target misfolded proteins are proving to be useful tools to study the pathogenesis and as possible treatments of these diseases. we have previously shown that active immunization with tetrapalmitoylated aβ - peptide embedded in liposomes (aci- ) was capable of eliciting a conformation-specific immune response in mice. the antigenic peptide was shown to mimic the aggregated β-sheet conformation of the pathological a β peptide on the surface of the liposomes by cd, ssnmr and tht assay. we have also reported the modulation of the conformation of aβ - derived sequences by means of different lipidation patterns, lipid chain lengths and liposome surface charge. here we present the characterization of this panel of liposomal peptide constructs by atr-ir and correlate the results with previous studies by cd. in summary, atr-ir secondary structure analysis of a battery of amyloid-derived lipopeptides allowed to study the key parameters responsible for the adopted conformation on the lipid bilayer. the presented strategy to control the conformation of liposomal peptide immunogens could be applied to the rational design of vaccines against a range of protein misfolding diseases, such as alzheimer's disease. cyclic lipodepsipeptides (clps) are secondary metabolites produced by various bacteria, most prominently bacillus and pseudomonas. these often display potential antimicrobial properties, frequently ascribed to the ability to disrupt cellular membranes or form ion-pores. clps are a structurally diverse group of molecules always consisting of an oligopeptide chain cyclized by a lacton bond and a fatty acid moiety. they are divided into several groups based on sequence similarity. one such group is the viscosin group, which contains clps produced by pseudomonas that all feature nine amino acids and a conserved sequence pattern of hydrophobic and hydrophilic residues. recently, we have extensively described the self-assembly properties of one viscosin group member, pseudodesmin a. , we have shown that in non-polar organic solvents this clp forms anisotropic supramolecular structures of unrestricted size, reminiscent of the ability to form ionpores in the non-polar cellular membrane environment. here, we report on how the most prominent structural variations within the viscosin group affect the conformation and self-assembly properties. since the well-defined monomer conformation of pseudodesmin a is key to this self-assembly, the most striking variation within the viscosin group is the variability of the leu stereochemistry. for the first time, a conformational study will be reported for clps featuring an l-leu residue, such as viscosin and viscosinamide. the results of this study contribute to a deeper understanding of the structure-function relationship of clps in general. effective design of peptide mimicking protein helical segments is a great challenge. an intriguing approach to improve the helical content in a peptide sequence is achieved through the "peptide stapling" strategy using a hydrocarbon cross-linker (staple) . in this study we have investigated the effect of the introduction and location of staples on secondary structure of sequences derived from a reported peptide, mimicking cullin binding region to kctd , , to gain an insight into the designing of new molecular entities able to modulate the kctd oncosuppressor activity. indeed, kctd plays a crucial role in the ubiquitination of hdac by acting, in complex with cullin , as an e ubiquitin ligase, thereby affecting hedgehog activity and medulloblastoma growth. in the designed peptides the stapling has been achieved by ring-closing olefin metathesis between two s- -( 'pentenyl) alanine residues incorporated at i and i+ positions and into different sites within the peptide chains. the stapling position has been chosen on the basis of cullin -kctd complex model. circular dichroism studies have highlighted that the stapled peptides exhibit remarkable and different changes in secondary structure contents in comparison with the linear precursors and the unmodified sequence. a nmr conformational analysis will allow the determination of the conformational preferences of the stapled peptides at atomic resolution with also relevant implications for the kctd-cullin recognition. based on our findings from protease-mediated ligation of cleavage-sensitive peptides and proteins using ionic liquids (ils) as additives and from chemical acylation reactions of peptides in ils, we hypothesized direct and unique interactions between biomolecules and ils as the molecular reason for il-mediated structural effects. to verify this, we performed systematic structural investigations of suc-ala-xaa-pro-phe-pna and ala-xaa-pro-phe model peptides dissolved in aqueous imidazolium-based ils using high resolution magic angle spinning (hr-mas) nmr spectroscopy. highly resolved nmr spectra are obtained that allow for complete h resonance assignments of the peptides as solutes and the ils as solvents. the changes in the proton chemical shifts Δδ( Η) of a peptide on dissolution in ils arise from direct interactions between both, including il-induced structural/conformational changes of the peptide molecules. , the strength, position, and type of these interactions are influenced by the nature of the amino acid residues in the peptide sequence as well as the nature and type of the il anions. downfield shifts indicate strong interactions between the ils and the peptide backbone, whereas upfield shifts point to hydrophobic interactions and stacking effects with the il imidazolium ring. the pattern of Δδ( Η) along the peptide chain reveals the sites and the type of interactions. the extent of Δδ( Η) implies a significant effect on the structure of the peptides. different Δδ( Η) were obtained for cis-and trans-configured peptide isomers and point to a conformer-specific il/peptide interaction. calcitonin is a -amino acid polypeptide hormone, which takes part in calcium metabolism in bones. it belongs to a protein family whose members may form amyloid fibrils. these fibrils (or pre-fibrillar aggregates) are related to serious diseases known as amyloidoses, whereas amyloid-like fibrils may have natural functional roles in many organisms. the amyloid form of human calcitonin is related to medullary thyroid carcinoma. using our online consensus prediction tool for 'amyloidogenic propensity' of protein molecules "amylpred", a novel hexapeptide of human calcitonin was predicted as a novel, possible amyloidogenic peptide. we investigated experimentally its ability to form amyloid fibrils, utilizing tem, x-ray fibre diffraction, atr ft-ir spectroscopy and polarized light microscopy. analysis of the results shows that this peptide self-assembles into amyloid-like fibrils and that amyloid fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites. we thank the university of athens for the financial support. school of pharmacy, university of norwich nr tj ; inserm u , université de rouen, mont saint aignan rfa, a rfamide neuropeptide, is the ligand of gpr and the system rfa/gpr is involved in feeding behaviour [ ] . a structural study by nmr showed that rfa, in a membrane mimetic medium, possesses a helix p -r -linker k -g -turn f -r motif. sar studies on truncated fragments of rfa indicate that, in addition to the turn motif, additional residues are required to maintain a correct biological activity ( rfa ec = , nm; rfa - ec = , nm; rfa - ec = , nm; rfa - ec = nm) [ ] . our aim is to synthesize new gpr ligands using an iterative process combining rational conception, peptide and modified amino acid syntheses, biological and structural analyses. structural and biological results will allow us to find new modifications and conceive more potent analogues. using nmr, we demonstrated that rfa - encompasses a nascent helix and a turn while rfa - possesses only the c-terminal turn structuration. the destabilization of the helical structure on fragments with a shorter n-terminal region could explain the decrease of the biological activity of rfa fragments. we decided to focus on the importance of a stabilized helix on the biological activity. we chose the hydrogen bond surrogate method which stabilizes a helix by replacing the main hydrogen bond with a covalent link without modifying the side-chains [ ] . syntheses, nmr characterization and molecular modelling of analogues will be discussed. acknowledgments: this work is supported by erdf funding through the is:ce-chem project and interreg iv a france-(channel)-programme. firstly, it induces a constraint by restricting the ı o-dihedral angle to values around - °. secondly, the xaa-pro peptide bond is subject to cis-trans isomerization characterized by an increased cis population and an activation energy that is low when compared to the other amino acids. besides, the five-membered ring of the pro residue can adopt two main distinct conformations (up-puckered or downpuckered) that are almost equally abundant in peptides and proteins. a variety of mimics and analogs have been designed in order to control the conformation of the peptide backbone and/or to alter the cis/trans ratios and the rotational barriers for cis-trans isomerization. in this presentation, nmr studies and theoretical calculations have been performed on model peptides ac-ser(Ψpro)-nhme, (s,s)-ac-ser(Ψh, cf pro)-nhme, and (r,s)-ac-ser (Ψ cf ,hpro)-nhme. their thermodynamic and kinetic features have been analyzed in chloroform, dmso, and water, allowing a precise description of their conformational properties. we found that trifluoromethyl c δ -substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. in chcl , the configuration of the cf -c δ entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn like or ppi/ppii-like backbone conformations. moreover, in water and dmso, this c δ -configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the nmr observations. in contrast to formation of the native state, aggregation of misfolded protein molecules into so-called amyloid fibrils is a strongly irreversible, kinetically-controlled process which may lead to structural variants of fibrils composed of chemically-identical protein chains. this poorly understood aspect of polymorphism of amyloid fibrils has important implications in vivo in the course of amyloidassociated neurological disorders -e.g. in the form of prion strains in creutzfeldt-jakob disease. in this paper, we examine cross-seeding behavior of a model amyloidogenic polypeptide -bovine insulin (bov-ins) and recombinant lys b -arg b insulin (kr-ins). fibrils of bov-ins and kr-ins exhibit characteristic "fingerprint" features in the amide i' infrared region. these spectral traits are transferred to daughter generations of fibrils upon cross-seeding of native bov-ins with kr-ins fibrils and vice versa. this study highlights an interesting aspect of a non-anfinsenian behavior of aggregating insulin: the memory effect. our results indicate that fine infrared features in the amide i region of fibrils from closely related proteins may not reflect different amino acid composition but rather the fact that a particular amino acid sequence favors an amyloidogenic pathway that determines a certain and spectrally-distinct stacking mode of β-strands. an encounter with an alien seed on the amyloidgenic pathway would hijack the yetnative monomers and force them to follow the assembly pattern (and resulting ir characteristics) of the preformed fibril. phosphorylation and oglcnacylation are dynamic intracellular protein post-translational modifications that often are alternatively observed on the same protein serine and threonine residues. phosphorylation and oglcnacylation commonly occur on residues in natively disordered regions of proteins, and often have opposing functional effects. in the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of alzheimer's disease, whereas oglcnacylation is stabilizing. a series of peptides derived from the prolinerich domain (residues - ) of tau was synthesized, with the free ser/thr hydroxyls, with phosphorylated ser/thr, with oglcnacylated ser/thr, and with the dietylphosphorylated ser/thr. phosphorylation and oglcnacylation were found by cd and nmr to have opposing structural effects, with phosphorylation favoring polyproline helix (ppii) formation, with oglcnacylation opposing ppii, and with the free hydroxyls intermediate. phosphorylation of serine and threonine residues in prolinerich sequences was found to induce ppii in other proline-rich sequences, with significant conformational restriction at phosphoserine and phosphothreonine residues observed by nmr. notably, serine and threonine residues are common in proline-rich domains of proteins, and ppii is a common conformation observed for phosphorylated ligands in protein-protein interactions. the ser/thr diethylphosphate was found to be a practical mimic of oglcnacylation, as an easily synthesized neutral amino acid with similar sterics to oglcnac and strongly opposing ppii. dept. of biology, university of padova, padova , italy we recently established that designed hairpin peptides interfere with the amyloidogenesis of both human pancreatic amylin and α-synuclein ( α-syn), two 'unstructured' polypeptides associated with human diseases. in the case of α-syn, the most potent 'amyloid inhibitors' act by diverting α-syn to non-amyloid aggregates. the binding sites for these agents and nonhairpin peptides of similar sequence have been determined by d-nmr-monitored titration studies using universally n-labeled α-syn. in the case of the hairpins that produce non-amyloid aggregates from α-syn, initial binding occurs in the non-amyloidogenic c-terminal segment of α-syn and the hairpin peptide is incorporated in the resulting aggregates. nmr studies also serve to define binding interactions and conformational changes that may be essential early steps in aggregation and pre-amyloid state processes. mt-ii is a long acting, non-selective super-agonist of melanocortin gpcr receptors, characterized by lactam bridge between residues i and i+ stabilizing a type-ii β turn structure. the recently introduced cui-catalyzed azide-alkyne , -dipolar cycloaddition, presents a promising opportunity to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-toside chain cyclization. we introduced this [ , , ]triazolyl-mediated cyclization in a pthrp model peptide, and demonstrated that i-to-i+ side chain-to-side chain cyclization offers a powerful approach for generating stable helix mimetic structures. , , in the current study we explored the potential of the i-to-i+ side chain-to-side chain , -disubstituted [ , , ] triazolyl-bridge to stabilize -turn conformations. we designed a series of [ , , ]-triazolyl containing cyclo-heptapeptides, derived from the model peptide lactam mt-ii, differing in the size of the bridge, the location and orientation of the [ , , ]-triazolyl moiety. conformational and biological studies, performed on this series demonstrate that [ , , ] triazolyl-mediated side chain-to-side chain cyclization results in the conformational stabilization of type-i β turn generating heterodetic cyclopeptides with enhanced in vitro potency and improved receptor subtype selectivity. this study confirms the versatile nature of the [ , , ] triazolyl-mediated side chainto-side chain cyclization. moreover the chemical accessibility, functional compatibility, and metabolic stability suggest that the [ , , ] international associated laboratory samuel de champlain, regional platform for cell imaging of haute-normandie (primacen), institute for medical research and innovation (irib), university of rouen, mont-saint-aignan, france scorpion toxins are polypeptidic molecules capable of selectivity binding to ion channels with the highest affinities and represent natural peptide blockers that are good tools to characterize new or particular types of ion channels. astroglial cells appear to express all ion channels species even voltage-gated ion channels previously suspected to be present only in excitable cells; but these channels are poorly studied and their function are not still completely elucidated.thus, the purpose of the present study was to investigate for the first time, the global effect and the potential protective effect of one scorpion toxin acting on kv channels, the pi ,on h o -induced oxidative stress and cell death in rat astrocytes. pi is a -residue toxin crosslinked by disulfide bridges, isolated from the venom of pandinus imperator and is one of the most potent and selective inhibitor of shaker b and kv . channels . in our study, pi was obtained by solid phase peptide chemical synthesis(spps) and named spi . the identity and homogeneity of spi were also verified by hplc, mass spectrometry analysis(maldi-tof) and an enzyme-based cleavage to determine the assignment of spi half-cystine pairings.the synthetic spi was found to be indistinguishable from the natural toxin. incubation of cultured astrocytes with graded concentrations of h o for h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. interestingly, pre-treatment of astrocytes with very low concentrations of spi ( - to - m),dose-dependently prevented cell death induced by h o , and the effect of spi was accompanied by a marked attenuation of ros accumulation. also, spi stimulated sod and catalase antioxidant activities in a concentrationdependent manner,and blocked h o -evoked inhibition of sod and catalase activities. in addition, at low concentration ( - to - m) the toxin exhibits no toxic effect on astrocytes.taken together these data indicate that the spi acts as a protective agent on astrocytes from oxidative assault and then might have therapeutic value in the potential treatment of a number of neurodegenerative diseases. [ ] . in order to select small active peptides, the library was kept at the minimum size and was built using only selected d-amino acids to avoid structural redundancies. to further increase the chemical diversity [ ] a set of different carboxylic acids were coupled to the n-terminus. the iterative screening led to the identification of an n-terminally modified tetrapeptide with a very high radical-scavenging activity. by analyzing several peptide variants, we found that antioxidant properties were strongly sequence-and structure dependent, because scrambled or proline-scan variantswhose secondary structures are deeply altered -are much less effective. data on peptide structure-activity relationship will be presented. an investigation of the in vitro effects produced in cellular models of oxidative stress are also in progress. structural analysis of peptide-analogues of zp proteins with amyloidogenic properties: insights into mammalian zona pellucida formation. n.n. louros, v.a. iconomidou, s.j. hamodrakas* department of cell biology and biophysics, faculty of biology, university of athens, athens , greece a filamentous structure surrounding mammalian oocytes, called zona pellucida, is responsible for species-specific sperm binding. this extracellular porous structure is composed of - glycoproteins, termed zp - , which assemble into filamentous polymers that are cross-linked by disulfide bonds. all zp proteins present a unit with high sequence homology at their c-terminal end, designated as the zp domain. this structural module is found in a family of extracellular proteins with various functions and from a wide variety of organisms (zp domain proteins). it is a binary structure of approximately amino acids, divided into two domains, the n-terminal region named zp-n and the c-terminal region known as the zp-c domain. the zp-c block is associated with protein function, whereas the zp-n domain is responsible for protein polymerization. structural analysis revealed a possible polymerization model based on backbone interactions between specific parts of the zp-n units. peptide-analogues of the aforementioned segments were synthesized, since the ''amylpred'' application, an 'amyloidogenic determinant' prediction algorithm developed in our lab, predicted them as being potentially 'aggregation prone'. our experimental data clearly indicate that these peptides do actually fold and self-assemble into amyloid-like fibrils. therefore, all the derived data suggest that mammalian zona pellucida is a novel functional amyloid, similar to its biological equivalent, silkmoth chorion that has been established to be a natural, protective amyloid. we thank the university of athens for the financial support. biostrucutres and bioimmaging institute of national research council, naples, italy the definition of the molecular basis of human diseases is one of the most important goals of structural biology. nucleophosmin (nmp ) is an ubiquitously expressed protein is a nucleolar chaperon and has a key role in several biological processes from ribosome biogenesis, to cell-cycle regulation and is a frequently target of oncogenic mutations in acute myeloid leukaemia (aml) [ ] . these mutations occur predominantly at the c-terminal domain of npm compromising its stability and causing the aberrant translocation of npm to the cytosol. this domain is structurally characterized by the presence of three alpha helices, and the folding of cter-npm proceeds via an extended nucleus and the denatured state retains significant malleable structure at the interface between the second and third helices [ ] . here, we have further investigated the structural determinants in the folding process of the c-term npm domain and on the basis of available structural and composition data, several peptides covering the three helices were designed and synthesized. their conformational properties were investigated by cd and nmr spectroscopy: these studies demonstrated that once removed from the protein architecture helix and helix substantially miss fully ordered conformations while helix retain a high helical content also in mutated variants. our observations may help structure-based drug-design inserm u university of evry-val d'essonne, bâtiment maupertuis, evry, france sl (stop-like protein with mass of kda) is a neuronal protein with a calmodulin-binding and microtubulestabilizing activity. microtubule binding is inhibited by calmodulin , . the lack of the stops gives rise to defects in synaptic plasticity, as observed in schizophrenia . to study the sl protein's structure and its interaction with calmodulin, we have used a residue peptide derived from sl , containing the microtubule binding motif mn. the structure of the peptide has been analyzed using circular dichroism (cd) spectropolarimetry. the cd spectra showed that the peptide's secondary structure consists in a predominantly β-sheets, while the tertiary structure consists in rather folded d-structure. the temperature dependence of cd indicated that the secondary structure of the peptide remained very stable between °c and °c, while the tertiary structure changed at about °c. the interactions between the sl peptide and calmodulin have been studied using the isothermal titration calorimetry (itc). the results showed that calmodulin binds the peptide with the affinity of m- , in a ca + -independent manner, the reaction being driven by entropic contributions. our results might contribute to understanding the microtubule stabilizing activity of the sl protein. peptides , . based on the structure in polar environment a transition from -helical to an α-helical conformation at the water/lipid interface in bilayer membranes is hypothesized the efficient method development for the analysis of proteins, peptides, and synthetic oligonucleotides with a novel hybrid reversed phase column f. becker * , n. shoji * , a. matsui * , c. yokoyama * , t. sato * , t. takai * , and n. kuriyama * * ymc europe gmbh, * ymc co., ltd. the recent development of bio-pharmaceutical industry has been remarkable and an effective analytical method with higher sensitivity, superior selectivity, and increased speed has been required in the characterization of peptides, proteins and oligonucleotides by highperformance liquid chromatography (hplc). the method development of reversed phase hplc requires optimization of several conditions, such as bonded-phase, column efficiency, solvent type, ph and temperature. especially ph and buffer type is a most important parameter to control retention of biomolecules which have multiple ionic functional groups. furthermore, temperature often becomes a key tool to achieve better peak shapes and resolution for larger molecular-weight compounds. although silica based reversed phase columns have been widely used for separation of biomolecules, they have low stability under alkaline conditions and a limited usable ph range. to improve the chemical stability at an expanded ph and temperature range , we have developed a new type of organic/inorganic hybrid reversed phase column, ymc-triart c and ymc-triart c . the novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety of mobile phase conditions. in this poster, we will show characteristics of this new hybrid column, and some example cases of efficient method development in separation of the biomolecules such as peptides, proteins and synthetic oligonucleotides. the fully-extended peptide conformation ( . -helix) is the longest possible for a single α-amino acid as it is characterized by an axial translation per residue of about . Å. therefore, it is extremely attractive for its application as a molecular bridge. however, it is known that the stability of this type of helical structure (relative to the competing, much shorter -helix) appears to be dramatically governed by the choice of the c-terminal functionality (only esters seem to be appropriate). in this work, we examined the series pyrac-(deg)n-o-(pno )bzl [where pyrac is the fluorophore -pyrenylacetyl, deg is cα,α-diethylglycine, n= - , and o-(pno )bzl is para-nitrobenzoxy] with the deg homo-peptides as fully-extended bridges in a static fluorescence study. to perform a very stringent comparison with our ft-ir absorption and nmr results, the solvent used was chcl . the trend observed in the quenching efficiencies for the shortest members of the series, the (deg) - peptides, steadily decreases from . (n= ) to . (n= ), and . (n= ). this finding is compatible with the conclusion that these three peptides are all essentially fully-extended in chcl solution. however, the quenching efficiencies for the longest deg homo-peptides. . for n= and . for n= , are not further reduced. the inevitable conclusion is that the tetra-and pentapeptides populate at least two different conformations (most probably, the . -helix and the helix), in which the probe dyads are located at different distances. notably, these results fit well with those extracted from our ft-ir absorption and nmr studies in the same solvent of low polarity. we conclude that a combination of terminally aromatic groups, as in the deg homo-peptide series investigated in this work, is not eligible for the fabrication of a stable fully-extended conformation. the surfactant protein c (sp-c) is a -residue highly hydrophobic peptide with two covalently linked fatty acyl chains that adopts a mainly helix structure while associated with the membrane. however, it misfolds into a β-rich structure under certain environmental conditions, which suggests that sp-c is in a metastable state . the slow α→β transitions are likely to be caused by the membranedissociation, deacylation and exposure to the neutral ph of the alveolar subphase, leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. we have been studying the ph-dependent conformational behaviour of both the acylated and deacylated isoforms of sp-c in a membrane mimetics chloroform/methanol/water mixture using a constant-ph molecular dynamics method [ ] [ ] [ ] . our simulations identify the increase of ph as a promoter of peptide-peptide interactions, which may contribute to the α→β transitions observed at neutral and alkaline ph conditions in the experimental results . we also observe that the loss of structure is more prone to begin at the c-terminal region and that the β motifs start to form between the n and cterminal disordered regions of the sp-c. in addition, the presence of the acyl groups results in a slightly higher structural stabilization of the sp-c helix and in a decrease of the n-terminal plasticity at acidic and neutral ph. since a comprehensive conformational analysis and the elucidation of the mode of association between sp-c and the membrane are essential to understand the interactions involved in structure stabilization and in some specific functions of the peptide, we are now studying both sp-c isoforms in a membrane environment. these studies will not only be the most relevant for the in vivo situation but will also answer to some questions left open by the previous work. were rather small. vp plays a very important role in controlling resorption of water by the distal tubules of the kidney and in regulating the osmotic pressure of blood in mammals. vt, while exhibiting both oxytocin and vasopressin activities, has not changed over the last million years until appearance of the mammals. currently, it occurs naturally in the non-mammal vertebrates [ ] . here we report on studies of arginine-vasopressin (cyfqncprg-nh , avp) and arginine-vasotocin ([ile ]avp, avt) in a mixed dodecylphosphocholine (dpc) and sodium dodecylsulfate (sds) micelles. dpc micelles are considered good models of eukaryotic cell membrane [ ] , while the sds micelle improves peptide/micelle solubility [ ] . for these studies we use d nuclear magnetic resonance (nmr) supported with molecular modelling methods. conformation-activity considerations relationships and the role of the peptide-micelle interactions on the structure of the analyzed peptides will be described and discussed. vibrational and chiroptical spectroscopic investigation on diamide peptide models k. knapp, a m. hollósi, a e. vass, a zs. majer a a eötvös lor nd university, institute of chemistry, laboratory for chiroptical structure analysis, budapest, hungary understanding of peptide folding has great importance in many fields of chemistry. since amino acid residues are the simplest building blocks of peptides, the investigation of their conformational properties could help us in understanding the structures of peptides or in designing peptides of specific d structure. describing the conformational building blocks of even the simplest peptide models (e.g., amino acid diamides) can serve as a good starting point to decipher higher-level structural properties of their polymers. among the simplest compounds which include the essential structural elements of polypeptide backbone are the n-methylamides of n'acetylamino acids. these compounds have been the subject of extensive spectroscopic analyses (ftir, vcd, ecd, nmr) [ , ] . the conformational landscape of these model compounds were analyzed by solution phase vcd and ecd spectroscopy and density functional theory (dft) computations. this work reports systematic vcd and ecd spectroscopic studies in different solvents on synthesized diamide models (ac-α/βhomo-xxx-nhch , xxx = ala, pro, phe). vcd and ecd data, combined with quantum chemical calculations performed at the b lyp/ - ++g** and - ++g** level of theory was particularly useful for identifying the preferred dipartimento delle scienze biologiche, università di napoli "federico ii", napoli, italy c dipartimento di scienze ambientali, seconda università di napoli, caserta, italy β-hairpin is an important secondary structural element used by many proteins for biomolecular recognition, and thus is an attractive tool for mimetic design . aβ -hairpin motif consists of two antiparallel strands linked by a turn region. in this work we aimed to stabilize a β-hairpin conformation by an interstrand covalent bridge, made through a click chemistry reaction, which yields , -disubstituted , , , triazole, starting from alkyne and azide reactive groups. in particular, we explored the conformational stability of a series of β-hairpin peptides containing a , -disubstituted , , triazole bridge with variable length. we employed the well structurally characterized trpzip peptide as a convenient b-hairpin model system. as alkyne amino acids were introduced propargylglycine (pra), homopropargylglycine (hpg) or bishomopropargylglycine (bpg), while the following azide amino acids were inserted: lβ-azidoalanine (dap (n )), l-γ-azidohomoalanine (dab (n )), and l-δ-azidoornitine (orn (n )). all peptides were synthesized and purified. successively, the linear unprotected peptides were cyclized in solution by the cu(i)-catalyzed azide alkyne cycloaddition (cuaac) reaction and purified prior to the spectroscopic characterization. linear and cyclic peptides were analyzed by a combination of cd and nmr techniques. have been investigated. their behaviour has been compared with that of proline in homochiral and heterochiral dipeptide sequences including l-or dphenylalanine. nmr and ir studies as well as x-ray diffraction analysis provide evidence that the additional β-phenyl substituent does not disrupt the tendency of proline to occupy the i+ position of a β-turn. moreover, the β-turn conformation is stabilised, with reference to l-pro, when l-cis(βph)pro or l-trans(βph)pro are combined with d-phe, that is, in the heterochiral sequences. the pyrrolidine conformation is significantly affected by the presence of the β-phenyl group. the puckering modes that alleviate most the steric hindrance introduced by the bulky phenyl substituent are preferred. the l-cis(βph)pro residue shows a marked propensity for the cγ-endo/cβexo half-chair arrangement whereas the trans derivative exhibits a higher flexibility, with different pyrrolidine shapes being accessible. interactions between the aromatic ring of (βph)pro and that of the contiguous l-or d-phe residue are observed in all the dipeptides crystallised. in solution, they seem to occur only for the heterochiral sequences. this intramolecular aromatic-aromatic interaction may be responsible for the higher β-turn ratio observed for such sequences and also seems to affect the conformational preferences of the pyrrolidine ring. in fmoc chemistry the trt and acm groups are widely accepted as a protecting group for cys. upon incorporation of these cys derivatives onto the growing peptide chain, however, considerable base-catalyzed racemization of cys is known to always occur at the activating and coupling steps with phosphonium or uronium reagents such as pybop or hbtu, respectively. in addition to this, racemization of the c-terminal cys esterified to resins also occurs during the repetitive n α -fmoc deprotection using piperidine. in order to find an s-protected cys derivative applicable for fmoc chemistry that can efficiently suppress racemization of cys both when its incorporation is conducted by phosphonium or uronium reagents and when it is linked to a resin via ester linkage, we introduced the methoxybenzyloxymethyl (mbom) group into the thiol function on cys. the mbom group was found to substantially supress racemization of cys ( . %) during its incorporation mediated by phosphonium or uronium reagents in comparison with the trt and acm groups ( . % and . %, respectively). even after a -h treatment of the peptide resin, in which its c-terminal cys was linked to a trt-type resin, with % piperidine in dmf, the mbom group caused a significant reduction in the level of racemization with the c-terminal cys ( . %) while trt and acm groups led to a considerable extent ( . % and . %, respectively). next, we demonstrated the usefulness of the mbom group on cys by applying it to ncl chemistry involving the coupling of a peptide thioester and a cys-peptide. in the synthesis of the latter peptide, however, a diastereoisomer with racemization of the nterminal cys tends to be difficult to separate from the intact peptide as its chain length increases. thus, this measure using fmoc-cys(mbom) can facilitate the avoidance of ambiguities in the quality of the synthesized peptides. labeled peptides are important tools in chemical biology to obtain information on their biological function in, for example, cellular communication processes as well as to study the molecular basis of diseases. the functionalization of bioactive peptides with biophysical reporter groups like strongly absorbing chromophores, is most often performed via post-synthetic chemoselective biocojugation reactions, involving amino, thiol, or azide specific reactions. an alternative approach for the installation intense chromophoric organic molecules, is the coupling of nonchromophoric precursor molecules to obtain chromophoric properties in the final product. recently, we have shown that the [ + ] cycloaddition-cycloreversion reaction between peptide-functionalized electron-rich alkynes and electrondeficient cyanovinyl derivatives results in the formation of a new class of π-conjugated peptidic donor-acceptor chromophores. , the chromophoric properties of the final product can be tuned by varying the p-electron conjugation pattern. herein, we describe the synthesis of the versatile building blocks, n-a-( -ethynylphenyl)-n-a-(methyl)-glycine and n-a-( -( , , -tricyanovinyl)benzoyl)-glycine, their subsequent application in spps to functionalize peptides with d-p-a chromophore precursor molecules, and their uvvis absorption characteristics. as a typical example, alkyne-functionalized aβ peptides, val-ala-ile and lys-leu-val-phe-phe-ala-glu, have been reacted with a series of cyanoolefins to obtain far-red intense imaging chromophores for amyloid-rich peptide aggregates. a novel method for regioselective disulfide formation in mini-proteins by kinetic oxidation control t. lindner, u. haberkorn, w. mier department of nuclear medicine, university hospital heidelberg, im neuenheimer feld , heidelberg, germany rigid frameworks are essential for the development of peptide-based alternatives to immunoglobulins. [ ] disulfideconstriction covalently stabilizes tertiary structures, which allows further miniaturization of protein-derived drugs by chemical synthesis. while the amino acid assembly steps in solid phase peptide synthesis are highly efficient, regioselective formation of multiple disulfide bonds is still a challenging and time demanding task. even after applying laboriously optimized folding conditions and extensive purification, a successful synthesis of the correctly folded isomer is not guaranteed. [ ] to circumvent these problems, a procedure that takes advantage of the different reactivity of free vs. acmprotected thiols during iodine oxidation was developed. this protocol to sequentially form two disulfide bridges in a one-pot reaction was applied to different peptides, such as (a) min , a mini-protein scaffold for phage-display assisted drug design; [ ] (b) α-conotoxin imi, a neurotoxic peptide isolated from the venom of marine cone snails; [ ] and (c) endothelin- , a peptide involved in blood pressure regulation. [ ] the presented technique tolerates precarious residues within the to-be-folded-peptide, such as fluorescent dyes or metal chelators as used for bioactivityassays. compared to the individually optimized literature procedures, this novel approach offers significant improvements: overall yields > % are obtained in first attempts and stepwise formation of the disulfide bridges is performed within a few hours instead of days. in recent thirty years, c-terminal modified peptides have been proved to have greater potential as apis (active pharmaceutical ingredients) due to their increased chemical and enzymatic stability and improved pharmacodynamic properties - . a prominent example, octreotide - , an octapeptidoalcohol, has witnessed as a potent anti-cancer agent targeted for gastro-entero carcinomas. in view of synthetic methodology, peptidoalcohol can not be directly prepared by standard spps protocol becouse of the c-terminal structure released from resin are not alcohol but always peptidoacid or peptidoamide. to overcome this problem, a novel protocol of shortened n- coupling cycles on merrifield resin and then the ammonolysis of peptedyl resin by an aminoalcohol as the c-terminal residue getting peptido-alcohol as targetting product has been devoloped in our lab. because of the cleavage treatment of peptidyl merrifield resin is not under acidic condition, such as hf or tfmsa, but ammonolysis, some side-chain producting groups(spg) related to boc chemistry like bzl, clz, tos…; must be avoided in sequence assembly. therefore a hybrid orthogonal protection (hop) of boc/fmoc protocol was adopted for the sake of producing naked peptidyl (without any spg) resin before ammonolysis. fifteen peptidoalcohols with different terminal alcohols were conveniently prepared, most of them released form resin with very good yields. due to its cyclic structure, proline is the coded amino acid with a more restricted conformational flexibility. the incorporation of additional groups into the pyrrolidine ring is a useful means to produce new amino acids that combine the conformational properties of proline with sidechain functionality. this is the case of β-phenylproline, (βph)pro, that can be regarded as a proline-phenylalanine hybrid in which the orientation of the aromatic substituent is dictated by the conformation of the five-membered ring and the cis or trans configuration of the phenyl group relative to the carbonyl moiety. accordingly, cis(βph)pro and trans(βph)pro combine the conformational properties of proline with an aromatic side-chain functionality that is rigidly oriented with respect to the peptide backbone, and this may be useful in the design of biologically active peptides and other applications relying on specificallyoriented side-chain moieties. we have developed synthetic procedures for the preparation of the cis(βph)pro and trans(βph)pro stereoisomers in enantiomerically pure form. the methodology is based on the preparation of racemic precursors of each amino acid and their subsequent hplc resolution on chiral columns. multigram quantities of the target amino acids have been isolated in optically pure form and suitably protected for use in peptide synthesis. the importance of peptide cyclization for studying peptide conformation, creating new structures, or for developing peptide therapeutics is well established. in particular, sidechain lactam bridges linking two amino acid residues that are several residues apart in the linear sequence or headto-tail backbone peptide cyclization enable rigidification of the structure and improvement of in vivo stability. native chemical ligation (ncl) is now an established method for producing backbone-cyclized peptides or proteins. the application of ncl to the synthesis of sidechain cyclized peptides is less frequent. head-to-side-chain cyclization by ligating a c-terminal thioester with a cys residue located on a lysine side-chain was used by few authors. the alternative tail-to-side-chain cyclization mode is rare, probably due to the difficulty of installing a thioester group on amino acid side-chains such as aspartic or glutamic acids the reaction of a bis( -sulfanylethyl)amido (sea on ) group with an n-terminal cysteine residue in water and at neutral ph results in the formation of a native peptide bond. [ ] oxidation of sea on results in a cyclic disulfide called sea off having a , , -dithiazepan- -carbonyl structure. [ ] sea off is a self-protected form of sea on . we show here that bis( -sulfanylethyl)amido side-chain acid(dab), ornithine and lysine were selected as building block; a and n,n'-cbz- -amidinopyrazole ( b) were selected as guanidinylating reagents for specific situation. for synthesis of n-terminus local cyclo-guanidine peptide, designated peptides were assembled on acid labile solid support such as rink amide resin by fmoc strategy. then either fmoc-dab(boc)-oh, fmoc-orn(boc)-oh or fmoc-lys(boc)-oh was incorporated respectively at n-terminus. fmoc was removed followed by guanidinylating by b and then peptide was cleaved by acid. by neutralizing with nmm in acetonitrile solution, side chain amino group and a-guanidine would form , or membered local cycloguanidine. the remaining cbz could be removed by hydrogenation. for synthesis of backbone side chain cyclic peptide, bis-fmoc-daa was introduced in the peptide previously on resin followed by removal of fmoc. selective guanidinylate side chain aminogroup by a followed by peptide assembling with an insertion of orthogonal protected daa at - aa apart from first daa. for synthesis of a-nh sidechain cyclic peptide, first daa should be introduced with orthogonal protected form. b was used to guanidinylate a-nh . after cleavage and neutralization of those two kinds of intermediates, guanidine-bridged marco-cyclic peptide was formed. the resin is also a multipurpose tool for the synthesis of carboxylic acids, esters and thioesters. when the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either n-bromosuccinimide (nbs) or copper(ii) acetate in pyridine. the resulting acyl diazene resin is then cleaved by peptide displacement at the c-terminus with amine. the fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (tfa). in our approach, we used a -fmoc-hydrazinobenzoyl am novagel resin to synthesize a peptide-substituted amide in the c-terminus. first, the oxidative cleavage was carried out with nbs in pyridine and a nucleophile [a protected (aminomethyl) benzimidamide (amba)]. however, the yield of the reaction was very poor. in the next step, we applied copper(ii) acetate in the presence of pyridine and amba. following optimization, the efficiency of the process was significantly improved. herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of our c-terminal modified peptides using the aryl hydrazine resin linker. blood vessels on tumor tissues, similarly to integrin receptors. this observation suggests cd as a selective target for targeted delivery of drugs and nanoparticles to tumor neovasculature using ngr peptides as homing motif. in our work, new cyclic-ngr peptides containing a thioether linkage were prepared. the influence of their structure on the speed of succinimide ring formation and deamidation was evaluated and compared with the previously published data on cyclic-ngr derivatives containing amide bond or disulfide bridge in the cycle (c[kngre]-nh and c[cngrc]-nh ). to avoid the deamidation under the conditions used for cyclization, the synthetic routes were optimized. the influence of the ph, ionic strength and temperature of the solution on their chemical stability was investigated. the structure of the cyclic peptides was investigated by circular dichroismand nmr-spectroscopy. receptor binding ability and the influence of the cyclic peptides on the cell adhesion and motility were also evaluated. this work was supported by grants from the hungarian national science fund (otka nk and k ) and the national innovation office (bio_surf, om- / ). clickable peptides and their attachment to oligonucleotides m. wenska, m. alvira, r. strömberg department of biosciences and nutrition, karolinska institutet, novum, se- huddinge methodology for the ready conversion of peptides into "clickable" azido-peptides with the possibility of selecting either n-terminus or c-terminus connection is presented. synthesis of peptide-oligonucleotide conjugates (poc's) include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. a general procedure, based on a new activated alkyne linker, for the preparation of poc's has been developed. with this linker, conjugation is effective at room temperature in mm concentration and submicromolar amounts. this is made possible since the use of a readily attachable activated triple bond linker speeds up the cu(i) catalyzed , -dipolar cycloaddition ("click" reaction). the main scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of i) an h-phosphonate based aminolinker ii) the triple bond donor p-(npropynoylamino)toluic acid (pata) and iii) azido-functionalized peptides. the method gives excellent conversion of oligonucleotide to the poc on solid support, and only involves a single purification step after complete assembly. the procedure which makes use of a low concentration of copper ions leads to a product with very little copper left (similar or less than in drinking water). the synthesis is flexible and can be carried out in non-specialist laboratories without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. comparison of alternative deprotection reagents to piperidine for the synthesis of a poly-alanine peptide on the tribute® peptide synthesizer m.a. onaiyekan,* j.p. cain, c.a. chantell, m. menakuru protein technologies, inc. tucson, az, usa in peptide synthesis, piperidine is a common agent for fmoc removal. however, piperidine is a controlled substance which requires special handling and cannot be used in some countries. therefore, it would be useful to identify alternative deprotection reagents to piperidine for fmoc removal. it is well known that poly-alanine sequences have a high propensity to aggregate after the fifth residue. in this application, (a) k-oh was synthesized using the tribute®'s intellisynth uvmonitoring and feedback system to compare the efficiency of fmoc removal by piperidine vs. three alternative bases (pyrrolidine, cyclohexylamine, and tertbutylamine) in the last cycles of the synthesis. it was found that pyrrolidine produced a higher purity product with fewer deprotection repeats and shorter deprotection times per cycle than piperidine, proving it to be a highly efficient, viable alternative to piperidine for fmoc removal. the endogenous tripeptide gpe also nammed "glypromate" is made up by the three n-terminal residues (glycine-proline-glutamate) of the insulin-like growth factor (igf ). this tripeptide is a partial glutamate antagonist and showed good results in different neuroprotective in vitro and in vivo experiments. , gpe also binds to glial cells regulating neurotransmitter levels in the brain. , however, gpe suffers from poor lipophilicity and a short half-life in vivo. that's why there is a need for more lipophilic and protease resistant analogues of gpe. in this poster we present the synthesis of trifluoromethylated analogues of gpe based on the or -cf -pseudoproline residues. introduction of fluorine atoms on bioactive compounds is known to deeply modify their physico and biochemical properties increasing lipophilicity and resistance to protease. thus, developing a trifluoromethylated analogues, we intend to increase the bioavailability of gpe, keeping the benefit of its neuroprotective properties. our research team is strongly involved in the synthesis of trifluoromethylated alpha-amino acids. recently we published the synthesis of -trifluoromethyl- , oxazolidines derived from fluoral and (l)-serine and we demonstrated that these five membered ring -cf pseudoprolines are hydrolytically stable and can be considered as proline analogues. that's the reasons why we are interested to replace the proline residue of gpe by those trifluoromethylated compounds. the development of original coupling conditions and the detailed synthesis of two pseudoprolines analogues of gpe will be presented in this poster. modifications. in combination with automated spps, unprecedented access to large peptides and small proteins for biological research has been achieved. we demonstrate the application of this methodology to the synthesis of a variety of peptides on the prelude® peptide synthesizer. exploring the space of fluorine-labeled α-amino acids for solid state f-nmr structure analysis of peptides: rational design, synthesis and applications p. solid state f-nmr is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. for this purpose the native peptide has to be selectively labeled with a suitable f-containing amino acid at several different positions. the resulting battery of singly f-labeled analogues is then analyzed by solid state f-nmr. the main limitation to this approach currently lies in the poor arsenal of available f-labels. we have therefore rationally designed and synthesized several specific amino acids bearing a cf -reporter group, which fulfil all strict criteria to a "proper" f-label. , to allow a geometry-based structure calculation, the cf group has to be rigidly attached to the peptide backbone. we thus rigidified the side chain using either a [ . . ]bicyclopentane moiety, a cyclobutane ring, or the intrinsic proline framework. this way, suitable cf -labeled analogues were created as substitutes for bulky hydrophobic amino acids (leu/ile/val/met), for aromatic residues (phe), for polar side chains (ser/thr), and for proline (pro). by now we have applied the developed f-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (gramicidin s, pgla, mag , kigaki, sap, temporin a, bp , etc). recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times on the symphony® peptide synthesizer. it was found that hdmc, pyclock, comu, hctu, and hatu worked well at shorter reaction times ( x min), but pyoxim and tffh only worked well at longer reaction times. the performance of pybop at shorter reaction times was poor only for more difficult sequences. these results are important for selecting an appropriate activator for fast spps applications. the plant cyclotides form the largest family of cyclic peptides . they contain a signature motif referred to as the cyclic cystine knot, which is derived from the cyclic backbone and three inter-knotted disulfide bonds. intriguingly, cyclotides can be boiled, treated with chemicals or enzymes without disrupting their overall fold. thus, they are sometimes labeled as ultra-stable proteins. in addition, cyclotides are tolerant to mutations, and as a scaffold they can successfully accommodate foreign bioactive epitopes of variable sequences . cyclotides share many of these properties with another disulfide containing cyclic plant peptide, the sunflower trypsin inhibitor (sfti- ) . emerging evidence indicates that cyclotides and sfti- are valuable not only as peptide stabilizing scaffolds; in combination with their cell penetrating properties, these disulfide rich cyclic peptides have significance as intracellular drug carriers. although both peptides are genetically encoded, studies to ascertain the exact mechanisms of their biosynthesis are currently on going. thus, the synthesis of cyclotides and sfti- are currently restricted to chemical means. we have recently adapted a fmoc-spps method for cyclic peptide synthesis, via n-acylurea intermediates with the assistance of microwave irradiation. this method is a safe and convenient alternative to boc-spps and has the ability to be automated conveniently. using this method, parent scaffolds as well as several cyclotide and sfti- analogues with potential antimicrobial and matrix metalloprotease activities were synthesized. with the rising interest in the cyclization concept as a tool to impart stability on unstable peptides, the cyclic peptide synthesis method adapted herein is anticipated to have numerous applications. fixed configuration. the nonnatural oligomers have an extended conformational space and are supposed to adopt non-canonical secondary structures . in addition, the backbone modification makes these molecules more stable towards proteolytic degradation. the majority of proteins in nature are post-translationally modified, and the most abundant modification is the protein glycolysation, which introduces wide structural variety to proteins. glycoproteins have an important role in the biological recognition process, such as immunodifferentiation, cell adhesion, cell differenciation and regulation cell growth . new aza-β -amino acids bearing either an azide instead of amine on lys and orn chain or an alkyne group will be described and used in solid phase synthesis to finally performed a click chemistry to cyclize pseudopeptides or to introduced a glycosylated function . true for a series of peptides that display strong corticotropin releasing factor (crf) antagonistic activity. seminal studies by rivier et al. have shown that the incorporation of a lactam bridge in the crf-sequence resulted in an enormous increase in activity and potency, due to stabilization of the bioactive a-helical conformation of the peptide; and the newly designed peptide was called astressin. based on the astressin sequence, we started a truncation and deletion study to arrive at astressin analogs with a reduced size but still remain active as crf antagonists. this study resulted in the smallest active crf antagonist, astressin( - ). this sequence was further optimized by the introduction of novel covalent constraints, other than the well-known lactam bridge. as a first approach, the alkene/alkane bridge, which can be introduced via ring-closing metathesis via alkenesubstituted amino acid side chains, and as a second approach, the triazole bridge ('click' macrocyclization), via either a cu(i)-or a ru(ii)-catalyzed cycloaddition reaction between azide-and alkyne-derivatized amino acid residues were explored. herein, we will present the details of the synthesis of the alkene-, azide-, and alkyne-functionalized amino acids, their use in spps, and the optimized approaches for macrocyclization. furthermore, the peptides have been characterized by hplc, nmr, lcms, and studied by circular dichroism spectroscopy to obtain insight into the helical propensity of the peptides in relation to the cyclic constraint. the synthesis of a nitronyl nitroxide, c α -tetrasubstituted αamino acid (a class of sterically restricted amino acids that promote the formation of peptide β-turns and helical structures) was achieved by derivatisation of racemic amino- -cyano-indan- carboxylic acid [aic(cn)]. racemic boc-aic(nn)-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phase transfer conditions with , -(bis)bromomethyl benzonitrile as alkylating agent, followed by acidic hydrolysis, n α -boc protection, and saponification of the ester function. resolution was achieved through formation of the diastereomeric amides of (s)-phenylglycinol with chromatographic separation and mild acidic hydrolysis. reduction of the nitrile group to an aldehyde was carried out with raney nickel in the presence of sodium hypophosphite. condensation with , -diamino- , -dimethylbutane gave the corresponding tetramethylimidazolidine, which was oxidised with chloroperbenzoic acid to the desired nitronyl nitroxide. the uv-vis absorption and epr spectra of the amino acid were recorded and its magnetic properties were examined. in order to develop the synthesis of this peptide using the fmoc solid-phase peptide synthetic methodology, orthogonally protected β-hydroxyaspartic acid was needed. more precisely we wish to dispose of ( r, r)-n -fmoc- -tbdm-silyloxy-aspartic acid α -allyl ester instead of the recently reported dmab ester - indeed, in preliminary assay using this protective group we experienced difficulties during the final cyclisation step . the synthesis was developed starting from inexpensive l(+) dimethyltartrate and extended to the others stereoisomers of the β-hydroxyaspartic acid. structure. for that, we chose to replace proline by silaproline to afford polysilaproline. this study shows the comparison of two polyamino acids: polyproline and polysilaproline polymers. homopolypeptides were synthesized by polymerization of corresponding amino acid n-carboxyanhydride . multicomponent reactions (mcrs) represent a chemical process involving at least three reactants for the formation of several covalent bonds in one operation . by definition mcrs are chemo-and regioselective, convergent stepefficient procedures and take place with high atom economy. the copper(i)-catalyzed , -dipolar cycloaddition of organic azides and terminal alkynes (cuaac) reported by meldal and sharpless has been involved in various fields of chemistry and biochemistry research. however only few reports describe the implementation of cuaac and mcrs. , recently our research focused on a novel threecomponent reaction based on a cu(ii)-triggered aminolysis of peptide hydrazide resin and an azide-alkyne cycloaddition sequence. copper(ii)-induced oxidative aminolysis of hydrazides generates cu(i), catalyst of the azide-alkyne cycloaddition. this feature was exploited to design a solid phase detaching three-component reaction. the mcr process requires a peptide hydrazide resin, an amino azide linker and an alkyne, resulting in the formation of peptide modified at the c-terminus through an amino , , -triazole linker. this method can potentially be applied to the synthesis of a large variety of peptide derivatives starting from fmoc-spps assembled peptidyl resins. furthermore, it is not practical to compare hplc spectra from different resin samples (e.g., before and after reaction) directly. a comparison by analyzing the same (mg) amount of resin would involve tedious sample preparation that is extremely error-prone and would be impractical because factors resulting from the increase or decrease of the molecular weight of the resin-bound compounds may have a significant influence on the results. the use of internal reference compounds allows rapid assessment of reactions performed on solid supports. the internal reference compound is bound to the resin together with the substrate and cleaved with the products after completion of the reaction. commercially available compounds can be used for this purpose, or likewise, the reference compound can be generated from the substrate by partial capping of a functionality. the peak integration of the reference compound in the hplc-uv spectra can be correlated directly to those of the rest of the compounds present in the reaction mixture and therefore a quantitative interpretation of the spectra with respect to conversion and yield is possible. here we demonstrate the proof of principle as well as the accuracy of this method. modifier proteins such as ubiquitin are conjugated to protein substrates in cells and thereby mediate various biological processes. of high interest, is the ubiquitin fold modifer (ufm , residues) which has structural similarity to ubiquitin but has no sequence similarity. unlike ubiquitin, ufm has not been extensively studied and little is known about its biological role. to understand ufm 's biological functions, access to pure, homogeneous natural and modified ufm protein is essential. chemoselective ligation techniques are suitable for providing such proteins. recently, a variation of the α-ketoacid-hydroxylamine (kaha) ligation was developed, which utilizes the chemoselective reaction between a c-terminal peptide αketoacid and a n-terminal -oxaproline. this modified form of the kaha ligation furnishes a native peptide bond and a homoserine residue. this ligation is useful for the synthesis of proteins from two unprotected protein segments in aqueous buffers. for the synthesis of larger proteins, a sequential ligation strategy is necessary. using ufm as the model system we have developed a sequential ligation procedure using kaha ligation with -oxaproline. applying the new sequential ligation strategy we have prepared ufm by total chemical synthesis. we have also prepared a cterminal thioester surrogate of ufm protein, which is suitable for conjugation to proteins of interest. the syntheses required the development of a bifunctional peptide segment bearing an α-ketoacid and an orthogonally protected -oxaproline. the preparation of the protein segments, their intermediates, the deprotection, and sequential kaha ligations towards the syntheses of ufm protein and c-terminal modified thioester ufm protein will be discussed. affinity and biological activity, we have designed and synthesized new analogues by multiple n-methylation of hut-ii( - ) backbone amide bonds. all the peptides were performed by a novel synthetic approach, in which the introduction of n-methyl groups occur during regular solidphase peptide synthesis. on these new ligands we evaluated the binding affinity and biological activity at the ut receptor and performed preliminary nmr conformational studies. since that time a number of different machines have been used to automate peptide synthesis. modern machines are following two general setups; the so called "single approach" and the "parallel approach". in the single approach, the machine is developed to synthesize one or few peptides simultaneously. the user is able to optimize the synthesis conditions on each single peptide and each single coupling step. the maximum product quality regarding purity and yield is the major task of this approach. in the parallel approach, the machine is developed to synthesize a huge number of different peptides in the same single setup and time-frame. the user always has to find a synthesis protocol appropriate for the needs of each peptide to reach the maximum quality, knowing that there will be always a number of failed peptides. as a result you will find both types of peptide synthesizers in laboratories all over the world: the single machine, for the complicated peptides, and the parallel machine, allowing generation of multiple peptides with standardized protocols for each. the tetras is the first instrument combining the advantages of both machine types and allows the user to synthesize up to different peptides in parallel. each peptide can have its own individual synthesis protocols, separate of all others. the user can combine different synthesis scales, peptide lengths, and activator reagents in one run. finished peptides can be removed and new peptides can be started while the tetras is still running. the tetras allows the user to establish an uninterrupted production shop using one instrument only. siemion, i. z.; peptide res the peptides: analysis, synthesis and biology monitoring peptide folding in membrane-active peptides: a time-resolved spectroscopic study e. gatto a cordopatis p. st european peptide symposium sar studies of triazolyl-containing cyclopeptides: a defined -turn structure increases potency and selectivity to melanocortin receptor subtypes c. testa, a,b proc. natl. acad. sci proc. natl. acad. sci usa multicomponent reactions microglobulin: a "difficult" protein s. abel, m. beyermann the protein ß -microglobulin constitutes the noncovalently bound light chain of the major histocompatibility complex class i (mhc) and plays an essential role in the dialysisrelated amyloidosis. [ , ] to examine the amyloid fibrils of the ß -microglobulin (ß -m) via infrared spectroscopy we intended to synthesize -c-labeled ß -m. [ ] due to the two cysteine residues in positions and we used the native chemical ligation (ncl) strategy for assembling the -mer protein. this necessitates the synthesis of three segments which was accomplished on solid phase using the fmoc/t-bu chemistry. the preparation of the segments had to be optimized with respect to aggregation, aspartimide and piperidide formation, trifluoracetylation, and s-tert-butylsulfonium formation. additionally, ncl steps had to be optimized, because of "internal" thioester formation, dimerization and the formation of side-products of the activated n-terminal segment peptaderm inc., krakowskie przedmie cie str. , warsaw, poland immunosuppressors, such as cyclosporine a (csa) and tacrolimus®, are routinely used in prevention of graft rejection after organ transplantation and in therapy of some autoimmune diseases, including skin inflammation. a naturally occurring in linseed oil cyclolinopeptide a (cla, c(-pro-pro-phe-phe-leu-ile-ile-leu-val) possesses a strong immuno-suppressive activity, comparable at low doses with that of csa , but is much less toxic. we synthesized new cla analogs, containing instead of one proline residue its six-membered mimics, pipecolic acid (pip): c(-pip-pro-phe-phe-leu-ile-ile-leu-val) ( ) and c(-pro-pip-phe-phe-leu-ile-ile-leu-val) ( ). the incorporation of pipecolic acid residue led to different conformational behavior of the nonapeptide cycle. nmr experiments in cdcl solution showed that cla analogue with the pipecolic acid residue in position was much more flexible than cyclopeptide . the new peptides were devoid of toxicity up to μg/ml with regard to human peripheral blood mononuclear cells (pbmc), did not inhibit tumor necrosis factor alpha production in blood cell culture, but exhibited dosedependent, anti-proliferative actions for phytohemagglutinin a-activated pbmc. since peptide was more potent it was tested for growth inhibition of l- lymphatic leukemia. the peptide was found to strongly inhibit the cell growth even at low concentration ( % inhibition at μg/ml). hiv- has emerged as the largest and the most devastating public pandemic in our days, affecting approximately million people worldwide . development of an effective, safe and preventive hiv vaccine remains an urgently needed priority. epitopes for hiv-specific antibodies in elite controllers, a subgroup of long term non progressors, encompassing segments of mper of gp and for the v loop of gp were identified using the phage display technology . immunization experiments with epitopes conjugated to an artificial sequential oligopeptide carrier (soc ), formed by four repeats of the tripeptide lys-aib-gly in tandem, or to the palmitoyl group are currently in progress. all syntheses were performed on a rink amide resin following the fmoc technology. conjugation of epitopes to the soc carrier was realized via a chemoselective ligation approach, which generates an oxime bond between the h n-o-groups of the modified lysine residues and the aldehyde group of each epitope institute of immunology and experimental therapy, polish academy of sciences, - wrocław, poland peptaderm inc., krakowskie przedmieście , - warszawa, poland nonproteinogenic amino acids have been a tool to modify the structures of natural peptides since a long time . bioactive peptides involved in a physiological and biochemical processes cannot be applied in the therapy because of their instability in physiological conditions. that's why the synthesis of their stable active analogues is a challenge for medicinal chemistry nowadays. -trans-hydroxyproline (hyp) is an important building block of natural collagen. it is responsible for the stabilization of collagen super helix, forcing the trans amide bonds configuration with preceding amino acids . at the same time the impact of trans- -hydroxyproline on the conformation other than the collagen peptide chains of biologically important compounds is little known. it is known that immunosuppressive activity of cla is comparable with cyclosporine a and is associated with the presence of the tetrapeptide fragment pro-pro-phe-phe containing pro-pro cis amide bond. now we present synthesis, conformation and biological activity of new analogues of cyclolinopeptide a (cla), containing -transhydroxyproline instead of proline residues in position or . we expected that the introduction of the hydroxyl group in the pyrrolidine ring might influence the biological activity and conformation of the native peptide due to its hydrophilic character and hydrogen bonding ability. the linear precursors of modified cla analogues were prepared manually by standard solid-phase procedure "step by step" on wang resin using fmoc/tbu strategy and tbtu as coupling reagent. the cyclizations of linear peptides have been made under high dilution conditions by means of edc/hobt coupling reagents. the biological activity of newly synthesized compounds as well as the conformational study will be evaluated. dip. di scienze ambientali, seconda università di napoli, caserta, italy nmr spectroscopy is a powerful method to perform structural studies on peptides. to completely fulfill the potential of nmr, peptides labeled with stable isotopes ( n, c, h) are essential. peptides are easily prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. an alternative cost-effective strategy is the recombinant expression of peptides in e. coli as fusion constructs with carrier proteins. the main problem of this approach is the need of chemical reagents or proteases to cleave the target peptide from its fusion partner after purification. proteases may determine the heritage of extrasequence amino acids at the peptide n-or c-terminus, while chemical reagents require harsh reaction condition that may modify target peptides. an interesting solution is represented by the use of inteins as fusion partner. inteins are protein elements that can catalyze their self-excision from a flanking sequences in mild conditions, by adding nucleophilic agents such as thiols or simply by shift of ph and temperature, bypassing the use of proteases or chemical reagents. we used the self-cleaving mxegyra mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, hplw and qk. , the two peptides target vascular endothelial growth factor receptor (vegfr) and have been described to modulate vegf-dependent angiogenesis. our expression and purification scheme allows to obtain homogeneously isotope labeled peptides. the availability of isotope labeled hplw and qk opens the way to nmr studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with vegfr. an nmr method to discriminate between the fullyextended and different helical conformations in a spacer peptide c. peggion*, m. crisma, f. formaggio, c. toniolo icb, padova unit, cnr, department of chemistry, university of padova, padova, italy the ideal fully-extended, α-peptide conformation, also known as . -helix, is characterized by φ = ψ = ω = °t orsion angles. the repeating motif of this foldamer is a pentagonal (pseudo)cyclic structure (called c ), stabilized by an intraresidue h-bond. the n-h and c=o groups in the . -helix are not involved in intermolecular h-bonds. multiple c conformations were observed in homopeptides made up of c α,α -dialkylated glycines with both side chains longer than a methyl. this is the case for c α,αdiethylglycine (deg), the residue studied in this work. it is known that deg homo-peptides can adopt the . -helix or the -helix depending on environmental factors and nand/or c-terminal moieties. , in this communication, we introduce an nmr method to discriminate between the . -helix and the -helix based on the observation of cross-peak intensities in the noesy human serum amyloid a (saa) is a highly conserved apolipoprotein produced by the liver under inflammatory conditions accompanying e.g. atherosclerosis, cancer and amyloidosis [ ] . it is also known that saa α isoform has the amyloidogenic properties [ ] . till now it is little known about structure of human saa, as it hampers structural studies due to its facile aggregation. the analysis of protein sequence and cd data together with theoretical studies revealed a typical globular structure of the protein [ ] . the c-terminal sequence of saa contains three proline residues, which probably are responsible for the unordered structure. recent in vitro studies involving saa and human cystatin c (hcc) revealed direct interactions between the ( - ) fragment of saa and the ( - ) sequence of hcc. the results of elisa test for the ( - ) saa fragment have shown that it binds to hcc very well. the nmr studies for the wild ( - ) sequence found an unordered structure in phosphate buffer. based on these data we decided to check how the point mutations pro→ala in ( - ) saa fragment could influence the peptide's structures. we synthesized four peptides with pro→ala point mutations and we performed cd experiments at different conditions. the results show that two of them contain disordered structure and two α-helical structures. in this project we analyze the solution structures of these peptides at the atomic resolution using d nmr supported with molecular dynamics. design and conformational analysis of stapled peptides mimicking cullin binding region to kctd . i. de paola, a l. pirone, a e. pedone, a s. di gaetano, a l. vitagliano, a r. fattorusso, b g. malgieri, b l. zaccaro*, a acknowledgement: this study was supported by eu within the european regional development fund (poig. . . - - / - ). model of angiotensin ii bound to the at receptor in the lipid bilayer environment m.t. matsoukas, t. tselios* department of chemistry, university of patras, gr- , patras, greece the renin-angiotensin system () plays a major role in blood pressure regulation. a sequence of enzyme reactions leads to the release of angiotensin ii which interacts principally with the type- angiotensin ii receptor (at ), a -residue, which belongs to the g protein-coupled receptor family. in the present study, the human at d model was constructed using modeler for the sequence alignment and loop refinement tools. on this basis, the crystal structure of bovine rhodopsin, (pdb code u ), was used as a d template. the gromacs software and amber sb forcefield were utilized for molecular dynamics calculations [ ] in order to evaluate the binding mode of angiotensin ii. the role of the critical amino acids of the binding site v , n , l , a , k , s , h , n and y is being studied. moreover, newest information on the role of the nd extracellular loop by unal et. al. [ ] have been implemented on the model, therefore we propose the contribution mechanism of the residues f -q for binding of angiotensin ii to the at receptor for activation and signaling. a. stavrakoudis department of economics, university of ioannina, greece one key step in the immune response against infected or tumor cells is the recognition of the t-cell receptor (tcr) by class i major histocompatibility complexes. it has been found [ , ] that such peptide/mhc complexes can interact with antibodies as well. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the cterminal of class ii complexes [ ] . in some cases, the same peptide/mhc complex has been found to interact with both tcr and antibodies [ ] . in these study a series of supermolecular complexes have been studied with stateof-art molecular dynamics simulations [ ] the dipeptide kyotorphin (tyr-arg, kyo) plays a role in pain modulation in the mammalian central nervous system (cns), and is one of the most investigated neuropeptides. the tyr-arg motif exists widely throughout the brain not only as kyotorphin, but also as the n-terminal part of several endogenous analgesic peptides , . also, this peptide is very rapidly degraded by aminopeptidases . one of the successful strategies in the design of neuropeptides with enhanced stability and improved delivery to the cns is that with the use of non-protein amino acids, like canavanive (cav), a structural analogue and antimetabolite of arginine (arg trichogin ga iv (noct-aib-gly-leu-aib-gly-gly-leu-aib-gly-ile-lol, in which noct is n-octanoyl and lol is leucinol) is an antimicrobial lipopeptaibol, a unique group of membraneactive compounds of fungal origin, characterized by a high content of the nonproteinogenic ca,a-disubstituted glycine aib (a-aminoisobutyric acid). owing to the gem-dimethyl substitution on the c a atom, aib exhibits a strong propensity to induce β-turns and /α-helical conformations in peptides. we have previously reported on a fluorescent analog of trichogin ga iv, the primary structure (and acronym) of which are: fmoc-aib-gly-leu-aib-gly-gly-leu-toac-gly-ile-leu-ome (f t ) where fmoc is fluorenyl- -methyloxycarbonyl, toac is , , , -tetramethylpiperidine- -oxyl- -amino- -carboxylic acid, and ome is methoxy. the double substitution of an energy donor (fmoc) at the n-terminus and an acceptor (toac) in the trichogin sequence enabled us to make use of time-resolved optical spectroscopies, spanning from the nanosecond to the microsecond time regime, to investigate the conformational propensity and the dynamical features of f t . experimental and computational results indicated that the d-structural and dynamical properties of f t are characterized by a transition from an elongated helix to a more compact conformation mimicking a helix-turn-helix motif. to further investigate the role of the flexible gly -gly central motif we synthesized a new trichogin analog having the gly residue substituted by aib: fmoc-aib-gly-leu-aib-gly-aib-leu-toac-gly-ile-leu-ome (f a t ) experimental and computational results indicated that also the f a t peptide populate two conformations, the dynamics of which were studied at different temperatures using time-resolved spectroscopic measurements. this replacement was demonstrated to stiffen the peptide backbone by reducing the flexibility around the crucial -gly -gly -dipeptide unit. the antigen α β , a member of the integrin family, is involved in the migration of lymphocytes through endothelium to the site of inflammation. thus, α β antagonists may be useful tools for the treatment of various inflammation disorders such as asthma and inflammatory arthritis. in addition, recent studies indicate that α β integrin promotes angiogenesis by allowing the invasion of myeloid cells into tumors, while α β antagonists prevent monocyte-induced angiogenesis, macrophage colonization of tumors and tumor angiogenesis. aiming to the discovery of novel α β antagonists, a series of new peptide analogues cyclized through cysteine disulphide bonds were synthesized and tested in vivo against angiogenesis in chicken embryo chorioallantoic membrane (cam model) . sar results indicated that: yr-c(cdpc)-conh promoted angiogenesis at the higher studied concentration and showed slight inhibition at the lower one, sal-r-c(cdpc)-oh, sal=salicylic acid, showed important inhibition of angiogenesis at dose-dependent manner, yr-c(cdpc)-oh and sal-yr-c(cdpc)-oh both showed no activity on angiogenesis. nmr spectroscopy was applied for the sequential assignment as well as for the elucidation of specific conformational features. experimental noe data were further imposed as distance constraints to a thorough conformational search by applying molecular dynamics simulations. energy refined produced conformers were used as template for the generation of the pharmacophore model associated with the antagonistic activity. such studies are intended to drive a rationalized design and development of this class of inhibitors. hynes r. o. cell, , , - . scaffold discovery by phylomers: a novel cd l specific scaffold derived from glycyl trna synthetase s.r. stone [ ] , k. hoffmann [ , ] , n. milech [ , ] , p. t. cunningham [ ] , m. kerfoot [ ] , s. winslow [ ] , y-f, tan [ ] , m. anastasas [ ] , c. hall [ ] , m. scobie [ ] , p.watt [ ] , and r. hopkins [ , ] [ ] drug discovery technology unit, telethon institute for child health research, roberts road, subicao, , western australia [ ] phylogica pty ltd, roberts road, subiaco, , western australia biopanning of phylomer phage display libraries against human cd l yielded a cluster of highly specific overlapping peptide fragments, from three bacterial genomes, corresponding to the highly conserved catalytic domain from the tetrameric gα β class of glycyl trna synthetases. structural analysis of the overlapping peptide fragments described a scaffold consisting of a central βsheet, comprising anti-parallel β-strands, flanked by nand c-terminal α-helices. further structural analysis revealed that these key structural features, which also encompass the crucial atp-binding motifs of the catalytic domain, are conformationally conserved across both tetrameric gα β and dimeric gα glycyl trna synthetases, yet importantly, there is only limited sequence conservation across these classes. given the identical function of the described domain and it's structural conservation, we postulated that members of the dimeric gα class would display similar cd l specific binding as the tetrameric gα β class, despite the sequence dissimilarity. to test this hypothesis, structurally equivalent peptide fragments of representative bacterial, archaeal and eukaryotic genomes comprising the dimeric gα class were tested for cd l binding in a process we termed ortholog scanning. the results showed that both archaeal (p. horikoshii) and eukaryotic (h. sapiens) structurally equivalent peptides bound to cd l with reasonable specificity and inhibited the cd :cd l interaction with comparable ic 's to the primary gα β class sequences. similar results were also observed for the representative bacterial gα class peptides. that the sequentially diverse orthologous peptides display cd l specific binding has important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its "drug-like" properties. we have initiated an investigation related to the effect of radical species upon structures of some peptide segments. in the proposed experimental protocol, aqueous peptide solution was submitted to gamma ray irradiation in controlled - kgy doses. the generation of peptide analogues, possibly induced by reactive oxygen species were examined by electrospray triplequadrupole tandem mass spectrometry (collision induced dissociation approach) and amino acid analysis of crude and/or purified by-products. noteworthy, the gamma irradiation process induced, regardless of the peptide sequence, a non-linear and progressive degradation of all peptides assayed. furthermore, these peptides could be classified in some different classes according to their halflife dose. for instance, the vasoactives angiotensin ii (aii), ang ( - ), bradykinin (bk) and some related peptides were more stable than the melanocyte-stimulating hormone α-msh, substance p or the bk 's ( - ) b receptor fragment (lvyvivgkrfrkksrevyqai). usually, the most prominent derivatives generated from this experimental protocol revealed that they are likely induced by oxidation process, yielding a variation of + da in their molecular weight. the main source of peptide modifications seems to lie either on the phe (hydroxyl group insertion at o-, m-or p-positions of its aromatic side chain) or met oxydation. in the former case, only phe and not phe is oxidized in the bk structure whereas substance p generates an analogue bearing metsulfoxide without modifying its phe , residues. thus, collectively, these findings clearly stress the complexity of factors involved in peptide structural modifications induced by gamma ray-type strong electromagnetic irradiation experiment. an additional target of this approach lies indeed, in the production of unusual peptides for further structure-function investigations. university of bern, bern, switzerland linear peptides are typically poor drug candidates due to their low bioavailability and rapid proteolysis. these limitations can be overcome by rigidifying their structure through head-to-tail or side chain-involving cyclizations. cyclic constraints may also increase biological activity by stabilizing secondary structures and by reducing the entropic penalty of binding to a protein target. the use of multiple branching amino acids in a peptide sequence, like diamino acids (as used in peptide dendrimers ) or amino diacids, allows to design peptides resembling polycyclic alkanes, a type of topology only rarely found in nature (e.g. amatoxins and lantibiotics). bicyclic homodetic peptides such as "norbornapeptides" (bicyclo[ . . ]heptapeptides) were prepared using an orthogonal protection scheme: the first cyclization is performed on resin after selective deprotection of a glutamic acid residue, whereas the second ring closure is achieved by amide bond formation at high dilutions. these peptides are structurally well-defined and cover an almost pristine area of peptide topological space. their conformational rigidity was investigated by means of d-nmr and x-ray crystallography and may offer a platform to design drugs tackling protein-protein interactions. the interaction of peptide ligands with protein receptors face peculiar challenge in recognizing binding surfaces due to availability of a multitude of conformations. therefore it is essential to constrain the peptide conformations for the recognition of receptors and thus finding the bioactive conformation. the cell surface receptor protein family integrins recognize "rgd" sequence which is present in different proteins. to determine the bioactive conformation required to bind with receptor αiibβ , the peptide sequence "riprgdmp" from kistrin was inserted into cdr loop region of rei protein (rei-rgd ). it helps out in finding the possible bioactive conformation of peptide by restricting the sampling space. the activity of rei-rgd was studied and found that as the temperature increased rei-rgd showed a higher affinity towards the receptor αiibβ . the proposed mechanisms for the increased activity of rei-rgd at higher temperature were justified in either of two ways. the modified complex forces the restricted peptide to adopt a bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. in this study we model the conformational preferences of "rgd" sequence in octapeptide "riprgdmp" at two different temperatures ( o c and o c) using multiple md simulations. we found that at higher temperature "rgd" sequence from "riprgdmp" adopt turn conformation, while a bend conformation was observed at low temperature. the analysis of various pharmacophoric parameters hint that the turn conformation of "rgd" sequences adopted at higher temperature could be the potential bio-active conformation, and helps out in designing of antagonists for cell surface receptor αiibβ . the -residue peptaibol antibiotic trichovirin i- a (tv) of the linear, covalent structure ac-aib-asn-leu-aib-pro-ala-val-aib-pro-aib-leu-aib-pro-leuol (ac, acetyl; aib, α-aminoisobutyric acid; leuol, l-leucinol) has been synthesized and very thin (~ μm) hair-like crystals were obtained from a methanolacetonitrile-water mixture. diffraction data were collected at k at the diamond light source england, using the microfocus beamline i and a x-ray beam focused to a size of μm full-width-half-maximum. two independent molecules (a) and (b) were located in the crystal's asymmetric unit . both chains assume complete turns of a curved right-handed helical conformation stabilized by intramolecular hydrogen bonds. up to now tv represents the longest right-handed -helix of a natural peptaibol sequence complementing those of synthetic, protected homooligo-aib- insulin is a protein hormone that plays a key role in regulation of blood glucose levels and, thus, has widespread impact on lipid and protein metabolism.insulin is known to act through binding to the insulin receptor (ir); however, the structure of the insulin-ir complex is not known. the crystal and nmr structures of insulin represent only inactive storage forms. it is widely acknowledged that insulin must undergo structural changes in the c-terminus of the b-chain upon binding to the ir. in addition, the n-terminus of the bchain may adopt two different conformations in hexamer, known as t-and r-states. the r-state of the n-terminus of the b-chain creates a long b -b central a-helix. the t-state of n-terminus is in an extended conformation. however, the biological relevance of the t/r forms remains elusive . in this study, we have focused on the synthesis of new insulin analogues modified at the nterminus of the b-chain and subsequently correlated their biological activities with their d-structures. the invariant residue glyb seems to be critical for the t/r transition. glycine can adopt wide range of dihedral angles (φ/ψ) and it occupies significantly diverse dihedral angles in t-and r-states. a-aminoisobutyric acid (aib) is an amino acid with a high helical propensity, which often folds into right-or left-handed α-helix. we have introduced aib at position b , b and b with the aim to induce the r-state of the hormone. in contrast, as d-pro and nmeala are not able to adopt the φ/ψ angles of the right-handed α-helix we have introduced these amino acids at position b to obtain the t-state of insulin. peptide dendrimers are tree-like molecules formed by alternating functional amino acids with branching diamino acids such as lysine. unfortunately these molecules have not yielded to structural characterization and little is known about their molecular-level structure. computational methods seem to be an adequate tool to address these issues.herein we present a comprehensive structural characterization of peptide dendrimers using molecular simulation methods. multiple long molecular dynamics (md) simulations were used to extensively sample the conformational preferences of several third-generation peptide dendrimers, including some known to bind aquacobalamine. we used several conformational analysis procedures (clustering, energy landscapes and multivariate analysis) to analyze conformational changes that can be correlated with particular structural trends.the results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviours were identified. some dendrimers favour mainly loose conformations, while others prefer more compact configurations. through a series of computational mutations we investigated the influence of the presence and placement of charged residues in dendrimer topology, finding that electrostatic interactions among charged residues are a major determinant in structure acquisition by peptide dendrimers. these conclusions bring new insight into the conformational behaviour of these systems and may provide better routes for their functional design. acid-mediated prevention of aspartimide formation in solid phase peptide synthesis t. michels, a r. dölling, b u. haberkorn a , w. mier*, a a department of nuclear medicine, university hospital heidelberg, heidelberg, germany; b biosyntan gmbh, robert-rössle-straße , berlin, germany aspartimide formation is one of the major obstacles that impede the solid phase synthesis of large peptides and proteins. the main reason for aspartimide formation is the piperidine-catalyzed fmoc cleavage of peptides containing aspartic acid. several side chain protecting groups have been developed but the complete prevention of aspartimide formation can only be achieved using n-( hydroxy- -methoxybenzyl) (hmb) as backbone protecting group. however, hmb-protected building blocks are difficult to synthesize and only the dipeptide containing glycine (fmoc-asp(tbu)-(hmb)gly) is commercially available. until now, no cost effective strategy to suppress this side reaction has been developed. formally, aspartimide is the result of an attack of an amidate species at the carbonyl carbon of the otbu protected side chain carboxylate of aspartic acid, which might be prevented by protonation of the amidates with piperidinium ions. in this work the suppression of aspartimide formation by adding small amounts of organic acids to the deprotection agent piperidine was studied. this procedure was shown to efficiently prevent the formation of aspartimide side products in several peptides, i.e. pres - -y, a -mer peptide derived from the hbv surface antigen and a peptide parathyroid hormone (pth) fragment. the testing of a series of different acids covering a broad range of pka values showed that this effect is virtually independent of the acid strength. since aspartic acid is found in most oligopeptides, the authors recommend to generally add % (v/v) formic acid to piperidine based fmoc cleavage mixtures. decomposition of the resin linkers during tfa cleavage of peptides in fmoc-strategy leads to alkylation of sensitive amino acids . this side product formation is a crucial drawback, especially during the synthesis of biologically important cys-containing peptides on wang support. through a battery of approaches ( h-nmr, uv and lc/esi-ms) we detected an unexpected alkylation of the sulfhydryl group of cysteine side-chain residues by the phydroxyl benzyl group from the wang resin linker. herein, we present the feasibility for s-alkylation of cys-containing peptides from wang linker decomposition. this sidereaction occurs during the final tfa cleavage of the peptide from the solid support, while the position of the cysteine residue within the peptide sequence as well as the resin's substitution influence the extent of cys-alkylation. the stephan angeloff institute of microbiology, bulgarian academy of sciences, sofia, bulgaria influenza viruses cause epidemics and pandemics all over the world. therefore, the development of virus resistance to drugs, leads to search for novel derivatives and approaches to chemotherapy for human influenza infection. antioxidant therapy is known to be one potential approach. the application of combination therapy of antioxidants with antiviral drugs could reduce the complications and lethal effects, caused by an influenza virus . in our study, amino group of neuraminidase inhibitor -oseltamivir, which belong to second generation anti-flu drugs, was covalent conjugated with known antioxidantscysteine, histidine. tryptophan and etc. the study of the role of the modified by antioxidants oseltamivir on proliferation of influenza virus is in progress. recently we reported a short synthesis of or -membered cyclic guanidine via intramolecular reaction of alkyl diamine with n,n'-cbz-methylisothiourea( a). here we report a further application of synthesis two types of cyclo-peptide guanidine-bridged cyclopeptides utilizing this mechanism -n-terminus local cyclo-guanidine peptide and backbone guanidine-bridged marco-cyclic peptide. three n,n'protected diaminoacids (daa) including , -diaminobutyric p . antifreeze glycoproteins (afgps) are found in the deep sea teleost fish in arctic and antarctic oceans. these biomolecules are able to inhibit the growth of ice crystals and depress the freezing temperature of the blood serum in fish enough to keep them from freezing in their sub-zero environments while the melting temperature remains unchanged . despite afgps have been consider as a potent cryopreservation, obstacles to develop afgps as medicinal and industrial application are mainly due to the lack of access to pure form from natural sources and the problem of understanding how afgps inhibit ice crystal growth. as a result, a considerable progress toward the design and synthesis of afgp analogues has been made several groups . in the course of the studies on the structure-activity relationships of afgps, we are interested in peptoids as mimics of α-peptides and synthesized monoglycosylated peptoid analogues by substituting the glyco-thr residue as afgps mimics. in this presentation, we will show our studies on how the insertion of peptoid residue into afgp backbone affects the afgp activity by measuring both thermal hysteresis (th) and ice recrystalliztion inhibition (iri). [ ] , both for diagnosis and endoradiotherapy. for this application the peptides can be attached with chelating agents that bind radioactive metals such as ga, ga or in for imaging or therapeutic radiometals such as y and lu. the chelating agent most frequently applied is the macrocyclic ligand , , , -tetraazacyclododecane-n,n',n'',n'''-tetraacetate (dota), it is commonly introduced as the tris(tbu ester). the cleavage of the tbu protecting groups on dota is known to be sluggish [ ] . several attempts have been made to synthesize dota with protecting groups that can be removed under mild conditions. however, these derivatives have not yet found widespread application. our new approach was to prepare a protecting group for dota-based prochelators that is convergently cleaved under the cleavage conditions of the amino acid protecting groups of the peptide. o-phenylisopropyl (opp) esters are more sensitive towards acid than tbu esters. deprotection occurs with % trifluoroacetic acid in dichloromethane [ ] . therefore, a synthesis of the prochelator dota-tris(opp ester) was developed. the copper-catalyzed azide-alkyne cyclization (cuaac), the most commonly recognized variant of "click chemistry," has emerged as a powerful technique for ligation, conjugation, and cyclization reactions of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhancing potency or selectivity by stabilizing an active conformation. one application of the cuaac that has generated interest is the use of this reaction to replace a disulfide bridge with the product triazole, which among other complementary properties may prevent in vivo redox chemistry. in this poster, we synthesize a new analogue of the cyclic cancertargeting peptide cngrc where we replace the disulfide bond with a triazole linkage using click chemistry and a fully automated, on-resin method using the single-shot delivery feature on the prelude® peptide synthesizer. unnatural amino acids including d-amino acids are manufactured mainly by the enzymatic process. however, one enzyme can produce only one amino acid due to its high specificity and it takes a long time and a lot of expenses to develop the appropriate enzyme itself. arca (alanine racemase chiral analogue) is an organic catalyst which can overcome these drawbacks and can produce almost all kinds of amino acids efficiently. the amine functionality of l-threonine is freely reacted with the aldehyde group of arca to form the corresponding imine, which is easily epimerized in the presence of organic base due to the acidity of the alpha proton of imine. the difference in the stability between the imines of the optical epimers rendered them to be shifted to d-allo-threonine derivative dominantly. once the epimerization reaction reached equilibrium, the reaction mixture was hydrolyzed under acidic condition to give d-allo-threonine and arca, which could be recycled repeatedly without significant loss in yield or purity to produce more d-allo-threonine from lthreonine in excellent yields. optimization of the reaction conditions with various bases and solvents is discussed and mass production of optically active d-allo-threonine including optical purification is described. the manufacuring process for the preparation of arca will be shared as well. our group is interested in the development of efficient synthetic routes for the preparation of enantiopure atrifluoromethylated amino acids (a-tfm-aas) starting from chiral cf -oxazolidines or imines and their incorporation into a peptide chain. these non-natural amino acids are very attractive compounds for the design of biologically active molecules, particularly peptides, due to the unique physical, chemical and biological properties impart by the cf group. as conformationally constrained cyclic amino acids have recently gained considerable interest, we are particularly focused on the preparation of pyrrolidine-type a-tfm aas. , incorporation of proline derivatives is known to restrict the amino acyl-proline cis/trans isomerization, to limit the protein folding and consequently to modulate the biological activity of peptides. based on these observations, mutter's group introduced pseudoproline building blocks (ψpro) into a peptide sequence as reversible protecting groups for ser, thr and cys. the ψpro residues proved to be versatile tools for overcoming the aggregation caused by hydrophobic interactions encountered during solid-phase peptide synthesis (spps). they also turned out to be inducers of βturns containing predominantly cis-amide bond and useful tools in peptide cyclization. here, we report the results obtained for the preparation of various hydrolytically stable trifluoromethylated pseudoprolines (cf -ψpro) as well as the methodological studies developed to optimize the synthesis of various c-and n-terminal cf -ψpro containing dipeptides. rennes, france protein strructure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. many attempts have been undertaken to design peptide-peptide interaction pairs and synthetic receptors de novo by using special building blocks. a rational approach starting from hydrazine to create new building blocks based on a tailored metalchelating amino acid analogues was envisaged. to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, several supramolecular entities containing one to three nitrilotriacetic acid analogue (ynta) moieties were synthesized. these new building blocks additionally contained an amino group or an acido group, which can be flexibly introduced into peptide in n or c-termini or into the peptidic chain by solid phase chemistry in fmoc/t-bu strategy. these multivalent chelators were characterized and the corresponding metalchelating peptides could act as metal sensors and synthetic receptors for histidine-tagged proteins. the potential of peptides as drug candidates is often limited by their pharmacokinetic properties. structural modification of the peptide backbone via n-methylation is a powerful medicinal chemistry tool that confers oral bioavailability to these molecules. n-methylation exerts a strong effect on the backbone conformation and, as a result, many n-methylated peptides show enhanced biological activity and higher receptor selectivity. another approach to increase the solubility of peptides is by conjugation of peg to a derivatizable functionality. by combining these two approaches we have developed n-oegylation. this novel form of peptide modification consists of the attachment of oligoethylene glycol (oeg) chains to the amide bonds. many bioactive peptides comprise one or more n-me amino acids which are essential for their activity. thus, we consider that replacement of a backbone n-me group by an oeg chain may imply a minimal structural perturbation and may lead to n-oegylated peptides with preserved biological activity. furthermore, our strategy is a promising way to improve the bioavailability of cyclic peptides that do not have any site where a peg could be attached. as a proof of principle, several n-oeg analogs of two bioactive cyclic peptides were synthesized in spps. first, we performed a full n-oeg scan of the sansalvamide a peptide. next, several analogs of cilengitide were prepared by replacing the n-me of valine by oeg chains of different length. depending on its size, the oeg residue was incorporated by using an n-oeg derivative as building block or, alternatively, using an n-substituted amino acid bearing an attachment site where a peg was conjugated post-synthetically.the biological activity of all the n-oegylated peptides was evaluated. some of the sansalvamide a peptide analogs exhibited cytotoxicity within the same range as the original peptide, which suggests that backbone amide groups may be useful oegylation sites in bioactive cyclic peptides. the modification of peptides is an important step in pharmacology to vary the affinity and the stability of peptidic drugs. whereas a wide range of strategies exists for the functionalization of the n-terminus and the side chains, facile variation of the c-terminus remains an important challenge. we consider peptidyl-phosphoranes as a promising platform to enable orthogonal and mild introduction of a great variety of chemical functionalities at the c-terminus. a convenient method for the synthesis of soluble peptidyl-phosphoranes has been presented by our group recently. in this, -bromo-acetyl bromide was coupled to a wang-resin followed by alkylation of triaryl-or trialkyl phosphine moiety. deprotonation to the phosphorus ylide and subsequent acylation with an fmocamino acid created the basis for assembly of the peptide by spps. final acidic cleavage produced a decarboxylated and unprotected, soluble peptidyl-phosphorane. from this point, a variety of orthogonal modification reactions at the peptides c-terminus is possible, e.g. click reaction with azides allow for the incorporation of triazoles as peptide bond mimetics. the wittig reaction opens up another interesting portal for c-terminal modification, as vinyl ketones are formed by reaction with aldehydes. the described chemistry was applied to modify caspase- inhibitors. in order to address the s site of caspase- , the commonly known devd inhibitor was varied at the c-terminus by introduction of different residues. the devd motif was synthesized as peptidyl-phosphorane and modified in wittig reactions. the resulting c-terminal vinyl ketones were obtained by the reaction of aliphatic and aromatic aldehydes. a small library was generated, and novel compounds were tested for their potential to inhibit caspase- . semmelweis university, department of biophysics and radiation biology, budapest, hungary considering the impact of uv irradiation on the structure and function of proteins , it is a matter of utmost importance to resolve the conditions of photolysis more deeply. we think that a protein, as a complex unit, gives multiple responses to all impacts therefore the analysis of these responses is a rather complex problem. the main goal of our research is the deeper understanding the tryptophan-mediated photolysis of disulphide bridges in bio-active proteins upon near-uv irradiation using cyclic peptide models, as small protein units, to define the caused functional damage. cation -pi interaction is increasingly recognised as an important noncovalent binding interaction which plays a role in establishing the final structures of proteins. within a protein, cation -pi interactions can occur between the cationic side-chains of either lys or arg and the aromatic side-chains of phe, tyr or trp . our earlier results with gla indicate that new covalent bonds are also formed between cys and lys during illumination, which is also a reason why the lys residue is planned to be included in the sequence of the models. our aim is to study whether the cation -pi interaction can have an influence on the ss-bridge splitting in small cyclic pentapeptide models. here we report about the conformational analysis, synthesis and spectroscopic investigation of lys-and arg-containing model peptides. the azide functionality is very popular mainly due to azidealkyne click chemistry used in many peptide ligation strategies. azido-peptides are usually prepared by incorporation of azide containing residues or azide functionalization of aldehyde resins affording c-terminal azido-peptides. alternatively, the n-terminus can be converted into an azide via a cu(ii)-catalyzed diazotransfer reaction using triflyl azide. recently, a number of safer, shelf-stable and easily prepared diazotransfer reagents has been developed, of which imidazole- -sulfonyl azide has been used to introduce azide moieties in proteins under copper-free conditions. typically, it has not been reported to be used on the solid phase. we provide a very easy, fast and efficient method for conversion of amines into azides on a solid phase support which in our opinion has major benefits over earlier reported methods making using of less stable reagents that require a metal ion catalyst. we demonstrate how the diazotransfer reaction can be performed on a solid phase support using the imidazole- sulfonyl azide reagent without the need for a metal ion catalyst. using a model peptide we studied the effect of stoichiometry, added base and solvent. in addition we examined the effect of the nature of the n-terminal residue on the efficiency the diazotransfer reaction. finally, we found that the optimal conditions to perform the reaction also depend on the nature of the solid phase support the reaction is performed on. the novo nordisk foundation center for protein research, university of copenhagen, copenhagen, denmark site-selective strategies for post-translational modification of peptides and proteins are essential tools for many areas of research in the life sciences, yet remain a chemical challenge due to the multiplicity of functional groups present. there are powerful chemoselective reactions, however, they aim at introducing only one functionality at each reaction site. here we present a one-pot, threecomponent dual-functionalization of peptides or proteins based on a , -dipolar cycloaddition between a functionalized malemide, an n-hydroxylamine and a peptide or protein with an n-terminal serine residue at the n-terminus, which is selectively oxidized to a -oxoaldehyde. most common moieties for labeling, e.g. fluorophors and peg-chains, are commercially available as maleimides. nitrones were easily obtained by condensation of peptide-aldehydes and primary nhydroxylamines under aqueous conditions. the chemoselective , -dipolar cycloaddition reaction between the peptide-nitrone, and a functionalized maleimide proceeded in aqueous solution at room temperature or with gentle heating, which provided the stable isoxazolidine product. we envision that this 'one site -two functions' method can be used widely to introduce two separate moieties. the method was used to introduce two separate ligands in a range of other peptides. for example, new multimodal molecular imaging techniques depend on facile chemical methods for site-selective dual-functionalization. we used our new methodology to synthesize a cyclic rgd-peptide for combined pet and optical molecular imaging. finally, the small protein ipb was successfully n-terminally modified, including with a peg-chain, using this new, general method. multiple sclerosis (ms) is the most known chronic, inflammatory, demyelinating disease of the central nervous system (cns), characterized by a progressive neurodegeneration, caused by an autoimmune response to self-antigens in genetically susceptible individuals. it is nowadays known that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neoantigens; in particular aberrant glycosylations affect various parts of the immune response and have profound effects on immune tolerance. in previous studies we demonstrated the value of the glycopeptide csf (glc) which, by virtue of the particular type i' β-turn structure, optimally exposes the minimal epitope asn(glc) to autoantibody recognizing in elisa in multiple sclerosis patients' sera . elisa assays allowed to conclude that the ability in detecting autoantibodies in multiple sclerosis sera was stricktly linked to saccharidic moieties and to conformation around minimal epitope of the antigenic glycopeptide. herein, taking advantage of such considerations, we focused our attention on the synthesis of a little library of lysine branched multiple antigen peptides (maps), containing the minimal epitope asn(glc), in an attempt to increase the antigenicity of linear peptide sequences . with this aim, we performed the spps of glucosylated maps via the building block approach, studying the role of different long spacers on the dendrimeric core, and the role of different peptide sequences around the sugar moiety, in order to optimize the synthetic process and to evaluate the influence on the affinity and specificity in sp-elisa. environmentally induced co-or post-translational modifications of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc. it has been previously reported that the use of synthetic post-translationally modified peptides, introducing fmoc-l-lys(nε-(±)-α-lipoic acid)-oh, as peptidomimetics of natural neoantigens allowed to detect autoantibodies in the sera of patients affected by pbc, and they might be useful diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. only the r-(+)-enantiomer of α-lipoic acid exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. but it remains unclear if the tridimensional structure of the lipoic acid is of any importance in the interactions antibody-peptide during the indirect elisa tests. therefore, it is necessary to synthesize each peptide separately with one absolute configuration of the lipoic acid. herein, we describe the synthesis of the two diastereoisomers fmoc-l-lys(nε -(r)-α -lipoic acid)-oh and fmoc-l-lys(nε -(s)-α-lipoic acid)-oh that have to be used in fmoc/tbu spps as building blocks for the synthesis of post-translationally modified peptides. recently it has been reported the introduction of a new generation of cd diagnostics based on a unique antigen approach, consisting on human ttg cross-linked with gliadin peptides coated on the elisa plates . on the basis of experimental data obtained by mass spectrometry and indicating which are the fragments of these two proteins that are supposed to be involved in the antibody recognition, we were able to select the most representative ttg and gliadin fragments , to design and synthesize by fmoc-spps nine cross-linked eoepitopes. aim of our study was the characterization of autoantigenic epitopes by testing, in celiac patients' sera, the reactivity of these nine synthetic peptides. these neoepitopes were tested in elisa to evaluate the iga and igg response against ttg-gliadin adducts in celiac patients' sera in order to develop a new elisa test based on peptides as an even more powerful diagnostic tool in terms of specificity and sensitivity. more than analogs have already been described, wherein the hydroxy acid and the amino acid constituents were replaced by d-amino acids and/or n-methyl amino acids with preserved or altered side chains. for certain types of cancer cells, several of these analogs were found to be more active than the natural product itself. however, it does appear that many of these compounds have limited solubility in water. b here we report the synthesis of novel analogs of the sansalvamide a peptide bearing an n-oligoethyleneglycyl (oeg) chain attached to the different backbone positions. attachment of this chain is aimed to enhance the hydrophilicity of the original peptide. our synthetic strategy to modify the backbone with the n-oeg group relies on the use of n-oeg amino acids, which were synthesized in solution and then used as building blocks in spps. as expected, couplings to the n-oeg residues were found to require special conditions. methods for the coupling to nmethyl amino acids were applied and this enabled to obtain the different linear pentapeptides, which were cyclized in solution. both the synthetic strategies of these demanding peptides as well as the preliminar evaluation of their biological activity will be deeply discussed. glycoconjugates such as glycoproteins and glycolipids have important roles in cell functions, for example, intercellular recognition, cell proliferation control, and information transmission. in order to study the structurefunction relationship, synthesis of these glycoconjugates is essential. glycoproteins and glycopeptides are classified into two categories: n-and o-glycosylated derivatives. the n-acetyl-α-d-galactopyranosylated ser or thr derivatives [ser/thr(α-d-galnac)] are important intermediates for o-glycopeptide synthesis. however, the synthesis of ser/thr(α-d-galnac) derivatives by chemical glycosylation is difficult because of the decreased nucleophilicity of hydroxy function in the glycosyl acceptor due to an unfavorable hydrogen-bonding pattern between the oh and α-nh groups . several approaches to overcome this problem have been reported , . in addition, the o-glycosidic bond is cleaved easily in acidic conditions. in this study, we assumed that the formation of a cyclic structure containing an α-nh group would increase the reactivity of oh function. thus, we focused on the n, n'isopropylidene derivatives of ser/thr containing dipeptides . we found the reaction of mannopyranosyl trichloroacetimidate and the n, n'-isopropylidene dipeptide in the presence of tmsotf in dichloromethane produced the desired glycosylated dipeptide in good yield. however the selective intermolecular disulfide bond formation is a very difficult and complicated synthetic problem. in this work we report on synthetic approaches for the formation of conjugates with intermolecular thioether or disulfide bonds. for the disulfide bond formation, we use two activation approaches: i) activation of the four cys residues of the carrier testing two activating reagents, in both solid and liquid phase respectively and ii) activation of the cys containing bioactive molecule. as bioactive molecule we selected the r ppleed sequence derived from the intracellular part of the αiibplatelet integrin receptor. this region is critically involved in platelet aggregation and is a target of intervention for developing antithrombotic agents . the ac-[lys-aib-cys(ch co-αiib - )] -nh and ac-[lys-aib-cys(cys-αiib - )] -nh conjugates were synthesized and examined for their ability to inhibit platelet aggregation. the biological assays indicated that the synthesized conjugates penetrate the platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. the molecules were reported to exhibit broad-spectrum cytotoxicity against the tumor cell lines. although these peptides contained the novel β-methoxytyrosine, lipton et al. reported the synthesis and cytotoxicity of desmethoxycallipeltin b, in which substitution of d-tyrosine for β-methoxytyrosine did not substantially affect the cytotoxicity of callipeltin b , . however, a structure-activity relationship study of the molecules has not been shown to date in detail. in the course of our recent research regarding the synthetic study of cyclic depsipeptides, we conducted studies on the synthesis of callipeltins supposed to be efficient structures for ccr inhibitors as anti-hiv drugs or anti-cancer agents. in the present study, we report the synthesis of cyclic depsipeptides of callipeltin b analogues consisting of l-, d-amino acids and/or n-methyl amino acids, for a structure-activity relationship study of linear-and cyclic depsi-peptides against hela cells . in the assay of synthetic peptides, all of the synthetic callipeltin b analogues exhibited no cytotoxicity. we supposed that dimethylpyroglutamic acid of callipeltin b was essential structure to show the cytotoxicity against hela cells. monash university, melbourne, australia protein-protein interactions represent a significant portion ( - %) of all interactions within the cell; as such these interactions are ideal targets for drug discovery. while difficult to target using small molecules, these interactions can be disrupted using a small section of the protein's binding partner. these short peptides must retain the defined secondary structure associated with the protein binding interface in order to inhibit their protein targets. as the secondary structure adopted by the parent protein is not always exhibited by its derived peptides, constraints are introduced as necessary to help define the structure of the peptide. inducing secondary structure reduces the energy required for organisation, decreasing the energy of binding and has the potential to increase stability with respect to degradation by proteases. solid phase peptide synthesis was used to make several small peptides corresponding to the structured sections of the binding partners for three protein-protein interactions. these peptides were designed to target heart disease, prostate cancer, and liver cancer respectively. secondary structure was introduced using lactam bridge constraints. for the αhelical peptides, a side chain constraint approach was used to nucleate helix formation. as hydrogen bonding between the c=o of the i th residue and the nh of the (i+ ) th residue stabilises native α-helices, constraints were introduced linking the side chains such that the residues were held in close proximity. for β-pleated peptides, an antiparallel β-sheet arrangement was achieved by introducing turn regions into the peptide in such a way that the β-strands were aligned. constraints were again introduced using lactam bridges between lys and arg or glu side chains. these peptides were characterised by nmr and cd spectroscopy to verify the correct secondary structure had been induced. göttingen, germany different properties can be combined in a single molecule by using a scaffold arranging functional groups in a predefined topology. the tasp (template-assembled synthetic proteins) concept describes templates to reinforce and direct the folding of designed molecules into a predetermined topology. [ ] due to their resistance to proteolytic degradation and their rigid basic structure, cyclic β -tripeptides are suitable carrier molecules for bioactive compounds; they are further known to form tubelike structures by stacking of the peptide rings leading to higher organization of functionalized peptides. [ ] with this scaffold different inhibitory systems were synthesized that feature cell penetrating and fluorescent properties. the signal transducer and activator of transcription (stat ) protein, which has been described as an oncogenic protein, was selected as the first target. [ ] a peptide sequence which targets the sh domain of stat was used in two different approaches. it was either directly attached to the cyclic β-tripeptide via a huisgen [ + ]cycloaddition or the peptide was incorporated into the inhibitor loop of the cystine knot microprotein omcoti-ii, which was also attached to the cyclic-β -tripeptide. [ ] further, sodium channels are addressed usingconotoxines. first, an alkyne functionalized conotoxin siiia was synthesized applying different folding methods. the alkyne linker will be used to attach a fluorophore or to functionalize a cyclic-β-tripeptide. using single molecule imaging the spatial distribution, local concentration and organization of the ion channels in neurons will be imaged. further, the cyclic-β -tripeptide templating effect will be used functionalizing with μ-conotoxines. those proteins are folding helper proteins. together with chaperones, they form receptor complexes. they catalyze the isomerization of prolyl bonds in various folding states of target proteins. indeed, their role has been implicated in refolding of denatured proteins, de novo protein synthesis and the biologically active conformation of proteins . among them, the fkbp subclass comprises the small ppi calstabin and . it is of interest to try to understand the way those proteins act, in order to help the overexpression of various types of membrane proteins, aiming at the renaturation, purification and crystallization attempts of receptors. we chose to work on calstabin because this short ( aa) protein has been described as a sub-family comprising isoforms (from ~ to ~ aminoacids), some of them not being fully described to date. the relative shortness of those proteins together with the fact that the two higher molecular weight ones are catalytically active as prolyl isomerases, facilitate the characterization of the synthetic proteins.in order to obtain the full length calstabin , a native chemical ligation (ncl) approach was chosen . an optimized stepwise elongation allowed the obtention of the c-terminal segment up to the cys . moreover, several methods were compared for the synthesis of peptide - opportunely functionalized at its c-terminus for the ncl.the ligation at thr site between peptide - featuring a bis( -sulfanylethyl)amino the chemical diagnostics of paintings is a relevant topic in the field of chemical sciences applied to the conservation and safeguard of cultural heritage. chromatography is a highly sensitive and suitable technique for accurate methods of analysis of the limited amount of sample material typically available from works of art. paint media deriving from proteins traditionally include egg, milk, animal and fish collagen glue. egg yolk (egg tempera), egg albumin (glair) and casein (a blend of related phosphoproteins commonly found in milk) are traditionally used as pigments binders. we propose the uplc-based amino acid analysis as diagnostics technique on non pre-treated or submitted to extraction processes model samples, showing that good results can be achieved with very scarce sample manipulation and great advantage. we applied the amino acids analysis carried out by the accq•tag™ ultra performance liquid chromatography to the standard and model samples. in particular, after protein hydrolysis ( h, °c, m hcl) of the samples, the amino acid derivatization by -aminoquinolyl-n-hydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis characteristic of the protein type. the results obtained confirmed the reliability of the data achieved and demonstrated that the accq•tag™ ultra uplc method could be a powerful technique to be applied to the relevant field of protein binders diagnostics for paintings conservation. moreover a multivariate analysis that offers a wide variety of tools and methods mainly concerned with mathematical models for the representation of multidimensional data has been proposed and the high model efficiency has been established for sample containing mixture of proteins. reactions performed on solid supports, such as resin, are commonly monitored by hplc-uv after cleaving the products from the support. however, uv-absorption coefficients may differ between compounds, and therefore the relation of the area percentage values of the peaks may not directly reflect the molar concentrations of the corresponding compounds. it is for this reason that, for example, in solid-phase peptide synthesis it is difficult to calculate the yield of the coupling of a fmoc-amino acid or the removal of the fmoc-group because of its high absorbance. recently, we reported the identification of minimal phosphopeptides that specifically interact with the pbd of human plk , but not those of the closely related plk and plk . comparative analyses of the crystal structures of the plk pbd in complex with the minimal phosphopeptides revealed that the c-terminal spt dipeptide functions as a high-affinity to the interaction. in an attempt to obtain the adequate cellular permeability and stability in vivo, we have accomplished the peptide-peptoid hybrid or peptomers cyclization using various methods like formation of amide, thioether and triazole and screened the plk inhibition activity on the first cyclic peptomers liibrary using pbd-binding assay. based on our first screening results, we also carried out the detailed investigation to further increase the activity and also to understand the significance of peptoid mimics as plk inhibitors. the mode of interaction between the cyclic peptomers and pbd might provide a template for designing therapeutic agents that target plk . a synthetic amino acid long peptide corresponding to the minimal metacaspase catalytic domain induces cell death in leishmania major c. servis, h. zalila*, i. gonzalez, l. lozano, n. fasel department of biochemistry, university of lausanne, epalinges, switzerland despite a lot of controversy during the last decade, there is increasing experimental evidence that cell death (cd) is genetically programmed in lower eukaryotes.in the cd proteolytic cascade of plants and protozoa, caspases are likely replaced by metacaspases that are cysteine peptidases recognizing arginines or lysines in p position. metacaspases have been found to control cell death in plants. the human protozoan parasite leishmania major expresses a single metacaspase (lmjmca) harboring a central domain with the catalytic dyad histidine and cysteine as found in caspases. metacaspase could therefore be one of the executioners of the death pathway in leishmania.in this work we showed that, in stress conditions, lmjmca precursor forms were extensively processed into soluble forms containing the catalytic domain and this domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion function. we tested different lengths of the lmjmca catalytic domain and found that the overexpression of the polypeptide corresponding to amino acids - was sufficient to sensitize l. major mitochondria to oxidative stress.we synthetized an aa long peptide corresponding to the minimal metacaspase catalytic domain (aa - ) and showed that it has specific metacaspase activity in vitro.we are currently investigating its activity on possible target proteins, which have been identified in a yeast two-hybrid screen. identifying proteins involved in the metacaspase signaling pathway will shed light on the understanding of cd in leishmania and open new perspectives in drug target investigation to fight leishmaniasis and other major infectious diseases. s. alasibi, g. ashkenasy department of chemistry, ben-gurion university of the negev, beer-sheva, israel various factors can affect the conformations and folding states of protein molecules and as a consequence their activity. these factors include amino acid mutations, interactions with other macromolecules, binding to regulatory molecules, and also external changes such as ph jump or shining light. in order to control the folding states and to modulate the functions of peptides and proteins by light, photocleavable groups are usually incorporated into specific residues to mask critical interactions. for example, introducing caging groups into coiled-coil proteins recognition interface affects complex formation and template-assisted ligation reactions, in which the coiled-coils serve as templates to catalyze the condensation reactions between two short peptide fragments . our research group has been studying peptides replication networks, which were made of coiledcoil peptides and analyzed the response of such networks to light as external trigger . it was shown that even replicating networks made up of a small number of molecules can possess complex behavior, considering the wealth of catalytic pathways and transformations. hence, boolean logic operations can provide valuable means to analyze and interpret their behavior . herein, we describe the use of chemical inputs and uv light to manipulate peptides folding and functionality within new synthetic networks. these networks perform complex behavior and, as a result, selective product formation is used to implement boolean operations that have not been achieved before. institute of bioorganic chemistry of ras, moscow, russia earlier, we have shown that n-acylated amino acid nitriles and amides react with ethylene derivatives forming the amino-and -hydroxypyridines and pyrroles [ ] . a possible reaction mechanism is the geterodienic condensation of aminooxazole derivatives to dienophiles. the higher yields were observed when used the dicarboxylic acids as dienophiles and -amino or alkoxyoxazole as geterodienes. while the same reactions with the fullerene derivatives, as dienophiles, gave low yields. the nitrile groups of specified pyridines possess ability to react with amino groups of peptides and proteins even at room temperature. in view of high activity of nitrile groups such pyridines can form tetrapyridotetrazoporphyrins and self-condensation products giving appropriate dendrimers (possible due to mobile hydrogen in the th position). high molecular weight dendrimers were identified by massspectrometry, gel electrophoresis and dynamic light-scattering. catalytic oxidizing properties of tetrazaporphyrin derivatives and phtalocyanin were used in synthesis of cyclic peptides and for the s-s bonds formation. the transformation of peptides into heterocycles via an intramolecular reaction of nitrile groups was used to determine the sequence of some peptides, which favored the resistance of transformed compounds to hydrolysis and to the electron impact at mass spectrometry. diazotization of peptides and their derivatives facilitates identification of amino acid sequence by mass spectrometry due to the peculiarities of their fragmentation. in addition, an amino acid analysis of the diazotized peptide makes it easy to determine the n-terminal amino acid. we present here a multi-disciplinary approach combining x-ray crystallography, computational analyses, and immunological tests to identify epitopes of the oligopeptide-binding protein a (oppa bp) from the gramnegative pathogen burkholderia pseudomallei, the etiological agent of melioidosis. computational analysis on oppa crystal structure was used to design potential consensus epitopes, that once synthesized as free peptides (comp - ) were found to be immunoreactive against sera from melioidosis patients. notably, one of the predicted peptides allowed to distinguish between seropositive, seronegative and recovered groups, underlining its potential for diagnostic purposes. parallel experimental epitope mapping, based on proteolysis and mass spectrometry, allowed us to identify linear peptide epitopes (exp - ) localized in similar protein regions as comp - . moreover, the match between theoretical and experimental mapping of epitopes was improved by expanding our computational approach, i.e. including an energy based decomposition procedure to divide oppa bp into separate fragments. overall, our results illustrate the successful development of a novel integrated structurebased approach for the discovery, design and preparation of epitopes. nonetheless, given antigen crystal structures, our method is expected to be broadly applicable in the design and generation of new epitope candidates, as being confirmed by on going experiments on different antigens. the application of peptide thioacids as reactive intermediates and building blocks has received considerable attention recently. the chemical ligation reaction between thioacids and azides has been reported for the synthesis of small to larger peptides as well as for the modification of proteins. fmoc based methods for the preparation of peptide thioacids have to our knowledge not been extensively researched and a facile approach to their synthesis is desirable.we have recently shown that t-butyl thioesters are robuster than previously reported, and can be used for the fmoc based solid-phase preparation of peptide thioesters being also easily cleavable with thiolates. peptides attached via a -mercapto -methylpentanol (mmp) resin can be cleaved using -mercaptopropionitrile to obtain protected thioacid peptides with a ß-elminable cyanoethyl group.the thioacid peptides could then be obtained in situ after treatment with dbu ( . % in dmf) and further reacted with sulfonyl azides in the presence of , -lutidine in a one pot reaction. by treating the cyanoethyl peptide thioesters with (nh ) s in a sodium phosphate buffer (ph = ), various model penta-peptide thioacids could be obtained cleanly at room temperature in up to % overall yield based on initial coupling. these peptides were then further ligated with electron deficient sulfonyl azide functionalized peptides.larger peptide thioacids could also be obtained using this protocol. a mer derivative of penetratin- , a cell-penetrating peptide from the third helix of the homeodomain of the antennapedia protein, was prepared as a peptide thioacid in a % yield (based on coupling of the first amino acid). in this report, a sensitive, selective and rapid uplc-ms method was developed for the determination of the [lys-gly] -mog - peptide in order to control the conjugation of mannan with the [lys-gly] -mog - peptide. the separation was performed on an acquity uplc system with a beh c column packed with . μm particles. the total run time was min. calibration curve based on peak area ratio was linear at the concentration range of - μg/ml, with a detection limit of μg/ml. the method showed satisfactory reproducibility and confirmed the entire conjugation between oxidized mannan and peptide sequence. the development of simple, low-cost and fast methods for protein purification is of increasing importance both for academic and industrial applications. a very promising approach is inverse transition cycling (itc) that exploits the temperature dependent aggregation properties of elps. elastin-like polypeptides (elp) are artificial polypeptides composed of pentameric repeats (val-pro-gly-xaa-gly) derived from mammalian elastin. elps are characterized by a specific transition temperature (t t ) that depends on the amino acid composition of the pentarepeat; they are water-soluble below and aggregate reversibly above this narrow temperature range (t t ). these properties are transferred to target proteins by n-or cterminal fusion with elps. during itc these fusion proteins precipitate, while other components remain in solution. repeated cycles of heating and cooling allow simple recovery of the target protein.we synthesized various elps consisting of to pentameric repeats and including different guest residues. the transition temperature of all synthetic elps was determined using photometric assays and measuring turbidity. in order to test if elp properties can be efficiently transferred, we fused elp to a small recombinant protein (ras-binding domain, rbd) by expressed protein ligation. this approach will allow the incorporation of elps with unnatural amino acids and other chemical modifications into target proteins. currently we are focusing on biotechnologically relevant enzymes that constitute a major cost factor in industrial processes. the authors thank süd-chemie/clariant for their financial support. department of pharmacy, university of patras, rio, greece peptides penetrating the cell membrane, known as cell penetrating peptides (cpps), as well as their mimics, used as delivery agents to cells have been reported , . cpps can be natural sequences or artificial constructs designed to capture the features of natural formations. cpps are particularly important in the delivery of peptides, proteins, nucleic acids, small molecule drugs or imaging agents. incorporation of a heterocyclic motif into a peptide or peptide-like backbone introduces conformational constraints and/or latent reactivity related to the heterocycle's structural profile. heterocycle-based cpp mimics are, thus, promising candidates for therapeutics protected synthetic non-ionic peptides, which are for example synthetic intermediates for the production of api's, are often very hydrophobic and not soluble in most common solvents. they are thus difficult to purify by preparative rp-hplc, classically used for industrial production. it is then challenging to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. the technique of support free liquid-liquid chromatography , including both its hydrostatic (centrifugal partition chromatography or cpc) and its hydrodynamic (counter-current chromatography or ccc) declensions, are mainly involved in phytochemical studies but has also been applied to peptide purification . the previously developed biphasic solvent systems are not adapted to the purification of highly hydrophobic protected peptides. to overcome this problem, two new scales of biphasic solvents systems and a ternary biphasic solvent system were developed to overcome solubility problems often encountered with those peptides. the new systems composed of heptane/thf/ch n/dmso/water, heptane/me-thf/nmp/water, and cmpe/dmf/water were efficiently used for the cpc purification of a mer protected exenatide and a mer protected peptide intermediate of bivalirudin synthesis. the developed scales show a wide range of polarity and should be useful for general use in cpc for the separation of hydrophobic synthetic free or protected peptides. the progressive aggregation of β-amyloid peptide (β-ap) into insoluble amyloid fibrils ultimately leading to formation of toxic amyloid plaques is widely considered to be the central pathogenic cause of alzheimer's disease. in the last decade accumulating evidence suggests that soluble oligomeric non-fibrillar forms of β-ap are neurotoxic as well. consequently, inhibiting the aggregation of β-ap is one of the therapeutic strategies against alzheimer's disease and a number of small molecules have been identified as inhibitors of β-ap aggregation and neurotoxicity. among these, curcumin, the phenolic yellow pigment and active ingredient of the turmeric herb, is receiving special attention because of its rich pharmacology that includes in vitro and in vivo inhibitory action against alzheimer's disease insults. in the current work the interaction of β-ap( - ) with curcumin is investigated with fluorescence, cd, and nmr spectroscopies in water and water-methanol mixtures and at various β-ap( - ):curcumin ratios. in nmr studies in % methanol curcumin behaves like a macromolecular species with a change in the sign of its noe signal providing direct indication of its association with β-ap( - ). in % methanol the presence of β-ap( - ) results in great broadening of the h peaks of curcumin, indicative of a complete change in its solution state. additionally, the fluorescence of curcumin in % methanol shows a blue shift with enhanced intensity, observations consistent with a hydrophobic modification of curcumin environment upon interaction with β-ap. finally, in water the induced circular dichroism spectrum of curcumin in the near uv region provides clear evidence for the loss of symmetry of curcumin molecule due to changes in its microenvironment generated by interaction with β-ap( - ). our experimental findings support the direct interaction of β-ap( - ) with curcumin and establish its importance as a potential aggregation inhibitor of β-ap. [ ] . based on its sequence, we synthesized h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and reported they had potent antiproliferative activity on cancer cells (a- and sw ), which were comparable to tt- and cycloheximide. a structure-activity relationship analysis revealed that lipophylicity of yo- and - could be responsible for their antiproliferative activity. now, we described the substitution of tyr of yo- and - by tyr(bzl), phe, -nal( -naphthylalanine), -nal ( -naphthyalanine) and the anticancer and dna polymerase inhibitory activities in order to explore the effect of hydrophobic substituent. among the compounds, yo- and - had the highest lipophilicity judging from their retention time and lipophilicity index (yo- : . min, . ; yo- : . min, . ). yo- and - exhibited strong dna polymerase inhibitory activity as well as antifroliferative activity on hct cells at m. these activities were greater than those of yo- and - . antiproliferative activity of the compounds containing -ada such as yo- , - , - , - and - , was comparable to that of the compounds containing -ada such as yo- , - , - , - and - . these findings suggest that the lipophilicity well correlates with dna polymerase inhibitory activity and antiproliferative activity on hct cells. further structureactivity relationship study is progressing in our group. multiple sclerosis (ms) is a chronic autoimmune disease of the central nervous system (cns) , . our aim was to immunologically control the attack of the myelin sheath in ms patients without the total suppression of the immune system. anthraquinones (mitoxantrone, ametantrone) are widely used in cancer therapy as immunosuppressants.mitoxantrone is also used to treat several forms of advancing ms, including secondary progressive ms, progressive relapsing ms, and advanced relapsingremitting ms . more specifically, mitoxantrone is an inhibitor of the type ii topoisomerase, which disrupts dna synthesis and dna repair in both healthy cells and cancer cells. herein, we report the synthesis of an anthraquinone type compound conjugated to the immunodominant - myelin oligodendrocyte glycoprotein (mog - ) for the selective immunosuppression of the encephalitogenic t cells in ms patients. the anthraquinone was synthesized by a friedel-crafts acylation of hydroquinone from phthalic anhydride, followed by reduction of the resulted quinizarine to its leuco form, addition of the appropriate diamine and air oxidation . the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. the synthesis of the mog - was performed under microwave irradiation and its conjugation with the anthraquinone was performed in solution. the final analogue was purified by rp-hplc and identified by esi-ms. benzopyrans, diketopiperazines and , benzodiazepin- , -diones are well-known and widely investigated scaffolds, e.g. the latter showing anxiolytic and antiarrhythmic effects. now, we propose a new potential "privileged structure" containing a triazole moiety mimicking the cis-amide bond within the , -benzodiazepin- , -dione motif .molecules based on this [ , , ]-triazolo [ , -d] benzo- , diazepin- -one scaffold are synthesized and decorated via a modular approach on wang resin using α-amino acids, -ethynylaniline building blocks and n-alkylating agents resulting in five points of diversity. the methodology involves the attachment of α-amino acids onto a solid support, subsequent removal of the fmoc group followed by an optimized diazotransfer reaction of the resulting amine yielding a resin-bound azide. conversion of the latter into a , -disubstituted , , -triazole moiety is achieved quantitatively by addition of a range of -ethynylaniline building blocks using a ru(ii)-catalyst. the desired scaffold can be obtained in high crude purities (> %) in solution via an acid catalyzed one-step cyclisation-release strategy. solution-phase n-alkylation finally affords the fully diversified scaffold. interestingly, n-alkylation induces atropisomeric effects which can be studied via h nmr spectroscopy.taking into account future screening results of the synthesized libraries, a well-thought decoration of this scaffold leading to discovery of new lead molecules is within reach. peptide symposium in the wonderful small seaside town of porto carras. maurice manning and lajos balaśpiri were nominated as captains of the two teams, the rest of the world and europe. similar matches were organized at subsequent european peptide symposia. now, in greece, the two captains would like to hand over their roles to younger scientists [professors gabor mezö(hungary) and laśzlóÖtvös (usa)] to continue this tradition at the coming european and possibly american peptide symposia. it seems best to play in the free time (in the evenings after the excursions). necessary conditions: good weather; a nice large soccer field; a soccer ball, preferably new; and jerseys and shorts (different colours), organized as always by the organizer commettee. the captain of the winning team will receive a trophy at the end of the nd european peptide symposium. the teams will remember two earlier excellent referees: professor lajos kisfaludy in porto carras [ ] and the soccer professor + ferenc puskaś (hungary) in budapest ( ). in the poster session, the results from the past years will be presented in about - pictures. these pictures may possibly be bought free, % can be saved at the poster session. all participants are welcome at the new party in athens. conclusion will be presented by the players and fans in athens. the human lactoferrin-derived peptide, hlf - , was proven to be highly active against antibiotic-resistant bacteria . however, the clinical use of this antimicrobial peptide (amps) is hampered by the peptide low stability due to fast degradation or to peptide aggregation, as the use of higher peptide concentrations results on higher toxicity levels. amp immobilization onto a biomaterial surface could be the pathway to overcome these difficulties . the aim of this work is the development of an antimicrobial surface by covalent immobilization of hlf - onto the surface of chitosan thin films. chitosan ultrathin films were prepared through the spincoating of a . % chitosan solution in gold substrates. hlf - immobilization was performed through an ss bound between hlf - terminal cysteine and an n-acetyl cysteine previously coupled at chitosan films. surfaces were characterized using ellipsometry (thickness), infrared reflection absorption spectroscopy (irras) and x-ray photoelectron spectroscopy (xps). bacterial adhesion studies were performed using methicillin-resistant s. aureus (atcc ). chitosan films were incubated with this bacterial suspension at ºc for h and h. the viability of the attached bacteria was evaluated using live/dead® bacterial viability kit (baclight tm ) and fluorescence microscopy. hlf - peptide was successfully covalently immobilized onto chitosan thin films. both soluble and attached peptide presented a higher antimicrobial activity than the control chitosan. identified as a potent vasoconstrictor that binds with high affinity to ut receptor. the cysteine-linked cyclic region, hut-ii( - ), is responsible for the biological activity and has been widely used to elucidate the structure-activity relationship of hut-ii. with the aim to investigate the role of hydrogen bond and the effects of a peptide backbone constraint on binding key: cord- -n sih authors: villard, viviane; agak, george w.; frank, géraldine; jafarshad, ali; servis, catherine; nébié, issa; sirima, sodiomon b.; felger, ingrid; arevalo-herrera, myriam; herrera, socrates; heitz, frederic; bäcker, volker; druilhe, pierre; kajava, andrey v.; corradin, giampietro title: rapid identification of malaria vaccine candidates based on α-helical coiled coil protein motif date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: n sih to identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. the corresponding synthetic peptides are expected to mimic structurally “native” epitopes. indeed the chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. these antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. this strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. human plasmodium falciparum (pf) infection is a dramatic public health problem. today approximately forty percent of the world's population is at risk of malaria. malaria causes more than million acute clinical cases, and at least one million deaths annually. ninety percent of malaria deaths occur in sub-saharan african countries mostly among young children and pregnant women (http://rbm.who.int). thus, there is an urgent need of a malaria vaccine. however, vaccine discovery, in general and particularly in malaria, is still a very empirical process. in fact, protective antigens do not bear any structural, physico-chemical or sequence-related characteristics that would allow their identification. for protective humoral responses, the only recognized characteristics of antigens are their antigenicity/immunogenicity and accessibility. in addition, the lack of surrogate markers of protection renders the vaccine discovery process difficult and time consuming. hence, in spite of a constant and impressive progress in molecular biology techniques and antigen identification and expression [ , ] , vaccine discovery is still labor-intensive, making the approach fastidious, costly and poorly adapted to highthroughput screening. proper protein folding and solubility remain a limitation in numerous cases. thus, overcoming the bottlenecks of manufacturing and identification of fragments/proteins, as possible targets of a protective immune response still constitute a scientific and technical challenge. we addressed this challenge by combining bioinformatics, chemical peptide synthesis and functional protection assays. we focused on the search for a-helical coiled coil motifs that, in general, do not exhibit a folding problem, and are a target of effective antibodies for several current malaria vaccine candidates (eg. lsa- [ ] , lsa- [ ] , msp- [ ] , and msp- [ ] and other pathogens [ ] . msp- , another leading malaria vaccine [ ] , also contains predicted a-helical coiled coil regions (unpublished results). our choice was based on the following considerations. first, the a-helical coiled coil motif bears a characteristic seven amino acid residue repeat (abcdefg) n with hydrophobic residues located in a and d positions and hydrophilic residues generally elsewhere. this motif can be easily identified by bioinformatic analysis. secondly, an important known characteristic of the ahelical coiled coil domains is that, taken separately from the whole protein, they frequently and readily fold into the same stable oligomeric structure [ ] . thirdly, for this reason, the a-helical coiled coil fragments are frequently recognized by conformational dependent antibodies, and can similarly elicit antibodies reactive with structurally ''native'' epitopes. in addition, these domains are short (about residues) and can be rapidly produced by chemical synthesis. furthermore, when the antibodies have an anti-parasite biological activity, this designates the corresponding proteins/ fragments as potential, novel vaccine candidates to be further developed and assessed. here, we focus on the pf parasite erythrocytic stage, a target of protective antibodies and describe a straightforward, rapid procedure based on bioinformatic analysis of a-helical coiled-coil motifs and peptide synthesis. the screening of the pf genome [ ] using generalized sequence profiles [ ] identified several hundred proteins containing putative a-helical coiled coil motifs. through proteome and transcriptome data [ ] [ ] [ ] we assessed which of these molecules are expressed in the pf parasite erythrocytic stage. the combined analysis/assessment identified over segments associated with this stage and displaying the putative a-helical coiled coil motifs with high probability score (table s ) . out of these a-helical coiled coil fragments, in general - amino acids long, present either in the same protein or in different ones, were chemically synthesized and hplc purified. among them, longer peptides (up to amino acids), which contained one or more a-helical coiled coil domains, were also synthesized (antigens , and ; table s ). the selected antigens were then tested in elisa assays for reactivity with three panels of sera obtained from adult donors from burkina faso, tanzania and colombia, respectively. to our surprise, all of the a-helical coiled coil fragments were antigenic, though the prevalence of responders varied greatly (tables and s ). in this manner, proteins were identified whose lengths varied from to , amino acids. twenty-one peptides with the highest prevalence of responders and elisa mean od value were selected for further studies. variation in recognition among the three panels of sera may be due to differences in the genetic background of the hosts, of the parasites and, most likely, to distinct malaria transmission conditions in the three regions. the high level of recognition of the a-helical coiled coil motifs may be explained by the fact that taken separately from the whole protein these fragments readily fold into the same stable structure in aqueous solution. indeed, circular dichroism (cd) studies of selected peptides associated with biological activities (tables and ) indicate that they predominantly assume an a-helical conformation in water. peptides , and ( figure s a ) exhibit a cd pattern characteristic of a high a-helical content, whereas the remaining peptides show cd profiles similar to that shown for peptide ( figure s b ) or intermediate between those shown in figures s a and s b characteristic of a partial a-helical organization. when analyzed by size exclusion chromatography on fplc columns, peptides presented elution profiles between those exhibited by chymotrypsin and ribonuclease (mw and kda, respectively). the cd and size exclusion chromatography results suggest that peptides adopt an a-helical coiled-coil structure, which need to be unambiguously ascertained by nmr and ultra-centrifugation studies. to test the biological activity of peptide-specific antibodies, the latter were purified by affinity chromatography using three serum pools obtained from papua new guinean adults. the serum pools were first tested in elisa assays against peptides that were the most antigenic (table ) ; from these, peptide-specific antibodies were purified from the most positive serum pool and tested again in elisa. these antibodies all reacted with parasite native proteins in infected red blood cells as shown by ifat ( figure a ; table ). reactivity was restricted to blood stages, since the antibodies did not react with sporozoites stages (data not shown), and this reactivity was also peptide-specific as shown by ifat competition assays with the corresponding peptide ( figure a ). the specificity of the antibodies obtained was investigated in detail, particularly since several peptides contain glutamic acid (glu)-rich sequences which are known to generate cross reactivity among several malarial glu-rich proteins [ ] . cross-reactions were systematically investigated using each of the affinitypurified antibodies on each of the peptides. results show thatwith few exceptions-each antibody preferentially recognizes the peptide against which antibodies were affinity-purified, i.e. they are specific for the corresponding peptide (table s ) . to determine if non-specific antibody binding to solid phase-adsorbed antigens could be responsible for the rare cross-reactivities detected, elisa competition assays were performed. to this end, binding of antibodies to the solid phase-adsorbed antigen was competed against increasing concentrations of the homologous or cross-reacting peptides. only homologous peptides competed best whereas peptides having sequence similarity did not (figures a, b , and s b), or at a much higher concentration ( figure s a ) including the shorter glu-rich peptides derived from the cterminus of peptide and (figures a and b) . finally, the pattern of recognition of peptides by the various sera tested, which differ markedly from one to the other (data not shown), confirms the above results i.e., specificity of antibodies to the corresponding peptide. antibodies corresponding to the selected peptides were tested for direct and cell-mediated anti-parasite activity. clinical experiments have shown that the antibody-dependent cellmediated inhibition (adci) of p. falciparum malaria represents one of the mechanisms controlling parasitemia and thereby clinical manifestations in humans [ ] . twelve peptide-specific antibodies proved able to induce a strong (more than %) and intermediate (lower than %) monocyte-dependent parasite killing (table ) , whereas, in the absence of monocytes, no direct effect of antibodies on parasite growth was observed. the effects were in the range observed with antibodies from african adults who have the highest natural protection known against malaria. therefore peptidespecific, human affinity-purified antibodies were functionally effective as shown by their ability to react with parasite proteins and to inhibit parasite growth. thus, in vitro functional assays show that peptide-specific antibodies elicited by natural exposure to the parasite can induce protective mechanisms effective against malaria. sixteen peptides -twelve targeted by adci positive antibodies and four controls-were used to immunize cb f mice ( table ) . eleven of them elicited an intermediate or high antibody response, four of which also recognized the parasite protein in infected erythrocytes as determined by ifat ( figure b ; table ). as seen before for human antibodies, recognition was restricted to blood stages since sporozoites were negative in ifat assays (data not shown) and by ifat competition assays with the corresponding peptide ( figure b ). anti-peptide mouse antibodies, which are positive in ifat, are also specific for the homologous peptide but not for the sequence related peptides , and (table s ) . thus, peptides, which were chosen for their propensity to form ahelical coiled coil, can induce the production of antibodies that recognize epitopes present in the native protein. improvement of the immunogenicity and structural specificity of the remaining peptides might be achieved in the future by a) a short elongation at the n-and c-terminal ends, b) stabilizing the a-helix as suggested by cooper et al. and lu and hodges [ , ] and/or c) use of other adjuvants. genetic polymorphism in current vaccine candidates is a major limitation to vaccine development. however available genotyping studies of our peptide sequences in parasite isolates of worldwide origin indicate very limited polymorphism (plasmodb . , and unpublished sequencing data). a few peptide dna sequences show deletion of one entire heptad repeat so that the shorter region still preserves its potential for the a-helical coiled coil formation. with regard to the structural features and cellular location prediction of the proteins corresponding to the peptides selected for adci assays ( table ) , of the proteins contain a pentapeptide conforming to the pexel consensus [ , ; , ] , but that none of these have a position within the amino acid sequence that conforms to the location of known active pexel motifs (see materials and methods and membrane segments, and none of them has a gpi anchor. only one protein contains a signal sequence. fourteen proteins are predicted to be in the cytoplasm, one in the nucleus, one in the mitochondria, and one in the peroxysomes (table s ). the prediction of the sub-cellular localization of these proteins should be taken with caution because gene annotation is being constantly updated and/or protein trafficking of the parasite is complex and not fully elucidated [ ] . further investigations will be required to determine the actual localization of the corresponding antigens. the predicted localization is a priori surprising for molecules able to trigger an adci activity. however, recent studies have shown that in addition to merozoite surface proteins, soluble proteins released at the time of schizont rupture were equally effective at triggering adci provided they defined at least two epitopes [ ] , which is the case for a-helical coiled coil heptad repeats. therefore, molecules expressing a trans-membrane domain that can be exported to the parasite or host cell membrane, as well as molecules present in the cytoplasm of maturing schizonts and released by bursting schizonts can trigger antibodies to cross-link fc-c receptors on monocytes to achieve pf parasite killing. in conclusion, an approach combining a genome-wide search by bioinformatics of a-helical coiled coil protein motifs and chemical synthesis can lead to the rapid identification and development of new malaria vaccine candidates. in fact, this approach is straightforward and easy to scale up; vaccine formulations may comprise mixtures of peptides or single constructs made up of several epitopes. in principle, this strategy can be extended to the discovery of proteins and vaccine candidates in other complex pathogens. the pf d genome [ ] was used for the bioinformatics analysis. the generalized sequence profile method and the pftools package [ ] were used to search for the short a-helical coiled coil domains. the coiled coil profiles were constructed using an alignment of several amino acid sequences corresponding to the known coiled coil domain. two profiles containing four and five heptad repeats were used for the analysis. the cut-off levels of the profiles were chosen by tests performed against sequence database of proteins with the known d structures. subsequently, the coiled coil figure . immunofluorescence microscopy analysis of pf d parasites with peptide specific antibodies. acetone/methanol-fixed schizonts and merozoites were reacted with a: human peptide specific, affinity purified antibodies obtained with peptides and (table ) and b: sera from mice immunized with peptide (table ) . grey: bright field images; blue staining: indicates dapi nuclear staining of schizont stage parasites; red staining shows labeling of peptide specific antibodies by cy -conjugated anti-human or anti-mouse igg specific antibody. merge picture is an overlay of the blue and red fluorescence channel. doi: . /journal.pone. .g regions selected by this approach were tested manually for the presence of the characteristic heptad repeats. these proteins were also analyzed by the coils program [ ] . the selected a-helical coiled coil containing proteins were further tested on their possible surface location and gpi anchoring by using the following programs: identification of potential signal peptides, secretomep and signalp (http://www. cbs.dtu.dk/services/) [ ] ; transmembrane spanning regions (tmpred http://www.ch.embnet.org/software/tmpred_ form.html and tmhmm http://www.cbs.dtu.dk/services/ tmhmm; [ , ] ), and gpi-anchored proteins (http://mendel. imp.univie.ac.at/sat/gpi/gpi_server.html; [ ] ) and prediction of sub-cellular localization (ptarget http://bioinformatics. albany.edu/,ptarget; [ ] ). to identify the pexel-like motifs in sequences of the selected proteins we used the following pattern [ [deq] that represents a combination of the pexel patterns indicated in recent papers [ , ] . the presence of the identified proteins in the asexual erythrocytic stages was also checked using the published data on the transcriptome and proteome of this stage of development of p. falciparum (www.plasmodb.org; [ ] ). peptides were synthesized on the advanced chemtech (hatley st george, uk) ac t omega multi channel synthesizer and the applied biosystem synthesizer a and a (foster city, ca) using solid-phase fmoc chemistry. crude peptides were purified by rp-hplc (c preparative column) and analyzed by mass spectrometry (maldi-tof; applied biosystem, foster city, ca). chemicals and solvents used for peptide synthesis were purchased from fluka (buchs, switzerland) and novabiochem (laufelfinger, switzerland). circular dichroism (cd) spectra of peptides were recorded on a jasco j- spectrometer (jasco corporation, tokyo, japan) equipped with a temperature controller and a . cm path length cuvette. the measurements were made in water at ph . and uc and at a peptide concentration of . mg/ml. the sera from burkina faso were collected in the village of goundry located in the central mossi plateau, between and km north of the capital ouagadougou, in the province of oubritenga. the climate is characteristic of areas of sudanese savannah, with a dry season from november to may and a rainy season from june to october. malaria transmission is very high during the rainy season and markedly seasonal. ethical clearance was obtained from the ministry of health, burkina faso. after obtaining informed consent from parents and caretakers, heparinized venous blood samples were collected during a crosssectional survey during the malaria low transmission season . the tanzanian sera came from a large-scale community based study undertaken in kikwalila village, kilombero district, morogoro region from to . blood samples from adults (. years) were taken by finger prick and the serum was kept at - uc until use. research and ethical clearance for the study was obtained by the tanzanian commission for science & technology. the colombian sera were collected in buenaventura the main port on the colombian pacific coast after human informed consent, during a cross sectional survey carried out from february to may within the framework of a project supported by the colombian research council, colciencias. the area has unstable transmission of both p. falciparum and p. vivax malaria. ethical clearance to draw blood from human volunteers was human purified antibodies were used at mg/ml; ifat was not performed on ring stages. valle. blood was taken by venipuncture into tubes containing edta and sera fractionated and stored frozen until use. the sera from adults from papua new guinea (png) pooled for affinity purification were collected in the maprik district of the east sepik province, during a cross sectional survey in july within the framework of the malaria vaccine epidemiology and evaluation project (mveep) supported by the united states agency for international development [ ] . the area is highly endemic for malaria. ethical clearance for mveep was obtained from the png medical research advisory committee. blood was taken by venipuncture into tubes containing edta. the pool of immune african globulins (piag) used for adci was prepared from immune individuals living in endemic areas and negative control igg (n-igg) was obtained from a pool of more than french adult donors with no history of malaria. briefly, the igg fractions from both positive and negative controls were purified using a size exclusion trisacrylh gf m (pall bioseprah; pall life sciences, ny) column followed by an ionic exchange deae ceramic hyperdh f column (pall bioseprah). purified igg were then extensively dialyzed against rpmi and kept at uc until use. cb f mice were injected times with mg of the indicated peptide in montanide isa at the base of the tail on day , and . bleeding was performed days after the second and third immunization. elisa was performed according to lopez et al. [ ] and anti human igg-or anti mouse igg conjugated to alkaline phosphatase was used (sigma, st louis, mo) as second antibody. individual human sera from , and adults donors from burkina faso, tanzania and colombia respectively were used at : dilution. serum was considered positive if the optical density (od) reading was higher than the mean od value+ standard deviation (sd) of the negative controls (individual serum samples from to naïve swiss donors) or if the od ratio of the mean of duplicate experimental values to the mean od of the negative control was higher than . for mouse sera, the end point value was determined as the last dilution of the mean od value+ standard deviation (sd) of the negative control (non immune sera). elisa competition assays were performed by incubating either each of the selected human affinity-purified antibodies, or antibodies elicited in mice, together with each of the antigens over the indicated range of concentrations for minutes at room temperature prior to addition to the elisa peptide-coated plate wells. antigen-sepharose conjugate preparation: mg of antigen was dissolved in ml of coupling buffer ( . m nahco containing . m nacl, ph . ). the cnbr-sepharose b (amersham bioscience ab, uppsala, sweden) was activated by swelling in mm hcl and then washed with coupling buffer. the antigen solution was added to the gel and the mixture was stirred for h at rt. after the coupling reaction, excess antigen was washed away with coupling buffer. the unreacted activated groups were blocked by treatment with ethanolamine ( . m; ph . ) for min at rt. the gel was then washed with sodium acetate buffer ( . m; ph . ), followed by coupling buffer. the antigensepharose beads were either used or stored at uc in pbs ( x) containing mm azide. isolation of specific antibody: pooled human serum was diluted five times with pbs ( x) containing . m sodium chloride and mixed with antigen-sepharose conjugate. this mixture was then stirred gently on a wheel o/n at uc. after centrifugation the supernatant was collected and stored at uc for further use. the antigen-sepharose beads were then washed with ml of trizma base tris ( mm containing . m nacl, ph . ) then with ml of tris ( mm, ph . ). the elution of bound antibody was achieved with glycine ( . m, ph . ). the fractions obtained were instantly neutralized with tris ( m, ph . ), dialyzed against phosphate buffer ( . m, ph . ) and the antibody concentration was determined by the absorbance of the solution at nm. slides coated with pf sporozoites were dried at rt for minutes, fixed with % acetone at uc for minutes, washed times in pbs- . % tween , dried carefully and blocked with ml/ well of pbs- % bovine serum albumin (bsa) for minutes at rt. slides coated with pf merozoites were fixed with % acetone at uc for minutes and dried o/n at rt. the appropriate antibody or serum dilutions prepared in pbs- % bsa were distributed ( ml/well) and incubated for h at rt in a humid chamber. after washing with pbs- . % tween- , goat anti-human or goat anti-mouse polyvalent immunoglobulins conjugated to cy (molecular probes) diluted / in pbs- % bsa or anti-human igg (fc specific) fitc conjugate (sigma) diluted / in evans blue solution ( / ) was added ( ml/slide) and incubated for h at rt in a humid chamber in the dark. slides were washed as above, covered with % glycerol, sealed and read using a fluorescence microscope (leica dmirb dc ). the uganda palo alto strain (fup/c) was cultured in rpmi- supplemented with . % albumax i (gibcobrl-invitrogen, san diego, ca). for adci assays, blood stage parasite cultures were synchronized by at least two successive sorbitol treatments followed, after maturation over h, by floatation on % porcine skin gelatin type a (sigma). blood monocytes (mn) were prepared from cytapheresis samples obtained from healthy blood donors with no previous history of malaria (lecourbe blood bank, paris, france). peripheral blood mononuclear cells (pbmc) were separated on ficoll density gradients j prep (techgen, les ulis, france) and washed in ca + and mg + free hbss buffered with mm hepes (both from gibcobrl-invitrogen). cells were then distributed on polystyrene -well flat-bottomed culture plates (tpp, trasadingen, switzerland) and adherent mn were selected by incubation for h at uc, in a humidified % co atmosphere. more than % of the adherent cells obtained in this manner were mn as estimated by the non-specific esterase test (a-naphtyl acetate esterase; sigma). mn from each donor were tested prior to adci assays and only those without direct inhibitory effect were used in assays. to wells containing mn purified as described above, ml of an asynchronous parasite culture at . % parasitemia and % hematocrit were added. wells were then supplemented with test or control antibodies (ab) and the total volume adjusted to ml with culture medium. after h and h, ml of culture medium were added to each well and after h the adci assay was stopped and the final parasitemia was determined by light microscopy on giemsa-stained smears by counting $ , red blood cells. for each ab tested, duplicate wells included the following controls ) non-specific monocytic inhibition, both mn+parasite, and mn+n-igg+parasites and ) direct inhibition by control or test igg, both n-igg+parasites, and test abs+parasites. piag and n-igg were used at a final concentration of mg/ml as positive and negative controls respectively. immunopurified tests abs were used at mg/ml. the specific growth inhibitory index (sgi) which considers the parasite growth inhibition due to the effect of test abs cooperating with mn was calculated as follows: sgi = [ (% parasitemia with mn and test abs/% parasitemia test abs)/(% parasitemia with mn and n-igg/% parasitemia n-igg)]. figure s cd spectra of the peptides (s a) and (s b) found at: doi: . /journal.pone. .s ( . mb tif) figure s elisa inhibition assay using anti-human peptide specific antibodies. binding of peptide specific antibodies to peptides (s a) and (s b) absorbed on elisa plates was inhibited by incubating specific antibodies ( - mg/ml) with peptides , and (s a) and peptides and (s b), respectively (see material and methods). peptides , and share nnm or mnn as sequence similarity while peptides and do not exhibit any apparent sequence similarity. table s structural feature and cellular location prediction of the proteins containing the peptides whose specific antibodies were tested in adci (table ) . found at: doi: . /journal.pone. .s ( . mb doc) identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing reverse vaccinology and genomics plasmodium falciparum liver stage antigen- is well conserved and contains potent b and t cell determinants protection against plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein antigen plasmodium falciparum merozoite surface protein displays multiple targets for naturally occurring antibodies that mediate monocyte-dependent parasite killing template-based coiled-coil antigens elicit neutralizing antibodies to the sars-coronavirus phase randomized double-blind safety and immunogenicity trial of plasmodium falciparum malaria merozoite surface protein fmp vaccine de novo design of alpha-helical proteins: basic research to medical applications genome sequence of the human malaria parasite plasmodium falciparum a flexible motif search technique based on generalized profiles transcriptomics and proteomics: tools for the identification of novel drug targets and vaccine candidates for tuberculosis a proteomic view of the plasmodium falciparum life cycle the transcriptome of the intraerythrocytic developmental cycle of plasmodium falciparum crossreactive antigens between life cycle stages of plasmodium falciparum antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes mapping of conformational b cell epitopes within alpha-helical coiled coil proteins a de novo designed template for generating conformation-specific antibodies that recognize alpha-helices in proteins targeting malaria virulence and remodeling proteins to the host erythrocyte a host-targeting signal in virulence proteins reveals a secretome in malarial infection proteomic analysis identifies novel proteins of the maurer's clefts, a secretory compartment delivering plasmodium falciparum proteins to the surface of its host cell multi-character population study of the vir subtelomeric multigene superfamily of plasmodium vivax, a major human malaria parasite a maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells a novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes fcgammarii and fcgammariii predicting coiled coils from protein sequences improved prediction of signal peptides: signalp . tmbase-a database of membrane spanning proteins segments predicting transmembrane protein topology with a hidden markov model: application to complete genomes prediction of potential gpimodification sites in proprotein sequences ptarget [corrected] a new method for predicting protein subcellular localization in eukaryotes plasmodb: the plasmodium genome resource. a database integrating experimental and computational data the malaria vaccine epidemiology and evaluation project of papua new guinea: rationale and baseline studies a synthetic malaria vaccine elicits a potent cd (+) and cd (+) t lymphocyte immune response in humans. implications for vaccination strategies the authors wish to thank luis rodrigues and florela penea for the synthesis and purification of peptides and thomas smith for discussion and all of the blood donors. key: cord- - owvqw authors: saunders, jaclyn k.; gaylord, david; held, noelle; symmonds, nick; dupont, chris; shepherd, adam; kinkade, danie; saito, mak a. title: metatryp v . : metaproteomic least common ancestor analysis for taxonomic inference using specialized sequence assemblies - standalone software and web servers for marine microorganisms and coronaviruses date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: owvqw we present metatryp version- software that identifies shared peptides across organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. improvements include: ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (lca) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). major expansion of the marine database confirms low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. metatryp was designed for ocean metaproteomics and has been integrated into the ocean protein portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. we describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. a coronavirus-focused metatryp database identified potential sars-cov- peptide biomarkers and indicated very few shared tryptic peptides between sars-cov- and other disparate taxa, sharing . % peptides or less ( peptide) with the influenza a & b pan-proteomes, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches. statement of significance when assigning taxonomic attribution in bottom-up metaproteomics, the potential for shared tryptic peptides among organisms in mixed communities should be considered. the software program metatryp v and associated interactive web portals enables users to identify the frequency of shared tryptic peptides among taxonomic groups and evaluate the occurrence of specific tryptic peptides within complex communities. metatryp facilitates phyloproteomic studies of taxonomic groups and supports the identification and evaluation of potential metaproteomic biomarkers. we present metatryp version- software that identifies shared peptides across organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. improvements include: ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (lca) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). major expansion of the marine database confirms low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. metatryp was designed for ocean metaproteomics and has been integrated into the ocean protein portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. we describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. a coronavirus-focused metatryp database identified potential sars-cov- peptide biomarkers and indicated very few shared tryptic peptides between sars-cov- and other disparate taxa, sharing . % peptides or less ( peptide) with the influenza a & b pan-proteomes, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches. when assigning taxonomic attribution in bottom-up metaproteomics, the potential for shared tryptic peptides among organisms in mixed communities should be considered. the software program metatryp v and associated interactive web portals enables users to identify the frequency of shared tryptic peptides among taxonomic groups and evaluate the occurrence of specific tryptic peptides within complex communities. metatryp facilitates phyloproteomic studies of taxonomic groups and supports the identification and evaluation of potential metaproteomic biomarkers. in metaproteomics the mixture of a large number of organisms within each sample collected from a natural environment creates challenges in the attribution of peptides to specific proteins. this is especially problematic in instances where exact tryptic peptide sequences are shared between two or more organisms. this potential for shared peptides across proteins can create uncertainty in protein inference and taxonomic attribution. in bottom-up proteomics, the primary method used in metaproteomics to date, whole proteins are typically digested into smaller peptides with the enzyme trypsin. since bottom-up metaproteomics directly measures these short tryptic peptides, as opposed to entire protein sequences, it is essential to understand the degree of shared peptides across proteins and taxonomic groups when assigning attributes of diverse environmental communities. previously, we described the development of the metatryp software which evaluates multiple organisms for shared peptides [ ] . metatryp takes the full predicted proteome of an organism based on its reference genome, performs an in silico tryptic digestion of the proteins, and then stores the tryptic peptides of that organism within a single sql database. multiple taxa proteomes are stored within the sql database. using metatryp tools, the database can be queried to identify how many taxa share a specific peptide (or list of peptides), and it can also identify the total number of specific tryptic peptides shared across multiple organisms and for other phyloproteomic analyses. the former application has aided in the development of targeted metaproteomic biomarkers for assessing environmental changes in space or time [ ] [ ] [ ] [ ] . a useful result of the latter application was the observation that the percentage of shared peptides between distinct marine microbial taxa was low, often in the single digit percentages, implying that the design of biomarker targets for species or even subspecies level analyses was tractable if sufficient care was taken. in this manuscript, we describe version of the metatryp software (https://github.com/whoigit/metatryp- . ). we have added additional features to improve its usability and performance. a major improvement was the addition of new data categories for different sequencing assembly methods, specifically those associated with assembled metagenomic and metatranscriptomic data as well as single cell amplified genomes (sags). metatryp v now supports these three specific data categories: "genomes" for reference cultured isolates, "specialized assemblies" from sags and mags, and "meta-omic assemblies" from metagenomic and metatranscriptomic assemblies. this greatly expands the utility of metatryp since cultured genomes are often unavailable from natural environmental populations due to many organisms being difficult to culture with classical microbiological techniques [ ] , or being only recently identified taxa. as a result, the availability of single cell genomes amplified and sequenced from the ocean environment (sags), metagenome assembled genomes (mags), and assembled metagenomics and metatranscriptomic data can contribute greatly to the identification and interpretation of metaproteomic data. yet because these metagenomic and metatranscriptomic resources have varying levels of completeness and confidence in their functional and taxonomic assignments, maintaining them as separate categories of tryptic peptides within the database structure is particularly useful. in addition, metatryp v now supports the calculation of a least common ancestor (lca) among shared tryptic peptides. for comparison, the unipept web portal [ ] has some similar functionality in identifying shared tryptic peptides and interpreting the least common ancestor; however, unipept relies on the uniprot database which does not incorporate the wealth of environmental meta-omic sequencing available. also, the unipept portal does not support local curated database construction where users can evaluate unpublished sequencing resources like those of newly sequenced organismal genomes or novel environmental sequencing not yet available in the uniprot curated database whereas metatryp can be installed locally for use with custom curated databases. the addition of these new sequence assembly data categories enables better prediction of shared peptides through enhanced representation of environmental sequence variability. the metatryp marine web portal currently contains a total of , , unique peptides from , , submitted protein sequences combined across all three data categories. multiple improvements to the metatryp software architecture and additional features were added to v . in order to improve performance speed and support these larger data categories (especially, metagenomic and metatranscriptomic assemblies), the metatryp sql backend was converted from sqlite in metatryp v to a postgresql backend in metatryp v . additional software and postgresql implementations support the lca analysis. metatryp v uses the same tryptic digest rules applied to metatryp v , following trypsin-based digestion rules for proteins with peptides - amino acids in length [ ] . here we describe the technical improvements within metatryp v , then demonstrate how metagenomics resources allow increased understanding of least common ancestor interpretations of metaproteomic results. additionally, we provide an overview of the metatryp web portal for marine microorganisms and the rapid deployment of a coronavirus-specific metatryp web portal demonstrating the application of metatryp to various research fields. finally, a private api was added providing lca analysis functionality to the ocean protein portal [ ] , enabling metatryp to be inserted into other pipelines in the future. metatryp is built upon a relational database management system (rdms). version was built using a sqlite database. while this database management system was sufficient for single reference genomes, it was lacking in speed needed for expanded sequencing data categories. in order to increase the speed of database construction (ingestion of proteomes), database searching, and expanded functionality, we have upgraded the database management system to a postgresql backend which is an object-relational database. the postgres backend has provided improvements in speed, as well as enhanced flexibility in searching. the advent of environmental de novo sequencing and assembly has identified an entire realm of microorganisms previously unknown to the scientific world, as many environmental microorganisms are not readily isolated using classical microbiological techniques [ ] . metatryp v focused on the construction of a search database using reference organismal genomes, metatyrp v is capable of handling newer types of sequencing and assemblies thus opening the search space to a much greater range of organisms likely found within the environment of interest. within the field of metaproteomics, there has been great emphasis placed on the need for curated and appropriate search databases to be utilized for peptide-to-spectrum matching (psm) [ , ] ; this also holds true for the construction of a metatryp database for evaluation of shared tryptic peptides in an environmental sample. metatryp relies upon protein sequences predicted from genomic sequencing and does not currently take into account any post-translational modifications (ptms). in order to expand the environmentally-relevant search space, metatryp v now handles newer assemblies from sources like metagenome and metatranscriptome assemblies, metagenomeassembled-genomes (mags), and single cell amplified genomes (sags). the incorporation of these newer data categories, in addition to the traditional single organism reference genome, greatly expands the environmental variability (and therefore, potential for shared tryptic peptides) within an environmental sample. in order to manage the larger sequencing databases generated by incorporation of this environmental data, and thus the greater burden of a larger search space, improvements were made to the back-end search database (see section "database back-end upgrades"). the database schema ( figure ) for metatryp was expanded to not only include these different data categories, but to identify them as separate search spaces as the uncertainty of taxonomic identity among the sequencing categories is variable and should be taken into consideration during interpretation. the new data categories roughly mirror the original reference genome schema. however, additional tables are required to properly map meta-omic data as multiple taxa are contained within a single meta-omic assembly file. environmental sequencing data is also more likely to have more frequent occurrences of ambiguous bases in assemblies. these are base locations where it is uncertain what the correct amino acid should be, sometimes a result of low-quality base calling by the sequencing technology or due to a single location where there are multiple possibilities for the amino acid at that single location in the assembly that cannot be determined. metatryp v will recognize ambiguous bases, specifically the base symbol "x", which represents the presence of an unknown or ambiguous amino acid during the ingestion phase. as the specific amino acid represented by "x" is unknown, the exact tryptic peptide cannot be predicted. during ingestion, metatryp will identify proteins which contain an "x" and report to stdout the affected protein. if there is a tryptic peptide containing the "x", that peptide will not be included in the metatryp peptide table; however, all other tryptic peptides from that protein will be included in the peptide table. sequence homology is conserved among more closely related organisms. however, it is possible that tryptic peptides, especially shorter ones around amino acids, may occur by chance across multiple taxa without a direct shared ancestry. in order to identify the occurrence of shared tryptic peptides either through shared evolutionary history or through stochastic variance in sequence, we have added least common ancestor (lca) analysis to metatryp v . the lca analysis incorporates the phylogenetic lineage of the sequences imported into the metatryp databases ( figure ), then calculates the "least common ancestor" by finding the unifying phylogenetic point for all the organisms containing the tryptic peptide queried. in order for metatryp to identify the common point in the taxonomic lineage, it requires a consistent taxonomic lineage to be used across the database for each proteome submitted. for metatryp the shared phylogeny of the taxonomic groups is identified by pulling the taxonomic lineages from the national center for biotechnology information (ncbi) taxonomy database [ ] . for the creation of a user-generated metatryp database capable of lca analysis, the user can submit the ncbi taxon id number (taxid) for the input sequence files, and metatryp will pull the taxonomic lineage information for each organism using biopython [ ] and pandas [ ] libraries in python . this lineage information is then used to calculate the lca among the organisms with shared peptides via the postgresql longest common ancestor function, which metatryp uses to return the lca for each sequencing data category. a primary goal of releasing the metatryp software originally was to enable other users to create and curate customized databases for searching tryptic peptides, specifically with a focus on marine microbial communities. metatryp v expands on this goal through the creation of a web server and api which can be queried easily by users, without the need to install and run the software locally. the metatryp v site can be found at https://metatryp.whoi.edu/. this web server takes as input a peptide sequence, multiple peptides, or a full protein sequence submitted into a text box by a user which is then in silico digested into tryptic peptides. metatryp then searches for the occurrence of these peptides across three different marine-specific data categories: an organismal reference genome data category ("genomes") same as metatryp v (si table ), and new data categories for "specialized assemblies" (si table ) which currently contains , archaeal and bacterial mags [ , ] assembled by binning metagenomic sequences [ ] [ ] [ ] from the tara oceans sequencing project [ ] , and a metagenomic & metatranscriptomic assembly data category ("meta-omic assembly") [ , , ] (si table ) which currently contains , , predicted proteins. ideally, additional mags and sags will be added to the metatryp web portal database in an effort to broaden taxonomic coverage in the marine environment. the addition of eukaryotic sags [ ] would significantly extend the diversity of the current database. results from a metatryp query are then returned to the user in an interactive drop-down genomes & microbiomes (img) genome ids are also shown, where available, with links out to img as the predicted proteomes for all those in the "genome" category were collected from in the current version of this database [ ] . metatryp can also compare entire organismal proteomes to identify the frequency of shared peptides across taxa within a given sequencing data category. those more familiar with nucleic acid sequencing often incorrectly imagine that because translation to amino acid space results in loss of variable dna codon information, peptides will lack the ability to taxonomically resolve species or subspecies. this feature previously existed in metatryp v for generating peptide redundancy tables within the "genome" sequencing data category and was used to show the relatively low occurrence of shared peptides across disparate taxa in the open ocean microbiome [ ] . this feature can now be implemented within metatryp v on all three major data categories: "genome", "meta-omic assemblies", and "specialized assemblies". within the web portal, there is now a visualization tool for creating ordered heatmaps of shared peptide frequencies among taxa for the genome and meta-omic data categories. this visualization page, "peptide redundancy heatmaps", was built in python . using the jupyter environment [ ] , pandas [ ] , and seaborn [ ] . users can select what taxa they wish to compare within a given data category, and a heatmap is generated and displayed on the page below in a a specified data category where the percentage is calculated as the number of shared peptides between taxon a and taxon b divided by the total number of peptides in taxon a. given the varying levels of genome completeness for a specific taxon in the "specialized assembly" data category, this percentage should be viewed with more caution. due to the aggregate nature of meta-omic assemblies, where many taxa of highly variable coverage depth are present within each dataset, this heatmap visualization feature is not currently supported in the web portal for this data category. the capabilities of the metatryp software make it applicable to scientific domains outside of respiratory syndrome-related (mers) coronavirus strains [ ] , and strains associated with the common cold [ ] like human coronavirus strains nl [ ] , hku [ ] and e [ ] for a total of coronavirus taxa in the database. it also contains the human proteome, the african green monkey (chlorocebus aethiops sabaeus) proteome as it is the taxonomic source of the vero cell line commonly used in virus replication studies and plaque assays [ ] , common oral bacteria [ ] , six lactobacillus strains associated with the human microbiome [ ] , the most common influenza strains (influenza a: h n & h n ; influenza b) as well as other influenza strains, and common proteomic contaminants in the crapome [ ] . all taxa included in the coronavirus database and their associated ncbi taxonomy ids (si table ) are listed on the databases page in the web portal (https://metatrypcoronavirus.whoi.edu/database). in order to capture the variability of sequences, we pulled all the proteins (aside from those in the crapome) for each taxon from the ncbi identical protein groups (ipg) database using taxon sequence identifiers (si table ). the ipg database enables collection of a single non-redundant entry for each protein translation found from several sources at ncbi, including annotated coding regions in genbank and refseq, as well as records from swissprot and pdb [ ] . one sequence for each identical protein group was collected for ncbi taxa with >= identical protein groups, collecting proteins from the specified taxon and all its children from the ncbi taxonomy database. however, for the taxon "severe acute respiratory syndrome-related coronavirus" (ncbi txid: ), only protein groups from that specific txid level were recruited (using the flag "txid [organism:noexp]" in the query). this taxon should only contain sars-cov- related proteins; however, it may contain some non-sars-cov- sequences due to inconsistent nomenclature during the emergence of this relatively new pathogen. proteins were collected using biopython [ ] and the ncbi entrez [ ] e-utilities api [ ] . by using the identical protein groups, the database captures sequence variability while reducing redundancy in database construction. this non-redundant collection of protein sequences per taxon is essentially the collection of the known pan-proteomes for each taxon; it is the representation of the predicted proteome of a single organism's genome plus all the sequence variation captured from sampling a population of organisms within a taxonomic group. for example, the taxon homo sapiens pan-proteome from ipg has > , , proteins capturing sequence variability from the population of human sequences in the ncbi database, whereas an individual human genome contains < , protein coding genes [ ] . due to the varying sequencing efforts of among some taxa, the length of the proteomes in this database may vary. for example, the taxon severe acute respiratory syndrome coronavirus (sars-cov- , -ncov, covid- virus; taxid ) has , unique identical protein groups whereas the taxon bat coronavirus isolate ratg (taxid ), sars-cov- 's closest sequenced animal virus precursor [ ] , has unique identical protein groups as of this writing (may , ) even though the genomes of these viruses are roughly the same length. since the population of sars-cov- has been sequenced more frequently, more sequence variability has been captured in the ncbi databases. due to the collection of the pan-proteomes for the coronavirus metatryp database, the peptide redundancy heatmaps need to be viewed with caution, as taxa which have received a higher degree of sampling effort will have more peptides associated with them. therefore, it is important to take into consideration both combinations of pairwise taxa comparisons for heatmap calculations (both sides of the heatmap separated by the diagonal). for example, one should evaluate the percentage of shared peptides between sars-cov- and ratg where the total number of tryptic peptides for sars-cov- is in the denominator and also where the total number of tryptic peptides in the denominator is for ratg . when calculating peptide redundancy between organisms, metatryp reports this calculation as "individual percent". the percentage of shared peptides across taxa can also be calculated by the number of peptides shared between taxa with the combined total number of peptides for both taxa in the denominator, metatryp reports this as "union percent". however, this also needs to be interpreted with care as when comparing a taxon that may have only been sequenced once (few total peptides) with a broadly sampled taxon (many peptides due to environmental variability), the signal of the rarely sampled taxon may be reduced due to the large n of the heavily sequenced organism. in addition, viewing "union percents" when comparing an organisms with a small proteome vs an organism with a large proteome would also skew any signal of shared peptides, for example sars-cov- only encodes proteins/genome [ ] , whereas the human genome encodes ~ , proteins. even though the ncbi ipg database is not used in the marine metatryp web portal, this same effect may be observed when comparing organisms with highly uneven proteome sizes, say if marine phages are added to the database, or with taxa that have varying levels of genome sequencing completeness, such as with specialized assemblies like mags and sags. backend upgrades for metatryp v provide improved performance: the switch from a sqlite database backend in metatryp v to a postgresql database backend has resulted in significant improvements in performance and functionality of metatryp. in particular, this transition has resulted in improved computational times and facilitated the addition of lca analyses to the software package. to test clock times, a database was constructed based upon the reference genomes from marine microbial taxa (si table ) in both versions of metatryp. si table shows the benchmarks associated with the construction of these genome-only databases for comparison, with metatryp v taking only % of the time it took metatryp v to construct the same database. by converting to a postgresql backend, the computational time to query the example database with the example peptide "lshqaiaeaigstr" was reduced over , x, dropping from . seconds using metatryp v to . seconds with metatryp v . the cpu utilization for metatryp v was also lower than for v . it is noted that setting up a postgresql server on a local machine requires administrative permissions and is more complex than sqlite which is more user-friendly and requires fewer system dependencies. therefore, for users without local administrative permissions, metatryp v with its sqlite backend remains a good lighter weight option (albeit with slower performance and without the capacity to handle diverse data categories and lca analyses table ) and the base schema for all three data categories is included in the metatryp v code repository (https://github.com/whoigit/metatryp- . ). using the results of the peptides set as the default example search in the metatryp web portal (lshqaiaeaigstr, vnsvidaiaeaak, vaaeavlsmtk), we see the results for these three peptides have varying levels of taxonomic lcas, ranging from species-to phylum-levels of taxonomic specificity (figures & ) . for peptide vnsvidaiaeaak, the lca across all data categories is the genus prochlorococcus, indicating that this peptide appears unique to this genus in the marine microbial community and is therefore a potential biomarker for targeted metaproteomics that allows species level specificity. for peptide vaaeavlsmtk, the lca for all categories is the order synechococcales as this peptide is found within the genus prochlorococcus and its sister genus synechococcus. taxonomic assignment of the original sequences of predicted proteins for the different data categories ranges in uncertainty. from reference genomes from cultured isolates being the most certain, to metatranscriptomic & metagenomic assemblies providing the least certainty in taxonomic assignment of the source proteins. the "specialized assemblies" of sags and mags exist somewhere in between on this taxonomic assignment uncertainty spectrum. peptide lshqaiaeaigstr (figure ) demonstrates this level of uncertainty within the "meta-omic assembly" category as a source protein for this peptide in the gos/omz (jcvi) metagenome cannot be identified with below the phylum level of cyanobacteria ( figure ) and in the "specialized assembly" category containing thousands of mags, this peptide is found in cyanobacterial mags and one verrumicrobial mag (verrucomicrobiales_bacterium_strain_np ), resulting in a lca of "bacteria". notably, this peptide is from the global nitrogen transcriptional regulation protein (ntca) which is highly conserved across the cyanobacteria [ ] . while this peptide has been previously identified as a potential biomarker for cyanobacteria [ ] , the addition of the "specialized assembly" data category identified the possible presence of this peptide in another phyla warranting further investigation. interestingly, upon further investigation, the protein in mag verrucomicrobiales_bacterium_strain_np , identified from metagenomes in the red sea [ ] , is > % identical to an ntca in the cyanobacterium genus synechococcus (ncbi accession wp_ . ). this verrumicrobial ntca may occur within this genome as a result of horizontal gene transfer or as an artifact of the mag binning process. either way, it indicates that peptide lshqaiaeaigstr should be used with caution as a cyanobacterial biomarker in environments with abundant verrumicrobia. however, this is an unlikely scenario as cyanobacteria tend to be far more abundant in marine environments than verrumicrobia. query results from a full sequence ntca from prochlorococcus med (ncbi genbank accession cae . ) show that other peptides, such as "lvsflmvlcr", may be more appropriate if targeting prochlorococcus only (si figure ). by separating sequence types into different categories, metatryp allows the user to balance the varying levels in confidence of taxonomic attribution, where reference genomes from cultured isolates are the best in taxonomic quality but more incomplete in environmental coverage, and vice versa for environmental sequences. the addition of , mags to metatryp has provided further insight into the prevalence of shared peptides across taxonomic groups in marine microbial communities. an analysis with metatryp v using a database of single reference genomes from common pelagic marine microorganisms demonstrated very little overlap in shared peptides across different taxonomic groups [ ] . expanding this analysis to the , mags shows a similar pattern of a very low occurrence of shared tryptic peptides across disparate taxa. figure shows the individual percentages of shared peptides across a random selection of mags. in general, taxa share < % of tryptic peptides, with a few clusters of more closely related organisms sharing more tryptic peptides --for example, gammaproteobacterial and euryarchaeotal clusters highlighted with red outlines --where the taxa share between - % or - % of tryptic peptides in each cluster, respectively. a similar pattern is shown when the cross-wise comparison of taxa is expanded to mags (si figures & ) . these results demonstrate that there should be sufficient resolution to discern between taxa using tryptic peptides identified by metaproteomic analyses, especially when coupled to lca analysis tools like metatryp to confirm peptide taxonomic origin. analysis of the coronavirus-focused metatryp instance is similar to the observations of marinefocused metatryp where there is a rather low frequency of shared peptides across disparate taxa with a higher frequency of shared peptides across more closely related taxa ( figure ; si figures & ) . within the broader group of the taxa associated with severe acute respiratory syndrome (si table ; infections [ , ] , as a combination of the coronavirus-specific database with other environmental databases (like marine microbial metatryp) may provide insight into potential tryptic peptide biomarkers in sewage effluent. this manuscript announces the release of version of the metatryp software package for assessing shared tryptic peptides in complex communities. coronavirus has the most shared tryptic peptides with its closest bat precursor virus, has some shared peptides with sars-cov- , and is very different from the "common flu". metatryp is a flexible software package to assess taxonomic occurrence of shared peptides applicable to proteomics studies of complex systems valuable for the identification of biomarkers and phyloproteomic analysis of complex communities. figures: figure . core elements of the metatryp v database schema. an entity relationship diagram depicting the core tables for the three sequencing categories (orange tables: "genome", green tables: "specialized assembly", and blue tables: "meta-omic assembly" data categories). the purple tables are shared tables among all three data categories containing information about the tryptic digestion rules (protease and digest tables) as well as the amalgamation of all unique tryptic peptide sequences found across all three data categories (peptide). the lines connecting the tables represent links between the data tables. the three data categories are stacked where tables represent similar information for each category. the metagenome data category requires two additional data tables as there are multiple taxa stored within a single meta-omic assembly sequencing file which requires an additional metagenome_annotations table for parsing; the blue connecting lines represent meta-omic specific data linkages. the lca results for these three separate peptides indicate the varying degrees of taxonomic uniqueness among the peptides in the "genome" and "meta-omic assembly" data categories. in these two data categories, lshqaiaeaigstr is unique to the phylum cyanobacteria. vnsvidaiaeaak is unique to the genus prochlorococcus. vaaeavlsmtk is unique to the order synechococcales. all of these peptides show potential as biomarkers at these varying taxonomic levels according to evaluation by the "genome" and "meta-omic assembly" databases. in general, there is a very low occurrence of shared tryptic peptides (< %) across disparate taxa. higher frequencies of shared tryptic peptides are shown among more closely related taxonomic groups. severe acute respiratory syndrome-related coronaviruses form a cluster in the top left corner. mers-related coronavirus shares < % of tryptic peptides with all taxa depicted here. the "common cold" strains of coronavirus (hku , e, and nl ) form a separate cluster in the bottom right corner. these coronavirus clusters are distinct and very different from the influenza a cluster in the middles, as the severe acute respiratory syndrome-related viruses share < % of shared tryptic peptides with influenza a and b. homo sapiens and chlorcebus sabaeus form a distinct group, sharing more tryptic peptides with each other than with any other taxonomic groups. needles in the blue sea: subspecies specificity in targeted protein biomarker analyses within the vast oceanic microbial metaproteome methionine synthase interreplacement in diatom cultures and communities: implications for the persistence of b use by eukaryotic phytoplankton multiple nutrient stresses at intersecting pacific ocean biomes detected by protein biomarkers abundant nitrite-oxidizing metalloenzymes in the mesopelagic zone of the tropical pacific ocean metagenomics: application of genomics to uncultured microorganisms. microbiology and molecular biology reviews : mmbr unipept . : functional analysis of metaproteome data development of an ocean protein portal for interactive discovery and education critical decisions in metaproteomics: achieving high confidence protein annotations in a sea of unknowns progress and challenges in ocean metaproteomics and proposed best practices for data sharing the ncbi taxonomy database biopython: freely available python tools for computational molecular biology and bioinformatics proceedings of the th python in science conference nitrogen-fixing populations of planctomycetes and proteobacteria are abundant in surface ocean metagenomes the reconstruction of , draft metagenome-assembled genomes from the global oceans binning metagenomic contigs by coverage and composition anvi'o: an advanced analysis and visualization platform for 'omics data binsanity: unsupervised clustering of environmental microbial assemblies using coverage and affinity propagation ocean plankton. structure and function of the global ocean microbiome functional tradeoffs underpin salinity-driven divergence in microbial community composition single cell genomics yields a wide diversity of small planktonic protists across major ocean ecosystems img: the integrated microbial genomes database and comparative analysis system a new coronavirus associated with human respiratory disease in china mechanisms and enzymes involved in sars coronavirus genome expression euro surveillance : bulletin europeen sur les maladies transmissibles = european communicable disease bulletin human coronavirus circulation in the united states identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus the genome landscape of the african green monkey kidney-derived vero cell line. dna research : an international journal for rapid publication of reports on genes and genomes deep metaproteomic analysis of human salivary supernatant anti-infective activities of lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents the crapome: a contaminant repository for affinity purification-mass spectrometry data entrez gene: gene-centered information at ncbi. nucleic acids research the e-utilities in-depth: parameters, syntax and more. entrez programming utilities help multiple evidence strands suggest that there may be as few as , human protein-coding genes a pneumonia outbreak associated with a new coronavirus of probable bat origin nitrogen control in cyanobacteria cryo-em structure of the -ncov spike in the prefusion conformation how sewage could reveal true scale of coronavirus outbreak the potential of wastewater-based epidemiology as surveillance and early warning of infectious disease outbreaks. current opinion in environmental science & health metaproteomic analysis using the galaxy framework we would like to thank a. murat eren, tom delmont, ben tully, elaina graham, and john heidelberg for graciously providing mag sequences and additional taxonomic information facilitating incorporation into the metatryp database. this work was made possible by grants from the national science the authors declare no conflicts of interest in the publication of this manuscript. key: cord- -un ysc v authors: nan title: poster presentations date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: un ysc v nan the boron neutron capture therapy (bnct) based on the interaction between b isotope and thermal neutron has been highly noted in recent years as one of promising techniques for treatment of cancers. p-( b)borono-l-phenylalanine (l- bpa), in which boron atom is enriched with b isotope, is now using clinically as an effi cient b carrier for treatment of patients in particular with malignant brain tumor and melanoma ( ) . in order to develop a practical method for the synthesis of l- bpa, we have recently examined the enantioselective method utilizing the negishi reaction based on the coupling of -( b)boronoiodobenzene derivative with -iodo-l-alanine derivative ( ) . from the standpoint of diagnosis of cancers, the magnetic resonance imaging (mri) is noted as one of common techniques. in particular, mri based on the measurement of f atom is becoming a remarkable one. to create practical materials utilizing as not only the b carrier but also mri probe, we had already synthesized the compounds containing both b and f atoms in a single molecule such as - [ -( b)borono- , -difl uorophenyl]-dl-alanine [dl- bpa( , f )] ., - [ -( l-proline is conformationally unique among coded amino acids in that its torsion angle is blocked (- ± ) by its characteristic fi vemembered pyrrolidine ring structure and the preceding torsion angle (tertiary amide) can undergo cis ( ) <-> trans ( ) isomerization much easier than the secondary amides of the usual peptide bonds. in addition, its torsion angle is commonly found either in the right-handed -/helical region (- ÷ - , or cis' conformation) or in the left-handed, semiextended, region [- ± , or trans', or poly-(l-pro) n conformation]. methylation at the c -position of a pro residue was suggested to block the preceding tertiary amide ( ) torsion angle of the resulting ( me)pro to the trans disposition and to restrict the , surface to the single region where the / -helices are found. we have synthesized a large set of n -blocked, ( me)pro-containing, dipeptide n'alkylamides having the general formulas p-d-( me)pro-xxx-nhipr and p-xxx-d-( me)pro-nhipr, where p is ac or boc and xxx is d-ala, l-ala, aib, gly, d-( me)pro, or l-( me)pro. the results of the present x-ray diffraction analysis clearly show that the region of the conformational map overwhelmingly preferred by ( me)pro is indeed that typical of / -helices, but the semi-extended, type-ii poly(pro) n helical region can exceptionally be explored by this extremely sterically demanding c -tetrasubstituted -amino acid. in addition, the known high propensity for -turn formation of the pro residue is even enhanced in peptides based on its c -methylated derivative. to complete the picture of the preferred conformation of ( me)pro, the synthesis and crystal-state investigation of a series of terminallyprotected homo-peptides from d-( me)pro are currently in progress in our laboratory the interest in guanidine-containing compounds is due to their important role in biological recognition processes. five-member cyclic n-amidinoamino acids represent hybrid structures, combining both proline rigidity and high positive charge of arginine guanidine group. structurally similar n-amidino-proline, n-amidino-pyroglutamic acid and cyclocreatine ( imino- -imidazolidine acetic acid) were chosen as objects of our study. the problem of their incorporation into peptide structure requires use of substituted derivatives or effective guanidylation technique. recently we have presented the synthesis of mts-protected n-amidinoproline. nevertheless, its application in n-termini modifi cation of peptides shows slow condensation rate in classical as well as solid phase synthesis for short peptides ( - residues) or completely fails in case of longer ones ( residues). on the other hand, polymer supported guanidylation of proline by bis-boc-protected carboxamidine- hbenzotriazole seems to be preferred route to n-amidino-proline containing peptides. three methods of n-amidino-pyroglutamic acid synthesis have been investigated and model dipeptide has been acquired on wang polymer by the cyclization of guanidine-glutamic acid. for selective boc-deprotection on wang resin the literature technique has been utilized. however, in this case esi-ms and nmr analyses evidence for side-reaction of triethylamine alkylation by polymer linker fragment. hplc analysis shows n-amidino-pyroglutamyl-phenylalanine stability at acidic and physiological ph but fast ring opening in water solution at ph . for cyclocreatine application in peptide synthesis we suggest the use of its p-toluenesulfonate having good solubility in dmf and showing effective coupling in cl-hobt/dic condensation. the examples of namidino-amino acids practical application in peptide synthesis will be presented. tryptophan is often a key pharmacophore which determines the affi nity of peptide ligands for their receptors. cyclic analogues of tryptophan which introduce local constraints and reduce the fl exibility of the indol moiety are very valuable tools to probe the bioactive conformation of the peptide ligands. one of the possibilities to freeze indol moiety of tryptophan is the synthesis of the additional -member ring by the formation of , , , -tetrahydro--carbolines. we report the synthesis of , -disubstituted , , , -tetrahydro--carbolines via the pictet-spengler reaction. methyl ester of tryptophan or dipeptides with n-terminal trp (trp-ala-ome, trp-leu-ome) were used as arylethylamine substrates and -amino aldehydes derived from l and d-amino acids were used as carbonyl components. we determined that there were no differences of the stereoselectivity of the pictet-spengler reactions in the case of trp-ome or trp-dipeptides. we also investigated the conformation of the newly created -membered ring in cis and trans diastereomers. the roesy spectra were used to assign the more stable conformation for each isomer. the conformations of cis isomers derived from tryptophan and dipeptides were the same and substituents on c- and c- were in both cases equatorial. the conformation of trans isomers were depended on the carboxyl part of trp. for methyl ester of tryptophan the ester group was axial, whereas for dipeptides we observed the opposite conformation with equatorial substituent on c- . our results show that the pictet-spengler reaction can be successfully performed for amino acids and peptides and the conformation of the newly created -membered ring depends on the substrates and the size of c- and c- substituents. helical-screw handedness of peptides composed of diastereoisomeric cyclic amino acids nagano helical-screw handedness in proteins is believed to result from thecarbon chiral center of l--amino acids. recently we have reported that the helical-screw sense of oligopeptides can be controlled without a chiral center on the peptide-backbone but by chiral centers at the side chain. that is to say, we designed and synthesized an optically active cyclic , -disubstituted amino acid (s,s)-ac( )c(dom), in which the -carbon atom is not a chiral center but chiral centers exist at the side chain. conformational analysis of the (s,s)-ac( )c(dom) peptides revealed that the hexapetide formed left-handed -helices both in solution and in the solid state, and the octapeptide assumed a left-handed -helix. herein we designed new two diastereoisomeric cyclic , -disubstituted amino acids; ( s, s)-and ( r, s)- -amino- -(methoxy)cyclopentanecarboxylic acid (ac( )c(om)) having chiral centers both at the -carbon atom and at the side chain. the amino acids ( s, s)-and ( r, s)-ac( )c(om) were synthesized starting from l-(-)-malic acid. that is to say, at fi rst, the malic acid was converted to diiodide compound, and bisalkylation of dimethyl malonate with the diiodide gave a cyclic diester. monohydrolysis of the diester, followed by curtius rearrangement produced separable mixtures of ( s, s)-and ( r, s)-ac( )c(om). we prepared both diastereoisomeric ( s, s)-and ( r, s)-homooligomers by solution-phase methods, respectively, and studied their preferred secondary structures using h nmr, ft-ir, cd and x-ray crystallographic analysis. zobel, ansgar; gaus, katharina; wollschläger, katrin; nieß, anke; juodaityte, jovita; sewald, norbert organic and bioorganic chemistry, bielefeld university, germany novel highly active and specifi c dna binding molecules have a considerable potential particularly with regard to applications in medical science. the major and minor grooves of the dna serve as recognition areas. it is of high interest for chemical biology to control dna-binding abilities of synthetic molecules. a possible modulator is light in combination with photoswitchable molecules. triostin a is a natural bisintercalator which belongs to the quinoxaline antibiotics originally isolated from streptomyces s- - . it consists of a bicyclic depsipeptide with n-methylated amino acids and a cystine bridge. its heteroaromatic quinoxaline moieties are able to intercalate gc-specifi cally into the dna inducing a change in conformation and therefore inhibit the transcription by blocking spezifi c enzymes. tandem is the n-unmethylated derivative of triostin a which binds at selectively to the dna due to the change in the hydrogen bonding pattern. the exchange of the cystine bridge of tandem with substituted azobenzene units leads to photoswitchable triostin a analogs. the synthesis of the depsipeptidic basic structure which contains the quinoxaline moieties is introduced as well as the coupling with azobenzene amino acids. the double cyclization step is accomplished under pseudo high dilution conditions. in order to differ between cisand trans-confi gurations of the molecule, nmr-and ir-spectroscopy was carried out as well as the photoswitchability of different analogues was tested by irradiation and rp-hplc analysis. for synthetic peptide vaccine prototype development: (pc) , which has an heteroatom in its structure and their bioconjugates obtained by microwave and carbodiimide methods were explained. for pc obtaining, the synthesis of the monomer, azabicyclo[ . . ]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [ ] [ ] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as uv, atr ft-ir, sec with four detectors and zeta sizer. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. atr ft-ir spectra of pc was recorded and molecular weights, polidispersity values of polymers, mark-houwink constants and molecular diameters of the polymers were measured with size-exclusion chromatography with four-detectors (light-scattering, refractive index, viscosity and uv). with zeta sizer, size-analysis of polymer chains were done and their zeta potentials were measured. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. after synthesis and modifi cation of pc, its conjugates with antigenic peptides like avian infl uenza hemagglutinin (ha - ) ypydvpdya and rgdsggc cell receptor peptide were obtained. their characterization was also performed. japan; in alzheimer fs disease research, sparing water-solubility and uncontrolled self-assembly of amyloid peptide (a ) - are signifi cant obstacles to establish an experimental system that clarifi es a pathological mechanism of a - . to solve these problems, we herein disclose water-soluble "click peptides" based on an o-acyl isopeptide method. these peptides contain an o-acyl instead of n-acyl residue at the gly -ser of a - , and are converted to a - via an o-n intramolecular acyl migration triggered by phchange (ph-click) or photo-irradiation (photo-click). these peptides had remarkably higher water-solubility than a - . in addition, these peptides clearly adopted monomer state with a random coil structure, which were verifi ed by various physicochemical assays. importantly, we could establish an in situ system predominantly comprised of monomer a - as a result that the monomer click peptide was converted to a - quickly and quantitatively in accordance with ph-change. both selfassembly and conformational change of the produced a - in situ were observed with time. similarly, the photo-triggered click peptide afforded a - by uv-irradiation. because the in situ production of intact a - from the click peptides could overcome the handling problems of a - , this strategy would provide a reliable experimental system for investigating a pathological function of a - in alzheimer fs disease. the antioxidant effect of introducing phenylpropenoyl moiety in either in n-terminal group of analgesic oligopeptides or in c-terminal end of opioide active amino acid, modifi cated with polyamines have been evaluated. the series of hydroxycinnamoyl peptide and amino acid amides were synthesized by standard method used in peptide chemistry. obesity is a major public health problem associated with morbidity and mortality and continues to increase worldwide. the gastrointestinal tract and the pancreas release hormones regulating appetite and body weight. obestatin is a recently described amino-acids peptide derived from preproghrelin. it was identifi ed by bioinformatic prediction ( ) . obestatin decreases food intake and body weight gain, decelerates gastric emptying, promotes sleep in rat, inhibits water drinking. it has been described that obestatin effects on feeding behavior and an u-shaped dose-response relationship was found: low doses ( . - nmol/kg) and high doses ( - nmol/kg) were ineffective. the amino-acid carboxy-amidated human obestatin nalapropheaspvalglyilelysleuserglyvalglntyrglnglnhissergln alaleunh and corresponding rat obestatin hpheasnalapropheasp valglyilelysleuserglyalaglntyrglnglnhisglyargalaleunh were synthesized by different schemes. synthesis was developed on the basis of solid phase synthesis of protected peptide fragments followed by their assembly into the fi nal product. fragments - , - , - , - , - were built on the acid-labile -chlorotrityl chloride resin using the orthogonal fmoc/tbu strategy. fragment - (hcl•hargalaleunh ) was obtained in solution. the assembly of the full-length peptide was carried out by fragments coupling in solution or using solid phase method. the free peptide was obtained by treating the protected peptide with mixture of tfa: h o:tis ( : . : . ). the peptide was purifi ed with hplc and isolated by lyophilization with %+ purity according to analytical reversed-phase hplc. the mass of molecular ion determined by maldi-tof spectrometry was in fi ne agreement with calculated value. the inverstigation of biological actions of obestatins is in progress. straightforward synthesis of enantiopure tfm-amino acids from chiral cf brigaud, thierry; chaume, grégory; huguenot, florent; caupène, caroline university of cergy-pontoise, france trifl uoromethylated amino acids (tfm aas) are current synthetic targets due to their unique properties and their synthesis in enantiopure form remains a challenge. we will report that chiral -trifl uoromethyl- , oxazolidines (fox) are highly versatile synthons for the stereoselective synthesis of various functionalized -trifl uoromethylamino compounds such as -amino nitriles , -and -amino acids, diamines and amino alcohols. , moreover, trifl uoropyruvate-based oxazolidines proved to be very valuable building blocks for the stereoselective synthesis of both enantiomers of -trifl uoromethyl proline and -tfm-pyroglutamic acid, in enantiopure form. moreover, the synthesis of the (s)--tfm--allylglycine and the novel (s)--tfm norvaline were achieved in a few steps from this starting material. we will also report a recent straightforward synthetic route to tfm-dihydroxyprolines in enantiopure form from trifl uoropyruvate-based chiral oxazolidines. chitinases catalyse the hydrolysis of chitin, the natural homopolymer of ( , )-linked n-acetyl-d-glucosamine. chitin is a key structural component of the cell walls, exoskeletons, and eggshells of pathogenic fungi, insects, and nematodes, respectively, which all rely on the ability to hydrolyse chitin at specifi c points in their life cycles. chitinase inhibitors are now attracting considerable interest as novel fungicides and insecticides, as well as chemical tools to study human diseases as diverse as asthma and malaria. in this context, the cyclic pentapeptide natural products, argifi n and argadin, are two exciting inhibitors which pose some interesting synthetic challenges. the argifi n structure includes two sensitive -linked asp residues, as well as an unusual carbamoylated arg side chain, while argadin contains a unique aspsemialdehyde residue, that is cyclised to the peptide backbone to generate a potentially labile hemiaminal. we will describe improved routes to both compounds that allow us to avoid signifi cant side reactions such as aspartimide formation and homoserine-mediated backbone cleavage that are observed in our previously reported syntheses. [ , ] this is achieved by carrying out the assembly and cyclisation of both peptides, including key side chain derivatisation steps, entirely on solid phase. the application of the strategies devised to the automated synthesis of argifi n and argadin and related natural products will be described. during the last two decades combinatorial chemistry has become an important tool for the quick synthesis of large numbers of small molecules used, for example, in the generation of the new lead structures for medicinal applications. additionally, it allows rapid access to diverse chemical libraries with novel structures and properties. small molecules libraries are generally prepared on solid support simplifying tedious purifi cation steps of the intermediates and facilitating the entire synthesis. piperazines and keto-piperazines are amongst the important backbones in today's drug discovery. the piperazine framework has been defi ned in medicinal chemistry as a "privileged scaffold". it is a molecular backbone with versatile affi nity properties representing a frequently-occurring binding motif, and providing potent and selective ligands for a wide range of biological targets. the high number of positive hits revealed in biological screens with the piperazine scaffold urged chemists to develop plenty of different synthetic methods that allow fast and effi cient building of these heterocyclic systems on solid support as well as homogenous chemistry. however, the majority of these methodologies is not stereospecifi c and the complex mixtures of the stereoisomers are generated. in order to preserve important chiral centers in potential hits we propose to use pipearzine backbone, initially prepared in optically pure form, bearing various tethers with orthogonally protected groups applicable via solid phase organic chemistry (spoc). we will describe a novel synthesis of keto and diketopiperazine building blocks. these chiral building blocks are applied in "around-the-scaffold" modifi cation strategy by spoc, introducing valuable physico-chemical properties in independent diversity points. solid-phase strategies for constraining peptide structures can serve for elucidating the relationships between conformation and activity. for example, the systematic substitution of natural amino acids by their aza-amino acid counterparts has been accomplished using a fmoc strategy featuring couplings with aza-amino acid chlorides to provide insight into the biologically active conformers of the melanocortin receptor agonist ac-his-d-phe-arg-trp-nh as well as the calcitonin gene-related peptide antagonist [d , p , f ]cgrp - ( ) ( ) ( ) . employing cyclic sulfamidates, we have now developed fmoc strategies for the effective solid-phase introduction of alpha-and beta-amino gamma-lactam constraints into peptides. since their pioneering use in a somatostatin analog with about fold greater activity ( ), lactam restraints have been used to study a variety of relevant targets ( , ) . our strategies now provide effective means for performing lactam scans of biologically active peptides as demonstrated using the growth hormone secretagogue ghrp- , and an allosteric modulator peptide antagonist of the il- receptor. our presentation will focus on recent developments in the solid-phase chemistry for synthesizing such constrained peptide analogs and the relationships between peptide conformation and biology, elucidated by these novel methods. references: barcelona science park, university of barcelona, -barcelona, spain; linaclotide is a -residue peptide currently undergoing phase ii clinical trials for the treatment of gastrointestinal diseases such as chronic constipation (cc). linaclotide, which can be administered orally, is an agonist of the guanylate cyclase type-c receptor found in the intestine. from a structural point of view, this small peptide presents a constrained structure with the presence of three disulfi de bridges between cys -cys , cys -cys , and cys -cys . h-cys( )-cys( )-glu-tyr-cys( )-cys( )-asn-pro-ala-cys( )-thr-gly-cys( )-tyr-oh in order to reach the large amounts required for a marketed peptide, an effi cient synthesis needs to be attained. to optimize the synthesis, its fundamental limitations need to be determined and addressed. in the case of linaclotide, the key points are related to the numerous cys (some of them consecutive) present in the peptide, for two reasons: the potential risk of racemization upon assembling the linear chain, and the misfolding of the three disulfi de bridges. for that, the concourse of different protecting groups and folding conditions as well as the analysis of the disulfi de bridges in the fi nal folded peptide has been studied and will be discussed in this presentation. balalaie, saeed ; arabanian, armin ; mohammadnejad, mahdieh ; gross, juergen h. university, iran (islamic rep.); university, germany gnrh analogues have been used for the treatment of steroid-dependent tumors, such as prostate and breast cancers. the development of more potent gnrh analogues depended largely on the important made in the science of peptide chemistry. ugi- mcr reaction is known for the synthesis of amide bond. we wish to report herein an effi cient method for the synthesis of some gnrh analogues based on ugi reaction using fourcomponent reaction of n and c-terminus peptides, aromatic aldehydes and isocyanides. this ugi- mcr could describe to build up novel gnrh analogues deriving from triptorelin and gonadorelin. all of the products were purifi ed using preparative hplc and the structures were assigned according to maldi mass spectrometry data. peptide synthesis is based in a proper combination of protecting groups and in the right choice of the right coupling method. nowadays, almost all peptide bond formed are carried out in the presence of hydroxybenzotriazole (hobt) or its derivatives (hoat, cl-hobt). thus, hobt derivatives are used in combination with a carbodiimide or another coupling agent or built into a standalone reagent such as immonium ( thus, a replacement of hobt should be found for preparation of peptides for research purposes and, more important, for the production of peptide based apis. herein, several alternatives to hobt will be discussed taking into account the explosivity properties. furthermore, a new family of immonium salts, which incorporates an proton acceptor in its carbocation skeleton. the novel proton acceptor coupling reagent has shown superiority to the described previously. an oxygen in the carbocation moiety confers to the reagent more solubility, enhances coupling yields and decreases racemization, allowing the use of just one equivalent of base. examples on the use of the non-explosive replacement for hobt together with carbodiimides or built into phosphonium and immonium salts, with the proton acceptor, will be discussed. thiocarbamate-linked peptides by chemoselective peptide ligation using phenylthiocarbamate chemistry peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide-based macromolecules. native chemical ligation (ncl) ( ), staudinger ligation ( ) lead to the formation of a native peptide bond at the ligation site. other methods result in the formation of unnatural covalent bonds such as oxime ( ), thioester ( ) linkages. these methods are of great interest when native peptide bonds are not absolutely required. in this context, we examined the potential use of the phenylthiocarbamate group in ligation chemistry. the phenylthiocarbonyl group can be easily introduced into peptides on alpha or epsilon amino group using phenylthiochloroformate and standard solid-phase method ( ) . it reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. s,n-shift of the alkylaminocarbonyl group from the cys side chain to the alpha-amino group did not occur. the method was used for linking two peptides chain through their n-termini, for the synthesis of a cyclic peptide or for the synthesis of di-or tetravalent multiple antigenic peptides. synthetic peptide derivatives which contain secondary amide moiety at c-terminus are inhibitors of serine and cysteine proteinases and thus could be effi ciently used as ligands in affi nity chromatography in order to isolate these enzymes. there is a number of chemical syntheses of amidous peptide derivatives in the literature, but the reaction yields are usually low and reaction conditions and purifi cation procedures are too complicated. it is also known that the application of enzymatic catalysis allows to obtain high reaction yields with simultaneous simplifi cation of synthetic and purifi cation procedures, and at the same time preserves the optical purity of target compound. subtilisin sorbed on silochrome could be effi ciently used as catalyst of acylation of secondary amides by the esters of acylpeptides. subtilisin's wide substrate specifi city allows to carry out the syntheses with peptides which contain hydrophilic, hydrophobic, dicarbonic-and diamino-acids at c-terminus. piperidine, morpholine, indole, diethylamine and other secondary amines are used in the syntheses as the amino-components. the reaction yield equals about - % in dependence of the nature of c-terminal amino acid of acylating peptide, pka of amine and the hydrophobicity of the fi nal compound. the distribution of the reaction product in liquid and solid reaction phases is investigated, the optimal reaction conditions for enzymatic acylation of secondary amines are established. all the compounds obtained are characterized by its nmr and mass spectra. the inhibition constants for some proteolytic enzymes with the compounds of research are defi ned. unnatural amino acids are becoming increasingly important substrates in modern drug design, synthesis and discovery research. in particular, arylhistidines naturally occur in the active site of the heme-copper oxidases as well as in cytotoxic and antifungal marine peptides ( ) . a method of choice for the preparation of unsymmetrical biaryl systems is the suzuki-miyaura cross-coupling of an aryl halide with an arylboronic acid. it has been shown that microwaves signifi cantly enhance this reaction leading to higher overall yields and purities as well as shorter reaction times. although a great variety of biarylic compounds have been prepared following this approach, so far, it has not been applied to the arylation of the histidine imidazole ring. initially, we studied a methodology for the synthesis of -arylhistidines via a microwave-assisted suzuki-miyaura cross-coupling reaction in solution ( ) . taking into account the advantages of the synthesis on solid support, such as the avoidance of tedious work-up which are particularly valuable for palladium-catalyzed reactions, we studied the application of the above methodology on solid-phase. here, we report the suzuki-miyaura reaction between a -bromohistidine and an arylboronic acid on solid support. the reaction conditions were optimized by varying several parameters such as the solvent, the reagent concentrations and the reaction time. the optimized conditions were subsequently applied to the synthesis of peptides containing a -arylhistidine residue. this work constitutes the fi rst suzuki-miyaura coupling involving the imidazole ring of a histidine on solid support. microwave-assisted attachment of fmoc-amino acids to resins via triazine "superactive esters". the substitution level of the new functionalized resin was calculated following a standard protocol based on the spectroscopic measurement of the soluble chromophore piperidine-dibenzofulvene. coupling reactions to the resin proceeds at room temperature in hours. moreover we carried out the time-consuming attachment of fmoc-amino acids to the resin by an automatic monomode microwave (mw) instrument (liberty, cem), in fact microwave is proposed as a valid alternative to enhance effi ciency of coupling reactions and it has been widely applied to spps. novel peptide-hetorocycle conjugates: derivatives of -(benzimidazol- -yl)alanine ( ) . considering the biological activity and complexing abilities of benzimidazoles we developed a direct solid-phase synthesis of benzimidazole-peptide conjugates, expecting these new compounds to express novel biological properties ( ) . the peptide-heterocycle conjugates are obtained by on-resin reaction between aldehydes and peptides containing a specially designed -( , -diaminophenyl)alanine residue ( ) . the stoichiometric amount of aldehyde leads to '-substituted -( h-benzimidazol- -yl)alaninecontaining peptides, the increase in aldehyde content results in `, `disubstituted derivatives. the reaction with dialdehydes gives novel amino acid residues containing tricyclic systems, in the case of ophthalic aldehyde -the pyrido-[ , -a] benzimidazole. the compatibility of our method with the fmoc solid phase peptide synthesis protocols was proven by the synthesis of analogues of immunosuppressory fragments of ubiquitin and hla-dq [ , ] . the biological activity of the modifi ed oligopeptides was compared to that of original fragments . as well as to similar quinoxaline-peptide hybrids. the collision-induced dissociation of substituted benzimidazole-peptide conjugates leads to characteristic fragmentation ions, which may be used as a diagnostic tool in the fragmentation of the benzimidazole-peptide hybrids. cardona, valérie; oswald, benoit genzyme pharmaceuticals, switzerland dehydroamino acids are an important class of compounds due to their presence in many biologically active natural products including the antrimycins, tentoxin, phomopsin a, or the phosphatase inhibitors like microcystin and nodularin. in the last decade an increasing interest in -branched dehydroamino acids has been developed based on their importance as commodity chemicals and value as tools in structure relationship studies. the incorporation into a peptide of such unsaturated amino acids introduces an element of conformational rigidity, as well as changes in reactivity, allowing for the development of high affi nity ligands for receptors. a variety of methods exist for the synthesis of dehydroamino acids. some of these approaches rely on thermodynamic control to dictate the alkene geometry. if there is no strong thermodynamic preference, or if the desired product is not the thermodynamically favoured isomer, the existing synthetic methods are often ineffective. we describe here an effi cient stereoselective method for the synthesis of , -branched dehydroamino acids (iii) from -aryldehydroamino acids (i). the -aryldehydroamino acids (i) were prepared from commercially available z/boc-phosphonoglycine trimethylester and aldehydes using the schmidt protocol. the transformation of (i) into their -bromodehydroamino acid derivatives (ii) is performed by a hoerrner reaction in a very good yield giving the trans derivatives. suzuki cross-coupling on (ii) with a boronic derivative generated the high value building blocks (iii). a variety of side chains can be incorporated using this type of reaction. the stereochemistry of these compounds has been determined using noe enhancement experiments. we report here a scalable and high yielding synthesis of stereospecifi c , -aryldehydroamino acid derivatives. versatile methods for synthesizing acyl-tetramic acids peptide analogs. davidov, gali; mozes, tamar; khandadash, raz; byk, gerardo bar ilan university, israel acyl-tetramic acid derivatives have been identifi ed as potential antiviral and antibacterial compounds. in this work we have improved their synthesis starting from tetramic acid lactams using peptide coupling reagents such as bop under microwave heating for generating the carbon-carbon bond of the acyl-tetramate. using this procedure we have synthesized a number of new tetramic acid derivatives in solution and generated a series of acyl-tetramic acid building blocks that were introduced into peptides using conventional spps methods. additionally, we present here a new solid phase microwave assisted synthesis of acyltetramates on pre-synthesized peptides and amino acids. antibacterial and antiviral activity of the products will be presented. peptide sequence and include all side-chain groups. in practice this is diffi cult to achieve. the task of making a turn mimetic can be regarded from the perspective of the backbone dihedral angles -to make a beta turn mimetic the phi and psi angles of two consecutive residues have to be controlled. limiting the dihedral angle space is most effectively accomplished with a cyclic constraint -one or more may be used. in this case a single medium ring cyclisation from the (i) carbonyl to the (i+ ) amine -in place of the classical hydrogen bond -forces the four backbone dihedral angles into a turn conformation. due to the polyfunctional nature of peptide systems the introduction of unnatural constraints can be synthetically challenging. in the present case a number of side reactions were encountered and had to be overcome. some were well known such as diketopiperazine formation and others involving transannular interactions were unexpected and gave rise to interesting byproducts. ultimately the side reactions were overcome by choice of cyclisation position and synthetic modifi cations. auto-assembling antimicrobial cyclic pseudopeptides including aza- -amino acids antibiotic resistance of pathogens against conventional antibiotics is increasing at a rate that far exceeds the pace of new development of drugs. so, antimicrobial peptides, both synthetic and from natural sources, have raised interest as potential useful drugs in the future. however, due to proteolytic degradation, peptides are not ideal candidates for pharmaceutical development. that is why numerous researches try to develop non natural peptidic analogues for enhancing metabolic stability, bioavailability, and biological absorption. in this class of peptidomimetics, pseudopeptides consisting exclusively or including aza -amino acids have emerged as a promising new class of compounds that favour hydrogen bond formation and can enhance biological activities when compared to natural parent peptides ( ) . we have designed "mixed" cylic pseudopeptides composed of -and aza- -amino acids that target bacterial cell wall and induce the death of the pathogens. particularly, some of these cyclic pseudopeptides have broad spectrum antibactericidal activities on gram positive and gram negative bacteria with low minimum inhibitory concentrations (mic). on the other hand, this type of molecules is not haemolytic and cytotoxic at antimicrobial activity levels( ) now, we try to explain the mechanism of action of our pseudopeptides that act on the microbial membranes. with nmr studies we have demonstrate that in solution cycles auto-associate at high concentrations and we investigate their behaviour in presence of small unilamellar lipidic vesicules (suv) ( ) . this phenomenon of auto association seems to be facilitated at the lipid interface. liu, hongqiang ; pattabiraman, vijaya r. ; vederas, john c. university of alberta, canada; lantibiotics are a class of antimicrobial peptides containing lanthionine ( ) and/or -methyllanthionine ( ) residues in cyclic moieties, and are currently used for food preservation. they also have potential as human therapeutics. lacticin is a two-peptide lantibiotic produced by l. lactis subsp. lactis dpc , and its components (a and a ) are active against a wide range of gram-positive bacteria by a synergistic mechanism in sub nm concentrations. however, the oxidation of the thioether of lanthionine ( ) and -methyllanthionine ( ) is a major source of lantibiotic instability and loss of activity. to investigate structure-activity relationships and determine whether sulfur can be replaced, an analogue of lacticin a in which oxygen atoms replace sulfur was synthesized by solid phase peptide synthesis. utilizing the stereochemicaly pure oxa-lanthionine ( ) and oxa--methyllanthionine ( ) with orthogonal protecting groups, the conformationally constrained tricyclic moiety in oxa-lacticin a was constructed. the results of biological evaluation of the oxidatively stable analogue will also be described. controlling -helical secondary structure of oligopeptides and its use as a chiral catalyst replacement of -hydrogen atom of -amino acids results in ,disubstituted amino acids. as an , -disubstituted amino acid, achiralaminoisobutyric acid (aib) is well-known, and widely used to construct helical secondary structures of oligopeptides. the helical secondary structures constructed by using aib usually show -helices, but nothelices in the case of short oligopeptides. recently we designed and synthesized a chiral cylic , -disubstituted amino acid (s,s)-ac c dom , in which the -carbon atom is not a chiral center but chiral centers existing at the side chain. homooctapeptide composed of (s,s)-ac c dom formed a left-handed -helix both in solution and in the solid state. furthermore, in the case that the (s,s)-ac c dom was incorporated into l-leuhexapeptide, the hexapeptide cbz-{l-leu-l-leu-(s,s)-ac c dom } -ome preferentially formed a right-handed -helix in the crystal state, whereas the hexapeptide cbz-{l-leu-l-leu-aib} -ome formed a right-handed -helix. the fi nding that the propensity of cyclic amino acid ac c dom is to form -helix over -helix, stimulated us to use the -helical oligomer containing the cyclic amino acid as an asymmetric catalyst. we prepared several l-leu-oligopeptides containing , -disubstituted amino acids, analyzed their preferred secondary structures, and studied a enantioselective reaction of prochiral substrate using -helical oligopeptides as a chiral catalyst. fbp domains: spps using pseudoproline and depsipeptide approaches, stability and structure of glutamine-rich analogs (nature biotech., ) . one peptide will soon be tested in clinical studies. the success depends on effi cient synthetic access to large amounts of the peptide. systematic modifi cations of the lead structure hbvpres/ - have to be performed resulting in a panel of peptide variants to be studied with respect to their applicability, pharmacokinetics and serum stability. we selected a series of peptides carrying deletions, point mutations, d-amino acid exchanges and sequence permutations. the syntheses of derivates were performed by fmoc-solid-phase synthesis on a rink amide am resin using hbtu/dipea activation on an applied biosystems a peptide synthesizer. after completion of the peptide sequence, stearic acid was attached to the n-terminus. the products were deprotected and detached from the resin by tfa treatment and subsequently purifi ed by hplc. the stability of the peptides was determined in human serum. we were able to synthesize all hbvpres lipopeptides in acceptable yields ( - %). preparative hplc was used to obtain intended compounds in high purity as determined by hplc and esi mass spectrometry. the serum stability studies revealed exceptionally long biological half-lives (t / > h for all lipopeptides) mainly due to the fatty acid residue. the peptides belong to a class of intrinsically unfolded peptides and are therefore not prone to aggregations within the synthesis. consequently solid phase peptide synthesis provides a suitable access to the peptides described. it can be speculated that the peptides are protected against degradation due to an association via their lipophilic end to serum proteins. sugar derivatives for self-assembling -cyclic peptides brea last years, numerous inorganic and organic nanotubes have been developed. self-assembling peptide nanotubes (spn) made from cyclic peptides have structural and functional properties that may be suitable for various applications in biology and material science. recently, our group have reported , -cyclic peptide ( , -cp) that forms highly stable homo-and/or heterodimers with partial hydrophobic cavities. in the present communication we will describe the design, synthesis and applications of a new class of self-assembling -cyclic peptides containing sugar derivatives that modify both the inner and the outer surfaces of the resulting supramolecular entities. peptido .rotaxanes: can a tetramide macrocycle travel to a station by wrapping up around a helical peptide thread? we are currently expanding this fi eld by synthesizing and studying the properties of new sets of peptido .rotaxanes. the initial set examined includes symmetrical, achiral compounds with a fmoc stopper at each terminus, threads built up with a central fumaric diamide station and two helical aib ( -aminoisobutyric acid) homo-peptides of different lengths (from to residues), and an aromatic tetramide macrocycle. these supramolecular systems have been characterized spectroscopically and two of them by x-ray diffraction as well. the fundamental interactions between macrocycle and thread (the same interactions offering the major contribution to the template-directed preparation of this family of molecules) are intercomponent h-bonds comprising the four amide groups in the ring and the two amide bonds in the fumaric derivative. the second, more complex, set of peptido .rotaxanes examined is represented by non-symmetrical compounds involving a thread based on a central helical -(aib) -peptide linker and two stations of opposite chirality (a -d-leu-gly-gly-tripeptide at the n-terminus and a fumaric diamide-l-leu moiety at the c-terminus). as stoppers and the macrocycle, we selected two diphenylacetyl groups and the usual aromatic tetramide, respectively. as spectroscopically assessed, the macrocycle initially positions on the fumaric diamide-l-leu station. subsequently, by using photons as stimuli to induce the fumaric<->maleic equilibrium, we were able to switch partially the relative macrocyclebinding affi nity in favor of the -d-leu-gly-gly-tripeptide station. this is the fi rst example of a rotaxane where the ring makes a journey to one of the stations by wrapping up around a helical peptide thread. synthetic novel n phenyl and bi-phenyl tetracarboxamides bis peptides. an approach to dna threading intercalators as expected potential pharmaceutical carriers for catatonic agents. naphthalene diimides were reported to bind to dna opposite grooves via the threading intercalation mode ( , ) . on the other hand, peptides constitute an excellent class of molecules for rapid drug discovery and lead optimization. the title bis dipeptides may, consequently, offer signifi cant dna biological probes. potential anticancer drugs or drug carriers could thus be presumed. herein, the title bis peptides a and b were suggested and synthesized as new prototype candidates of such class compounds. pre-assembled and purifi ed peptides via the conventional methods of peptide synthesis are, subsequently, coupled to either , , , -benzene tetra-carboxylic dianhydride or , , , -naphthalene tetra-carboxylic dianhydride. the expected structures of obtained a and b as preliminary candidates were confi rmed via the chemical, chromatographic and spectroscopic methodologies. the chemistry of both a and b will be discussed. synthesis of other candidates, the corresponding biological, pharmacological and physicochemical investigations are under realization. x = phenyl or bi-phenyl ring [compound a and b respectivel the fragment pth( - ) is suffi cient to bind and activate the pth type i receptor (pth r). the molecular mechanisms by which pth binds to and activates the pth r have been extensively investigated. recent investigations focusing on the interaction of n-terminal modifi ed fragments pth( - )nh with pth r showed that certain modifi cations can increase signalling potency and that enhancement of the -helicity in the pth( - ) sequence yielded potent analogues of pth( - )nh . the design of cyclic analogues represents a widely used strategy to increase peptide stability and potency. the structural constraint induced by cyclization reduces conformational fl exibility and may enhance potency, selectivity, stability and bioavailability as well as membrane barrier permeability. initially, the work was concentrated on conformational constrains in n-terminal such as the introduction of ctetra-substituted amino acids. global restrictions in the conformation of a peptide are possible by limiting the fl exibility of the peptide strand through cyclization. to this purpose, the amino acid side chains that are not involved in receptor recognition are connected together or with the peptide backbone. previous works on the role of side chains of aa determined through d-scan, analogues which maintained better -helical structure, contained d-gln in position and . recently gardella and co-workers reported that an analogues of pth( - ) cyclized between and residues exhibited almost the same activity of linear analogues. so, in this work two new potent cyclic analogues in position and were synthesized directly on spps, using side chains of lys and glu or ser. then they were biologically tested and analyzed by cd, according to our previous work. boudreau, marc; vederas, john university of alberta, canada the neopetrosiamides a and b are two diastereomeric tricyclic peptides that inhibit amoeboid invasion of human tumor cells. they were isolated by anderson and co-workers from the marine sponge neopetrosia sp. collected in papua new guinea, with their subsequent structure elucidation by the same group in . the peptides are residues in length and contain three disulfi de bonds, as well as the unusual amino acid methionine sulfoxide at position . the two peptides differ only by being epimeric at this sulfoxide functionality. we report the total synthesis of neopetrosiamides a and b as well as an analog wherein the methionine sulfoxide has been replaced by norleucine, the carbon analog of methionine. the strategy involved solid phase peptide synthesis to generate the linear species, and selective formation of the disulfi de bonds from orthogonally protected cysteine residues. synthesis of mimetics of the antibody d a : a new class of antitrombotics. in the lab for tromboses research of the kulak (belgium) an antitrombotic antibody (ab) called d a has been characterized ( ) . the ab inhibits the interaction between the plasma peptide von willebrand factor (vwf) and by injury exposed collagen, a prerequisite interaction in thrombusformation in arteries (high blood shear). it has been shown in vivo that the ab d a has an antitrombotic effect without showing the bleeding complications that known antitrombotics exhibit. this makes the ab an attractive lead for antithrombotic drug design. by x-ray and mutagenesis studies, the paratope (active site) of the antibody has been determined. amino acids, discontinuous in the primary structure of the antibody but a quasi linear unit in space, play an important role in the interaction. incorporation of the functional groups of the amino acids and fi xation of the secondary structure of the paratope will be the aim in the design of the paratope mimetics. with this rational design we will try to develop an orally available, synthetic paratope peptidomimetic with the same antitrombotic effect as the antibody. the development (solid phase and solution peptide synthesis) of the mimetics is a stepwise process. in each step conformational restrictions are introduced. in this we hope to divert away from the peptide and go to a drug like molecule. peptide ligands for sh -and ptp domains containing phosphotyrosine are of great interest to infl uence the activity of kinases, phosphatases and other functional proteins. backbone cyclization can help to stabilize these ligands against proteolytic degradation and to form their bioactive conformation. till now backbone cyclization was performed with bifunctional and in few cases with trifunctional amino acids but not with phosphotyrosine. the assembly of such peptides requires preformed building units. because the necessary reductive alkylation of phosphotyrosine derivatives leads only to very low yields we used n-functionalized pseudodipeptides with the unprotected phenolic hydroxyl group. for fragment condensation we synthesized building units of the common structures: fmoc-aaØ[co-n(x)-(ch )n-nh-alloc)tyr(oh)-oh and fmoc-aaØ[co-n(ch )m -cooall)tyr(oh)-oh. depending on the steric hindrancy of the n-terminal amino acid these pseudodipeptides were synthesized in solution or at sasrinresin. they were purifi ed by fl ash chromatography and analytically characterized by hplc, esi-ms and nmr. we tested our strategy on the synthesis of an octapeptide-ligand for the n-terminal sh -domain of the phosphatase shp- : glu-gly-leu-asn/abu-ptyr-nle-asp-leu-nh . couplings of the dipeptide units were performed with pybop, stepwise coupling to the peptide fragments with unprotected phenolic hydroxyl group with pentafl uoro phenylesters. after fi nishing the assembly the obtained polymer bound octapeptides were consecutively cyclized, phosphorylated, removed from the resin and purifi ed by hplc. based on elucidated side reactions the synthetic strategy was optimized. the obtained backbone cyclic and phosphorylated ligands were tested for their infl uence on the phosphatase activity of shp- . the found enzymatic activities are correlated to size, direction, hydrophobicity and conformational fl exibility of the lactam bridges. biology and medicine. in this context, we have devised a new family of compounds named « polyamide amino acids » (paas), constituted by a pna (peptide nucleic acids) backbone mimicking rna sugarphosphate backbone, onto which aminoacid residues are linked. therefore, these paas could constitute a new type of rna ligands, liable to specifi cally interact with an rna target through an original interaction mode. to assess the ability of paas to be potential rna binders, we fi rst prepared tetra-paas (t -t ) via solid-phase synthesis, starting from paa monomers deriving from alanine, phenylalanine, lysine and arginine residues. interactions of the tetramers with a hiv- tar rna fragment, taken as a target model, was investigated by fl uorescence spectroscopy and circular dichroism. to give insights about specifi city, binding affi nities to tar were also assessed using an excess of a trna mixture. results showed kd values varying from . to m, indicating the importance of the aminoacid side chains in the interaction. thermodynamic analyses revealed that even if electrostatic interactions play a part in the complex formation, the binding is enthalpy-driven, highligthing the importance of non-electrostatic interactions in the recognition process. these results are of special interest since rna/ ligand association specifi city is typically assumed to be due to shortrange non-electrostatic interactions. moreover, t -t were shown to be specifi c to tar in the presence of an excess of trna. all together, these results are encouraging and they highlight the potential of paas as rna ligands. indeed, the use of only four paa monomers as building blocks leads to tetra-paas displaying both affi nity and specifi city for their rna target. studies on the solid phase synthesis and selective detection of peptide derived amadori products by mass spectrometry stefanowicz mass spectrometric analysis of glycation products of proteins and peptides attracts increasing attention ( ) . peptide-based amadori products could be used as markers of diabetes mellitus, which makes them the subject of interest in clinical chemistry. recently, several procedures of siteselective synthesis of amadori-modifi ed peptides has been published [ , ] . in this communication we will present the synthesis of a new, fully protected derivative of glycated lysine which was successfully applied as a building block for incorporation of the glycated lysine moiety into peptide chain according to the standard fmoc-solid phase synthesis protocol. we will also present a new and straightforward method of selective detection of peptide-derived amadori products by esi-ms basing on characteristic neutral losses in the sugar moiety. the proposed approach in contrast to the neutral loss scanning, which requires triple quadrupol mass spectrometer, can be performed on the instruments with higher resolution and sensitivity, like q-tof . new apa inhibitors interacting with the s subsite bring new insights in the apa substrate specifi city mediated by the calcium ion. aminopeptidase (apa, ec . . . ) is a membrane-bound zinc metallopeptidase involved in the maturation of brain angiotensin iii, a peptide which exerts a tonic stimulatory action on blood pressure in hypertensive animals ( ) . therefore, developing inhibitors of this enzyme should result in new antihypertensive agents with possible new application in the treatment of certain forms of hypertension ( ) ( ) . we and others have previously reported the design of such inhibitors ( ) ( ) . nevertheless, the improvement of the affi nities of the inhibitors is relatively impaired since the three dimensional ( d) structure of the enzyme is not available. with the aim of getting insight into inhibitors optimization, a d model of apa was constructed in our group as an alternative ( ) . furthermore, it is well established that apa substrate specifi city and enzymatic activity are calcium dependent. in order to render this model more accurate and so on to identify the amino acids residues involved in calcium binding, we have designed new inhibitors able to explore the apa s subsite to better understand its specifi city. we will report the synthesis of these new inhibitors, as well as an inversion of the specifi city of apa s subsite in absence of calcium. these results and the identifi cation of the amino acids implicated in calcium binding will be discussed on the basis of site-directed mutagenesis studies. one of the designed inhibitors, ni (ki = nm) is to date the more potent inhibitor of apa activity measured without calcium. altogether, these data should allow the improvement of apa inhibitors by structure aided design. synthesis, resolution, and absolute confi guration of bpaib, a benzophenone-containing c -tetrasubstituted -amino acid photoreactive amino acids with benzophenone side chains, the prototype of which is bpa ( -benzoyl phenylalanine), have found numerous applications as photo-probes for covalent modifi cation of enzymes and receptors, as well as in intramolecular quenching by a nitroxide free radical in trichogin peptide analogs. however, the remarkable fl exibility of the bpa side chain may question the extrapolation of results of photo cross-linking experiments and photophysical data to protein mapping and intramolecular distances, respectively [m. saviano et al., chembiochem, , , - and references cited therein]. to overcome this problem, we designed a new "constrained bpa" amino acid, bpaib, belonging to the sub-class of the ci <->ci cyclized, c -tetrasubstituted -amino acids (strong -turn and helix inducers in peptides). racemic boc-bpaib-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phasetransfer conditions with -benzoyl- , -(bis)bromomethyl benzene as alkylating agent, followed by acidic hydrolysis, n -boc protection, and saponifi cation of the ester function. resolution was achieved through poster abstracts the terminally-blocked dipeptide bz-bpaib-l-phe-nhchx with chromatographic separation of the diastereomers and acidic hydrolysis. x-ray diffraction analysis of a crystal of a z-bpaib-l-phe-nhchx diastereomer allowed the assignment of the absolute confi guration of the bpaib enantiomers. photo-crosslinking studies with this residue are currently in progress. n-methylation of n -acetylated, c -ethylated, fullyextended homo-peptides: synthetic and conformational aspects moretto peptides characterized by single or multiple n-methylated, ctrisubstituted (protein) -amino acids are one of the subject of increasing interest in medicinal chemistry. several naturally occurring peptides, remarkably stable to proteolytic attacks, are based on n-methylated peptides. n methylation of the -conh-function is a useful tool for discriminating solvent exposed from intramolecularly h-bonded secondary amide groups in peptides. we are currently extending this reaction to linear peptides based on c -tetrasusbtituted -amino acids. after having investigated synthesis and conformation of the n-methylated homo-peptides from the c -methylated, helicogenicaminoisobutyric acid and c -methylnorvaline residues [a. moretto et al., biopolymers (pept. sci.) , , - ], in this work we examined the n-methylation reaction on homo-peptides from c , -diethylglycine (deg), known to overwhelmingly adopt the fully-extended, multiple c , conformation. we studied the following peptide series: ac-(deg) n -n(et) , with n = - . under the experimental conditions used, only monomethylation (on the n-terminal, acetylated residue) takes place. our ft-ir absorption, nmr, and x-ray diffraction analyses support the view that the fully-extended conformation preferred by the original peptides is dramatically perturbed in all of the derivatives mono-methylated at position . computational study on secondary structure of oligopeptides containing chiral , -disubstituted -amino acids we have shown mcmm conformational search method and amber* force fi eld using macromodel is useful to predict secondary helical structures ( -helix, -helix) of oligopeptides prepared from ,disubstituted -amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral , -disubstituted -amino acids to predict the helical screw sense of helical structures. we calculated , -disubstituted peptide using mcmm conformational search with various force fi elds (amber*, mmff, opls) . in the case of using amber* force fi eld the results were in agreement with those of x-ray and were most stable conformation evaluated by - g level molecular orbital calculation. these results indicated that computational simulation using conformational search calculations with amber* force fi eld is most useful for conformational analysis of oligopeptides containing , -disubstituted -amino acids. a new colorimetric test for solid-phase amines and thiols. simple colorimetric tests still remain the most convenient tests for providing information on the course of solid-phase reactions. a colour test, which indicates the presence or absence of a functional group by visual detection, is a simple, practical tool to monitor the completeness of the reaction. although the variety of colour tests for amines, there is still a need for a reliable and sensitive test in some synthetic approaches. here, we wish to report a new simple, fast and sensitive colorimetric assay for the visual detection of solid-phase bound primary, secondary amines and solid-phase bound thiol groups using -alkyl- -aryl-imidazo[ , -a]pyrimidinium salts. in the search for a new synthetic approach to polysubstituted -aminoimidazoles ( ), we have reported a new procedure, employing substituted -aminopyrimidines and -bromocarbonyl compounds. the intermediate imidazo [ , a] pyrimidinium salts in the synthesis of substituted -aminoimidazoles appeared to give a strong colour when reacted with primary and secondary amines. due to the strong colour change of amino functionalised resins after reaction with the "desc" reagent ( ), a protocol for the detection of resin bound primary and secondary amines has been developed. the "desc" test is a new, practically, simple, quick and reliable test for the visual detection of resin bound primary and secondary amines in a sensitive way. the "desc" reagent is accessible from the cheap starting materials and can be prepared in two steps. tetrafl uoroborates of chiral n-triazinylammonium salts were found stable and useful in enantioselective (ee up to %) peptide synthesis from racemic amino acids in solution. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents ( ) . broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. we have attempted to expand the scope of application of this family of reagents expecting their effi ciency in enantiodifferentiating reactions in solid phase peptide synthesis (spps). tetrafl uoroborates of chiral n-triazinylammonium salts were obtained by treatment -chloro- , -dimethoxy- , , -triazine with tetrafl uoroborates of appropriate tertiary amines in the presence of sodium bicarbonate ( ) . enantiodifferentiating reagents were used in synthesis on wang resin under classic condition for triazine based coupling reagents ( ) . the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the predictability of confi guration and repeatability of enantiomeric purity of incorporated amino acid. even if only threefold excess of racemic carboxylic acid, enantiomeric purity of coupling products in spps reached ee - . the demand for diversity of non-proteinogenic amino acids, willingly used as new building blocks, is severely restricted by laborious procedures leading to enantiomerically homogeneous individuals. typical sources such as isolation from natural products, biotechnological methodology, even the asymmetric syntheses posses a limited value because complex or tedious synthetic procedures or limited access to the pool of chiral auxiliaries. herein we present the novel, general approach allowing enantioselective incorporation of any enantiomer of amino acids directly from racemic substrate. according to our procedure, the chiral auxiliary is used only at the stage of enantioselective activation. the departure of chiral auxiliary yields in the traceless way, the activated derivative of n-protected amino acid identical with those which is obtained with the well known, classic, achiral triazine coupling reagent ( ) . therefore, the traceless chiral coupling reagents can be successfully applied directly in the coupling stage without additional studies of their reactivity. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents. broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the repeatability and predictability of enantiomeric purity and confi guration of incorporated amino acid. freeman, noam s.; hurevich, mattan; gilon, chaim hebrew university jerusalem, israel azapeptides are peptide analogues in which one or more of thecarbons, bearing the side chain residues, has been replaced by a nitrogen atom. azaamino acid residues conserve the pharmacophores necessary for biological activity while inducing conformational changes and increased resistance to proteolitic degradation. these properties make azapeptides an attractive tool for structure-activity relationship studies and drug design. a general approach for solid phase synthesis of azapeptides has been developed based on the in-situ activation of n- -( , dimethoxyphenyl)propan- -yloxycarbonyl (ddz), n €™-substituted hydrazines, with phosgene, followed by introduction to n-terminus of resin-bound peptide. the ddz-aza-amino building units include aliphatic, aromatic and functionalized side chains, protected for synthesis by the fmoc strategy. solid-phase azapeptide synthesis is demonstrated including selective mild deprotection of ddz with mg(clo ) and coupling of the next amino acid with triphosgene. the mild ddz deprotection is also orthogonal with boc chemistry. we describe the synthesis of n €™-substituted ddz protected hydrazines which have wide applications in the synthesis of azapeptides as well as in general synthesis of substituted hydrazines and aza containing peptidomimetics. ddz protected hydrazines offer the possibility to construct novel and unique drug-candidate structures such as branched and cyclic azapeptides. fast improved synthesis of insulin-like peptides barlos the a-and b-chains of insulin-like peptides were synthesized by various methods. the best results were achieved by dividing the sequences of the a-chain into three protected fragments and that of the b-chain into two fragments. the fragments were prepared on -chlorotrityl resin and/ or -methoxybenzhydryl resin using fmoc-amino acids. condensation of the fragments was carried out either by solution phase or solid phase techniques. the joining of the a and b chains was also studied using either biomimetic or chemoselective oxidative folding methods. a new approach for amides and peptides chemical synthesis by means of phosphonic acid/alkylene oxide chemistry few different derivatives of l-phe were synthesized, using originally discovered method of phosphonic acid/alkylene oxide chemistry. this method provides the successful synthesis of variety amides and dipeptides without preliminary protection of alfa-nh group of lamino acids. the supposed mechanism displays the formation of nphospholane carboxyanhydride ( -oxy- -aza- -phospholane- -one), or an o-hydroxypropyl h-phosphonate salt of an amide of l-phe. the nphospholane carboxyanhydride is protected at the n-terminus, as well as is activated at the c-terminus. this pathway favors the desired reaction with a variety of nucleophiles from the n-to c-terminus. this method also allows the synthesis of different esters of amino acids employing alcohols as nucleophiles previously described by us ( ). formation of disulfi de bonds in synthetic peptides is one of the more challenging transformations to achieve in peptide chemistry, in view of possible formation of oligomeric by-products and other side reactions, as well as occasional solubility problems in aqueous oxidizing media. the recently introduced polymer-supported oxidant, clear-ox™, has proven to be a very valuable tool in the preparation of disulfi de-bridged peptides. [ , ] oxidations using clear-ox were carried out at ph ranging from to in water/acetonitrile solutions, with concentrations - times higher than comparable solution oxidations. the latest progress in the preparation of various monocyclic and bicyclic peptides will be presented. the amyloid -peptide (a ) is believed to play a causal role in alzheimer's disease (ad). one of the hypotheses of a neurotoxicity is that it induces the generation of reactive oxygen species (ros) and hydrogen peroxide (h o ) formation by binding to metals such as copper and iron. it is hypothesized that the sole tyrosine residue plays an important role in a -mediated toxicity, due to its ability to form a dityrosine cross-link from tyrosyl radicals generated in the highly oxidative environment in the brain. hence, studies of the dityrosine cross-linked a peptide dimers would increase our understanding of the oxidative alteration and dimerisation of a in amyloid formation and ad related neurodegeneration. we are investigating the synthesis of the a peptide dimers containing the dityrosine cross-link. dityrosine, suitably protected for solid-phase peptide synthesis (spps), has been prepared from iodotyrosine using a miyaura borylation-suzuki coupling method. studies on the synthesis of dityrosine-linked peptide dimers through incorporation of dityrosine in spps are underway. initial model studies employing , -diaminopimelic acid (dap) have validated this approach. preparation of the model daplinked a peptide dimers will be discussed, as well as progress towards the dityrosine-linked a peptide dimers. nepomniaschiy, natalia; brik, ashraf ben-gurion university of the negev, israel the staudinger reaction, discovered nearly a century ago, occurs between a phosphine and an azide to form an aza-ylide. this transformation was further explored and developed, leading to several reactions of highly synthetic importance. traceless staudinger ligation is one example of such modifi cations, in which an ester moiety is placed within the phosphine structure to capture the nucleophilic aza-ylide, by intramolecular cyclization, leading to a stable amide bond. while the aza-ylide intermediate is known to be stable in organic solvents, it tends to hydrolyze rapidly under aqueous media to furnish the primary amine product. in the traceless staudinger ligation, however, the reduction of the azide to amine is a competing side reaction, as it would reverse the capture step leading to two peptide fragments. we have found that such reduction step would be benefi cial if an electrophile and an azide (rather than the phosphine) are placed within the switch peptide system. investigating various reducing reagents led us to the discovery that tris( -carboxyethyl) phosphine hydrochloride (tcep) is excellent azide reducing reagent. both the reduction and the acyl transfer steps are rapid and occur in a few minutes. applying the tcep-mediated triggering in switch peptides, derived from the a to understand the currently unclear processes of pathological folding, self-assembly, and aggregation of amyloid peptide will also be reported. fast fmoc-deprotection reagent for peptide synthesis diffi cult sequences have long been known in solid phase peptide synthesis, theses sequences can experience sever aggregation both during synthesis and under purifi cation. the aggregation is believed to depend mainly on -sheet formation or/ and hydrophobic properties of the peptide. now a days there are several strategies to circumvent theses problems, one of the most successful is to incorporate a backbone protective group as the hmb group, develop by johnson and co-worker( ), and thereby prevent aggregation due to -sheet formation. an additional charge will increase the solubility of peptides towards aqueous solutions and thereby facilitate the purifi cation by hplc (using acetonitrile and water system) and analysis (by maldi-tof mass spectrometer) of the peptide. here we present the nmec (n-methyl-n-[ -(methylamino)ethyl] carbamoyl) group( ) that is a orthogonal protective group for tyrosine side chain and the hydroxyl moiety of the hmb group. the nmec group is fmoc compatible and is protected during the synthesis with a boc group. the fi nal cleavage from the resin with tfa renders the amine of the nmec group protonated and will increase the solubility of the peptide during purifi cation and analysis. the nmec group can be easily removed with a mild alkaline treatment by a cyclization elimination reaction. the nmec group has been employed in the synthesis of diffi cult and hydrophobic peptides as the membrane spanning sequence of the calciton receptor, amyloid ( - ) and amyloid ( - ) with success. [ , ] . for example, infl uenza a virus-related peptide (h-gilgfvftl-h) with a diffi cult sequence was synthesized using o-acyl isodipeptide unit. analysis of the crude peptide revealed high purity of the product with no by-product derived from the diffi cult sequence or epimerization. using ch cl as solvent in coupling the isodipeptide unit, a - was also synthesized with almost no major side reaction .. racemization-free segment condensation method was developed by employing n-segments possessing a c-terminal urethane-protected o-acyl ser/thr residues .. the synthesis of long peptides/proteins by racemization-free segment condensation has thus become possible at ser/thr residues and not only c-terminal gly/pro residues. growing interest to peptide substrates, inhibitors and other peptidomimetics required new highly effective synthetic techniques. the important goal of enzymatic peptide bond formation is the optical purity of the peptide, which facilitates the product isolation. thus using enzymatic condensation as a last step in peptidomimetics production is preferable. fixing of enzymes on/in suitable insoluble supports has many advantages: high operational stability, ease of separation, possibility of recycling and improved activity in low water media. chitosan, natural hydrophilic polysaccharide, was used as a matrix as it has distinct advantages over the other supports due to (a) its renewable nature -it is available in large quantities as a waste product from fi shing industry and (b) its excellent fi lm-forming ability, allowing attachment to reactor walls for advanced processing. serine proteases subtilisin and chymotrypsin, and cysteine protease papain were immobilized onto chitosan. the fi lms of biocatalysts were prepared by drying the mixed solution of chitosan and enzyme in acetate buffer ph . . treatment with glutaraldehyde was found to give material with high stability and good mechanical properties. the obtained biocomposites showed high amidase activity against specifi c chromogenic peptide substrates and protein substrate azacasein. immobilized subtilisin and chymotrypsin also possess esterase activity against p-nitrophenyl acetate. the longterm storage in aqueous buffer and acetonitrile had a little effect on the hydrolytic activity of subtilisin-based biocomposite. the dependence of subtilisin/chitosan hydrolytic activity on temperature and ph was studied. obtained samples possessed high synthetic activity and were capable to catalyze peptide bond formation in dmfa/mecn mixture in reaction zaalome+fpna zaalfpna for subtilisin, zaaome+lpna zaalpna for papain. the product yield reached - % in h. acknowledgement: this work was supported by rfbr - - synthetic chemistry or on solid phase and b) the chemical ligation of the [cys (acm)]-( - )-thioester with the [cys (acm)]-( - ) segment, both deprotected and hplc purifi ed. the required intermediate peptides were prepared by optimized fmoc-based methods either stepwise or by convergent synthesis. for the formation of the two disulfi de bridges a two step procedure was investigated, involving a dmso oxidation step to form the cys -cys linkage, followed by iodine oxidation to form the cys -cys bond. native chemical ligation at valine haase, christian; rohde, heike; seitz, oliver humboldt-universität zu berlin, germany native chemical ligation is perhaps the most useful technique for peptide segment coupling in water. ( ) a c-terminal peptide thioester reacts with an n-terminal cysteine. the requirement for this rare amino acid represents the major bottle neck to the synthesis of native proteins. several strategies have been developed to overcome this limitation. in the extended ligation, n-terminally attached auxiliaries allow access to other ligation junctions by mimicking the cysteine-thiol moiety. the ligation-desulfurization approach employs -thiol amino acids for fragment coupling followed by sulphur removal. herein, cysteine acts as precursor of the abundant alanine( ) and phenylalanine can be obtained by using its -mercapto derivative. ( ) . we demonstrate the application of penicillamine in the generation of valine junctions employing the ligation-desulfurization approach. the , -dimethylcysteine building block is commercially available with various protecting group patterns suitable for routine solid phase synthesis of peptides. the ligation at penicillamine proceeded surprisingly fast despite the steric demand at the thiol group. even leu-val ligation sites, which appear in hydrophobic peptide segments, are accessible. we also present an improved method for achieving metal-free desulfurization and show applications in the synthesis of valine-containing peptides.( ) references: . p. e. dawson the application of n-alkyl cysteine (nac)-assisted thioesterifi cation reaction to the synthesis of polypeptides by the thioester method azide as a protecting group for lysine side chains on the solid phase peptide synthesis oriented toward the peptide condensation by the thioester method peptide ligation chemistry has been developed based on the use of peptide thioester as a building block in the thioester method ( ) and native chemical ligation ( ) . we found that a cysteine-containing peptide is transformed into the corresponding s-peptide (peptide thioester) by the n to s acyl shift reaction ( ). on the other hand, in , zanotti et al. reported that a diketopiperazine thioester, cyclo(-cys(coch ph)-pro-) ( ) was formed when a dipeptide p-nitrophenyl ester, phch co-cys(st-bu)-pro-onp ( ), was treated under reductive aqueous conditions ( ). thioester would be formed via intramolecular n-s acyl shift reaction followed by diketopiperazine formation. based on these observations, we designed a cysteinyl prolyl ester (cpe) autoactivating unit for preparation of the peptide thioester and for the peptide ligation [ , ] . a peptide containing the cpe unit, peptide-cpe , was transformed into a peptide thioester of diketopiperazine, cyclo(-cys(peptide)-pro-) , then thiol-thioester exchange reaction with an external thiol produced the peptide thioester and a diketopiperazine, cyclo(-cys-pro-) ( ) . the poster abstracts peptide-cpe was also able to ligate with a cysteinyl peptide , via the thioester in one-pot, to give a polypeptide . ( ) has enabled the synthesis of entire proteins including those carrying posttranslational modifi cations. one of the most frequently encountered modifi cation of eukaryotic secretory and cell surface proteins is the attachment of oligosaccharides to asparagine residues (n-glycosylation). despite many efforts in this fi eld the function of nglycosyation is poorly understood, which is mainly caused by the lack of pure glycoproteins. since purifi cation of natural glycoproteins is quite tedious due to heterogeneity in the sugar part, the total synthesis of homogeneous glycoproteins has become an attractive target ( ) . we have addressed this topic by choosing rnase c as a model glycoprotein. instead of the oligomannosidic type rnase c is containing a complex type n-glycan. for synthetic reasons ligations had to be conducted in a sequential manner ( ) by fi rst reacting the recombinant - cys-segment ( ) with an n-terminally protected and glycosylated thioester - .. selective deprotection of the ligation product - and ligation with synthetic thioester - in a one pot manner was accompanied by unexpected side reactions. these drawbacks were fi nally overcome leading to full-length glycosylated rnase c displaying enzymatic activity. a new pseudo-native ligation: multiple, successive azidealkyne cycloadditions. ( ) the reaction has become a classics. as triazoles are stable to acid and basic hydrolysis, and reductive and oxidative conditions, the reaction has been widely used in chemistry and biochemistry, proving to be an useful tool in combinatorial, bioconjugate or medicinal chemistry. the reaction has been applied in the peptide fi eld as an easy way to synthesize cyclic, labelled and side chain modifi ed peptides including effi cient synthesis of pseudo-glycopeptides. conformational and structural studies prove that the triazole ring is an excellent trans-amide surrogate and thus, can be considered as a new pseudo-native linkage. ( ) in order to explore the potential of the reaction as a way to successively assemble several peptide chains to generate mimics of proteins, we have elaborated a new strategy for consecutive triazole formation based on a semi-orthogonal alkyne protection scheme. ( ) so far, we have improved our fi rst one-pot successive cycloaddition approach performing the fi rst three successive cuaac in mild conditions thanks to a highly selective deprotection of two different alkyne groups. the application of this strategy represents a new promising method for the chemoselective pseudo-native ligation dedicated to the synthesis of protein chimera or for the decoration of molecular templates. solid phase synthesis using lightly crosslinked polystyrene supports has proved to be a successful route to the manufacture of peptides. the methodology introduced by merrifi eld nearly fi fty years ago has remained largely unchanged. during the last twenty years or so, it has been argued that incorporating a hydrophilic polymer within the conventional hydrophobic polystyrene matrix is benefi cial. others have suggested that polyamide or polyether-based resins may prove to be superior to polystyrene. despite this, large-scale peptide manufacture has generally relied on traditional polystyrene supports. this is due in part to the diffi culty in producing composite supports economically in large volumes, but also due to the handling diffi culties that are often associated with very high swelling polar polymers. other problems arise from the fact that many of these types of resin are relatively low loading and so yields are greatly diminished. in this poster we offer a solution to these problems: a polystyrene derived support which is modifi ed to create economical, amphipathic resins suitable for large scale peptide synthesis. attachment of appropriate handles or linkers enables both peptide acids and peptide amides to be produced. the synthesis of a variety of peptides is demonstrated. peptide synthesis in water using boc-amino acids nanoparticles reactants. here, we studied in-water solution-phase method using water-dispersible nanoparticulate boc-amino acids, which are the most common building blocks but are diffi cult to use for peptide synthesis in water. water-dispersible boc-amino acid nanoparticles were prepared by pulverization using a planetary ball mill in the presence of peg, and the size of the resulting water-dispersible nanoparticles was determined by dynamic light scattering analysis. the scanning electron microscopy image of water-dispersible boc-phe-oh nanoparticles also revealed nanosize particles. we studied in-water coupling reaction using waterdispersible boc-amino acid nanoparticles, and leu-enkephalinamide was successfully synthesized in water according to the boc chemistry. an alternative way for conopeptide formation kádár, kinga ; panyi, györgy ; tóth, gábor k. university of szeged, hungary; university of debrecen, hungary the chemistry used to oxidize the free thiol bonds to the corresponding disulfi de bond in a controlled fashion remains a signifi cant challenge in spite of many advances in peptide chemistry. the primary reason of this lies in the diffi culties involved in the formation of multiple regioselective disulfi de bonds. in this work we focused on the elucidation of synthetic strategies for the preparation of multiple disulfi de containing peptide venoms regulating the ion channels of immune cells-anuroctoxin and tc -, and a neuropeptide with regulatory functions-orexin a. for the synthesis of these naturally occuring cys-rich peptides we had chosen the oxidative folding being the most simple of the methods available. in the case of anuroctoxin we isolated the native form of the peptide toxin with high selectivity and we optimized the folding conditions, so within an hour the majority of the linear peptide folds in the cyclic form with all four disulfi de bonds in the correct form. the regioselectivity of the isolated isomer was verifi ed with biological measurements. for the synthesis of orexin a we optimized the folding conditions and, using a polymer-supported oxidant-the clear-ox resin-, within two hours we could isolate the correctly folded isomer as the major reaction product. the correct structure was proven by coelution of the isolated isomer and the commercially available orexin a. but oxidative folding does not always give the correctly folded isomer. the best proof for this is the tc scorpion toxin which albeit our tryings always gave the misfolded isomers if we used the linear, unprotected peptide. therefore a new chemical synthesis using different side-chain protection needs to be taken into consideration. the synthesis of orthogonally protected tc and its use for the preparation of the correctly folded peptide to be underway. poster abstracts have fundamental importance in biological recognition processes. one of the most challenging task among of them the rational preparation of the glycosylated peptides especially having oligosaccharide moieties. there are two main strategies for the synthesis of glycopeptides: the synthon and global (convergent) method. both of them can be implemented in liquid or solid-phase. since the glycosylation could appear on o and n atoms of the amino acid side-chain, due to the different reactivity of the glycosidic linkage different chemical strategies will necessitate. in this presentation we compare several chemical strategies for the preparation of two model peptides (leu-lys-asn*-gly-gly-pro, gly-val-glu-asp-ile-ser*-gly-leu-pro-ser-gly,*site of glycosylation). as glyco-part several mono, di and trisaccharide including chitobiose, galactosilxilose, mannosil-n-acetyl-glycosil-n-acetyl-glucosamine were used and several of the used strategies led to successful preparation of these glycoconjugates. site-specifi c pegylation of human igg -fab using a rationally designed trypsin variant in the present contribution we report on a novel, highly selective biocatalytic method enabling c-terminal modifi cations of proteins with artifi cial functionalities under native state conditions. the approach is based on the fourfold trypsin variant k e/n h/e h/d k which was generated by site directed mutagenesis and initially specifi ed in terms of selectivity and activity using a peptide library of the general structure bz aax aa x bb x cc aag-oh. the trypsin variant was found to bear a restricted proteolytic activity towards the rare recognition sequence yrh with an occurance of less than . % in native proteins. the specifi city is zinc ion dependent due to an artifi cial metal binding site in the s ´-subsite ( ). placing of the recognition sequence at the cterminal region of respective proteins via standard mutagenesis protocols provides suitable precursor targets for site-specifi c modifi cation via a transamidation reaction. due to the great demand for polymer-modifi ed antibody fragments for pharmaceutical purposes we evaluated the function of the approach on example of the c-terminal pegylation (peg...polyethylene glycol) of the human igg fab fragment. the fragment itself was expressed with the recognition sequence and an additional strep-fusion enabling effi cient purifi cation. the derivatization of the antibody fragment with kda peg via the trypsin variant (mw ~ kda) proceeds effi ciently with a total yield of isolated modifi ed protein of %. interestingly, no undesired cleavage reactions could be detected leading to fully active modifi ed antibody fragment. references: . willett, w.s., brinen, l.s. et al. ( ) . "delocalizing trypsin specifi city with metal activation." biochemistry ( ): - . -chloro- , -bis-( , , -trifl uoroethoxy)- , , -triazine and n- , -( , , -trifl uoroethoxy)- , , -triazin- yl)ammonium tetrafl uoroborates as highly effi cient coupling reagents jastrzabek, konrad; kolesinska, beata; kaminski, zbigniew, j. we expected that modifi cation of substituents in the triazine ring improve activity, stability and solubility of new triazine based coupling reagents. in order to increase activity we prepared -chloro- , -( , , trifl uoroethoxy)- , , -triazine by treatment of cyanuric chloride with , , -trifl uoroethanol. taking advantage of modular structure of triazine coupling reagents the entire family of n-( , -( , , -trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates have been obtained. effi cient coupling reagents should be useful for amide bond formation between broad range of substrates, work in stoichiometric quantities, be soluble and stable in most of the solvents. it should function effi ciently in solution as well as in spps, to get high purity crude products and minimize racemization of the products. we found n-( , -( , , trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates useful for activation of carboxylic components. the participation of triazine "superactive ester" as intermediate in the condensation has been proved in the model experiments. utility of reagents n-( , -( , , trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates were confi rmed by peptide synthesis in solution in high yield. in our study we focused our attention on the performance (in terms of purity of the crude and extent of racemization) testing the solid phase synthesis of acp( - ) and model peptides with aibaib fragment, which are a good example of diffi cult peptide sequence. purifi cation of chemically synthesised proteins by use of cleavable imac-based n -terminal protein protecting groups (tags) ( ), there remains a challenge in terms of time and cost, for the separation of impurities from the chemically synthesised product in spps. this is particularly acute in the case of protein synthesis where the crude product mixture contains capped (acetylated) truncates of comparable size and structure to the protein product. such a common situation results in extensive, and expensive, purifi cation techniques such as large scale hplc. to this end we designed a hydrophobic tag, tbfmoc ( ) , which incorporated the base-labile characteristics of the fmoc ( ) group. the tbfmoc group causes retention on hydrophobic columns and hence separation from untagged impurities. although this was effective, we were of the opinion that a complementary tag was required which would have the characteristics of metal-complexation and cleavage by -elimination for subsequent removal of the tag from the protein product after imac. considerations implicit in the design of such an n -terminal protecting group, or tag, will be discussed , and applications to the chemical synthesis of chemokines will be dealt with in the presentation. in last few decades, acridines which are known as anticancer, antimicrobal and antiviral agents have been in the center of interest of scientists. the heterocyclic molecules demonstrate a noteworthy group of compounds which interact with biological targets e.g. topoisomerase i, ii [ , ] . we wish to pay our attention to a new group of tuftsin-acridine conjugates. tuftsin offers a wide range of biological activities such as supporting of immune system, bactericidal, tumoricidal activity. however tuftsin is unstable in plasma which reduces its effi cacy. that is the reason for search of new analogues that were more resistance to proteolysis degradation ( ). tuftsin-acridine conjugates were synthesized using a fmoc solid phase strategy. the elongation of peptide chain was based on twostep procedure: deprotection and coupling reaction with dic and the additive of hobt in dmf/dcm/nmp mixture. tuftsin analogues were modifi ed at -amino group of lysine via introduction of the simple amino acids to obtain isopeptide bond. tuftsin analogues were conjugated to the acridine molecule via fl exible linkers. the carboxylic group of linker was connected to n-terminal group of peptide-resin using standard method for amide bond formation. the coupling reaction was performed with peptide-resin and the -fold excess of -nitro-acridine derivative using tbtu, hobt in the presence of diea in dmf for h. simultaneous deprotection of peptide side chain and cleavage from resin was done with the tfa cocktail. final products were purifi ed by spe and characterized by elemental analysis, ms and h-nmr spectroscopy. the effect of microwave irradiation under controlled temperature conditions on the solid-phase synthesis of peptides was investigated. for optimization studies a model peptide (h-gly-ile-leu-thr-val-ser-val-ala-val-oh) was selected which suffers from poor synthetic effi ciency under standard spps conditions. synthesis of the nonapeptide was performed using various combinations of solid supports (polysterene, tentagel, chemmatrix) and solvents employing fmoc/but orthogonal protection strategy. applying controlled microwave heating, the reaction times were signifi cantly reduced while maintaining a high purity of crude product with no racemization being observed. the optimized microwave synthesis method was succesfully applied for longer, aggregated peptides. microwave coupling and cleveage were accomplished in a dedicated reactor setup that allowed accurate internal reaction temperature measurment using a fi ber-optic probe system. comparison studies between microwave-and conventionally heated reactions will be presented. during the last few years microwave assisted peptide synthesis has became popular and it was reported to be useful in some cases. there is also an ongoing discussion about an additional "microwave effect". based on this background we have synthesized several model peptides on a microwave synthesizer and compared with the synthesis on conventional batch and continuous fl ow synthesizers with and without microwave irradiation. during this investigation we have also compared reaction rates and purity of the peptides synthesized under the infl uence of microwave irradiation or conventional heating on this different synthesizer systems by various conditions. in no case we can fi nd any additional postulated "microwave effect". the results of this investigation will be presented and discussed. conotoxins form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. the subgroup -conotoxins specifi cally and selectively bind to subtypes of nicotinic acetylcholine receptors (nachrs), which are targets for treatment of several neurological disorders. the aim of this work is to develop an improved method able to generate conotoxins in high yield and purity. this will overcome a key barrier currently preventing the effi cient synthesis of small focused libraries in order to investigate the structureactivity relationship (sars) of those peptides. the development of general synthetic strategies for the preparation of conotoxins and analogues are essential to effi ciently approach important questions within the area of neurobiology and for the development of novel drugs for treatment of various neurological diseases. a new and highly effi cient synthetic strategy for the synthesis of alpha-conotoxin-mii has been developed. this strategy combine solid phase synthesis with microwave assisted heating to produce the two disulfi de bonds peptide in high yield and purity. we fi rst report here the use of microwave assistance in order to form a disulfi de loop. this technique demonstrates the advantage of preparing the fi rst disulfi de bridge while the peptides are resin bound. this step is critical to provide the dicyclic native peptide, that was performed by a followed classical in solution oxidation by iodine strategy. an enhanced total procedure for the synthesis of ctxmii is recommended, which can be effi cient applied at several small disulfi de rich peptides of biological importance. solid phase peptide synthesis in aqueous environment using microwave assistance heating galanis, athanassios s. since the fi rst reports on the use of microwave heating more than years ago, microwave-assisted organic synthesis (maos) has become an important tool for rapid and effi cient synthesis of organic molecules. microwave assisted heating has been further applied to peptide synthesis in order to accelerate the rate of synthesis and to improve the yields for the synthesis of diffi cult sequences. as far as water as solvent is concerned, numerous recent publications report the combination of water as an environmentally benign solvent for chemical transformations with the use of microwave irradiation as an effi cient heating method. we report herein, the combination of the microwave-assisted heating and the use of water as solvent for solid phase peptide synthesis. a variety of common amino acids derivatives and coupling reagents have been studied in order to optimize coupling reactions in water by microwave-assisted heating. we also describe the total synthesis of a small peptide using water as solvent. the solid-phase synthesis using the environmental friendly aqueous medium dramatically reduces the cost of the synthesis and could be broadly applied in research or in industrial production of peptides. vanier, grace; collins, jr., michael; singh, sandeep; collins, jonathan cem corporation, united states the application of microwave energy for solid phase peptide synthesis (spps) represents a major breakthrough for overcoming incomplete and slow reactions typical of conventional spps. microwave energy has been applied successfully in a manual and automated approach for enhancing synthesis of peptides and peptidomimetics. common side reactions such as racemization and aspartamide formation have been studied and shown to be easily controllable with optimized methods that can be applied routinely. we will present the latest research on improving the synthesis of diffi cult peptides with microwave energy. synthetic antifreeze glycopeptides and analogues: synthesis, structural analysis, and functional studies norgren, anna s. glycosylated peptides are involved in various biological processes such cell adhesion and differentiation. in contrast to mucin-type oglycan peptides which are present on the membrane of mammalian cells antifreeze glycopeptides (afgps) are a little investigated example of glycosylated peptides containing similar components. afgps usually consist of a varying number of repeating units of (ala-ala-thr) with minor sequence variations and the threonine hydroxyl oxygen glycosylated with the disaccharide -d-galactosyl-( - )--n-acetyl-d-galactosamine. antifreeze activity has been proven by different experimental observations like suppression of recrystallisation and ice nucleation, thermal hysteresis and change of the crystal habitus. although it is known that the n-acetyl group at the c position of the galactosamine, the -confi gured glycosidic bond to the threonine hydroxyl group and the -methyl group are essential, the adsorption mechanism is not yet understood. ( ) . in contrast to fragment condensation, solid phase peptide synthesis gives the possibility to prepare one defi ned product and to introduce structure inducing amino acids such as proline. the disadvantage of spps with the bulky glycosylated amino acids is the low coupling effi ciency. this problem was overcome by using more active coupling reagents and microwave-enhanced methods during coupling leading to suffi cient coupling effi ciency without having to apply great excess of the glycosylated amino acid. after purifi cation the peptide structure was examined by cd and nmr in water and dmso at different temperatures. additionally, the substances were microphysically analysed according to their recrystalisation inhibition activity and their infl uence on the crystal habitus. microwave technology applied to spps has been recently proposed as valid support to the enhancement of coupling rates. we also used microwave energy in conjugation process between polyelectrolytepeptide bioconjugation with edc, hbtu. the use of peptide epitops of viruses particles in the composition of polymeric conjugates as vaccine has several potential advantages over whole viral or bacterial preparation. recently, our group report a novel approach to a totally synthetic vaccine which consists of fmdv (foot and mouth disease virus) vp peptides, prepared by covalent conjugation of peptide biomolecules with membrane active carbochain polyelectrolytes in the present study, peptide epitops of vp protein both - (p ) amino acid residues (ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) and triptophan (trp) containing on the n terminus - amino acid residues (trp- - ) (p ) were synthesized by using the microwave assisted solid-phase methods. synthesis of peptides were performed by microwave assisted spps. peptides characterized by lc-ms and purifi ed by rp-hplc. bioconjugation between polyelectrolytespeptide were synthesized by two different microwave assisted method. the fi rst one is classical carbodiimid activating method. the second one is hbtu activating method. the second method is a novel and effective method for bioconjugation process. peptides and polyelectrolytespeptide bioconjugates analysed and comparised by gpc system with four dedector (uv. refractive index, light scattering, viscosity). microwave-assisted solid-phase synthesis of peptide probes to detect specifi c biomarkers: shifting off limitations affecting conventional synthetic strategies biomarkers play a key role in development of diagnostic/prognostic tools. in fact, since their involvement in several diseases such as autoimmune diseases (i.e., multiple sclerosis, rheumatoid arthritis) and neurodegenerative diseases as alzheimer's disease, biomarkers represent a great promise for the development and set up of tuned therapies. for autoimmune diseases, we proposed the development of synthetic posttranslational modifi ed peptides as antigenic probes for characterization of specifi c and high affi nity autoantibodies as biomarkers. by an innovative "chemical reverse approach" we propose optimisation of antigenic probes by a statistically signifi cant screening of sera guided by autoantibodies circulating in patients' biological fl uids ( ) . spps via the building block approach is the principal strategy leading to modifi ed peptides. high purity is a condicio sine qua non for effi cient biomarker detection. these syntheses can present several diffi culties as result of internal aggregation of resin-bound peptides during elongation steps, reducing reagents penetration, and signifi cantly decreasing reaction rates in both acylation and deprotection steps. such events strongly affect purity of the crude peptides and therefore, fi nal yield. we demonstrated that application of microwave (mw) energy in spps of modifi ed complex peptide probes exhibits several advantages improving coupling rates, possibly because of the decrease of chain aggregation during the synthesis ( ) . we report the mw-assisted synthetic conditions of diffi cult peptides (i.e. glycosylated, citrullinated, multiple antigenic, amyloidogenic peptides, etc.) by which we strongly improved preparation of diagnostic/prognostic probes in terms of crude purity, fi nal yield, and time-consuming compared to conventional protocols. fidan, zerrin; volkmer, rudolf charité berlin, institute for med. immunology, germany the alpha-helical coiled-coil structure is a versatile protein-interactiondomain, in which the coiled-coil is composed of at least two righthanded amphipathic alpha-helices, which are coiled up around each other into a left handed supercoil. this widespread structural motif is involved in many biological procedures like transcription, scaffolding or signalling. in addition, the most characteristic and important identifying feature of a coiled-coil is the recurrence of a periodic heptad repeat sequence. coiled-coils are able to form complexes up to heptamers of different orientation. based on this signifi cant occurrence and also their structure coiled-coil peptides have the ability to function as molecular recognition molecules. our goal is to use self-associating coiled-coil sequences as molecular building blocks (lego brick). the ligation of desired functionalities on coiled-coil sequences opens up the opportunity to add different functionalities on artifi cial complex peptide molecules that stick together via coiled-coil moieties. this makes coiled-coil sequences to useful tools during complex oligopeptide assembly. we want to present novel chimeric oligopeptides which are build up by two segments, one responsible for association the other for functionality. as a model for the association segment several gcn -leucine zipper mutants ( ) and as functionality segments amongst others ww domains were used. both segments were linked by the native chemical ligation approach. we will present in detail the synthesis of a gcn -leucine zipper mutant and also the following c-terminal thioester formation for the subsequent ligation step. furthermore, we will present the synthesis of the functional device, a ww-domain with a n-terminal cystein residue and also the native chemical ligation of both synthesized peptide fragments supported by hplc and mass-chromatography. dialysis-related amyloidosis, a disease arising from long-term dialysis is characterised by gradual accumulation of -microglobulin ( m) amyloid fi brils in bones and ligaments. although -m is known to form amyloid fi brils in vitro under acidic ph conditions, seeding of preformed amyloid fi brils or as an effect of ionic strength, the mechanism underlying aggregation of soluble -m into insoluble fi brils under physiological conditions is largely unknown. interestingly, eakin and co. recently showed that the chemical basis of the amyloidogenesis process is a backbone isomerisation of the conserved pro . our aim is to understand the molecular mechanism associated with -m amyloidogenesis using fourier transformed infrared (ftir) spectroscopy and site-directed-isotope labelling, a method in which the vibration of the labelled residue is shifted from its original position in the spectrum. we already demonstrated that ftir spectroscopy along with site directed-isotope-labelling is a promising approach to obtain information on the microenvironment of tyrosine side chain residues. to use this approach in the case of -m amyloidogenesis, we decided to synthesise an isotopically labelled -m at crucial residues such as pro that should undergo drastic variations in their microenvironments during the misfolding. with its residues, -m is over the limits of a reasonable synthesis using spps but the two suitably positioned cysteines in its sequence offer the possibility of a chemical synthesis using native chemical ligation. thus, we were able to realise the chemical synthesis of an isotopically labelled -m in a good yield using a three segments strategy with disconnections at the two cysteines and thiazolidine as a masked cysteine for the middle segment. the synthetic -m obtained was in full agreement with the characteristic -m ftir spectra. a novel cyanophycin synthetase from thermosynechococcus elongatus bp- catalyzes non-primer-dependent cyanophycin synthesis. cyanophycin (multi-l-arginyl-poly[l-aspartic acid]) is synthesized by cyanophycin synthetase (cpha). it was believed that cyanophycin synthetase requires asp, arg, atp, mg + and primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of low-molecular mass cyanophycin. despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear. in the present study, we searched for a cyanophycin synthetase that synthesizes cyanophycin from asp and arg without added primer in vitro. cyanophycin synthetase from thermosynechococcus elongatus bp- (tlr protein) was produced by an escherichia coli geneexpression system as a c-terminal his-tagged protein. we found that tlr protein synthesized cyanophycin without added primer. the tlr protein had strict substrate specifi city and used only asp and arg as substrates. the optimal ph was . , and mg + or mn + was essential for cyanophycin synthesis. atp could not be substituted by gtp, ctp, or ttp. the molecular mass of the tlr protein as estimated by gelfi ltration chromatography was } kda. thus, the tlr protein appeared to be a homo-tetramer of -kda subunits including the his-tag sequence. the tlr protein had thermal stability and fully retained its activity after a -min incubation at ‹c. additionally, we examined cyanophycin synthesis at ‹c, ‹c, ‹c, and ‹c. sdspolyacrylamide gel electrophoresis showed that the molecular mass of cyanophycin increased with increased reaction temperatures. synthesis of peptide thioester by fmoc chemistry through hydroxyl side chain anchoring barta , ) . azapeptides are usually synthesized on solid phase. a method, permitting the convergent synthesis of azapeptides starting from unprotected fragments, would offer the possibility to study aza amino acids effect in complex polypeptides or proteins. we have focussed our study on ligation reactions leading to the formation of an azagly residue at the ligation point, as gly is a frequent amino acid in peptides or proteins. the fi rst synthetic strategy relies on the reaction between a peptide thioester and a n-terminal azaglycine peptide. experimental conditions were found which permitted the chemoselective formation of azapeptide but with racemisation of the c-terminal amino acid of the thioester fragment. alternately, a synthetic strategy based on the chemistry of the phenylthiocarbonyl group, permitted the successful synthesis of azapeptide without racemisation. ( ), native glycoforms are of therapeutic interest. our retrosynthetic strategy implies sequential native chemical ligation ( ) and the split of il- into three fragments. an n-terminal fragment il- ( - ) thioester and the cysteine fragment il- ( - ) were expressed in e. coli. the carbohydrate carrying fragment il- ( - ) was synthesized via spps. to obtain the recombinant fragments specifi c termini were required. the n-terminal il- ( - ) was fused between two inteins and after expression and refolding from inclusion bodies the target peptide was released as a c-terminal thioester after dual intein cleavage. the cysteine fragment il- ( - ) was expressed as a single intein fusion also leading to inclusion bodies. this enabled the release of il- ( - ) with an n-terminal cysteine after intein cleavage of the refolded fusion protein. the central segment il- ( - ) contains a sugar residue attached to the n-glycosylation-site at asn . this fragment features both a c-terminal thioester and an n-terminal cysteine protected as a thiazolidine. sequential ligations leading to the full length il- glycoprotein will be presented. synthetic chemistry syntheses of shorter fragments which are fi nally linked together ( ) . this "small building block approach" should also simplify synthesis of modifi ed prion protein. in our plan of mouse prion (moprp) synthesis, we have employed consecutive chemical ligations. we have started with moprp( - ) in which it is possible to study native chemical ligation between cysteine in the position of and methionine in the position of ( ) several moprp( - ) peptide thioesters with aryl and alkyl thiols were prepared for further study of the native chemical ligation of moprp( - ) and moprp( - ). the optimal ligation conditions were found by a kinetic study which was carried out in a range of ph and with various thiols. infl uence of electron withdrawing and electron donating groups was studied in both aromatic and aliphatic thiols and it was found that it is possible to affect the ligation by choosing an appropriate thiol. ligation optimal conditions, kinetic study results, and suitability of various thiols for the ligation are discussed. low-cost industrial chemo-enzymatic synthesis of pharmaceutical, nutraceutical and diagnostic oligopeptides the production costs for oligopeptides and derivatives thereof by chemical synthesis are extremely high. therefore dsm pharmaceutical products embarked on a research programme on chemo-enzymatic peptide synthesis. important advantages of this technology are that no expensive stoichiometric coupling reagents nor side-chain protection are required and that no racemisation occurs. focus is on elongation in the n ¨c terminal direction using novel enzymatic c-protecting group interconversion methods. one of the preferred building blocks are amino acid amides, but selective deprotection of peptidic c-terminal amides is a challenge. we discovered that using a peptide amidase c-terminal peptide amides can be directly converted to methyl esters in almost pure methanol. thus, separate enzymatic hydrolysis and reactivation steps are no longer required. other versatile building blocks are amino acid t-butyl esters. we discovered that peptide c-terminal t-butyl esters can be transformed, using commercially available proteases, to activated alkyl esters such as methyl-and benzylesters which can be directly used in another protease-mediated coupling reaction. furthermore, we found that using commercial enzymes side chain unprotected peptides with a free c-carboxyl terminus can be directly transformed to cterminal thioesters and c-terminal aryl amides in the presence of the corresponding thiol and aniline, respectively. finally, a real-life example of large-scale industrial chemo-enzymatic peptide production will be shown. it is known that chiral amino acids, as well as their dipeptides, may catalyze the asymmetric condensation of glycolaldehyde in water. on the basis of the particularly large erythrose enantiomeric excesses (ee) obtained when utilizing the chiral l-val-l-val catalyst and given the possibility of an abundant delivery of other types of amino acids to the early earth, we have studied the catalytic effect on this synthesis of the peptides based on c -methylated -amino acids, such as iva (isovaline or c -methyl, c -aminobutyric acid) and c -methylvaline, ( me)val, that are abundant in meteorites. results of the catalysis experiments showed the all c -methylated peptides to the tetramer level exhibit signifi cant chiral infl uence on the synthesis of tetroses and mimic the effect of the l-val-l-val catalyst in having a larger erythrose ee than threose ee, as well as in their confi guration relationship with the sugars (the product erythrose acquires ee of confi guration opposite to that of the catalyst in case of peptides, while it is the same for amino acids). interestingly, the largest ee ( % for erythrose) was obtained with the iva homo-tetrapeptide under mild conditions. the homo-dipeptides of both iva and ( me)val also produced a signifi cant ee ( % for erythrose) that appears to increase with time. because c -methylated amino acids are non-racemic in meteorites, do poster abstracts not racemize in aqueous environments, and are known to be ( )-helix formers in peptides with as few as four residues, these results suggest that meteoritic, c -methylated, -amino acids may have contributed to molecular evolution upon delivery to the early earth by catalytically transferring their asymmetry to other prebiotic molecules. synthesis' and application of peptide adhesives for wound healing natural polymers with repeating peptide sequences, like spider silk and mussel glue have interesting properties that can be used for biomedical applications. however, the isolation from their natural sources is rather diffi cult and the desire to introduce modifi cations necessitates the development of novel methods to synthesize such polymers with repeating peptide sequences. recently, we have shown that the microwave-assisted copper(i)-catalyzed , -dipolar cycloaddition can be used in the polymerization of the model dipeptide azido-phe-alapropargyl amide. by varying the reaction conditions we could either obtain small cyclic oligomers ( - aas) or linear polymers which consisted up to aas residues ( ). to broaden the scope of this polymerization reaction, two novel biodegradable monomers, azido-phe-ala-lys-propargyl amide and azido-phe-ala-glyc-lys-propargyl amide were polymerized. both monomers are water-soluble and contain recognition sites for the proteases trypsin and chymotrypsin; moreover, the tetrapeptide can be chemically hydrolyzed depending on the ph of the solution. the molecular weight of the polymers could be tailored between - kda ( to aas residues). the enzymatic degradability of the polypeptides was monitored by a ninhydrin-based colorimetric assay and by maldi-tof. the results showed that the peptidic polytriazoles were smoothly degraded by trypsin and chymotrypsin. from these data we can conclude that the microwave-assisted copper(i)-catalyzed , -dipolar cycloaddition reaction is an effective tool to synthesize biodegradable functional polymers which provides new opportunities to design (novel) biomedical materials. details of monomer synthesis, polymerization reactions and degradation studies will be presented. peptide synthesis on cellulose membranes, known as spot synthesis, is an ideal tool to determine biological interactions and/or key positions in proteins. in general we can differ in two kinds of synthetic peptides with different possibilities: over the past years synthetic peptide library techniques have emerged as powerful approaches to determine t-cell epitopes and to specify peptide binding to mhc-i and/or mhc-ii molecules. the sequence-based approach is a straightforward method to directly identify t-cell antigens belonging to a specifi c target (e.g. a virus of interest) without the need of any further deconvolution steps. the entire protein sequence is presented as a set of overlapping peptides (peptide scan), which are subsequently screened for t-cell stimulation. several cd- t-cell epitopes in selected cmv proteins have been identifi ed using standard spps techniques. however, the peptide sequences synthesized by standard spot synthesis can only be cleaved without authentic c-termini (ß-alanine or glycine as c-terminal amino acid). here, we summarized three different established methods to generate cleavable peptides with authentic c-termini in adequate amounts, with suffi cient purity and within a justifi able time-scale. the standard spot synthesis to generate membrane bound peptides use glycine or ß-alanine membranes to map epitopes of b-cells, allergenic proteins, antibodies etc. furthermore, we have also established the "method of inverted peptides" to screen protein domains requiring peptidic ligands with free c-termini such as pdz-or . . domains. another highlight of peptide libraries is the screen of posttranslational modifi cation in proteins by phosphorylation at serine, threonine, and tyrosine residues which plays a role in eukaryotic cell cycle regulation, cell differentiation, apoptosis, or cytoskeletal regulation. labeled peptide libraries, consisting of -helices, -loops and -sheets peptides, have been successfully prepared for use in peptide arrays that act as a protein-detection system and have been applied for proteinrecognition studies [ ] [ ] [ ] . it is known that more than half the proteins in a mammalian cell are post-translationally modifi ed by such processes as phosphorylation, glycosylation, and acetylation. such modifi cations play an important role in various bio-recognition, for instance glyco- proteins are involved in cell-adhesion, infection, and biological protection. hence the designed peptide library used for arrays has been expanded to include fatty acid attached peptides and glycopeptides. fatty acids were easily introduced by conventional fmoc-spps, while the synthesis of glyco-peptides was not easy. in the present paper we describe the effi cient construction of o-glycoside structured and labeled peptides by fmoc-spps using a building block strategy. glycosylated threonine has been used as a building block and was prepared in large amounts. glucose, mannose, galactose and lactose were acetylated and coupled to the hydroxyl group of fmoc-thr in the presence of bf oet . the resulting protected sugar-amino acids were manually assembled on to the peptidyl resin containing a fl uorescent dye using a peti-syzer® (hipep laboratories). after construction of the glycol-peptides the acetyl groups on the sugar were removed by hydrazine hydrate, and the peptides were then cleaved and purifi ed. the resulting glycopeptides were characterized by lcms. the present protocol has been used to prepare ca. one hundred glycopeptides that have been tested against various carbohydrate-binding proteins. parallel small scale peptide synthesis meets a fast, lowcost purifi cation method for the production of high quality peptide microarrays to analyze dna/protein interactions herein, a parallel synthesis in a small scale ( , mol) which takes place in -micro well plates is demonstrated. with regard to a fast but yet effi cient, low-cost purifi cation in a parallel manner a ''fmoc-on'' purifi cation method, which is integrated into the synthesis process, was developed. peptides were cleaved from the resin with their fmoc-group still on. the crude products were transferred by extraction into another -micro well plate fi lled with purifi cation material. purifi cation takes place due to the high affi nity of the terminal fmoc-group to this material. hplc measurement shows a high recovery ( %) and purity ( %). cleavage of the fmoc-group during the purifi cation process is also possible. here, hplc measurement demonstrated a recovery of % and a purity of %. a fully automated synthesis of more than peptides in a -micro well plate combined with a parallel ''fmocon'' purifi cation is shown. the peptides were used for the production of microarrays to analyze dna/protein interactions. the "fmoc-on" purifi cation allows easy and cost-effi cient purifi cation of peptides for all kind of biological applications. is an antibiotic-macrolide related to a group of tylosin (tyl) which structure is based on -member lactone with carbohydrate substitutes attached. macrolides are well known translation inhibitors, they bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft; hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. recently we have designed and synthesized a number of peptide derivatives of macrolides where the peptide part modeled the growing chain, while the antibiotic served as an "anchor" for positioning the peptide at the specifi c site of rt. these derivatives are of interest both as antibacterial agents and as potential probes for investigation of the interactions of nascent peptide chain with the specifi c sites of the rt and their infl uence on the translation. now we report a new type of peptide -macrolide conjugates in which peptide binds by its -amino function through a spacer to the '-hydroxyl group of the mycaminose residue of des. two proline-containing peptides are chosen: gp and ggp. proline residues of these peptides are supposed to interact with the peptidyl transferase center region when the complexes of these '-peptidyldesmycosins with bacterial ribosomes are formed. the fi rst step of the synthesis was acetylation of '-, "-, "and "'-oh-groups of tyl by ac o in pyridine following by hydrolysis in n sulphuric acid resulted in des with all protected oh-groups except '-oh-group of mycaminose. this free hydroxyl group was used for reaction with succinic anhydride leading to formation of reactive carboxyl group. the next step was a reaction between this carboxyl function with -amino groups of peptide methyl esters. the structures of new '-peptide derivatives of des were proved by ms maldi and nmr-spectroscopy. synthesis . ]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [ ] [ ] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as esi lc-ms, uv and atr ft-ir spectroscopy. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. atr ft-ir spectra of pc and pc-hemagglutinine peptide was recorded. for peptide synthesis we have used microwave assisted solid phase peptide synthesis method. lc-ms was used to determinate of molecular weight of hemagglutinin. after we have obtained m/z values of peptide we used preparative hplc for purifi cation of hemagglutinin. after synthesis and modifi cation of pc, its covalent conjugates with hemagglutinin (ha - ) were obtained by carbodiimide activation. its characterization was also performed. the peptide coupling reagent fi eld has clearly evolved in the last decade from carbodiimides to onium (phosphonium and uronium) salts. the era of industrial coupling reagents began in with the introduction of dicyclohexylcarbodiimide (dcc), which at that time was already known and well studied, as a reagent for the formation of amide bond. unfortunately, carbodiimides did not comply with the concept of ultimate coupling reagents because its high reactivity provokes racemization and side reactions during the coupling reaction. at the beginning of the 's, -hydroxybenzotriazole (hobt) was proposed as an additive to dcc to reduce racemization and from then on other benzotriazole derivatives such as -hydroxy- -chlorobenzotriazole (cl-hobt) or -hydroxy- azabenzotriazole (hoat) have also been used. the obt active esters are less reactive than the o-acylisourea, but are more stable and less prone to racemize. the use of the most reactive aminium salt, hatu , is inconvenient because of the price, which makes its use detrimental for industry. hctu/tctu, based on cl-hobt, are a good alternative to hbtu/tbtu, because of the presence of the chlorine atom that stabilizes the structure, hence, making these reagents less hazardous. herein, pyclock, the phosphonium salt of the cl-hobt is introduced p - n-methylation of biologically active peptides is of interest because it results in increased conformational integrity and stability against enzymatic degradation. furthermore, n-methylated peptides have a decreased ability to form h-bonds to water molecules and, consequently, a better ability to cross biological barriers. this is exemplifi ed by the naturally occurring peptide cyclosporine which is orally active. in an effort to improve the blood-brain barrier permeability of the cyclic enkephalin analogues h-dmt-c[d-cys-gly-phe-d(or l)-cys]nh (dmt = ', '-dimethyltyrosine), we prepared analogues that were n-methylated at phe and/or cys . n-methylated cys derivatives were prepared either by direct methylation (ch i)/nah) or by using oxazolidinone chemistry. single n-methylation of the two cyclic peptides at phe or d(or l)-cys produced four analogues that all showed very high mu and delta opioid agonist potencies in the guinea pig ileum and mouse vas deferens assays. the two analogues n-methylated at both phe and d(or l)-cys also retained high agonist activity at both receptors. results from molecular mechanics and molecular dynamics studies on the conformational behavior of these peptides will be presented. during the last years the number of deaths due to hemostatic impairments such as coronary angioplasts, coronary thromboembolisms, myocard heart attack, pulmonary embolism etc. has become equal to those caused by cancer formations. haemostasis is a key process whose correct functioning is an important defence mechanism of the human organism. it is a blood coagulation process activated in case of injury of the blood system. if it is functioning correctly vascular-motor and cell reactions are triggered and the blood coagulation cascade is activated. one of the most important enzymes in the blood coagulation cascade is factor xa. that's why its inhibitors are promising alternative against thrombotic disorders. in our previous work, we reported the synthesis of hybrid structure between isoform and of antistasin and the active sequences d-phe-pro-arg; d-arg-gly-arg; phe-ile-arg and tyr-ile-arg ( ). beside the analogues with c-terminal cooh group, the peptide d-phe-pro-arg-pro-lys-arg-nh was synthesized. the biological activity of the last one was times bigger than natural isoform of ats and some times more active than all other synthesized analogues. in the current work we described synthesis and biological activity of c-terminal amide analogues of all early synthesized peptides in order to deduce the structure-activity relationship. the anticoagulant activity according to the aptt and ic was determined. the neo-angiogenesis is the formation of new blood vessels from a pre-existing vasculature which enables the supply of the nutrients and oxygen necessary for tumor growth. consequently, inhibiting the blood vessels sprouting in order to starve the tumour constitutes an attractive anti-tumoral therapy. the vascular endothelial growth factor or their receptors (vegfr- and ) are key mediators of neo-angiogenesis and constitute nowadays validated targets for anti-angiogenic therapies ( ) . despite this fact, strategies aiming to develop pure receptors antagonists and not inhibitors of the tyrosine kinase activity remain scarce. we have previously designed antagonists that interact with the extra-cellular domain of vegf receptor and thus prevent vegf binding ( ) . these antagonists were proved to be potent and able to interfere with the vegf signalling pathway. because these compounds were rationally designed by using the co-crystallized structure of vegf with the immunoglobulin like fragment of vegfr- , we raise the following question: do these antagonists really target the immunoglobulin like domain of vegfr- ? in order to respond to this question but also with the future aim of optimizing our antagonist in a rational way we need to co-crystallize at least one of our antagonists with the d fragment of the vegfr- . furthermore, since human vegfr- d is a potential target in the development of angiogenesis modulators, the availability of this protein domain, and mutants, should be useful in the screening and development of novel specifi c ligands. up to now, the -amino acid polypeptide chain of vegfr- d has been only produced by gene expression ( ) . here, we report the fi rst solid phase peptide synthesis of the vegfbinding domain of the vegf receptor and the in vitro experiments which permitted to verify its biological activity. chung, nga n. different types of turns are important elements of secondary structure in peptides and proteins. the most common ones are different kinds of -turns involving four consecutive amino acid residues. the dipeptide unit containing the second and the third residues of such turns exists in the cis-conformation. our cispeptide bond motif -aminopyroglutamic acid promotes the -turn type vi/vi', and can be treated as a hybrid of glycine and alanine. this feature prohibits its utilization as a replacement for any possible dipeptide unit. in order to convert our compound into a more valuable tool for probing the existence of a particular peptide bond in the cis-conformation, we have elaborated the synthesis of nmonoalkylated derivatives of -aminopyroglutamic residue through a reductive alkylation reaction performed on solid support. following this idea, we have obtained analogues with n-benzylated and n-(phydroxy)benzylated -aminopyroglutamic residues as scaffolds for the phe-ala and tyr-ala dipeptides units, respectively. only one of enkephalin analogues, [n(bzl)-(r,r)-apy - ]-enkephalin amide, possesses similar agonist potency as leu-enkephalin in the gpi assay, indicating that the c-terminal part of this peptide may assume the cisconformation upon binding to the receptor. this is the fi rst active opiod analogue containing a -aminopyroglutamic acid residue. supported by kbn (poland) grant p f and nih (u.s.) grant da- mif- (l-prolyl-l-leucyl-glycine amide, plg) is a brain peptide, presented in hypothalamic tissue, as well as the c-terminal tripeptide of the neurohypophiseal hormone oxytocin and inhibits the release of melanocyte-stimulating hormone (msh) in some systems. recently, some chemical modifi cations of mif- to enhance opiate agonist/ antagonist actions as well as binding activity of analogues have been reported. on the other hand, the concept of structural modifi cation in peptide fragments to confer them specifi c properties is of current interest in the study and design of new bioactive targets. a well known example is the incorporation of unnatural amino acids into the molecule of natural biologically active peptides leading to analogues with signifi cant theoretical and practical importance. with this idea in mind we studied possibilities of introducing unnatural amino acids canaline (can), norcanaline (ncan), canavanine (cav), nor-canavanine (ncav) and slys into mif-moiety in order to achieve a better analgesic effect. to obtain the peptide mimetics, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the paw-pressure (pp) and hp tests. the experiments were carried on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the randall-selito paw pressure test using analgesimeter (ugo basile). it was found that substitution of leu in position of mif-molecule by unnatural amino acids increased the pain threshold. the analgesic effect with cav-substitution was highest, whereas parent mif- showed only a minute increase of pain-threshold. the compound m had the strongest effect on learning and memory and the compound p showed the strongest and fastest analgesic effect. the compound m and p were also able to modify the effect of the model cns-drugs (hexobarbital and pentylenetetrazole). theoretically and experimentally determined octanol/water logp values of the compounds correlate with their cns-effects. m and p had higher logp than m and p and showed better antinociceptive and anticonvulsant activity in vivo. compounds possessed also chelating activity towards fe ions. the stronger chelating activity of the compounds with the -spacer clearly correlated with their better neuropharmacological activity in vivo (in comparison to the compounds with the -spacer). the position isomery may also contribute for the variations in their pharmacological activity. the limited number of the compounds does not allow derivation of well-defi ned structure-activity relationships, however, their d models show possibility for many low energy conformers with different atoms involved in formation of fe chelating complexes. a major challenge in opioid peptide chemistry is the synthesis of novel compounds mimicking the endogenous peptide ligands. these new peptidomimetics should be biologically active and more stable against enzymatic degradation than their parent ligands. one of the possibilities is the introduction of -amino acids into the peptides sequence. monosubstituted -amino acids ( -or -) due to their similarity in structure to -amino acids, moreover their tendency to give folded structures even in short peptides and to the stability towards mammalian peptidases may be very useful in the creation of the new potentially active compounds. we have developed simple and effi cient two step conversion of the cyanoacetate into fully protected -amino acids. the procedure involves knoevenagel condensation of the methyl cyanoacetate and aromatic aldehydes (for aromatic path) or alkylation of the methyl cyanoacetate with various alkyl halides (for aliphatic path) at fi rst then reduction and boc-protection (performed in one pot) of the resulting fi rst stepproducts. we focused our attention on applying above method to the synthesis of different -amino acids (as homologues of -amino acids) as elements for the synthesis and structure -activity relationship study of endomorphin analogues. small library of analogues has been created in which -amino acids in every position with exception of pro were substituted by their respective -analogues. as templates, endomorphin- (tyr-pro-trp-phe-nh ), endomorphin- (tyr-pro-phe-phe-nh ) and its d-ala -analogue (tapp) have been used. in this communication the pharmacological consequences of such modifi cation in endomorphins will be discussed. solid-phase synthesis and effects of amino acid and peptide analogues of non-protein amino acid canavanine on nociception non-protein amino acids have been widely used as components of peptides to enhance biological activity, proteolytic stability, and bioavailability. it is well known that unnatural amino acids with guanidine functionality exhibit diverse pharmacological effects when introduced in biologically active systems. our previous efforts were focused on the preparation and the characterization of unnatural amino acids, particularly those containing a basic functionality in the side chain. we have synthesized numerous unnatural amino acids, structural analogues of arginine and lysine, which demonstrated certain biological effects. recently, as part of our ongoing research focused on the search of novel arginine mimetics, we developed an effi cient approach for solid-phase synthesis of unnatural amino acids nor-canaline (ncan) and nor-canavanine (ncav). next we studied the possibilities of introducing ncan and ncav into the molecule of biologically active peptides. we also studied their antinociceptive effects using the paw pressure (pp) and hp tests. in the last few decades, considerable attention has been devoted on the potential role and activity of certain environmental pollutants (edcs) in increasing anomalies that involve the endocrine system of wild species and man. ( )the development of a fast high-throughput detection system for the quantitative analysis of edcs requires the development of a novel sensitive solid phase edcs extraction method. because of the high affi nity of edcs with the estrogen receptor, this has been greatly investigated. this study has leaded the recognition of the amino acids located in the ligand binding domain of the receptor which interact with edcs. use of a dipodal scaffold molecule and different combination of the crucial amino acids, will allow the generation of a library of small to medium size biomimetic receptors. in this communication, we will disclose our fi rst results in the preparation of a small library of biomimetic receptors, with and without the dipodal scaffold, to evaluate their affi nity towards edcs and the role played by the scaffold structure. romeralo tapia, rosa ; van der eycken, johan ghnet university, belgium; ghent university, belgium endocrine disrupting chemicals (edc's) are an important class of pollutants, which have in common that they show affi nity for the hormone binding domain of the estrogen receptor, thus disturbing the endocrinal system. detection in waste water has not been succesful due to their low concentration. therefore, new sensitive screening methods are highly demanded. a possible solution is the use of estrogen receptor mimics for affi nity chromatography. to this end, scaffold will be synthesized and used for building a tetrapodal peptidomimetic library. amino acids known to be important for the estrogen-receptor interaction will be preferably incorporated. first of all, we set out to prepare one dipodal peptide library member . the orthogonally protected scaffold was synthesized in one single step starting from the commercially available amino- -nitrobenzoic acid. using fmoc solid phase chemistry on wang resin as the solid support, this dipodal scaffold allowed the attachment of two different oligopeptide chains.employing this methodology the synthesis of a small library will be performed, and the affi nity of the different peptidomimetics for edc's will be investigated. on-resin microwaves-assisted ring closing metathesis for the synthesis of octreotide dicarba-analogues di . octreotide is an antitumoral agent used mainly as a carrier of radionuclides for cancer diagnosis and therapy. it shows the same disulphide bridge of the parent srif that is prone to be opened by oxidizing and reducing agents. this prompted us to search a more stable tether bridging the active motif of the cognate molecule. the premier reaction of rcm was performed in an oil bath under severe experimental conditions i. e. anhydrous argon atmosphere and long reaction times [ ; ] . the microwaves assisted version was, instead, effi cient for the cyclopeptides yield and required very short reaction times. we also evaluated the effi cacy of different grubbs catalysts in the microwaves assisted rcm, operating in different conditions of temperature and time. in the search for potential antimicrobial drugs, we have been dealing with two microbial enzymatic systems: n á -succinyl-l-diaminopimelic acid desuccinylase (dape , ) and n á -acetyl-l-ornithine deacetylase (arge , ), which might be promising targets for potent and selective enzyme inhibitors based on the modifi cation of n á -succinyl-diaminopimelic acid (dap) and n á -acetyl-ornithine (orn). the inhibition of both the enzymes would possibly interrupt the pathways leading to development of bacteria due to a role of meso-dap as essential component of peptidoglycan based bacterial cell walls and orn, as well, being a component of biosynthetic pathway for arginine that can serve as a source of both the carbon and nitrogen in microorganisms. our effort was focused on the synthesis and characterization of two series of n á -substituted derivatives of dap and orn, potentially interfering with the hydrolytic action of dape and arge in the processes of bacterial growth. in this introductory study, the compounds prepared were also assayed against bacterial strains escherichia coli and bacillus subtilis and inhibitory activity of orn derivatives was found with regard to differences in the structure of n á -substituent. the fi rst report on practically effective bradykinin (bk) antagonists for b receptors was published in . the key to conversion of bradykinin into an antagonist was replacement of pro with an aromatic d-amino acid; d-phe was fi rst used. however, our studies demonstrated that the d-amino acid residue at position of the bradykinin antagonist, until recently considered to be necessary for b antagonism, can be replaced by suitable l-amino acid or achiral residue or, together with the amino acid occupying position , by a sterically restricted dipeptide unit. having all this in mind we synthesized and bioassayed two new analogues of bradykinin. the peptides were designed by substitution of position of bradykinin b receptor antagonist ([d-arg ,hyp ,thi , ,d-phe ]bk), previously described by stewart's group, with structural isomer of proline: -iso-pro or its homologue: -homo-pro. it's worth emphasizing that position in bradykinin molecule is occupied by proline residue. our previous results demonstrated the importance of the position in the peptide chain into which the sterically restricted -aminocyclohexane- -carboxylic acid residue (acc) was inserted. these fi ndings prompted us to investigate how introduction of l-pipecolic acid residue (l-pip) in position or of stewart's antagonist will affect pharmacological properties of resulting compounds. in comparison to the acc residue, the ring of l-pipecolic acid also consists of six atoms, but includes the nitrogen atom. bearing in mind that acylation of the n-terminus of several known b blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the aforementioned four analogues were also synthesized in the n-acylated form with -adamantaneacetic acid (aaa). the activity of eight new analogues was assessed in isolates rat uterus and in rat blood pressure test modifi cations of the myelin basic protein epitope mbp - divert th to th : immune responses in peripheral blood mononuclear cells (pbmc) from multiple sclerosis patients. postranslational modifi cations (citrullination, phosphorylation, deamidation, methylation, glycosylation) are common biological processes that alter specifi c parts of a protein after synthesis. nearly all known proteins undergo some form of postranslational modifi cation and almost all amino acids can be altered by one or more of these processes. the modifi ed protein thus, contains new or rare amino acids or new specifi c side groups that can have critical infl uence on the structure and function of the protein molecule. conversion of arginine to citrulline, an important postranslational modifi cation, was fi rst described by fearon. arginine residues in proteins can undergo this modifi cation and the resulting citrulline remains part of the protein in the position of arginine. citrulline is not a natural amino acid in proteins, and may induce immune responses. such responses have been recently implicated in the pathogenesis of autoimmune/infl ammatory diseases such as ms and rheumatoid arthritis . herein we investigated cytokine secretion in peripheral blood mononuclear cells (pbmc) of ms patients and controls, and attempted to correlate cytokine polarization with the nature of the antigenic stimulus. we synthesized peptide analogs that map to the myelin basic protein ( . analogs p and p resulted from the citrullination of the and arginine residues in epitopes p and p . we then tested ms and control pbmc with various concentrations of the peptides and investigated cell proliferation by the brdu proliferation assay and cytokine secretion by elisa. we suggest that citrullination of self-antigens maybe an important step in triggering disease in susceptible individuals. incorporation of aza- -amino acid into rfa( - ), the endogenous ligand of gpr : structural analysis. aza- -peptides, mixing -and aza- -amino acids (the aza analogs of -amino acids), represent a novel and exciting type of peptidomimetics. in particular, we have shown that aza- -amino acid induces a n-n or hydrazino turn, stabilized by an eight-membered-ring intramolecular hydrogen bond between the carbonyl acceptor group of the residue i- and the amide proton of the residue i+ . interestingly, this n-n turn promotes a well-defi ned -turn formation (hydrogen bond between the co of the residue i- and the hydrazidic proton of the aza- -moeity) when an -amino acid is foregoing. rfa, a novel neuropeptide of the rfamide superfamily, exhibits high affi nity for gpr and induces a potent orexigenic effect in mice. in biomimetic environment, rfa encompasses an -helix between pro and arg residues and a canonical -turn centered on ser . rfa( - ), whose sequence is strictly conserved across species, is about times less potent than rfa. this heptapeptide shows important distortions of the -turn that may be responsible for its weak potency. the aim of this study was to restore the -turn formation in rfa( - ) (ggfsfrf-nh ) by the presence of an aza- -amino acid. for this purpose, we have (i) synthesized the aza- -counterpart of each residue of rfa( - ), (ii) individually incorporate the surrogate into the heptapeptide and (iii) investigated the d structure under nmr restraints of the hybrid peptides. the results will be presented with a particular attention on the serine position. the analysis of the structure-antithrombotic activity correlation for peptide receptors of adhesive glycoproteins with the general formula arg-xaa-asp gribovskaya, olga; martinovich, vera; golubovich, vladimir institute of bioorganic chemistry, belarus structural analogues of the arg-gly-asp sequence with the general formula arg-xaa-asp, where xaa -ala, d-ala, -ala, -abu, -ak, pro, d-pro, asn-trp, were synthesized using classical methods of peptide chemistry in solution, the levels of their antithrombotic activity were determined and stable conformations were calculated using a pairwiseadditive approximation method. interatomic distances in the obtained conformers were calculated between various atoms in the functional groups, including carbons in the carboxylic groups, nitrogens in the -aminogroups, amino and imino nitrogens in the guanidine group, distances in total. the analysis of correlation of the calculated interatomic distances and measured values of antithrombotic activity was carried out using statistical methods. we show that the distance between the amino nitrogen of the guanidine group of arginine and the -carboxyl carbon of aspartic acid determines the antithrombotic activity. it has the optimum value in the arg--ala-asp peptide, which was the most active among the synthesized analogues of the arg-gly-asp sequence (ic - . mkm, adp, . mkm). microbial proteases with narrow specifi city as an instrument of peptide chemistry kotlova the problem of selective removal of short n-terminal peptides from the gene-engineered proteins is actual by many reasons. at fi rst it is connected with gene-engineering method of protein preparation in form of its precursors. the problem of n-terminal formyl-met removal from gene-engineered proteins, which are produced by bacillacae may be solved by introduction of specifi cally-cleaved insert after formyl-met. subsequent specifi c removal of such short n-terminal peptide results in mature protein. moreover, the analysis of cleaved short peptides could characterize the process of mature protein formation. we suggest microbial enzymes with narrow specifi city to solve the mentioned problem. for this aim we have isolated the trypsin-like enzyme and postproline-specifi c endopeptidase from aspergillus sp. and glutamyl-specifi c protease from b. intermedius. these enzymes have high specifi city approved by hydrolysis of short synthetic peptides and long polypeptides. for example, specifi city of postproline protease was established by hydrolysis of mellitin. the fi nal peptide mixture was analyzed by rp-hplc and mass-spectra. the use of enzymes with narrow specifi city is demonstrated in the analytic method of detection of formyl-met presence at the n-terminus of gene-engineered -interferon. trypsine hydrolysis of -interferon leads to more than peptides are formed including the -membered n-terminal peptide. being hydrolyzed with glutamyl-specifi c enzyme -interferon is converted to peptides, including -membered n-terminal peptide. application of postprolinespecifi c enzyme for -interferon hydrolysis leads to formation of short -membered peptide and long fragments. experimental data show that in case of analytic control of -interferon maturity the utilization of postproline-specifi c protease is preferable. in other cases the enzyme selection is ruled by the features of protein primary structure. bimodal action of cystatin related epididymal spermatogenic (cres) protein and its reactive site loop derived s-s bridge bicyclic peptides on proprotein convertase- (pc ) activity several precursor proteins found on sperm surface and reproductive tissues were proposed or confi rmed as substrates of pc . these include adam proteins, growth factors proigf and and hormonal protein pacap. lack of pc leads to impaired fertility in mice, suggesting its important role in reproduction. pc is thus considered an important target for development of nonhormonal contraceptive agents. pc -inhibitors are expected to fi nd therapeutic, clinical and biochemical applications. a lot of interest has grown to develop specifi c pc inhibitors. recently natural inhibitor of serpin family have been described for pc , , and furin, but not for pc . however, a new serpin, cres, has been reported from epididymis fl uid, where pc may be found. so far, cres has only been shown to inhibit pc , which is not present in reproductive tissues. we propose that cres may represent a natural regulator of pc , based on localization and other studies. in this study we generated recombinant human cres protein ( aa), and its reactive-site loop (rsl) derived acyclic and cyclic -mer peptides with various s-s linkage combinations. cres inhibited pc with ic ~ um, while rsl-peptides were found to be more potent with ic - nm, depending on the s-s linkage locations. furthermore, we noted that at lower concentrations, cres and its peptides fi rst enhanced pc activity before any inhibition occurred. this bimodal behavior may be due to cleavage. molecular modeling showed strong interactions between cres and pc via some of their key residues. overall, we noted that "rsl" is the most crucial element required for pc inhibition by cres. funds were from from cihr (ab). the de novo design of peptides and proteins has assumed considerable interest didehydroresidues, in particular alpha, in the recent years. alpha, beta-beta-didehydrophenylalanine (deltaphe) are being considered important conformational constraints inducing tools in de novo peptide design. deltaphe is a noncoded, achiral residue, an analog of the naturally occurring phenylalanine amino acid with a double bond between calpha and cbeta atoms. introduction of deltaphe in peptide sequences is known to induce conformational constraint, both in the peptide backbone as well as the side chain, and to provide the peptide with increased resistance to enzymatic degradation. in small peptides containing eta turn structure, and in peptides containing a single deltaphe, a type ii b more than one deltaphe residues, helical structures are stabilized. a number of deltaphe peptides varying in length, content and position of deltaphe residues have been found to contain helices of both screw senses. following these design principles, we were able to design, synthesize and characterize super secondary structural motifs like helix-turn-helix, helical bundle and glycine zipper. we have extended this work to the de novo design of peptides with antibiotic and anti-fi brillization activity. a series of cationic peptides containing deltaphe residues have shown remarkable antibiotic activity and are being developed further. deltaphe containing peptides may have longer in vivo half life owing to their ability to resist enzymatic degradation. more recently, we have observed that small peptides containing deltaphe self-assemble in nanotubular and nanovesicular structures. these nanostructures have also been used to entrap small drug like molecules. we have fully characterized these systems and are exploring their potential as delivery agents in biological systems. the synthesis and design of peptidomimetic oligomers that adopt designed, compact conformations (foldamers) have become a challenging task in recent years. among them, -peptide foldamers exhibit rich diversity of secondary structures. [ , ] a relationship has been established between the backbone chirality pattern and the prevailing secondary structure, which underlines the role of stereochemical control in the -peptide foldamer design.( ) an important challenge is to introduce proteinogenic side-chains to generate diverse anchor points on the molecular surface promoting their application in drug discovery. it has been proved that stable helices can be obtained when sequences were coupled with -amino acid enantiomeric pair motifs in a lego approach.( ) in this work, -amino acids and open chain functionalized -amino acids were inserted between the enantiomeric pair design element for and , respectively. confi gurations in the backbone were designed to promote helix formation. nmr and ecd measurements augmented with ab initio calculations revealed that forms a h - helix and forms a h / helix. these helices are unprecedented in the literature. to prove that the stereochemical patterning is crucial in the folding, we perturbed the confi guration motifs of the backbones by swapping the sequence of the central -amino acids. the peptides with exchanged residue order could not fold into helices. these novel structures can be useful scaffolds for biomedical applications. introduction small length synthetic peptides, based on sp c-terminal fragment, increase the secretion of tnf-and prevent the proliferation of several cancer cell lines. we have already shown the antiproliferative activity of tri-and tetra-peptoids in breast and prostate cancer cells. the aim of this study was the synthesis of hexa-peptoids, analogs of sp c-terminal region, containing the residues d-trp and tic and the peptoid ones nhn(r)ch co, nphe and nala in their sequence and their evaluation against cancer cells proliferation. methods and results all the syntheses were carried out stepwise using the fmoc/ but methodology on the solid support -cltr resin and dic/hobt as coupling reagent. all analogs were purifi ed (hplc) and identifi ed ( cyclotheonamides constitute a family of structurally related cyclic pentapeptides of marine origin that inhibit trypsin-like serine proteases. their binding mode has been elucidated by the x-ray structure of cyclotheonamide a in complex with trypsin. an extended peptide conformation which is stabilized by macrolactamization allows to address in a substrate-like manner beside the s pocket also the s ' and s pocket. the s ligand, (s)- -amino- -guanidino- -oxo-hexanoic acid, interacts via its guanido function with asp at the bottom of the s pocket. in addition, the ketone covalently modifi es the gammaoxygen of ser by hemiketal formation ( ) . recently, two novel cyclotheonamides have been isolated from a marine sponge of the genus ircinia. one of them, cyclotheonamide e , is a potent inhibitor of human beta-tryptase ( ) . in this study, cyclotheonamide e was modifi ed at two positions: (i) the s ligand was replaced by beta-homolysine or as betahomoarginine to obtain reversible acting tryptase inhibitors, and (ii) the alpha amino function of (s)- , -diamino propionic acid, which is not part of the cyclic backbone, was used as anchoring point for basic p residues to exploit interactions with the negatively charged glu of tryptase. these analogs were synthesized by a combination of solid phase and solution phase chemistry .. synthetic details as well as the inhibitory profi le of these novel cyclotheonamide e analogs will be discussed. oostatic peptides containing d-amino acids: activity and degradation in the fl esh fl y neobellieria bullata bennettová deteriorating effect of c-terminus truncated p and p analogues [ , ] of decapeptide h-tyr-asp-pro-ala-pro -oh ( ) on ovarian development (i.e. oostatic effect) of insect species diptera, orthoptera and hemiptera has stimulated an analysis of metabolic degradation of corresponding peptides after application to the insect body [ ] [ ] [ ] [ ] . radiolabeling in different positions of the peptide chain allowed determination of the degradation decisive steps -the fast splitting off the c-terminal pro from the p followed by successive cleavage of tyr and asp from the nterminus. after introduction of corresponding d-amino acids into peptide chain of p, we could see the same or even stronger oostatic effect of the analogues in comparison with parent peptide after application on the fl esh fl y neobellieria bullata. in the degradation assay, an elimination of enzymatic cleavage of peptide bonds pointing from the central labeled pro residue to either d-ala or d-asp residues in the neighbourhood was observed, resulting in total increase of the analogs stability. structure-activity relationships( sar) studies of camk ii inhibitors. in fact, in vitro it can phosphorylate up to proteins, including enzymes, ion channels, transcription factors and a number of these proteins appear to be physiological substrates. for example, cam-kii is highly concentrated in the postsynaptic density of glutamatergic synapses where it phosphorylates and potentiates current through the a-amino- -hydroxy- -methyl- -isoxazolepropionic acid-type glutamate receptor ion channel (ampa-rs). in fact, this family is encoded by four genes ( , , and ), whereas the and isoforms are expressed in diverse tissues and , isoforms are most prominent in neural tissues. the identifi cation of camk ii inhibitors is important to better defi ne its physiological. in literature is reported a -aa brain-specifi c protein that and potently inhibited kinase and bound the catalytic domain of cam-kii activity with an ic of nm. the inhibitory protein (cam-kiin), and a -residue peptide derived from it (camkiintide, krppklgqigrakrvvieddriddvlk), was highly selective for inhibition of cam-kii with little effect on cam-ki, cam-kiv, cam-kk, protein kinase a, or protein kinase c. cam-kiin interacted only with activated cam-kii (i.e., in the presence of ca +/cam or after autophosphorylation). here we report a structure-activity relationships study using as template the camkiintide. we synthesised a peptide contains all amino acids present in camkiin-tide, however, in random sequence (camkiin-tide scramble). then we operated a progressive deletion of amino acids from c-terminal and dall'n-terminal to detect minimum active sequence. moreover camkiintide and cankiintide scrumble was made cell-permeable by n-terminal addiotion of an antennapedia (rqikiwfqnrrmkwk). here we report the biological results of our study. ptprj: a receptor-type protein tyrosine phosphatase as a target of new peptides with antitumoral activity to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. it has been demonstrated that ptprj (also known as hptpeta, dep- , cd ) is able to inhibit cell growth promoted by specifi c ptk some of which are over-expressed in certain types of cancer. moreover a role for ptprj was also assessed in vasculogenesis; infact its reduced activity in enhanced vegf-induced vegfr activity leads to increased cellular responses, supporting a ptprj-vegfr interaction. on these basis it is important the identifi cation of molecules with agonist activity which are able to stimulate the function of residual ptprj in malignant cells and stopping the proliferation and possibly trigger programmed cell death. using as a template a peptide, already identifi ed, with agonist activity against ptprj(h-[cys-his-his-asn-leu-thr-his-ala-cys]-oh), here we report a structure-activity study carried out through endocyclic modifi cations (ala-scan, d-substitutions, single residue deletions, substitutions of the disulfi de bridge) and the preliminary biological results of this set of compounds. faulty of chemistry, university of gdansk, poland; faculty of chemistry, university of gdansk, poland isolated in from the sunfl ower seeds trypsin inhibitor sfti- is up to date the smallest naturally occurring peptidic proteinase inhibitor and therefore is an excellent starting structure to design peptidomimetic serine proteinase inhibitors. peptomeric library consisting of monocyclic analogues of sfti- was designed and synthesized by the solid phase method with the intension of select chymotrypsin and cathepsin g inhibitors. all peptomers contained in positions and nbenzylglycine (nphe) that mimics proteinogenic phe. in the synthesized library different peptoid monomers were introduced in the segment - . this is a turn region that makes an important contribution to structural integrity and rigidity of sfti- . deconvolution of the library against both proteinases by the iterative method in solution revealed that the highest chymotrypsin inhibitory activity displayed analogue with n-[ -( -aminoethyl)morpholyl]-glycine (naem) in position . this analogue was even more active than the one with pro in this position which is absolutely conserved in bownan-birk inhibitors. while, deconvolution carried out against cathepsin g indicated that analogue with npiperonylglycine (npip) in position and and with n-butylglycine (norleu) in position presents the highest inhibitory activity. acknowledgements: this work was supported by ministry of science and higher education (grant no. /h / / ). a platform for the design and optimization of new antimicrobial peptides effective against specifi c pathogens has been set up. the main features expected for these peptides are low environmental impact, broad spectrum of activity, reasonable bacterial selectivity, and low eukaryotic cytotoxicity. our approach also includes the use of a design of experiments protocol in order to fi nd peptide sequences that fi t these features. the obtained peptides would represent an alternative to currently used antibiotics or pesticides. this work focused on fi nding new control agents against economically important plant pathogenic bacteria such as erwinia amylovora, pseudomonas syringae and xanthomonas vesicatoria for which the available methods are not suffi ciently effective. nowadays, their control is mainly based on copper compounds and antibiotics. although antibiotics are highly effi cient, they are not authorized in several countries and resistance has been developed on plant pathogens. we have synthesized combinatorial libraries of cyclic decapeptides and linear undecapeptides. these libraries have been screened for antibacterial activity and eukaryotic cytotoxicity, and have led to the identifi cation of peptides with mic values of . - . microm. notably, cyclic peptides active against e. amylovora have been found, constituting the fi rst report of this type of peptides with activity towards this bacteria. the best peptides are bactericidal, display a low eukaryotic cytotoxicity at concentrations - times higher than the mics, and show a low susceptibility towards protease degradation. best peptides have been tested in vivo by evaluating their preventive effect of inhibition of p. syringae, x. vesicatoria and e. amylovora infections. the most active peptide is slightly less effective than streptomycin, currently used in fi eld. therefore, the best analogues can be considered as good candidates for the development of antibacterial agents for use in plant protection. selection of chromogenic and fl uorogenic substrates of neutrophil serine proteases using combinatorial chemistry approach human serine neutrophil and mastocytes proteases such as cathepsin g, neutrophil elastase, proteinase and -tryptase are involved in several physiological processes. unwanted activity of those enzymes yields to severe pathological states like infl ammation, wegener granulomatosis or various types of cancer. therefore monitoring of the activity of these proteases is crucial for the proper therapeutical treatment. the simplest method for determination of protease activity is the use of synthetic substrates. they are also useful for characterization of the enzyme specifi city. in this work we report selection of chromogenic and fl uorogenic substrates of human serine neutrophil and mastocytes proteases applying combinatorial chemistry approach. peptide libraries were synthesized by the portioning-mixing method. deconvolution of synthesized libraries was performed using iterative approach in solution. -amino- -nitro benzoic acid attached to the c-termini of synthesized peptides served as a chromophore released upon the interaction of peptide with enzyme. additional introduction of -methoxy- -coumaryl acetic acid or -amino benzoic acid on á-amino groups of the chromogenic substrates converted them into fret displaying compounds. the most active substrates were subjected to further modifi cation applying non-proteinogenic amino acids. as a result, one of the most selective substrates of these proteases was obtained. we also attempted to construct a simple tools to detect activity of human serine neutrophil and mastocytes proteases by immobilization, through amide and peptoid based linkers, of selected substrates on solid phase. angiogenesis modulation: identifi cation of tetrameric tripeptide as inhibitor of vegfr- by the screening of peptide combinatiorial libraries vascular endothelial growth factor receptor- (vegfr- , flt- ) and their ligands are involved in complex biological processes associated to severe pathological conditions, like angiogenesis, infl ammation and metastasis formation ( ). thus, the search for antagonists of flt- has recently gained a growing therapeutic interest. in order to identify new molecules able to selectively bind flt- and neutralize its activity, a screening of a combinatorial tetrameric tripeptide library built with nonnatural amino acids has been carried out by a competitive elisa-based assay. the library has been designed on a branched tetrameric structure in order to obtain molecules with a high recognition surface and, using building blocks, a complexity of . different peptides has been achieved. peptide mixtures composing the library have been utilized as competitors of the flt- /plgf (placental growth factor) interaction and the most active components have been isolated following an iterative deconvolution procedure. the selected most active hit shows a selective binding to flt- over kdr and inhibits in vitro its interaction with both plgf and vegf-a. the peptide is fully stable in biological environments, prevents flt- phosphorylation and blocks huvec capillary-like tube formation stimulated by plgf or vegf-a. in vivo the peptide inhibits the vegf-induced neoangiogenesis in chicken embryo chorioallantoic membrane assays and also stimulates cornea neovascularization (cnv) by displacing vegf from a complex with sflt- . all data suggest that the compound has potential applications in diseases characterized by pathological angiogenesis, such as tumor growth and ischemic retinopathy. heinlein, christian; unverzagt, carlo bioorganische chemie, universität bayreuth, gebäude nw i, bayreuth, germany since the purifi cation of homogeneously glycosylated glycoproteins remains a diffi cult task the synthesis of entire glycoproteins is a fi eld of emerging interest ( ) . especially the synthesis of the required protein fragments in high purity and good yields demands for effi cient methods. fragment condensation of protected peptides can reduce deletions in the crude product and thus facilitates purifi cation. condensation of peptide fragments in cspps ( ) is however subject to epimerization upon cterminal carboxyl activation. to overcome this problem the use of cterminal gly or pro is usually performed. peptides containing c-terminal pseudoprolines can also be coupled without stereomutation because the serine or threonine residue has been reversibly protected as a prolinelike oxazolidine ( ). this concept facilitates the synthesis of long peptides by epimerization-free fragment condensation at c-terminal ser/ thr residues in addition to gly/pro residues thus increasing the number of safe condensation sites. after solving several problems associated with the generation of peptide acids with c-terminal pseudoprolines the synthesis of the glycosylated rnase fragment was carried out as a racemization-free fragment condensation. tuftsin is liberated from fc-domain of the heavy chain of igg by two specifi c enzymes. being the tetrapeptide of biological origin is extremely important product because it can activate a few elements of immune system such as granulocyte and macrophage. tuftsin indicates not only immunological stimulating factor but also antibacterial, antivirial and antitumor properties. in spite of its wide range of activity, the peptide is unstable in plasma and it has become the aim of the formation of novel analogues more resistant to proteolysis degradation. the introduction of the additional residue at -amino group of lysine caused that new bond became stronger than peptide bond in central chain [ ] [ ] [ ] . we synthesized linear tuftsin derivatives that were prepared on the solid phase using a fmoc/tbu procedure. the method of elongation of peptide chain was based on two-step procedure: deprotection and coupling step. segment coupling reaction was carried out with tbtu, hobt and in the presence of diea in dmf/dcm/nmp mixture. the introduction of the simple amino acid (ala, -ala, val, ile or gly) at -amino group of lysine let us obtain the isopeptide bond. the modifi cation was achived by introducing lysine residue, protected at -amino group with mtt. the selective removal of mtt group was caused by % tfa treatment. peptides were cleavage from resin and purifi ed. tuftsin derivatives were confi rmed by ms, amino acid analysis, elemental analysis and rp-hplc analysis. peptides were sent to assay their microbiological properties and the results will be described as a structure-activity relationship. the synthesis and structural study of iso-aß( - ) the aß( - ) peptide is diffi cult to synthesize, because of its high tendency for aggregation during the synthesis. both the couplings and the fmocremoval can be troublesome, due to the steric hindrance. the incorporation of the ester-bond disturbs the structure, thus ease the synthesis after the th residue. if the peptide would be synthesized using boc-chemistry the removal of the -amino protecting group would be less problematic, because the % tfa/dcm mixture solubilizes well the peptide chain. thus a boc-strategy was devised for the synthesis of iso-aß( - ). the purifi ed peptide was studied with cd-spectroscopy and dynamic light scattering. these structural studies revealed that the isopeptide has some ß-sheet content even in a ph solution. it was realized also that small aggregates are present in the precursor peptide. both techniques mentioned above showed, that after altering the ph to . , the ß-sheet content of the peptide increases and aggregation takes place. with the use of the isopeptide, aß oligomers and -with prolonged incubation -fi brillar structures can be formed for biological studies. continuing our program of syntheses of muramyl dipeptide (mdp) and nor-muramyl dipeptide (nor-mdp) conjugates as potential immunomodulators, we designed novel conjugates of mdp or nor-mdp with tuftsin derivatives containing isopeptide bond between -amino group of lysine and carboxylic group of simple amino acids such as alanine, glycine and valine. the synthesis of a greater number of conjugates will enable structure-activity relationship studies. tuftsin analogues containing isopeptide bond showed increased chemical resistance and activity in relation to tuftsin. the introduction of the additional residue at -amino group of lysine by nhco-formation caused that isopeptide bond became stronger than peptide bond in central chain. the protected pentapeptides (h-thr-lys(y)-pro-arg(no )-obn, y= ala,gly,val) were synthesized by the conventional chemical procedure using mixed anhydride method. acylation of the thr amino group of partially protected pentapeptides by -benzyl-mdp or -benzyl-nor-mdp was performed using the mixed anhydride method with isobutyl chloroformate and n-methyl-morpholine (nmm) in dry dmf. the protected conjugates were isolated and purifi ed with a preparative tlc. the identities of the protected products were confi rmed by high resolution h-nmr ( mhz, cosy, tocsy, roesy, ghsqc, ghmbc) spectroscopy. the fi nal products were hydrogenated with h / pd/c in % methanol-acetic acid and purifi ed with preparative tlc. the identities of the conjugates were confi rmed by tlc qualitative amino acid analysis, and elemental analyses. finally, the combined use of muramyl peptides with other immunomodulators, e.g. such as tuftsin, retro-tuftsin other chemotherapeutics is promising in the therapy of different infections, autoimmunological diseases and anticancer therapy. effi cient microwave-assisted synthesis of myelin epitopes mog - and mog - using cltr-cl resin fmoc/tbu methodology. unlike conventional heating, microwave energy directly activates any molecule with a dipole moment and allows for rapid heating at the molecular level. the protected peptides were synthesized using the cem liberty automated microwave peptide synthesizer in and . hours respectively, with a -minute fmoc deprotection ( % piperidine solution in dmf) and -minute coupling reactions using dic/hobt in dmf solution. the maximum temperature reached during both the deprotection and coupling reactions was °c except for the coupling of fmochis(trt)oh ( °c). the fi nal crude products were of high purity as identifi ed by analytical rp-hplc. siah up-regulates the hif- alpha hypoxic response pathway: siah binding peptides coupled to cpps inhibit siah activity and demonstrate proof of concept that siah is a viable anti-tumor drug target vegf, a secreted downstream component crucial for angiogenesis, has been successfully targeted clinically using antibody therapy (avastin). the hypoxic response, however, activates other pathways that stimulate tumor growth, suggesting that inhibitors of upstream components in the pathway may be useful to give a broader spectrum of inhibition. we have focused on the siah proteins that regulate hif- ƒÑ levels by ubiquitylation and degradation of the prolyl hydroxylases (phds) immediately upstream of hif- ƒÑ in the hypoxic response pathway. in a proof-of-principle study, we have shown that siah inhibition by expressed protein fragments (from a drosophila high affi nity interacting protein, phyllopod) can inhibit the stabilization of mammalian hif- ƒÑ during hypoxia and limit tumor growth in a mouse tumor model. a amino acid peptide sequence (phyl) from the phyllopod protein has been identifi ed as the interaction site with siah. the aim of this work was to show that a small peptide could mirror the activity of the transfected recombinant phyllopod protein. to achieve this, phyl was covalently attached to cell penetrating peptides (cpps) and tested for its ability to inhibit hif- ƒÑ stabilization and hypoxic response in human u os cells. both the tat sequence and penetratin were utilized as cpps and attachment was via disulfi de, maleimide or through a pro spacer sequence. the cpp¡vphyl constructs were found to have varying activities but a penetratin-pro -phyl was found to be inhibitory in the u os cell-line hif- ƒÑ stabilization assay. this result demonstrated proof of concept that siah inhibition could be attained by targeting a restricted and specifi c protein-protein interaction site. ( ) . the principal event in the development of prion disease is the transformation of the normal cellular prion protein (prpc), which is highly helical in nature, into the pathogenic isoform prpsc which is insoluble and has an extensive ƒÒ sheet structure. the events surrounding this transformation are very poorly understood, but the mechanism of the detailed steps involved in this process is fundamental to the understanding of prion disease pathogenesis. in an endeavour to study the structure of polypeptide component of the n-terminal section of prpc, synthesis of a range of prp polypeptides were assembled on a cem liberty microwave synthesiser. these fragments ranged in length from to amino acids and span the protein sequence from position to . standard cem synthetic coupling cycles were used, except when peptides were greater than amino acids in length, whereby longer coupling cycles were employed. a number of purifi cation strategies were employed, such as c and c rp-hplc at either c or c and size exclusion chromatography. the purifi ed peptides were characterised by rp-hplc and esi-ms. in addition, they were analysed for secondary structure by cd spectrometry, and evaluated for cell toxicity and fi bril forming ability with tht. the synthesis of a mer peptide for investigating the mechanism of action of the hiv fusion inhibitor t however, the mechanism of action of t is still in debate. it is believed that peptides derived from gp chr region may share a common mechanism, by binding to gp nhr coiled coil and preventing formation of the fusogenic gp core-six helix bundle ( hb), thereby inhibiting fusion between the virus and target cell membrane. we have synthesized a -mer nhr peptide-n -covering all the binding sites for any length of the chr-peptide. synthesis of the polypeptide was carried out using conventional as well as microwave assisted solid phase synthetic methods. details, including comparison of the synthetic approaches will be presented. using c , another anti-hiv chr peptide containing the pocket-binding domain, as a control, we analyzed the activity of t to interact with n- to form hb and to inhibit the -hb formation between n- and c by cd spectroscopy and elisa. we found that t- , unlike c could neither form a stable hb with n , nor inhibit the -hb formation of the fusogenic hb core. our results thus suggest that t- and c- peptides inhibit hiv fusion by different mechanisms of action. the oligomerisation equilibrium of ts is modulated by a fi ne tuning; shifting this equilibrium towards the monomeric form would cause ts inactivity and low translation, overcoming resistance mechanisms encountered for (co)substrate-like inhibitors as the inactive monomer regulates its own expression. our group designed some small ligands to interfere with thymidylate synthase dimerisation, including short peptides taken from the interface sequence that represent the natural ligand for this region, mimicking the other subunit without forming a functional dimer. interesting biological data are arising from the activity tests but a rapid screening assay is necessary to prove they are really interfering with ts oligomerisation. fret is a spectroscopic phenomenon whose intensity depends on the distance between two fl uorophores; when this value varies due to conformational changes of the protein the probes are linked to, consequent fret variation can be used to sense the state of the protein(s) ( stromal derived factor- (cxcl ) is a amino acid cxcchemokine with critical role in homing, migration and guiding of different cell types including hematopoietic progenitor cells (hpc), stem cells, tumour cells and neuronal cells during embryogenesis and in adults ( ). these various functions in physiological as well as pathophysiological processes make this small protein interesting for regenerative medicine. to apply this chemoattractant in medicine, it is needed to form spatially and temporally controllable concentration gradients of active sdf- in response to a non-tissue damaging trigger like visible or near uv-light. after irradiation of an inactive prodrug dramatic change in conformation or lack of sterical hindrance should then lead to fully biological activity under physiological conditions within a few minutes. to prove the principle and assess its potential in photodynamic therapy of neuronal injuries a water-soluble, photosensitive sdf- analogue has been developed. for this the expressed protein ligation (epl) approach has been used, in which one segment has been recombinantly expressed and purifi ed using the impact ® -system to yield the corresponding peptide thioester ( ) . the modifi ed peptide fragment has been chemically synthesized on solid phase using fmoc-strategy. the photocleavable moiety, the -nitroveratryloxycarbonyl (nvoc) protecting group, has been introduced at a side chain amino group. furthermore, the analogue has been characterised by physico-chemical methods and has also been tested in vitro on transfected cos- cells in order to determine its biological activity after activation. sdf- is a chemokine that plays a major role in traffi cking of hematopoietic stem cells (hsc). thus it enables the formation of bone marrow during embriogenesis and later in adult life it supports retention and homing of these cells in the bone marrow. furthermore it is involved in organogenesis and regeneration, respectively. due to these promising features the subform sdf- could serve as a therapeutic target. for studies on the small protein concerning its molecular properties as well as its therapeutic potentials, it needs to be modifi ed chemically. in order to reach these goals, the n-terminus sdf- - has been cloned and expressed recombinantly in e. coli er as a thioester, while the c-terminus sdf- - has been synthesized via solid phase peptide synthesis. modifi cations are thereby introduced at the c-terminus at lys . up to now carboxyfl uorescein has been coupled to the -amino group of the lysine residue. the two fragments then have been ligated via expressed protein ligation (epl), a subform of the native chemical ligation (ncl). activity studies and fl uorescence microscopy on hek cells transfected with the sdf -specifi c g-protein coupled receptor cxcr have been conducted. stueber, werner; lewandrowski, peter; frisch, juergen; weinschenk, toni; singh, harpreet immatics biotechnologies gmbh, germany ima is a multiple peptide vaccine for the treatment of renal cancer (rcc). the tumor-associated peptides (tumaps) contained in ima were identifi ed by immatics directly from primary renal cells (= primary rcc tumor tissue samples), selected regarding their over-expression in rcc and proven to be immunogenic using in vitro t-cell assays. ima consists of individual peptides ( tumaps) and nonactive ingredients which are used as excipients of the pharmaceutical presentation of ima . all peptides are synthesized by conventional fmoc chemistry. the sequences of the peptides will be presented and technical issues will be discussed. in the fi nal formulation of ima g of each peptide plus excipients are fi lled into glass vials and lyophilized. the challenges of the production of multi peptide drugs will be discussed. such challenges comprise the synthesis, the production of the formulation as well as the analyses of the fi nal presentation of ima . results of the phase trial in vaccinated rcc patients showed that ( ) ima was safe, ( ) multiple t-cell responses to vaccinated peptides correlated with favourable clinical outcome and ( ) patients with a lower percentage of regulatory t cells (tregs) were more likely to develop a vaccine-induced multiple t-cell response. novel peptides -msh analogs with high candidacidal activity in an attempt to improve the candidacidal activity of -msh and to better understand the peptide structure-antifungal activity relations, we designed and synthesized novel peptide analogs. because previous data suggested that the peptide [dnal- , phe- ]--msh( - ) has greater candidacidal activity than -msh and is the most potent of the analogs tested in the past, this compound has became our lead ( , ) . from this lead compound we have synthesized a new library of peptides where we have replaced the glycine in position with unconventional amino acids. here, we report new analogs with a strong antimicrobial and candidacidal activity. the obtained results are very encouraging in that they show the great potential of these peptides as a truly novel class of candidacidal compounds. derivative were modifi ed by the same amino acid to establish infl uence of the adjacent amino acid on antimicrobial activity of the compounds studied. the activity of all obtained peptides was screened against model gram-positive (bacillus subtilis) and gram-negative (escherichia coli) bacteria whereas antifungal activity was tested against yeast pichia pastoris. all tests were performed using antibiogram method whereas the minimal inhibitory concentrations were determined using two-fold serial dilution technique. huang, yen-hua ; colgrave, michelle l. the cyclotides are a family of naturally occurring macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, which impart extraordinary stability to this peptide family. a recent study demonstrated that the prototypic cyclotide kalata b possesses signifi cant activity against two economically important sheep nematodes haemonchus contortus and trichostrongylus colubriformis. an alanine scan of the molecule highlighted the residues critical for activity. in this work, we explore the relative importance of positively charged residues in different regions of the molecule to aid the understanding of the structural and biological basis of its nematocidal activity. a lysine scan has been conducted, in which each of the non-cys residues in this amino acid peptide has been successively replaced with lysine and the suite of peptides have been assayed against the two sheep nematodes. substitution of residues in loop , v , g , n , t , w , v , l , p , and v decreased or completely abolished the activity, suggesting that these residues are critical to the nematocidal activity of kalata b . on the other hand, incorporation of a positive charge in positions g , t , t , n , and g signifi cantly enhanced the anthelmintic activity of the grafted peptides, up to fourfold, compared to native kalata b . these increases in activity after lysine incorporation into the kb scaffold raise the possibility of being able to engineer greater anthelmintic activity into the peptides, further highlighting their potential as anthelmintic agents. ivanova, v.p. interaction of cells with extracellular matrix (ecm) affects many aspects of cell behavior including growth, morphology, migration and differentiation. integrins are known to mediate cell adhesion to proteins of ecm. ligand-binding properties of integrins depend not only on composition of ecm ligands and level of integrin expression in cell, but also on spectrum and activity of soluble factors indirectly infl uencing cell-matrix contacts. interaction of cells with substratum may be divided into cell adhesion and spreading. following an initial cell attachment event, cell may or not spread depending on cell type and the nature of the molecular signals they receive. oligopeptides released from different proteins during their proteolysis in or out of cells may act as the such short-time existing signals. in the present study we investigated the effect of multiply repeated peptide fragment in different collagen types on cell spreading. murine embryonic fi broblasts ( /ml) were allowed to adhere for min at ° c with or without peptide (in various concentrations) to plastic surface pre-coated or not with gelatin. it was shown that the synthetic peptide increased the number of spread cells and caused shape changes in cells spread on different substrata. a -min pretreatment of cells with the peptide (before conducting of cell spreading assay) resulted in inhibiting of cell spreading on gelatin. our results suggest that the peptide regulation of cell spreading could be related to its effect on re-distribution of integrin receptors in sites of cell contacts with substratum. bioactive peptides from cyanobacteria cyanobacteria are versatile source of small peptides from which vast majority are cyclic. cyanobacterial culture collection in university of helsinki contains over strains and this collection is used for screening of new bioactive compounds. cyclic heptapeptides, microcystins are the best known cyanobacterial peptide family. over structural variants have been described from which most are strong hepatotoxins. our research group have participated to the determination of the structure of many novel microcystins and other cyclic and linear cyanobacterial peptides. in recent years we have found highly toxic and novel microcystins from lichen associated cyanobacteria ( ) and from anabaena strains of baltic sea ( ). in the structural analysis of new peptides from the known peptide families we have used liquid chromatography ion trap mass spectrometry which have proven to be very effective method and is in many cases the only method needed in the verifi cation of a new structure. with this lc-itms method we have found many new structural variants from anabaenopeptin, anabaenopeptilide and spumigin peptide families. in collaboration with many research groups we have found cyanobacterial compounds/extracts which inhibit protein kinase c activity, inhibit/activate boar sperm motility, disintegrate cell membranes or are antidotes for microcystin toxicity. structures of the compounds are under study except the microcystin antidote which structural analysis showed that it belongs to a rare peptide family containing imino bond in cyclic skeleton. the results of n-terminal modifi cation of arginine vasopressin with cis- -amino- -phenyl-cyclohexane carboxylic acid. the highly potent oxytocin receptor antagonists arginine vasopressin (avp) is a cyclic nonapeptide with multiple functions. the main peripheral physiological roles of avp are the regulation of water balance, the control of blood pressure, and the release of adrenocorticotropin hormone (acth). moreover, avp also exhibits to some extent typical oxytocin (ot, a closely related neurohypophyseal peptide) activities such as the galactogogic and the uterotonic effects. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. cis-apc modifi cation at position of avp is suffi cient to change the pharmacological profi le of the peptides. analogues i -iv were moderately potent antidiuretic agonists with prolonged action. in regard to the uterotonic activity, all peptides with cis-apc were highly potent antagonists, except compound i. it supports our earlier hypothesis that an amino acid residue in position has signifi cant impact on pharmacological activities. the incorporation of unnatural non-proteinogenic -amino acids into peptides has emerged as a novel and promising approach in peptide modifi cation. the conformationally restricted amino acid derivatives are of particular interest. this thesis are also supported by our already -years research focused on the effects of steric restriction and the presence bulky substituent in the n-terminal part of avp molecule on biological properties of the resulting analogues. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. all the analogues were devoid of the pressor potency and exhibited only negligible antidiuretic activity. interestingly, in regard to the uterotonic activity, the new compounds exhibited moderate (i) or high (ii -iv) antioxytocic potency. it should be point out that single substitution e.g. replacement of tyr in avp molecule with igl results in moderately potent and highly selective antagonists of oxytocin (i). our new igl substituted peptides proved that the presence of the sterically restricted amino acid residue may result in high active and selective antiuterotonic agents. these in our opinion interesting fi nding demonstrates the usefulness of our approach in the design of new highly active and selective analogues with desired pharmacological properties. arginine vasopressin (avp), a neurohypophyseal hormone and neuromodulator, is a cyclic nonapeptide with a disulfi de bridge between cys residues at positions and . as a hormone avp exerts its biological effects upon binding to three receptor subtypes termed: v a , v b (v ), and v . furthermore, avp to some extent can interact with the oxytocin receptor (ot). biological activity of peptides is determined by their structure and conformation. conformational restriction of bioactive peptides is therefore a well-established strategy to change their pharmacological profi le. peptide fl exibility can be restricted by a local constraint imposed, e.g. by introducing amino acids with limited conformational freedom, that has an impact on specifi c orientations of the peptide backbone and the side chains. in this work, we decide to check the infl uence of the bulky ( design, synthesis and biological activities of temporin a and temporin l analogues. temporins a (ta) and l (tl) are antimicrobial peptides isolated from the skin of red european frog "rana temporaria". temporins are active against a broad spectrum of microrganism: ta (flpligrvlsgil-nh ) is preferentially active against gram-positive bacterial strains; tl (fvqwfskflgril-nh ) has the highest activity against fungi, and bacteria, including resistent gram-negative strains, but it shows haemolytic activity too. ta exerts its antimicrobial activity by its ability to form a transmembrane pore via a 'barrel-stave' mechanism or to form a 'carpet' on the membrane surface via the 'carpet-like' model. recently we investigated the preferential conformation of tl and ta in sds and dpc solutions which mimic bacterial and mammalian membranes, respectively. in sds, the peptides prefer a location at the micelle-water interface; in dpc, they prefer a perpendicular location to the micelle surface, with the n-terminus imbedded in the hydrophobic core. tl shows higher propensity, with respect to ta, in forminghelical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles ( ). on these results we designed and synthesized new ta and tl analogues and found interesting differences in their effi cacy against microbial species, and fi nding a new potent antimicrobial agent without haemolytic activity. arginine vasopressin (avp), a neurohypophyseal nonapeptide hormone [cycle - (h-cys -tyr -phe -gln -asn -cys -pro -arg -gly -nh )], elicits a variety of responses both centrally and peripherally by acting on three distict g-protein coupled receptors: v a (vascular), v b (pituitary) and v (renal). it also binds to the oxytocin (ot) receptor. in addition to its well-known antidiuretic activity, avp has also complex cardiovascular actions and adrenocorticotropic hormone (acth) releasing activity. binding of avp to the v a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. it is generally accepted that the conformation of the n-terminal part of neurohypophyseal hormones analogues is important for their pharmacological activity. in continuing our work aimed at the design of selective avp analogues, we synthesized twelve new analogues of avp containing mercapto propionic acid ( we also studied the effect of modifi ed c-terminal amide on biological potency of the new avp analogues. the analogues were synthesized by fmoc/bu t solid phase methodology and were tested for their rat uterotonic in vitro activity, rat pressor activity and antidiuretic activity using conscious rats. the modifi cations performed had a signifi cant impact on pharmacological activities of the analogues. acknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii), and particularly the program pythagoras i, for funding the above work. it was also supported by the research project no. z of the academy of sciences of the czech republic. three-dimensional structure and mechanism of action of an antifungal peptide generated from hemocyanin cleavage in a penaeid shrimp an antifungal peptide, pvhct, which corresponds to the amino acid c-terminal sequence of the shrimp respiratory protein hemocyanin, has been previously identifi ed in the plasma of the penaeid shrimp litopenaeus vannamei ( ) . it is generated by proteolytic cleavage in response to a microbial challenge. similarly, a c-terminal fragment of hemocyanin displaying antimicrobial activity has been isolated from crayfi sh plasma ( ) . these peptides are believed to contribute to the crustacean defence. the phenomenon of in vitro antimicrobial peptide generation from a respiratory pigment already observed with hemoglobin thus appears not to be restricted to mammals ( ). pvhct displays a broad spectrum of antifungal activity with minimum inhibitory concentrations (mics) in the range - m ( . m against the shrimp pathogen fusarium oxysporum). its activity would be based on the inhibition of spore germination ( ) . to contribute to the elucidation of the mechanism of pvhct antifungal activity, we determined its three-dimensional structure by circular dichroism (cd), nmr and molecular modelling and examined its effects on the f. oxysporum spore ultrastructure by transmission electron microscopy (tem). cd and nmr data indicate that pvhct is unfolded in an aqueous environment but adopts a similar helical structure in methanol solution and in dodecylphosphocholine (dpc) micelles used to mimic biological membranes. the structure consists of an amphipathic -helix spanning residues to . tem shows that pvhct induces structural changes of the plasma membrane accompanied by a disorganization of the cytoplasm and a signifi cant decrease of lipid bodies. glucagon-like peptide- (glp- ), glucagon (gcg), and oxyntomodulin (oxm) are highly homologous peptide hormones derived from posttranslational processing of the preproglucagon gene. the sequence of oxm in particular, is identical to the sequence of gcg, with an amino acid extension at the c-terminus. pharmacological doses of oxm activate both the glp- receptor (glp r) and the glucagon receptor (gcgr) albeit with lower affi nity compared to glp- and glucagon, respectively. in order to understand the origin of this dual specifi city, we synthesized a number of oxm analogs in which one or more of the native amino acids were replaced. first, a chimera was produced in which all the residues differing between glp- and gcg were grafted into the oxm native sequence, yielding an analog with the same pharmacologic profi le as glp- . further studies showed that surprisingly, the switch from glp r/gcgr co-agonism to glp r-selective agonism could be obtained by a single amino acid substitutions at position of oxm. in particular, the analog with gln substituted by glu (oxm-q e) showed the same activity as native oxm on glp r, but complete loss of activity on gcgr. oxm and oxm-q e were compared in a hyperglycemic clamp study performed in diet-induced obese (dio) mice. due to the short half-life of the peptides, both were infused intravenously at ~ g/kg/ min in chronically catheterized mice. the amount of exogenous glucose required to maintain the hyperglycemic level was -fold greater with the selective glp r agonist oxm-q e than with the glp r/gcgr coagonist oxm, showing that abolishment of gcgr activity signifi cantly improves the glucose-lowering effect. we believe that these fi ndings could be useful for the development of a peptide therapeutic for the treatment of type diabetes. hospitals across europe and north america have lately been plagued with infections caused by clostridium diffi cile, a diffi cult to treat bacterium due to its resistance to many commercially available antibiotics. recently, we isolated a two-component bacteriocin, thuricin, produced by bacillus thuringiensis that exhibits activity against c. diffi cile. we found that these peptides, called trn and trn , operate together in a synergistic fashion to inhibit bacterial growth. the peptide combination was found to be highly active against a wide range of c. diffi cile strains, including the virulent epidemic strain of the o ribotype, as well as most other clostridia and some bacilli and listeria species. maldi mass spectrometry revealed that both peptides have molecular weights that are lower than those predicted from their genetic sequences, indicating that they are post-translationally modifi ed. sequencing of the mature peptides through tandem mass spectrometry showed that each peptide has three modifi ed amino acid residues near its c-terminus. these residues were found to be two units lighter than their expected natural amino acid masses, suggesting a loss of two hydrogen atoms through dehydrogenation or oxidation. in order to confi rm these proposed modifi cations, we are investigating the production of c-and n-labeled trn and trn for structure elucidation by nmr. isolation of labeled peptides will be achieved by growing b. thuringiensis on defi ned media containing [u- c] glucose and ( nh ) so . subsequent nmr experiments will enable us to determine the three-dimensional solution structures of trn and trn , individually and bound together. by elucidating thuricin's structure, we aim to better understand its mechanism of action against c. diffi cile. the most potent peptidic human bradykinin (bk) b receptor antagonist is hoe- (icatibant). in this study we present the synthesis of nine new analogues of hoe- substituted at position , or with chosen d-amino acid residues and/or nonproteinogenic amino acid residues, e.g. n-cyclohexylglycine (nchg), -aminocyclohexane- -carboxylic acid (acc), octahydroindole- -carboxylic acid (oic), piperidine- -carboxylic acid (nip) and -phenylpiperidine- -carboxylic acid (ppc). in the next nine peptides we combined the above mentioned modifi cations with the placement of -adamantane acetic acid (aaa) at position . all new analogues were tested on human umbilical vein for their antagonistic potency. only three compounds containing nchg , nchg or ppc exhibited noticeable antagonistic activities on human bradykinin receptors thus still less potent then original hoe- sequence. each of them with aaa showed lower activity then parent analogues. peptides: d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg and aaa-d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg, although strongly potent antagonists against bk-induced blood pressure lowering responses in rats, did not show noticeable antagonistic activity on bk-induced contraction of the isolated human umbilical vein, a well-established b r bioassay system, suggesting a species dependent activity of the compounds. due to advances made in the peptide fi eld during the last few years, peptides as therapeutics have continued to gain more interest. the interest in peptide therapeutics generally comes from their high specifi city to targeted sites, their diversity and their usually low toxicity. pt- , also known as bremelanotide, and mt-ii are potential future drugs and both are heptapeptides with a -membered cyclic monomeric lactam bridge. they are melanocortin receptor agonist and an analog of alpha-melanocyte stimulating hormone (a-msh). the pt- molecule has a c-terminal acid group while mt-ii has a c-terminal amide function. both neuropeptides have been tested in treating male sexual and erectile dysfunction as well as female sexual arousal disorder. pt- , patented by palatin technologies, new jersey, usa, is the only known synthetic aphrodisiac and unlike other erectile enhancers like viagra, it does not act upon the vascular system. instead, it directly increases sexual desire and is used nasally as a spray. a scalable method for the synthesis of both peptides using fmoc-chemistry and the problems involved during the synthesis process will be discussed in details. tateaki, wakamiya; takahiro, nishimaru; kiyoe, mori; yoshihiro, yamaguchi kinki university, japan l-glutamate (glu) is known to be major excitatory neurotransmitter not only in the mammalian central nervous system but also in the ganglia of arthropods. binding of spider toxins to glutamate receptors (glurs) results in the inhibition of glu-mediated neurotransmission. in order to elucidate the mode of binding between glu and glurs, we focused on the visualization of glurs by complex formation with fl uorescentlabeled analogs of nptx- ( ), a spider toxin with the structure of n -( , -dihydroxyphenylacetyl-l-asparaginyl)-n -l-lysyl- , -diaza- , -dodecanediamine [dhpa-asn-dada( lys)]. in the present study, the modifi ed nptx- , i.e., dhpa-asn-dada( abg) ( ) in which the lys residue of was replaced with the n-( -aminobutyl)glycine (abg) residue, was employed as a template compound to create the fl uorescentlabeled analogs of nptx- , since the biological activity of is three times higher than that of . we thus carried out the modifi cation of dhpa in the analog , i.e., ) dhpa was replaced with the coumarintype acyl residues (type- ); ) the phenylacetyl residues having alkyne side chains that can be converted into suitable fl uorophores based on the click chemistry (type- ); and ) the coumarin-type acyl residues having the mercapto group to form disulfi de bond with the cys residue in glurs (type- ). this paper presents the synthesis and biological activity of various nptx- analogs to adopt as probes for visualization of glutamate receptors. an investigation of the functional requirements of apidaecin ib c-terminal fragment by means of peptoidpeptide hybrids by acting on one or more intracellular targets, without damaging the cytoplasmatic membrane. their particular killing mechanism and their low toxicity against mammalian cells make them attractive for the development of new antibiotics. structure-activity relationships studies have been carried out on short pro-arg rich antimicrobial peptides isolated from insects and the characterization of various natural isoforms of the -residues peptide apidaecin ib allowed to identify an evolutionary conserved region in the c-terminal part of the molecule. even a single point mutation in this region results in reduction or loss of antimicrobial activity. we recently described the synthesis of some apidaecin ib peptoid-peptide hybrids in which each arginine was replaced by the corresponding n-alkyl glycine residue ( ). the afforded modifi cation made the resulting peptoid-peptide hybrids more resistant to proteolysis but moving the [narg]residue from the n-to the c-terminal end of the molecule progressively reduced the antibacterial activity. here we report the synthesis of a series of novel analogues containing a n-homoarginine or n-norarginine residue in position , or . the effect of the size of the side-chain of the peptoid residue on the antimicrobial activity is also reported. in order to strengthen the peptide resistance to proteolysis and by considering that enzymic cleavage of the arg -leu peptide bond yields a fully inactive compound, we also prepared the [nleu ]-apidaecin analogue. the conformational properties of the resulting peptoid-peptide hybrid, which is devoid of any antimicrobial activity, will be compared to those of apidaecin ib and the other [narg]peptoid-peptide hybrids. a wide variety of organisms produce antimicrobial peptides as part of their fi rst line of defense. however, antimicrobial activity is not the main function of some of these peptides. in the cuttlefi sh sepia offi cinalis we observed an antibacterial activity for the neuropeptide : h-alsgdaflrf-nh ( ). this decapeptide belonging to the fmrfamide family involved in regulation of reproduction and chromatophore function and is able to inhibit the growth of marine bacteria. circular dichroism studies have revealed for this amphiphilic peptide a helical structuration in sds micelles and in % tfe. to improve this antimicrobial activity, we fi rst introduce a lysine residue instead of aspartic residue, the antimicrobial activity of this new peptide has been improved by augmentation of the positive net charge (+ to + ). moreover preliminary results have shown that the incorporation of aza- -amino acids analogues could create original hybrid pseudopeptide with superior activity . the natural peptide is not effi cient against staphylococcus aureus whereas the pseudopeptidic analogue revealed a minimum inhibiting concentration (mic) between - m. therefore some -amino acids were replaced by aza- -amino acids. we will show in this communication that depending on the substituted residue and on the structuration, these modifi cations could lead either to no activity or to a drastic enhancement of the antimicrobial activity, demonstrating that aza- -amino acids can facilitate the burying in lipidic bilayer of antimicrobial peptides that acts on bacterial membranes( ) references: isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of burkholderia thailandensis e the poor overall fragmentation behavior in ms n studies suggested a branched cyclic peptide with a rigid lasso structure. optimization of the fermentation conditions increased the production by -fold and subsequent nmr structural studies proved the lasso structure of the peptide that was named capistruin. heterologous production of the lasso peptide in e. coli showed that the identifi ed genes are suffi cient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related burkholderia and pseudomonas strains. to our knowledge, this is the fi rst rational based identifi cation of a novel lasso peptide and the presented approach should be advantageous for the isolation of further lasso peptides in the future. endogenous antimicrobial peptides (amps) are the earliest molecular factors in the evolution of innate immunity. marine invertebrate animals have no acquired immunity with a system of antibodies diversifi cation. they are presumed to use an amps-based system as principal defense against potential pathogens. we have discovered a new family of small ( -residue) amps, termed arenicins, in coelomocytes of marine polychaeta lugworm arenicola marina. these amps exhibited activity against gram-positive, gram-negative bacteria and fungi. complete amino acid sequences were determined for each isoform. arenicins have one disulfi de bond (cys -cys ). arenicins have no structure similarity to any previously identifi ed antimicrobial peptides. a novel -residue antimicrobial peptide, aurelin, exhibiting activity against gram-positive and gram-negative bacteria, was purifi ed from mesoglea of a scyphoid jellyfi sh aurelia aurita. complete amino acid sequence of aurelin was determined. aurelin has cysteines forming three disulfi de bonds. the total rna was isolated from the lugworm coelomocytes and from the jellyfi sh mesoglea, rt-pcr and cloning were performed, and cdnas were sequenced. a -residue preproarenicin contains a putative signal peptide ( amino acids) and a long prodomain. a residue preproaurelin contains a putative signal peptide ( amino acids) and a propiece of the same size ( amino acids). aurelin reveals partial similarity both with defensins and k+ channel blocking toxins of sea anemones and belongs to shkt domain family. overlapping of biological properties of marine animal amps and toxins along with their sequence homology might be a consequence of divergent evolution from a common ancestor. antimicrobial peptides from marine organisms could afford design of new antibiotics manifesting broad-spectrum antibacterial activity. cyclic enkephalin analogs containing two alkylurea units we shall present some structure-activity results for enkephalin analogs, derived from the exhaustive combinations of d-lys and d-orn in position with lys, orn, dab and dap in position , both positions coupled ¦Ø-¦Ø¡¯ by means of the urea bridge. accordingly, they all are restrained by - -membered rings. in addition, we introduced the -nh-ethylurea unit instead of -nh of amide group in previously published analogs [ ] [ ] [ ] . their in vitro activities were determined in the gpi and mvd assays. the peptides are more active than enkephalin in the gpi while have similar activities to the latter in the mvd assay. the effect of the introduction of ethylurea unit at the c-terminus on the activities is also discussed. chemical shifts of the peptides in water were fully assigned and their sequences confi rmed. several cross-peaks between the protons of amidoalkylurea unit and preceding residues have been observed in each case, suggesting that the unit may be involved in specifi c interactions between residues and . understanding the structure/activity relationships of hepcidin iron is an essential element for nearly all living organisms and plays a key role in a range of processes including oxygen transport and storage, catalysis of redox reactions, production of metabolic intermediates and host-defence ( ). until recently, little was known about the regulatory elements involved in the control of iron uptake and distribution within the body. the recently discovered peptide hepcidin has been shown to be a key regulator of iron metabolism within the body in response to a range of conditions, including infl ammation, hypoxia and anaemia ( ) . this talk will focus on work towards elucidating structure/activity data for hepcidin with the aim of gaining a better undertstanding of the interaction between hepcidin and its receptor, ferroportin. we hope that this information will facilitate the design of synthetic agonists or antagonists of ferroportin to be used as potential drug leads. three-dimensional structures of investigated analogues were determined using two-dimensional nmr spectroscopy and molecular dynamics simulations with time-averaged restraints. the analysis of structural differences exhibited by different modifi cations provides the basis for understanding conformation -activity relationships and thereby the mechanism of interactions of the analogues with receptors. nmr structure of the micelle-bound rfa and rfa, two peptide ligands of the gpr receptor a novel rfamide peptide, named rfa and with no meaningful similarity with other members of this family, has been recently characterized. its precursor encompasses several potential cleavage sites and thus may generate various mature peptides including an nterminally extended form of rfa, termed rfa. both peptides act as endogenous ligands of the g-protein coupled receptor gpr . this receptor has been recently implicated in the bone metabolism regulation the determination of the d structure of such peptides is essential for the elucidation of their structure/function relationships and for the design of potent agonists or antagonists. although structure elucidation of a ligand in the absence of the target receptor can deliver limited insight into the bioactive conformation, there is emerging evidence that interactions with the cell membrane is a key step required for receptor recognition. in this context, we have investigated the solution conformation of rfa and rfa by cd, nmr and molecular modelling in different media, in particular in one miming the "free" form of the molecule (methanol or mixture tfe/water) and in a cell membrane mimetic medium (dpc micelles). in an organic solvent, both peptides adopt the same conformation, i.e. an amphipathic alpha-helical structure, fl anked by two n-and c-terminal disordered regions. when bound to dpc micelles, the n-terminus remains fl exible and an helix is present at the same position as in the "free" form for both molecules. in contrast, the cterminal extremity becomes structured adopting an inverse gamma-turn conformation. these data represent the fi rst step for the rational design of new molecules that might be used in the treatment of osteoporosis. acknowledgements: supports were obtained from inserm and ifrmp . the nmr spectrometers are supported by grants of the conseil régional de haute-normandie. nmr and molecular modelling facilities are provided by the centre de ressources informatiques de haute-normandie. antimicrobial activity of analogues of a peptide isolated from venom glands of social wasps polistes major major inhabiting the dominican republic recently we have described isolation and biological activities of several new peptides from the venom glands of social wasps polistes major major found in dominican republic. we have also reported the synthesis of their analogues in order to investigate structure-activity relationship with respect to the antimicrobial and hemolytic activities ( ). here we report the activities of a few further analogues of one of the peptides called pmm (h-ile-asn-trp-lys-lys-ile-ala-ser-ile-gly-lys-glu-val-leu-lys-ala-leu-nh ). the parent sequence or its truncated analogues were modifi ed on the n-terminus with -aminocaproic acid, glycolic acid, palmitoic acid, and -acridinyl and -( , , , -tetrahydro)acridinyl groups. the new analogues were tested for their antimicrobial activity (determination of minimal inhibitory concentration values -micusing broth dilution method) and hemolytic activity (determination of the ic value using suspension of rat erythrocytes). the palmitoylation unfortunately did not enhance antimicrobial activity against the tested microorganisms (bacillus subtilis, staphylococcus aureus, escherichia coli and pseudomonas aeruginosa). substitution of amino acids ser or glu subsequently for alanine, serine or lysine did not infl uence the activity of the peptides signifi cantly. gramicidin s, gs, c-(val-orn-leu-d-phe-pro) , was isolated from bacillus brevis. it forms a two-stranded antiparallel -sheet fl anked by two ii' -turns. it was found that the distribution of hydrophobic and hydrophilic residues on the opposite sides of the sheet is a structural feature required for gs antimicrobial (am) activity. despite its wide gram+ and gram-antimicrobial activity gs is useless in therapy because of its high hemotoxicity in humans. it was found, however, that the analogues of gs- (gs with lys-leu inserted into each strand) got more am selective, when their amphipatic moments were perturbed by swapping adjacent lys leu/val or confi guration reversal at lys ( ) . here, we report on effects of similar perturbations put on gs original c-decapeptide, using the following examples: c-(val-lys-leu-d-his-pro) , , . as the mother compound, and its three analogs, viz. c-(val-d-lys-leu-d-his-pro-val-lys-leu-d-his-pro) -lys converted to d, c-(lys-val-leu-d-his-pro-val-lys-leu-d-his-pro) -val -lys swapped, and c-(val-leu-lys-d-his-pro-val-lys-leu-d-his-pro) -lys -leu swapped, - ; all having reduced ring-sequence symmetry. the peptides were synthesized by solid-phase methods using -fl uorenylmetoxycarbonyl (fmoc) methodology. having solved their structures by d-nmr and having tested their activities/selectivities, we confi rmed that only had relatively favorable bio-profi le, as already published ( ) cereulide, a foodborne peptide highly toxic towards the insulin producing beta-cells of the pancreas cereulide is a heat stable cyclic, lipophilic (log kow . ) peptide ( g mol/ ) produced by certain strains of bacillus cereus, a bacterium connected to emetic food poisonings. it is insoluble in water, soluble in ethanol, methanol, dmso, food oils. cereulide exposure caused collapse of mitochondrial membrane potential in all tested human cells at low (ng/ml) exposure concentration (nk cells, t lymphocytes, caco , calu , neural paju, hela). toxicity is caused by its action as ion carrier with a selectivity of k + : na + = > : . in various foods connected to human illness, concentrations of . to g/g of cereulide were measured. in a case where the remains of a meal that had caused acute serious illness of two adult persons, were obtained for analysis, . g of cereulide was found /g of food. we undertook to explore the effects of purifi ed cereulide, and cereulide containing bacterial extracts, on porcine pancreatic islet cells in culture. foetal porcine islet cells were exposed to heat killed extracts from food-borne b. cereus strains producing or not producing cereulide and to purifi ed cereulide. effects were assayed using viability staining with fl uorochromes and cellular contents of dna and insulin. exposure to ng/ml of purifi ed cereulide caused necrotic cell death of the islet cells impairing their insulin content within days. cell extracts of cereulide positive b. cereus strains connected to food poisoning or isolated from food items were toxic, corresponding to their measured cereulide content. extracts of b. cereus strains producing or not producing the b. cereus diarrhoeal toxin, but not cereulide, were tolerated by the porcine islet cultures up to concentrations fold higher compared to extracts from strains containing cereulide, produced substance toxic towards porcine fetal langerhans islets and beta cells. application of non-sequential pharmacophore concept for design of antimicrobial peptide dendrimers unique structure of dendrimeric compounds consisting of a central core and several generations of branches provides opportunity of multiple practical solutions in the area of medicine. location of a high number of functional groups at the surface, allows to present multiple pharmacophoric units to the receptors, with immediate application in the design of a new generation drugs or vaccines, tools for studying autoimmune diseases or understanding gene delivery mechanism. here we present another possible application of dendrimeric compounds -preparation of drug molecules, which mimic active conformations of various macromolecular ligands. we focused on low molecular weight basic dendrimeric peptides, which mimic active conformations of recently discovered natural antimicrobial peptides. structurally, natural compounds are linear cationic peptides consisting of - amino acids that kill a broad spectrum of microbes destabilizing ordered structure of their cell membrane. it is generally accepted that positive charge and an induced amphipathic conformation are necessary for their antimicrobial activity. the project is related to multi-drug resistance of numerous bacteria against conventional antibiotics and involves de novo design of - generation dendrimeric peptides, which mimic sequencerelated active conformations of natural compounds (non-sequential pharmacophore concept). apparently, several groups of small peptide dendrimers were synthesized and structurally characterized (nmr, cd). they are potent antimicrobials, active against broad spectrum of species including mrsa and esbl strains. interactions between model membranes and peptide dendrimers of various structure will be discussed. seawater desalination is most commonly done today by reverse osmosis (ro) using thin-fi lm composite membranes. a major problem in ro desalination is biofouling, caused by adhesion and growth of bacteria to form biofi lm on the membrane surface. recently we proposed a new approach to reduce biofi lm formation on ro membranes that is based on immobilization of antimicrobial peptides (amps) onto the membrane surface. in this study we screen amps capable of reducing biofi lm growth under conditions simulating seawater desalination, and search for mode of binding to the membrane without affecting the peptides bioactivity. a specifi c bioassay was developed for screening peptides activity in high salinity conditions in order to evaluate the inhibition of biofi lm growth, based on growing biofi lmforming bacteria in a -wells microtiter plate. we prepared various amps known from the literature by solid phase peptide synthesis (spps) using fmoc-chemistry. the bactericidal activity of amps was examined in fresh water and compared to high salinity water. most amps lost their activity in high salinity conditions; yet, few peptides possessed their bactericidal activity and were used in subsequent experiments. searching for mode of linkage was performed by evaluating the bactericide activity of amps modifi ed with numerous types of linker molecules. based on literature studies that showed no decrease in bactericidal activity of amps upon n-terminal modifi cation, we prepared the corresponding peptides with modifi ed spacers on their amino-terminal and evaluated their antimicrobial activity in solution. indeed, we obtained gly -spacers that retained activity, which were used subsequently as linkers to ro membranes. the mode of binding, as well as bactericide activity of the peptides and of the membranes will be presented and discussed. this study will lay the bases for a novel approach to decrease biofi lm formation on the surface of ro membranes during ro desalination. bioactive tripeptides ile-pro-pro and val-pro-pro protect endothelial function in vitro in normotensive and hypertensive rats milk drink containing casein-derived bioactive tripeptides isoleucylprolyl-proline (ipp) and valyl-prolyl-proline (vpp) has been shown to decrease blood pressure both in animal models and clinical studies. this effect can be attributed to the tripeptides. it has been suggested that one possible blood pressure lowering mechanism of the tripeptides could be angiotensin-converting enzyme (ace) inhibition. however, not all studies support this fi nding. the effect of tripeptides ipp and vpp on vascular function was investigated in vitro using rat mesenteric arteries. superior mesenteric arteries isolated from male wistar-kyoto and spontaneously hypertensive (sh) rats were incubated in krebs solution containing mm of the peptide (either ipp or vpp) in + ºc for , , or h. after incubation mesenteric artery rings were mounted in an organ bath chamber and extensive vascular reactivity measurements were performed. acetylcholine-induced endothelium-dependent relaxation was better preserved (p < . ) in mesenteric arteries of both strains incubated with ipp or vpp compared to the control. clear differences were not observed in sodium nitroprusside-induced endotheliumindependent relaxation. the ace-inhibitory activity of ipp and vpp was studied by measuring the response to a single administration of angiotensin i and ii in organ chambers. proportioned to kcl-induced contraction, no clear reduction in angiotensin i -contraction was seen. thus, ace-inhibition may not be the main mechanism for the long-term effects of ipp and vpp in the protection of endothelial function. we suggest that the tripeptides do not affect smooth muscle but they protect endothelium during incubation indicated as preserved acetylcholine-induced endothelium-dependent relaxation. and directly and modulate other parts of host innate immunity. today, more than cationic peptides have been identifi ed. they all have certain conserved physical features including a net positive charge, contain approximately % hydrophobic amino acids and have sizes ranging from to amino acids. however, virtually any -sheet, loop including -helix, type of secondary structure can arise including -turn and extended. the multitude of cationic peptide sources, structures and a spectra of activity is matched by a number of complex and controversial models attempting to describe and explain their modes of action. little is known about the sequence requirements of short host defense peptides like bactenecin ( mer). with help of our novel technique using a artifi cially created luminescence producing gram negative bacteria and peptide synthesis on cellulose we can investigate the sequence requirements of such peptides. hundreds of peptides are tested for their ability to kill pseudomonas aeruginosa. complete substitutional analyses of different bactenecin variants as well as a semirandom peptide library with about members were measured. the complete substitutional analysis will give us information about the importance of each single position whereas the peptide library will give us broader information which composition of amino acids results in an active antimicrobial peptide. the data will be analyzed using the quantitative structureactivity relationship approach (qsar) to identify sequence patterns that discriminate between superior activity cf. equivalently active and inactive. this will give us mechanistic cues for a better understanding of the mode of action of the short antimicrobial peptides. the results of these measurements and analyses will be discussed in detail. here we propose a simple and rapid fl ow cytometric method to assess internalization of the peptides in bacteria. the method is based on the use of fl uorescently-labeled peptides and of the extracellular quencher trypan blue to discriminate between a cell surface and cytoplasmic localization of the tested molecules. to his aim, we used bodipylabeled peptides showing different modes of action. these included some fragments of bac , a proline-rich peptide known to penetrate bacterial and eukaryotic cells without membrane damage [ , ] , and polymyxin b, a peptide antibiotic that binds to lps and to the cell membranes. by using this approach coupled to fl ow cytometric analysis, we showed that the fl uorescence intensity of e. coli and s. typhimurium cells treated with sub-inhibitory bodipy-bac concentrations did not decrease despite extensive washing and addition of the quencher trypan blue. in contrast, the fl uorescence of cells treated with bodipy-polymyxin b, as well as that of bacteria treated with a fl uorescein-labeled anti lps antibody, were promptly and almost totally quenched by addition of trypan blue, indicating their accessibility on the bacterial surface. these results confi rm the suitability of this method to rapidly infer the localization of labelled molecules in an accessible or inaccessible compartment of the treated bacterial cells. hp ( - ) is an antimicrobial peptide derived from the n-terminus of helicobacter pylori ribosomal protein l (rpl ). in our previous study, several analogues of hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , with amino acid substitutions that increased or decreased net hydrophobicity, were designed and showed that an analogue, a designed by substituting gln and asp with trp at positions and , respectively, caused increased antibacterial activity in minimal inhibition concentration (mic) and minimal bactericidal concentration (mbc) without having hemolytic activity. the peptide a acted also synergistically with known antibiotics including chloramphenicol against bacterial cells. fluorescence activated fl ow cytometry showed that a -treated cells had higher fl uorescence intensity than untreated cells, similar to that of melittin-treated cells. the peptide a showed a strong antimicrobial activity against antibiotic-resistant pseudomonas aeruginosa from otitis media, clinically isolated mrsa and vrsa including biofi lm-forming bacteria in vitro and in vivo. the ototoxicity of a was studied in vivo by topical application to the middle ear in guinea pig model. twenty guinea pigs ( groups, each group n= ) were each injected by transtympanic approach with ul of ug/ml, ug/ml, ug/ml, ug/ml of a , and . % gentamicin sulfate was instillated as a control. auditory brainstem responses (abr) to click were measured between st and th days after injection. histologic investigation of cochlea was performed by scanning electron microscope and light microscope. the results showed that topical application of a to the middle ear is well tolerated without cochlear damage. the present study, therefore, demonstrates that the usefulness of antimicrobial peptides for multi-drug resistant bacteria including as a new ototopical agent for crpa otitis media. structure-activity relationship study of kiss- : identifi cation of an antagonist of gpr in the absence of ccda, ccdb inhibit the cell division and can kill bacteria by a mechanism that involves the dna gyrase. bacterial dna gyrase is unique among the type ii topoisomerase with ability to negatively supercoil dna. the enzyme consists of two subunits, a (gyra) and b (gyrb) and operates as an a b heterotetramer. the mechanism of the inhibition of the gyrase activity by ccdb is still an object of many debates, but is clear that r residue of gyra and the c-terminus of ccdb (w -i ) play a crucial role in the gyrase-ccdb interactions. as an approach for a better understanding of this mechanism as well as for development of new gyrase inhibitors, we have synthesized peptide analogues of the ccdb protein and studied its activity by supercoiling assays and bacterial growth. five fragments (ccdb et , ccdb et , ccdb et , ccdb et and ccdb ss ) of the natural ccdb were designed and synthesized by spps. for the design, we considered the residue c-terminal -helix (residues e to w ), the loop that connects two strands of the wing sheet (residues r to l ) and an n-terminal region that includes the fi rst of the fi vestranded antiparallel -sheet. all peptides, except ccdb et , showed inhibition of the supercoiling activity of the dna gyrase, especially ccdb et with a mic = m. free peptides not showed antimicrobial activity, but when encapsulated in liposome (suv) were able to inhibit the bacterial growth in liquid culture medium. the growth inhibition in vitro for ccdb et was about % for gram negative bacteria. our fi ndings revealed a novel synthetic inhibitor of dna gyrase and ccdb et analogue is a good starting point for the development of a new and specifi c class of antibacterial agents based in the dna gyrase inhibition. acknowledgements: support: fapesp and cnpq synthesis and neuroprotective properties of short peptides consisting solely from glycine and proline residues now appears more and more data on biological activity of short peptides consisting solely from glycine and proline residues. it is suppose, that these peptides, named as "glyprolines" (gps), can be formed from collagen, ÅÑÌ and related proteins. however an effect of these peptides on cns is not yet understood. the aims of this work were to improve a methodology of gps synthesis and to study cytoprotective properties of some new gps in culture of neuronal cells. gps with a common structure (gp)n, (pg)n, and (pgp)n (n= - ) were synthesised by consecutive growing of peptide chain and fragment's condensation. synthesised peptides were characterised by hplc, mass-spectrometry, element analysis etc. cytoprotective activity of gps was assessed on an increase of survival of cultivated ÐÑ cells after Í Î -induced oxidative stress. it was shown, that from all tested peptides only (gp)n and pgp reveal cytoprotective activity. at the concentration m these peptides reduced an amount of damaged cells . - . times in comparison to the control. thus preliminary data received show that some gps demonstrate a strong cytoprotective activity and therefore it is advisably to put them on further study on in vivo models. in preliminary investigations we found that all the peptides inhibited to a high degree the replication of hsv- in vero cells. moreover, these compounds did not show any cytotoxic activity against the vero cells. sexual dimorphism of hldf- peptide neuroprotective action in alzheimer's models in vivo and vitro we established that hldf- peptide, biologically active fragment of human leukemia differentiation factor (hldf), restores ability for learning, and also prevents loss of long-term memory and decrease in the exploratory behavior of wistar rat-males with experimental alzheimer's disease induced by intrahippocampal injection of betaamyloid peptide a ( - ) and ibotenic acid. the hldf- peptide was shown to render protective infl uence directly on primary culture of hippocampal and cerebellar neurones, isolated from newborn male brain, under conditions of the beta-amyloid toxicity. protective action of hldf- peptide on newborn rat-male neurons is connected with poster abstracts its ability to reduce -alpha reductase mrna expression in more than times, blocking testosterone metabolism into dihydrotestosterone (dht) and thus interfering hyper activation of nmda receptors in ca areas of hippocampus. the protective action of hldf- peptide was investigated also and on female-rats with experimental alzheimer's disease. it was shown, that in contrast to males at which both forms of memory are broken: and long-term and working ones, in the case of females, injections of a ( - ) + ibotenic acid result in infringement of only working memory, at safety of long-term memory. introduction of hldf- peptide restored the broken working memory at females as effectively, as at males. however the mechanism of protective action of peptide on primary culture of hippocampal and cerebellar neurones, isolated from newborns female-rat brain, is connected not with the decrease in hyper activation of nmda receptors, but with abad ( beta-hydroxysteroiddehydrohenase of the th type) mrna expression enhancement which is a target of beta-amyloid peptide action and blocking of progesterone conversion into alpha-dihydroprogesteron due to -alpha-reductase mrna expression inhibition. in vitro and in vivo studies of p- , an antimicrobial peptide active against multidrug resistant gram positive cocci we have designed octapeptide based on the sequence of sepecin b, an antibacterial protein of sacrophaga peregrina, effective against gram + bacteria. we systematically incorporated internal hydrophobic residues for a cationic, helical amphipathic structure effective for its high antimicrobial activity. it is also modifi ed c-terminally by a dehydro leucine residue effective for its structural stability. the synthesis of dehydro amino acid was done by solution phase and other amino acids were coupled by solid phase peptide synthesis method. anti-bacterial activity of the peptide was done by standard micro broth dilution technique against clinical isolates of gpc including methicillin resistant s. aureus (mrsa), methicillin sensitive s. aureus (mssa), hlar, group a and group b streptococci cultured from pus, wound and throat swab. all these isolates were known to be responsible for nosocomially acquired infections. s. aureus atcc was used as quality control strain. invivo effi cacy of this peptide was also tested in a mouse model with epidermal lesions caused by mrsa. the mic obtained for the peptide was g/ml (average) for the tested strains. the peptide showed no hemolytic activity against human red blood cell. in the invivo studies the healing was induced early (after hrs total healing was seen) in the experimental animals. this novel peptide has a potential to evolve as a therapeutic option in infections caused by resistant gpc. , an adhesive protein, is recognized by the activated platelet integrin á ÉÉb through the arg-gly-asp (rgd) sequence resulting to platelet aggregation and thrombus formation. inhibition of this process can be achieved by rgd peptide analogues that bind to á ÉÉb receptor. however, this class of inhibitors upon binding to receptor cause an outside-in signaling which induces a further activation of the platelet. in previous studies we presented cyclic (s,s)-cdc-containing compounds (ic ~ ìm) and á ÉÉb derived sequences (y mesradr , á ÉÉb - ) (ic ~ ìm ) that exhibit a non-rgd-like inhibitory activity [ , ] . this interesting aspect could be the basis for the design and development of a new class of anti-platelet agents that could overcame the drawback of platelet activation through the outside-in signaling. to this aim we designed, synthesized and tested for their inhibitory potency various á ÉÉb - hybrid analogues incorporating the (s,s)-cdc-motif. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. the inhibition assays on the adp induced platelet aggregation revealed that incorporation of the (s,s)-cdc-motif considerably increases the inhibitory activity of the á ÉÉb - analogue. the unique toxic effect of acrebol, a novel peptide from acremonium exuviarum a novel peptaibol, named acrebol, was isolated and purifi ed from the fungal strain bmb found in water-damaged wood-based indoor building material. the strain bmb was identifi ed as acremonium exuviarum based on morphology, its sequence, and cycloheximide resistance. ms/ms analysis showed that acrebol is a mixture of two almost identical peptaibols composed of - amino acid residues with masses and da, acephe-iva/val-gln-aib-ile-thr-leu-aib-pro-aib-gln-pro-aib and acephe-iva/val-gln-aib-ile-thr-leu-val-pro-aib-gln-pro-aib, respectively. the c-termini of the peptaibols was seroh however the sequence (mass of da) between b and seroh could not be interpreted. both isoforms of acrebol had a strong toxic effect on boar spermatozoa, feline fetus lung cells, murine neuroblastoma, and mouse insulinoma min cells. we found that, unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria. acrebol did not disturb the ionic homeostasis and osmotic balance of mitochondria and induced no release of apoptogenic proteins (cytochrome c) from the intermembrane space of mitochondria. acrebol strongly inhibited complex iii of the respiratory chain (ic ~ ng/ml), presumably, the outer quinone-binding center and, similarly to myxothiazol but in contrast to antimycin a, decreased the production of superoxide anion in the outer compartments of mitochondria. in the boar spermatozoa, acrebol, blocking the respiratory chain, caused the atp depletion due to the oligomycin-sensitive reversion of the reaction of atp synthesis, which resulted in the inhibition of progressive movement. acrebol induced necrosis-like death of mouse insulinoma min cells whose energetic metabolism is strongly dependent on oxidative phosphorylation in mitochondria. thus, acrebol is a unique peptaibol with a specifi c pattern of the toxic effect. delta sleep inducing peptide (dsip), its analogues and deltaran®: biological activity and mode of action for the last decade we have been engaged in studies on the endogenous neuromodulator dsip (waggdasge) and a large group of its derivatives in respect of both their physiological activity and mechanism of action. a wide range of evidences confi rmed benefi cial effects of dsip and some active analogues under experimental stress models. dsip has emerged as promising and potentially effective therapeutic agent due to strong and unique adaptive and stress protective activity revealed during the study. dsip related drug deltaran® registered in russia has also showed the signifi cant effi ciency in animal test models and clinic. the peptides of dsip family often do not demonstrate any effects under normal and comfortable conditions or even cause slight prooxidative and stress promoting effects. these properties and established wide profi le of biological activities of dsip complicate the work on dsip mode of action. cellular and subcellular effects of this peptide still remain poorly studied. previously we investigated some biochemical events underlying the stress protective effi ciency of dsip and its derivatives. in continuation of these studies we have attempted to evaluate the putative dsip infl uence on classical cellular processes utilizing stressprotective heat shock proteins (hsps) and apoptosis. effect of dsip on the level of hsp expression in human erythroleukemia cell line k was detected.. we have found that dsip down regulates the increase of intracellular hsp level during incubation of cells in high density cell culture. according to our preliminary data dsip increased hsp level in murine t-cell line ctll- similar to -and ß-adrenergic receptor agonists. in murine thymocytes dsip increased both apoptosis and hsp . we propose that registered effects of dsip are mediated through adrenergic receptors. cellular mechanisms of dsip and related peptides action are under way. identifi cation, chemical synthesis, and antimicrobial activity of tbd- -the fi rst -defensin isolated from reptiles antimicrobial peptides (amps) and proteins have been discovered in single and multicellular organisms indicating the importance of this peptide class for the innate immune system. amps are active against bacteria, fungi and viruses by affecting either the microbial cytoplasmic membrane, thus increasing its permeability or interacting with specifi c targets. defensins form one of the major subfamilies of amps, among which -and -defensins play a signifi cant role in bridging innate and adaptive immunity in mammals. the cationic -defensins are cysteine-rich and vary in length from to residues. the -stranded structure of -defensins is stabilized by a characteristic arrangement of three conserved disulfi de bonds between cysteines - , - , and - . although -defensins lyse the bacterial membrane at higher concentrations it has been shown that they modulate also the immune system, e.g. being chemotactic for t-cells. we have isolated a novel mer -defensin called tbd- from leukocytes of the european pond turtle and deduced its complete sequence de-novo by combining different tandem mass spectrometry techniques (maldi, esi; cid and etd) and edman degradation. it was also possible to identify the disulfi de pattern in a tryptic digest by maldi-mass spectrometry. the deduced peptide sequence was afterwards confi rmed by solid phase peptide synthesis. thus two fragments were synthesized by standard fmoc/ t bu chemistry using three orthogonal cysteine protecting groups (trt, acm, tbu) to selectively form the right disulfi de bridges. after native chemical ligation of the two peptide fragments the fi rst two cysteines were oxidized on air followed by iodide oxidation for the second and dmso oxidation for the third disulfi de bridge. in antimicrobial activity assays, tbd- synthesized in -conformation was active against gram-positve, gram-negative bacteria, and fungi similar to the native peptide. trichogin ga iv (trga), an antimicrobial peptide of the lipopeptaibol family, continues to reveal peculiar properties since the time of its former identifi cation by rebuffat and coworkers. x-ray diffraction studies showed that trga is folded in a mixed /a-helix conformation, while nmr, cd and ir absorption experiments proved that this structure is predominantly populated in solution, although more disordered structures contribute to the conformational landscape of trga. we have recently shown by time-resolved experiments, that an equilibrium between helical conformers and more compact, folded conformers takes place in solution, with interesting transition dynamics in the microsecond time scale. in our conformational studies on trga we realized that the d geometry of the peptide chain reproduces the structural environment of the ion coordination site of calcium-binding proteins. this fi nding urged us to investigate the binding properties of trga with respect to ca(ii), gd(iii) and tb(iii), because lanthanide ions have been widely employed in biochemical studies as best substitutes of ca(ii). the binding of ca(ii), gd(iii) and tb(iii) to trga gives rise to a conformational transition, monitored by cd spectra at different ionpeptide molar concentration ratios. at high r values, the cd curves of all the ion-peptide complexes show a positive maximum at ~ nm, typical of type ii turns, suggesting the population of bent structures. the quasi-isodichroic point found for all systems between and nm, indicates that an equilibrium between extended helical and bent conformations actually takes place. fluorescence experiments on the tb(iii)/trga adduct have shown that, upon ion binding, the fl uorescence quantum yield of tb(iii) markedly increases, due to the release of water molecules from the ion coordination inner shell. molecular dynamics calculations on the peptide/ca(ii) adduct were also performed to obtain structural and dynamical information. antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. castros group isolated and determined the sequence of the peptide hy-a (ifgailplalgalknlik) of skin secretion from the frog hypsiboas albopunctatus which showed antimicrobial activity. the aim of the present work was evaluated analogues to supply information poster abstracts about the relationship structure-biological activity. the peptides were synthesized by spps using the fmoc chemical approach. the biological activities were assayed by measuring growth inhibition of two types of gram-positive bacteria and others two types of gram-negative. the synthesis and purifi cation of peptides by hplc was effi cient and a high purity level ( %) was obtained. the peptide containing trp in position (for fl uorescent studies) replacing leu presented mic values comparable to wild type sequence: um, um, um and um for e. coli, p. aeruginosa, s. aureus and b. subtilis, respectively. two peptides with this modifi cation but containing at the n-terminal region one group acetyl or a residue of asp showed mic values of um for e. coli and p. aeruginosa, although um for gram-positive bacteria. different results were observed when the residue added was lys. in this case, the activity against whole bacteria was sustained or increased. conformational properties were investigated by cd techniques in water, tfe and in zwitterionic micelles (lpc). the cd experiments demonstrated that in water, the peptides have a random structure, but in tfe and lpc solutions they acquired an ordered structure, composed mainly by -helix. however, these data there is no relationship between the structure and activity against bacteria gram-positive. these results showed that the n-terminal region of the peptide hy-a develop key roles in its antibacterial action different types of bacteria. pexiganan is an antimicrobial peptide remarkably effective against bacteria causing skin infections, and has been commercially developed as a topical cream to treat infected diabetic foot ulcers. [ ] [ ] [ ] as peptide drug-based therapy often suffers with low drug stability in vivo, suitable delivery systems must be developed. with this purpose in mind, we have designed a pexiganan-chitosan conjugate to combine the exceptional bioadhesion and tissue regenerating abilities of chitosan [ ] [ ] [ ] with the excellent antibiotic properties of pexiganan. we herein wish to report our fi rst results on the successful synthesis, ft-ir and amino acid analysis of a pexiganan-chitosan conjugate prepared by covalent attachment of a cys-containing pexiganan analogue to the chitosan's amino groups, by means of the heterobifunctional cross-linker sulfo-emcs. . overexpression of e. coli oligopeptidase b confers resistance to the proline-rich antibacterial peptides scocchi, marco; de gobba, cristian; mattiuzzo, maura; gennaro, renato university of trieste, italy the proline-rich antimicrobial peptides (pramps) are a large group of cationic peptides isolated from mammals and insects, which show a spectrum of antibacterial activity limited to gram-negative bacteria and a remarkably low cytotoxicity towards eukaryotic cells. pramps are thought to act in a permeabilization-independent manner, via energydependent internalization into bacteria followed by recognition and inactivation of internal molecular targets. with the aim of investigating their mode of action, we have isolated a number of e. coli clones showing increased resistance to the bovine proline-rich peptide bac after transformation with a dna library from bac -resistant mutants. among the recombinant plasmids responsible for resistance, some of them harboured the gene coding for the oligopeptidase b (opdb), a serine peptidase belonging to the prolyl oligopeptidase family (pop) broadly distributed among unicellular eukaryotes and gram-negative bacteria, which has emerged as an important virulence factor. opdb was cloned and expressed under the control of an inducible promoter and the transformants tested for susceptibility to a panel of antimicrobial peptides, including pramps. olipopeptidase activity of purifi ed opdb was then tested in vitro against the same peptides. the results indicate that the clones overexpressing opdb are more resistant to the pramps and that the degree of resistance correlates with the expression level of opdb. in addition, in vitro incubation of pramps with the purifi ed peptidase, followed by mass spectrometry analysis, showed that it promptly hydrolyzes all the peptides assayed to short, inactive fragments. these results suggest that opdb may contribute to cleavage and inactivation of antimicrobial peptides that are internalized into the target cells and support the notion that opdb is a novel virulence factor. guan, shuwen; huang, lei; li, pengfei; wang, liping; li, wei college of life science, jinlin university, china great progresses have been made in the research on identifying molecules of therapeutical potential for delaying aging. using caenorhabditis elegans, we had tested the effects on stress resistance and life span of treatment with dhhp- (deuterohaemin-alahisthrvalglulys), synthetic mimetics of the antioxidant peroxidases, which neutralizes peroxide. to further test the mechanisms of dhhp- on life span extension, exogenous protein sod and catalase levels were measured. we show that dhhp- is able to elevate in vivo sod and catalase activity levels after two days administration. treatment with exogenous dhhp- affected endogenous protein sod and catalase levels and elevated the expression of sod- ::gfp in the head and vulva. on the other hand, dhhp- can extend life span of c. elegans lacking sod- or ctl- gene expression by rnai. this suggested that the antioxidant enzyme in c. elegans may not the only target affected by dhhp- . du, haidong; yan, yumei; wang, liping; li, wei college of life science, jinlin university, china high concentration of hydrogen peroxide (h o ) induces nuclear dna fragmentation, lipid peroxidation and has been implicated in many diseases including heart failure, parkinson's disease, and cancer. in a systematic attempt to develop an effective scavenger of h o , we have successfully synthesized an artifi cial microperoxidase, deuterohaemin -alahisthrvalglulys (dhhp- ) as core catalytic center to which amino acid peptide was covalently attached. dhhp- exhibited potent peroxidase activity, favorable membrane permeability and thermal stability.two experimental models of oxidative injury were established in order to investigate the anti-oxidative effect of dhhp- : one was induced by hypoxia-reoxygenation in cultured heart-derived h c cells bioactive peptides and another was caused by h o in cultured neonatal rat ventricular myocytes. dhhp- could protect cells against oxidative injury by determining mtt cell proliferation assay, ldh leakage and ca +-atpase activity. furthermore, dhhp- repressed the apoptosis gene expression, such as p , hsp and heme oxygenase¢ñ, that proved dhhp- had protective effect in cultured neonatal rat ventricular myocytes . taken together, this small microperoxidase exhibited excellent ¡°druggable¡± properties, and could be a promising agent for ros-associated diseases with unmet needs. trichogin ga iv is the most extensively investigated member of the class of lipopeptaibols that are linear peptide antibiotics of fungal origin, characterized by the presence of a variable, but remarkable, number of aib residues, a fatty acyl group at the n-terminus, and a , -amino alcohol at the c-terminus. several analogues of trichogin ga iv with amino acid substitutions or deletions were designed which allowed determination of the minimal inhibition concentration against gram-positive and gram-negative bacteria and various pathogenic fungal cells. the natural peptide exhibits a specifi c activity against s. aureus and only a marginal hemolytic effect. interestingly, trichogin ga iv is active also against several methicillinresistant s. aureus strains. studies on synthetic analogues demonstrated that substitution of the c-terminal leucinol by leu-ome, or substitution of one aib residues by the epr label toac do not perturb signifi cantly the biological activity of the peptide. on the other hand, removal of or n-terminal residues eliminated any antibacterial activity. finally, studies of proteolytic degradation on trichogin ga iv and analogues where the aib residues are replaced by leu demonstrated that the presence of several non-coded aib residues endows the natural peptaibol with remarkable resistance to proteolysis. the present results indicate that trichogin ga iv is a promising lead compound for the development of new, selective and protease-resistant, antibacterial drugs. the siderophore microcin family: from the genetic systems to the antimicrobial peptides vassiliadis, gaëlle; peduzzi, jean; rebuffat, sylvie muséum national d'histoire naturelle-cnrs, france microcins are low molecular weight antimicrobial peptides secreted by enterobacteria and involved in microbial competitions within the intestinal tract. they are synthesized by the ribosomal pathway. we have isolated the fi rst siderophore peptide ( ), a post-translationally modifi ed form of the chromosomally encoded microcin e (mcce ) from klebsiella pneumoniae, having a potent bactericidal activity mainly directed against escherichia coli. the post-translational modifi cation consists of a glycosylated catechol-type siderophore linked to the c-terminus. we have recently identifi ed the genes responsible for the acquisition of this modifi cation and proposed a model for its biosynthesis ( ) . in order to identify novel siderophore peptides, we analyzed the genetic systems of several microcinogenic strains. based on the genetic organization of the microcin gene clusters, three microcins which had preparation of a close mimic of the n-terminal part of the c a receptor (c ar) by selective introduction of sulfated tyrosine residues ( ) and chips in complex with our sulfated c ar-model by multi-dimensional nmr techniques. based on these interaction data and the solution structure of the complex, chips-based c ar inhibitors for use in anti-infl ammatory therapy might be designed. insulin-like peptide (insl ) was fi rst identifi ed through a search of the expressed sequence tags (est) databases. primary sequence analysis showed it to be a prepropeptide that is predicted to be processed in vivo to yield a two-chain sequence (a and b) containing the insulin-like disulfi de crosslinks. the high affi nity interaction between insl and the receptor rxfp (gpcr ) coupled with their apparent co-evolution and partially overlapping tissue expression patterns strongly suggest that insl is an endogenous ligand for rxfp . given that the primary function of insl /rxfp pair remains unknown, an effective means of producing suffi cient quantities of this peptide and its analogues is needed in order to systematically investigate its structural and biological properties. a combination of solid phase peptide synthesis methods together with regioselective disulfi de bond formation were used to obtain insl . both chains were identifi ed as being ¡ §diffi cult sequences¡¨ and were unusually resistant to standard synthesis protocols including those mediated by microwaves. the a-chain was also prone to signifi cant aspartimide formation. the b-chain, in particular, required the use of the strong tertiary amidine, dbu, for more effective nƒÑ-deprotection during its assembly. following chain combination and sequential disulfi de bond formation, the resulting synthetic insl was obtained in good overall yield and shown to possess a similar secondary structure to human relaxin- (h relaxin). the peptide was able to inhibit camp activity in sk-n-mc cells expressing the human rxfp receptor with a similar activity to h relaxin. in contrast, it had no activity on the human rxfr receptor. novel cyclic bacteriocin-like peptides from strains of anabaena (cyanobacteria) leikoski ( ) and microcystis aeruginosa ( ) . the genome sequence of fi lamentous and diazotrophic cyanobacterium anabaena revealed a putative gene cluster (acy) encoding a bacteriocin-like peptide. one of the acy genes encoded a prepeptide, which showed n-terminal homology to the known cyanobacterial prepeptides but the mature peptide product in anabaena could not be predicted. we sequenced prepeptide genes from several anabaena strains to fi nd a prepeptide containing either cysteine or methionine, since sulphur containing amino acids enable the detection of the mature peptide product through s-labelling and lc-ms. the nucleotide sequence of the prepeptide genes revealed enormous variety in closely related anabaena strains. one strain, anabaena b contained a methionine in the prepeptide, and a cyclic heptapeptide product corresponding to the precursor was discovered in this strain. since the cleavage sites were found to be conserved in all anabaena strains the products could be predicted from the amino acid sequence and identifi ed by lc-ms. in addition, the biosynthesis of the decapeptide anacyclin in anabaena was verifi ed by heterologous expression of the acy genes in e. coli and the structure of the peptide was confi rmed with a synthetic reference peptide. altogether, new cyclic peptides were found in strains of anabaena with very little sequence conservation. cyanobacteria seem to be versatile producers of peptides using both ribosomal and non-ribosomal biosynthetic pathways. it is well established that n-terminal fl ank of corticotropin-releasing factor molecule is crucial for its biological activity. little, however, is known about in vivo effects of crf-derived peptides. this study was aimed to investigate possible actions of a tripeptide crf fragment - pro-pro-ile (ppi). tripeptide ppi was found to exert effects similar to fullsize crf molecule after central administration. the tripeptide induces behavioral activation in home cage, but inhibits behavioral activity under stressful conditions. ppi increases blood pressure and heart rate, blood glucose level and body temperature, decreases pain sensitivity, increases eeg amplitude and large doses of ppi induce seizures. ppi inhibits sexual motivation and performance in mating tests in males. crf antagonist -helical crf - abolishes ppi infl uence on circulatory system, glucose metabolism, thermoregulation and pain sensitivity. adrenalectomy does not interfere with hyperglycemic and hyperthermic actions of ppi, while pancreatectomy prevents hyperglycemic effect. nonselective beta-adrenergic blocker obsidan prevents hyperglycemic and decreases hyperthermic effects of the tripeptide and ganglionic blocker hexamethonium abolishes both effects. taken together effects of ppi correspond to stress-reactions, involving corticotropin-releasing factor and evidence that ppi is either ) a part of a crf molecule active site, or ) physiologically active crf derivative, realizing its effects through activation of crf receptors, or ) an independent regulator, affecting crf-ergic neurons. acknowledgements: the work was supported by rfbr grant - - - -à huggins, kelley; andersen, niels university, united states amyloid fi bril formation is associated with at least human diseases; among these, the human-amylin(ham)-derived deposits in type ii diabetes were the discovery system and ham aggregation is one of the more thoroughly studied systems. to date, inhibitors of beta-aggregate and fi bril formation have been polyphenols, mutants of ham, or short peptide related to the ham( - ) sequence, nfgailss. we now report that stable beta-hairpin scaffolds displaying trp and tyr residues are effective inhibitors, delaying the onset of both the cd changes associated with beta structure formation and the nucleation time and net enhancement of the fl uorescence observed with added thiofl avin-t (tht). under our test conditions ( microm ham, % hfip in mm phosphate buffer, ph ), ham begins to display an increase in beta structure by cd at min with a constant maximal value from - min. tht fl uorescence also indicates a circa min onset time with a rapid (< min) rise to the full response. inhibition (delayed onset and reduction in the maximal fl uorescence enhancement) has been observed with a number of hairpins; those with two trp residues on a single face of the hairpin are more potent. of these, our best inhibitor to date, kkltvwipgkwitvsa (p = d-pro), increases the onset time more than -fold at equimolar concentrations. at molar equiv., the onset time is greater than min and tht fl uorescence levels out at < % of the control value. in analogy to the report by prof. ghosh (jacs , p. ) that a beta-sheet protein with added tyr and trp residues inhibits fi bril formation by the alzheimer-related abeta peptide; we expect that designed beta-hairpin scaffolds will be more generally applicable and will afford new insights into the recognition phenomena of amyloidogenesis. baabur, hemda; ashkenasy, gonen ben gurion university, israel the main advantage in utilizing proteins for the development of sensors and receptors, over the more often used approach that exploits small molecules, is the ability to manipulate large recognition surfaces, which expose toward the bulk solution number of different functional groups. we search for stable artifi cial proteins that can be utilized as 'universal' recognition entities. to that end, modular chemical syntheses are exploited for the preparation of repeats-proteins. leucine rich repeat proteins (lrrs) are - residue sequence motifs present in several proteins with diverse functions. internalin b (inlb), a surface lrr protein of the human pathogen listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. consensus design uses statistical analysis of sequence alignments of families of homologous proteins for protein engineering. we show here that using consensus design, series of protein mutants that differ in the recognition surfaces are synthesized and will be probed for their ability to bind different natural and non-natural ligands. comparative structural studies of potent neuroprotective peptides of the humanin family although the rescue activity of hn peptides has been linked to a number of signaling pathways and receptors, their mechanism of action remains unknown. in this work cd and nmr data on the above peptides are presented and compared in an effort to defi ne structural characteristics related to their function. evaluation of our fi ndings in combination with existing structure-function relationship data for this class of peptides, brings forth fl exibility as an important structural feature that may facilitate interactions with functional counterparts of the neuroprotection pathway. the ability to adopt a partial helical conformation in the presence of low concentrations of tfe is another common structural feature that may be defi ning the interactions of these peptides in the environment of cell membranes. desleu-aga(c r)hng and cl display a more complex behavior shifting from -helical to -sheet conformations depending on ph, peptide concentration, and % of tfe present in solution. this fact may be related to their high potency since in hn literature evidence for self-association ability has been linked with neuroprotection. our cd and nmr experimental data combined with theoretical modeling will hopefully provide important clues for the elucidation of the mechanism of action of the hn family of peptide the process of molecular recycling describes a dynamic mechanism by which individual amyloid fi brils are continuously dissolving and reforming ( ) . the present work aims at the study of the dynamic properties of the -amyloid, a ( - ), amyloid fi brils. since the formation of a aggregates has been suggested as a key process in the pathology of alzheimer's disease, the study of the dynamic nature of a ( the coiled coil structure is widely distributed in natural proteins, such as transcription factors, receptor proteins and enzymes. this motif has been recently used by chemists for the design of functional synthetic proteins. specifi c coiled coil sequences can be utilized as templates for self replication processes ( , ) . the stability and the activity of these replicators depend on the characteristics of the amino acid residues in the recognition interface. it has been suggested that it is possible to control protein structure and the replication process by external triggers such as light, and that the light can also be used to monitor the conformational changes and/or to follow the protein functionality( ). we show here our ability to control the folding stability of coiled coil peptides and their reversible or irreversible self replication effi ciency by light, and we demonstrate the possibility to exploit fret couples to facilitate in-situ monitoring of the folding and reactivity. we use 'caged' mutants with a photo-switchable molecule in the recognition interface of the peptidewhich disrupts its coiled coil structure -as inactive species. deprotection of the caged proteins is used as a mechanism to restore the self replication process. the ligation is followed by monitoring the changes in fl uorescence of either the donor or acceptor of the fret couple. we will describe synthesis and structural characterization of caged and cage-free peptides and measurements that show, as expected, that the cage free peptides are more stable as coiled coils and better catalyst for their own formation than the caged analogs. moreover, we discuss the reversible replication process and how we can shift and control its equilibrium staes. , a member of the rfamide peptide family, has been reported to have pronociceptive and analgesic activities as well as pro-and anti-opioid effects. these contradictory effects seem to result from npff capacity to bind two different gprotein coupled receptors (npff and npff ). although the exact role of npff and npff receptor is still unclear, this complex system appears to play an important role in pain regulation. structure-activity relationship (sar) studies using analogs of npff and derived peptides have shown that the c-terminal part of the molecule (i.e. pqrf-nh ) is crucial for affi nity and activity. however, some of the essential features for ligand recognition by npff receptors are still missing to design highly selective molecules. in order to provide further insight into ligand recognition, we have investigated the solution conformation of npff in different media using circular dichroism and nmr spectroscopy. our results showed that ( ) in the presence of methanol or trifl uoroethanol, turn-like elements are present in npff structure, and ( ) are not yet established, although genetic and animal models have shown a causal role of amyloid -peptide (a ) in ad. however, recent debate has focused on whether amyloid fi brils or soluble oligomers of a are the main neurotoxic species contributing to neurodegeneration and dementia. one approach for preventing aggregation would be the conversion of the peptide conformation. prior investigations indicate that polymeric nanoparticles offer strong advantages in modulating the secondary structure of the peptide ( , ) . these results have encouraged us to extend our work on polymeric nanostructures for conformational transformations contributing to the development of new therapies for these diseases. complexes of polyampholytes and dodecanoic or perfl uorododecanoic acid were prepared ( ) resulting in nanoparticles with hydrodynamic diameters ranging from to nm. the fl uorinated nanoparticles induced -helix rich structures in a peptide, whereas their hydrogenated analogues were less effi cient leading in most cases to aggregation orsheet formation, as determined by circular dichroism spectroscopy. the degree of fl uorination, the hydrophilic balance and the charge density of the fl uoropolymers, as well as the size of the nanoparticles in aqueous solution, are decisive for the interactions. the impact of these structures on the a -induced toxicity in cultured neurons was studied. we report that the fl uorinated nanoparticles increased the a -mediated mtt ( -[ , -dimethylthiazol- -yl]- , diphenyltetrazoliumbromide) reduction, which indicates a higher cell viability. the anti-apoptotic effect of the complexes was evaluated by determining the activation of caspase- . this assay also confi rms the decrease of a -mediated cytotoxicity in the presence of fl uorinated biocompatible complexes. alternative strategy to investigate enzymatic activity using peptides containing toac spin probe ( ), the present work extended the investigation of the specifi city of angiotensin i-converting enzyme (ace, ec . . . ), a dipeptidyl carboxypeptidase which cleaves the c-terminal dipeptide from angiotensin i (angi, drvyihpfhl) to produce the potent vasoconstrictor angiotensin ii peptide (angii). the use of paramagnetically labeled ai analogues attaching the toac ( , , , tetramethylpiperidine- -oxyl- -amino- -carboxylic acid) probe ( ) advantageous allows the monitoring of their conformation and its enzymatic hydrolysis specifi city through the epr and fl uorescent methods, the latter due to the quenching effect induced by the stable free radical toac probe upon the tyr residue of angi. the study of toacattaching ai analogues at positions , , , , , and indicated that the fi rst four analogues are substrates for ace in the decreasing order ~ > > , thus confi rming that greater the proximity of the unnatural probe to the cleavage site ( - ) of the sequence, the smaller are the substrate specifi city of analogues. otherwise the quenching effect of tyr fl uorescence by toac decreased with increasing distance between both residues, thus suggesting overall fl exible structures for most of analogues. these fi ndings were also corroborated in a combined cd and epr studies although some differences were detected among the derivatives either in the variation of ph or amount of the structuring tfe studies. finally, differences between epr spectral lineshapes of some labeled analogues and their corresponding cleavage products seems to allow a real time monitoring of the enzymatic reaction. the effect of the so called " -sheet breakers" (bsbs) on a - aggregates inhibition of aggregation of amyloid -peptide seems to be a critical step in the therapeutic approach to prevent amyloidosis in alzheimer's disease. the therm " -sheet breaker" (bsb) had been introduced by soto c. et. al. as a -residue peptide in , that inhibits amyloidprotein fi brillogenesis. we synthetized several derivatives of the "soto peptide" as well as a big number of peptidomimetics. their mechanism of action has been studied by nmr spectroscopy, cd and transmission electron microscopy (tem). all of these methods showed that the soto peptide lpffd and the similar peptides can not prevent a aggregation. these compounds bind to the surface of a aggregates and decrease bioactive peptides the specifi c surface area of a which accessible for the cell-membranebound receptors, can lead to a decreased toxicity. the -sheet breaking effect does not work up to an a peptide-small peptide ratio of : . as a consequence, these compounds are not -sheet breakers, only they modify the surface of a fi brils and rather speed up the formation of -sheet structure. designing trehalose-conjugated peptides for the inhibition of alzheimer's a oligomerization and neurotoxicity. interactions between cationic or anionic porphyrins and polypeptide templates with charges that are opposite to those of porphyrins have been extensively investigated for their possible applications in biomedicine and photodynamic therapy (pdt). the infl uence of porphyrin on the conformation of the peptide part of the complexes is studied in this work. non-covalent interactions of cationic tripeptide l-lysyl-l-alanyl-lalanine (kaa) with anionic meso-tetrakis( -sulfonatophenyl)porph yrin (tpps) and its copper(ii) (without axial ligand), iron(iii) (one axial ligand), and manganese(iii) (two axial ligands) derivatives were investigated in aqueous solutions by vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. although both the cd spectroscopies are sensitive to conformation, particularly vcd is extremely sensitive to subtle conformational changes. the vcd spectra of pure kaa in the amide i' (c=o stretch vibration) region showed the spectral patterns typical for left-handed polyproline ii helical conformation (ppii). interaction of kaa with non-metallated tpps was accompanied by change of the amide i' vcd patterns -loss of vcd intensity and arising of a new negative band at ~ cm - -that was interpreted as a partial change of ppii into less compact conformation as extended helix or -sheet segment. in case of cu(ii)-and fe(iii)-tpps, the loss of the amide i' vcd intensity of kaa was observed only. for mn(iii)-tpps having two axial ligands, the vcd pattern was unchanged compared to the pure tripeptide indicating that this derivative is not able to change the conformation of kaa in peptide-porphyrin complex. suitable models of biologically important small-sized proteins -histons -located in the chromosomes of eucariotic cells. they are able to interact with negatively charged functional groups of many biologically important molecules as dna, polyuronic acids, and porphyrins and thus infl uence a broad range of biological functions. in this work, the solution conformation of synthetic oligotripeptides (llysyl-l-alanyl-l-alanine) n [n = , , ] was investigated at the different temperatures and ph using combination of vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. vcd spectra of all the oligopeptides measured at room temperature show a negative couplet (positive to lower frequency) in amide i' region. this spectral pattern is indicative of left-handed polyproline ii helical conformation (ppii), which is stable at wide range of ph. the temperature dependence of the vcd spectra indicates that ppii conformation of all the oligopeptides remains stable even at °c, independently on length of oligopeptide chain. these results were confi rmed by temperature dependent ecd experiments, where the characteristic negative and positive bands at about and nm, respectively, were observed. taken together, vcd and ecd results suggest that ppii conformation of (lys-ala-ala) n sequence is the dominant conformation within the range of temperature from to °c. all the results including spectral data analysis are discussed in detail. acknowledgment: the work was supported by the research grant / /p from the grant agency of the czech republic. we thank miroslava Žertová, msc (iocb prague) for the oligopeptide synthesis. -peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the fi eld of -peptides towards the construction of possible new secondary structures, the replacement of the c and c atoms of the -amino acid with heteroatoms could be an attractive modifi cation, for example c -atom of -peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies about hydrazine peptides [ ] [ ] [ ] , and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of h-[(cis or trans)-acpc- s-aza-acpc] -nh peptides (figure ), their potential energy hypersurface were probed at the ab initio b lyp/ - g** level. the cis ( r, s) formed / -helices, while the opposite acpc enantiomer resulted strand. the trans ( s, s) formed -strand, while the opposite acpc enantiomer resulted -helix. the hybrid-peptides in question were synthetized on solid support, and their high-resolution d assignments were made by using nmr, which was supported by ecd, vcd, qls and tem methods. the hydrazino modifi cation resulted in better water solubility than that of the acpc homooligomers. this result is very important concerning the further biological applicability. mazur, adam; katarzy ska, joanna; zabrocki, janusz; jankowski, stefan technical university of lodz, poland cyclolinopeptide a, (cla, ), a cyclic nonapeptide cyclo(-pro -pro -phe -phe -leu -ile -leu -val -), possesses strong immunossupresive and antimalarial activity as well as the ability to inhibit cholate uptake into hepatocytes [ , ] . the mechanism of cyclolinopeptide a activity is similar to that of cyclosporine a. the object of our investigations are six cla analogues with pro and pro residues replaced by -isoproline or -homoproline. the immunosuppressive activity of these new cla analogues was evaluated on the basis of the mouse splenocyte proliferation assay .. nmr spectra analysis (chemical shifts assignment and structural constraints) was based on d and d nmr experiments at mhz. long ( ns) molecular dynamics calculations were carried out using gromacs program (gromos g a force fi eld) for isomers of at least % content. in woolley, andrew university of toronto, canada thiol-reactive azobenzene-based cross-linkers provide a straightforward means for introducing a photo-isomerizable unit into a peptide or protein structure. we have shown that the conformational response of the peptide in such systems is critically dependent on the manner of attachment of the cross-linker as well as the cross-linker structure. cross-linkers can be introduced in which trans-to-cis photoisomerization leads of formation of helical structure, or conversely to loss of helical structure. these effects can be understood in a semi-quantitative manner by calculating the degree of mismatch between the steric requirements of the linker and the conformational ensemble of the peptide. kinetic studies reveal that in general photoisomerization of the linker is fast (several ps) whereas subsequent peptide folding or unfolding occurs over hundreds of microseconds. such cross-linkers are thus potentially useful phototriggers for studying protein folding processes, as well as for controlling the equilibrium stability of different conformational states. applications to photo-control of bioactive peptides and proteins will be discussed. short corticotropin-like peptides with stress-protective activity it was synthesized linear è cyclic corticotropin-like peptides, which contained from to amino acid residues. the ability of each of the synthesized peptides to inhibit the specifi c binding of tritium-labelled corticotropin ( - ) to the adrenal cortex membranes of rat in vitro was investigated. on the base of the obtained results peptides with the highest inhibitory activity were selected for in vivo tests. the infl uence of the selected peptides on the level of -oxicorticosretoids and catecholamines in the adrenals and blood of rats in the experiments on acute hemorrhage and hypobaric hypoxia, cold and heat shock and low doses of g-radiation were studied. it was established that intravenous injection of short peptides at the dose of g/kg could correct disturbance of -oxycorticosretoids-catecholamines system in the adrenals and plasma of rats that were subjected to hemorrhagic shock and hypoxia. the rest of investigated peptides possessed lower stress-protective activity. it was also shown that under cold or heat shock, thrice-repeated intranasal injection of these peptides into rats at the dose of - g/animal abolished temperature induced changes in the level of -oxycorticosretoids and catecholamines in the adrenals as well as the content of free histamine and the activity of diaminoxydase in the myocardium. it has been shown that the stress-protective activity of corticotropin-like peptides is mediated by the corticotropin receptor in cortex of the adrenals. a study of immunobiological activity of the wrnwdyyk octapeptide the evaluation of the effect of japanese herbal in many cases, japanese herbal (kampo) medicines have been used in the empirical treatment of chronic hypofunction. however, the kampo medicines consist of several herbs, whose pharmacological mechanism is not clear. in western medical science, medical doctors usually treat patients according to disease diagnosis. in eastern medical science, treatment is based on diagnostics called gsho h, which is a unique concept in kampo medicines and quite different from diagnosis. the concept of gsho h is diffi cult for non-professionals to understand, furthermore, there are responders and non-responders when nonprofessionals prescribe kampo medicines. the concept of gsho h focuses on individuals, not diseases, therefore, it is diffi cult to gain a given effect for everyone by general clinical trials. in this study, we investigated the effects of prokinetic kampo medicines on plasma levels of gut-regulatory peptides (somatostatin, motilin and gastrin) and compared with that of dopamine receptor antagonists, the western prokinetics. the fi ve kampo medicines, including pinelliae tuber and zingiberis rhizoma, three dopamine receptor antagonists or placebo was orally administered. venous blood samples were taken before and till min after administration. plasma peptide levels were measured using a sensitive enzyme immunoassay. the dopamine receptor antagonists and kampo medicines caused signifi cant increase of plasma gut-regulatory peptide levels compared with placebo group. in recent years, chronic hypofunction without mechanical problem, such as non-erosive refl ux disease and functional dyspepsia, is diffi cult to cure using western medicines. the preliminary study indicated the plasma somatostatin levels of patients with any symptoms are high and plasma motilin levels of them are low compared with those of healthy subjects. to evaluate kampo medicines using bioactive peptides as biomarkers, it may be possible to cure diseases that is diffi cult to treat by western medicines. bapst, jean-philippe; calame, martine; eberle, alex n.; tanner, heidi university hospital basel, switzerland radiolabeled -msh analogs are potential candidates for melanocortin- receptor (mc -r)-mediated melanoma targeting. several short -msh peptides carrying a dota ( , , , -tetraazacyclododecane- , , , tetraacetic acid) metal chelator were designed and evaluated as potential diagnostic (e.g. with in, , ga) or therapeutic (e.g. y, cu) radiopharmaceuticals. the analogs tested to date showed high affi nity for the mc -r in vitro, excellent internalization into the tumor cells, as well as a good incorporation in tumor xenografts and a low uptake in normal tissues in vivo, except the kidneys where considerable uptake is observed. our current studies attempt to infl uence the pharmacokinetic parameters in order to address specifi c uptake (i.e. by melanoma) versus non-specifi c uptake (i.e. by the kidneys). as glycosylation had been shown to improve tumor-to-kidney ratios in the case of somatostatin and to reduce peptide re-uptake by the tubular system of the kidneys in general, we investigated glycosylated analogs of [nle , asp , d-phe ]--msh - (napamide). carbohydrate moieties such as glucose, galactose and maltotriose were introduced at various positions on the msh peptide carrying the metal chelator dota for labeling with in. the peptides were evaluated in vitro in both murine and human cell lines for mc -r binding and cellular localization, and in vivo in b f tumor-bearing mice for tissue distribution. the tumor-to-kidney ratio for gal-napamide ( - h aucs) was superior to any of the previously published msh peptides. other glycopeptides showed very good binding affi nities but lower selectivity in vivo. in additional, a series of non-glycosylated dimeric derivatives, bearing one or two moieties of the chelator complex, were developed which displayed excellent receptor affi nity but tended to result in higher kidney accumulation. by contrast, at least one negatively charged dota-napamide showed excellent tumor-to-kidney ratios. there is a critical lack of validated early biomarkers for most conditions and diseases. early diagnosis does enable treatment of less severe disease states, the use of less invasive techniques and could potentially reduce the costs of healthcare systems. however, it is most likely that peptides in tissue or blood -or their modifi cations -can be specifi cally associated with different disease states. the discovery and verifi cation/ validation of such disease markers are two distinct workfl ows. during the discovery phase, a relatively small number of samples with a high number of potential biomarker candidates are screened. the highthroughput provided by the itraq™ reagent strategy allows for simultaneous analysis of such samples. once biomarker candidates have been identifi ed with initial statistical signifi cance, these have to be validated. this validation workfl ow involves analyzing a large number of samples with a relatively small number of candidates to establish the biological signifi cance of the biomarker candidates. rather than switching to immunological techniques for this validation step, we suggest a mass spectrometry based approach. this orthogonal strategy is a novel targeted, high throughput quantitative multiplexed multiple reaction monitoring (mrm) approach. the approach relies on assay development using a combination of mrms to target specifi c peptides identifi ed in discovery, followed by ms/ms to confi rm that the quantitative mrm signal results from the target peptide. in addition to being a quantitative method, this validation approach is extremely specifi c and sensitive. several published examples of the application of this mrm-based approach utilizing the actual or hypothetical physical properties of peptides in complex mixtures will be presented. infl uence of the charge on the in vivo behavior of radiolabeled bombesin analogues prostate and breast cancers are the second leading cause of cancer death in men and women, respectively. the side effects related to the treatments that are available today have a great infl uence on the patients' quality of life. therefore, the development of new diagnostic and therapeutic strategies may have an important impact in the outcome of these cancers. gastrin-releasing peptide (grp) receptors are present in high quantities in a variety of cancers, prostate and breast tumors among them. targeting of over-expressed grp receptors with radiolabeled bombesin (bbs) analogues would offer an interesting tool for tumor imaging and therapy, depending on the radionuclide used. some analogues of bbs, based on the fragment - , were functionalized with the (n his)chelator for labeling with the m tc-and re-tricarbonyl-core. despite an increased metabolic stability, these analogues showed very low tumor uptake. additional insertion of a ala-ala linker led to increased tumor uptake but still unfavorable in vivo properties. in order to further improve the biodistribution, novel polar linkers with different charge were introduced in the molecule. a positive charge resulted in increased kidney uptake, whereas one single negative charge led to a signifi cant increase in the tumor uptake and also signifi cantly higher tumor-totissue ratios. co-injection with natural bbs importantly inhibited the uptake in the tumors and receptor-expressing tissues, which confi rmed the specifi city of the in vivo uptake. moreover, imaging of the tumor xenografts by spect/ct was also much clearer with the analogues bearing one negative charge. additional negative charges, however, resulted in a loss of binding affi nity and internalization, and unfavorable biodistribution. in conclusion, bbs analogues with one single charge in the linker hold a greater potential for imaging and therapy of grp receptor-overexpressing tumors peptide-macrolide conjugates as novel instruments for protein biosynthesis study bacterial ribosomes are targets of numerous therapeutic agents. macrolide (ml) antibiotics prevent nascent polypeptide growth by blocking the ribosomal exit tunnel (rt). availability of high-resolution structures of complexes of ribosomal s subunits with ml enabled developing novel ideas concerning their inhibitory activity of translation. formerly we obtained peptide derivatives of ml in which the peptide part modelled a growing chain, while an antibiotic moiety served as an "anchor" for positioning the peptide in the rt. the goal of this study was to synthesise tryptophan-containing peptide derivatives of -omycaminosyltylonolide (omt) to monitor location of the specifi c trp binding site that modulate the activity of the peptidyl transferase centre of the ribosome. the peculiarity of compounds designed in this study lies in that of a trp containing peptide fragment, connected to the primary hydroxyl group in position c of omt, is oriented in the rt towards the hypothetical trp binding site. the following peptides were used: boc-l-trp-gly-oh, boc-l-trp-ala-oh, boc-l-trp-abu-oh, boc-l-trp-ape-oh. variable distance between trp residue and macrolide ring was achieved by introduction of glycine, -alanine, -aminobutyric acid and -aminovaleric acid as c-terminal amino acids of the dipeptides. the peptides were obtained from boc-l-trp-oh and ethyl esters of the corresponding amino acids by condensation with bop followed by saponifi cation of the peptide esters. the reactions between peptides and omt were produced using dcc and dmap as condensation agents. as a result peptide-macrolide derivatives were obtained. all compounds were purifi ed by column chromatography on silica gel and characterized by hplc, mass-spectrometry and nmr. it was shown by means of chemical probing that trp containing omt derivatives specifi cally bound to rt of the ribosome. moreover these compounds displayed an antibiotic activity in testes with several staphylococcus strains. zwanziger, denise ; neundorf, ines ; schatzschneider, ulrich ; beck-sickinger, annette g. university of leipzig, germany; university of bochum, germany npy is a amino acid peptide amide that belongs to the pancreatic polypeptide-hormone family ( ) . it is the most abundant neuropeptide in the brain and triggers a number of central activities, such as regulation of food intake as well as stress induced reactions [ ] [ ] [ ] . npy forms a selective interaction with at least three receptors, which are called y , y and y . in the past it was shown that y -receptors are overexpressed in > % of all breast tumors as well as in % of the derived metastases. normal breast tissue expresses y -receptors, while the neoplastic tissue expresses y -receptors .. as a result, the npy-y -receptor system can be used for tumor targeting and therapy. in the fi rst step we synthesized the truncated and modifi ed npy analogue [pro ,nle ,bpa ,leu ]npy( - ) (nle-norleucine; bpa-benzoyl-phenylalanine) by solid bioactive peptides phase peptide synthesis using fmoc/tbu strategy. the npy analogue was characterized with respect to in vitro binding affi nity and selectivity at y -receptor expressing human breast cancer mcf- cells and the metabolic stability in human blood plasma, respectively. then we coupled different potential chelators to [pro ,nle ,bpa ,leu ]np y( - ), which was n-terminally modifi ed by two ß-alanines as spacer between the peptide sequence and the chelator. next, we determined their ability of metal conjugation of diverse metals, as cu + and re +. metal conjugated peptides were characterized by maldi-tof-ms (matrix assisted laser desorption/ionization mass spectrometry), rp-hplc (reversed phase high performance liquid chromatography) and ir-spectroscopy (infrared). after optimization of the metal conjugation fi rst binding affi nity studies were performed. chain is mainly expressed in skeletal muscles and peripheral nerves, and interacts with cell surface receptors such as integrin, dystroglycan, and heparan sulfate proteoglycans. biological functions of the laminin chain n-terminus including integrin binding and heparin/heparan sulfate binding were found previously. here, we focused on the n-terminal region of the mouse laminin chain (position - ) and screened the biologically active sequences using peptides. the synthetic peptides were generally amino acids in length and overlapped with neighboring peptides by amino acids. cell attachment activity of the peptides was evaluated using a peptide-coated plastic plate assay and a peptide-conjugated sepharose bead assay using ht- human fi brosarcoma cells. eleven peptides showed cell attachment activity on plate assay and fi ve peptides showed cell attachment activity on beads assay. previously we screened active sites on the laminin chain and identifi ed several active sequences. when we compared with active sequences of the and chains, a - (yydetvasrnlsln) and a - (ggklkyaiyfea) are unique active peptides only within laminin chain. these results suggest that the biological activity of a - and a - are chain specifi c and are useful for investigating the chain specifi c functions of laminin chain. novel peptide biopharmaceuticals by using phage display technology Štrukelj, borut; lunder, mojca; bratkoviè, tomaž university of ljubljana, faculty of pharmacy, slovenia libraries of random peptides displayed on the surface of bacteria, mammal cells or bacteriophages are an essential tool that enables systematic study of target molecule interactions. the identifi cation of ligands from large biological libraries by phage display has now been used for almost years. in a last few years several improvements have led to numerous high affi nity peptide ligands that express various biological activities. phage-displayed peptide libraries have been used successfully to isolate peptide ligands directed to a functional site for which the natural ligand is or is not a protein or peptide. by using a modifi ed, in-house developed selection proctocol, we successfully selected several phage clones with high affi nity to pancreatic lipase, ghrelin and cysteine protease cathepsin k as target proteins. based on their deduced animoacid sequences, twenty heptapeptides with the highest affi nity to target proteins were synthesized and characterized for their capacity to inhibit enzyme or acceptor function. the most succesful peptide candidates inhibited pancreatic liapase with the apparent inhibition constant of um, and cathepsin k with the apparent inhibition constant of , um. a set of peptidomymetic compounds were sinthesyzed based on the aminoacid sequence of selected peptides and their inhibitory activity was determined. the most potent candidates were selected for further development of peptide biopharmaceuticals aganist obesity (inhibitors of pancreatic lipase and ghrelin) and in the prevention of osteoporosis (cathepsin k inhibitors). novel approach to non-specifi c elution in phage display using ultrasound phage display is used to select and optimize peptides or protein domains binding to virtually any protein and sometimes even non-protein targets. several rounds of screening are performed, until the increase in phage output, or binding assays performed with phage pools, indicate that the population of binding phages has been adequately enriched. in cases where ligands of particular target are not known or available, targetbound virions are released by non-specifi c elution for example with acidic buffer, or competitively with free target molecule, or by addition of bacterial host directly to the target-bound phages. we used streptavidin, immobilized by adsorption, as a model target protein for affi nity selection of peptides from phage display library. the effi ciencies of a number of well-known typically used non-specifi c elution strategies in selecting and retrieving a phage clone displaying the tripeptide biotin mimetic (hpq) from a streptavidin coated surface were compared. all the commonly used elution strategies have failed to elute and select high affi nity hpq-bearing phage clones bound to streptavidin. the failure was shown to be due to the inability of eluants to break the interaction of high affi nity clones with the target, which is thus likely to be the cause for failed selection with other targets also. to surmount this, we have introduced a new elution strategy, combining low ph elution buffer with sonication which, in addition to loosening the peptidetarget interaction, also serves to detach the target molecule from the immobilization surface. this ultrasound-based method enabled single step selection of a high affi nity peptide from a library of diversity greater than , and thus represents a dramatic improvement in searching for novel, specifi c peptide ligands. apoptosis to various tumor cells but not in normal cells. using fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of trail scan was prepared and cell viability assay was directly performed on peptide disk with jurkat cells. six peptide sequences that could induce cell death were found, and particularly rnscwskd (trail - ) peptide was shown the strongest effects. then, peptides of stronger effects were found through amino acid substitution, and the cnscwskd peptide induced > % cell death in treated cells. features of apoptosis, such as dna fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with trail for binding to the death receptor (dr) or dr were observed, suggesting that this peptide is a trail mimic. caspase- activation was observed in various tumor cells treated with this peptide as well as with trail, while no activation was observed in human normal fi broblasts. the cnscwskd peptide is a potential candidate for use in cancer therapy. the being an incretin mimetic, exendin- exerts the same action as glp- including the glucoregulatory and the insulinotropic effects through glp- receptor. exendin- may be preferable to glp- on the aspect of stability. however it was comprised with amino acids and was not suitable for developing as an oral drug. recently, there was an upsurge in the development of exendin- mimetic as potential therapy for type diabetes. in this study, a receptor-binding region on the surface of rinm f cell was used as the target to screen peptide ligands for the receptor in a phage -mer peptide library. dna sequencing revealed a group of closely related peptides from the fourth round of selection. through the activity of decreasing blood glucose assay in vivo, the cell proliferation assay and capability against dppiv in vitro, one highest bioactivity peptide (qpsvgmkpsprh, ex- pa) was acquired. the bioactivity of ex- pa was almost identical with exendin- on the promoting cell proliferation in a dose-dependent manner. the decreasing blood glucose effect of ex- pa was up to min after administration. the stability against dppiv of ex- pa maintained good performance that % parent peptide remained after h. in summary, ex- pa as a shorter glp- receptor agonist mimicked the action of exendin- and built a good foundation for designing the oral diabetes drug. key words: exendin- , mimetic, phage display peptide library, screen over the last decades we have witnessed the emergence of bacterial resistance to virtually all clinically important antibiotic types. therefore, continuous development of antibacterial agents with completely novel modes of action accompanied by rationalization of chemotherapeutic prescription is the key strategy to adopt in a long-run struggle against the growing problem of pathogen resistance. numerous indispensable antibiotics interfere with peptidoglycan cell wall biosynthesis making this unique metabolic pathway a well validated target for antimicrobials. while nearly all of these antibiotics inhibit late stages of murein synthesis occurring on the extracellular side of plasma membrane, initial cytoplasmic steps have not been extensively exploited as drug targets. we have performed affi nity selections from random linear and conformationally constrained (disulphide cyclized) peptide libraries displayed on bacteriophage particles against two essential bacterial enzymes murd and mure, involved in the cytoplasmic synthesis of peptidoglycan monomer precursor. selected peptides were found to inhibit respective targets in an in vitro assay with ic values of m to . mm. reported inhibitory peptides should be regarded as templates for design of low-molecular-weight peptidomimetics. resulting murd and mure inhibitors with improved potency and/or physicochemical characteristics (especially membrane permeability) have the potential to act as broad spectrum chemotherapeutics. fidan, zerrin; ljeskovica, nizama; portwich, michael; volkmer, rudolf charité-universitätsmedizin berlin, germany coiled coil (cc) sequence motifs are common structural motifs and versatile protein interaction modules. the cc is composed of at least two right-handed amphipathic a-helices that wrap around each other into a left-handed supercoil such that their hydrophobic surfaces are in contact. cc can associate up to heptamers, form homomeric and heteromeric complexes at different stoichiometries, and be aligned parallel and antiparallel. a characteristic of all coiled coils is the presence of heptad repeat sequences [abcdefg]i, where i denotes the heptad number. although cc motifs can be predicted with a high degree of confi dence, predicting the association states and topologies is still a great challenge. we will report on synthetic peptide arrays useful to study cc associations at the amino acid level. in contrast to rational design, which mostly depends on short model peptides, our approach relies on the full-length homodimeric gcn -and the heteromeric cjun/cfos coiled-coil domain formation. the infl uence of amino acid substitutions on association is tested without restrictions and presumptions and the stoichiometry is examined by biophysical methods. furthermore, we generate arrays comprising hundreds of (putative) cc sequences and subsequently probe them for association to a set of several native cc sequences. an interactome can be drawn for each of the investigated cc sequences, enabling one to interpret the biological functions. we will present: (i) association analyses of all single substitution variants of the gcn -, cfos and cjun leucine zipper probed with the associated native leucine zipper; (ii) the exposure of a heteromeric cc-network deduced from gcn variants and the determination of the networkstoichiometry; (iii) the cc-interactom of several native coiled coils like the cc sequences of akap , pqbp , orai and others. almost two decades ago we used the spatial screening technology to optimize activity and receptor subtype selectivity among rgd recognizing integrins. this culminated in the avb selective superactive pentapeptide c(-rgdfv-) which gave rise to a number of peptidomimetic modifi cations. among them we studied all retro-, inverso-and retroinverso peptides of this structure. only one retro-inverso peptide exhibits full activity: the peptide c(-dgrvf-) strongly inhibits avb /vitronectin binding ( nm) but not aiibb /fi brinogen binding (> nm). all investigated retro-sequences show very low affi nity for avb . recently it was discovered that the ngr sequence in the fi bronectin domain fi after rearrangement into iso-asp-g-r exhibits high binding affi nity for integrin avb . this surprising result stimulated us to investigate cyclic peptides containing the iso-asp-gly-arg sequence .. after optimisation we obtained peptides which bind with high activity to integrin a b and lower, but signifi cant activity for avb . in the ongoing work we investigate if receptor modelling can help to understand these results. selectivity and activities for these two integrins have been obtained recently in our group using two different peptidomimetic scaffolds bioactive peptides (tyrosine based and diacylhydracine based) using a homology model of the integrin a b for which no x-ray structure is known. binding of integrin ligands involves binding of a carboxyl group to the metal ion (ca or mn) in the so-called midas region of the integrin. so far all integrin ligands contain a carboxyl group and any attempt to substitute this group by an isoster failed. we recently found that hydroxamic acids (-conhoh) allow for the fi rst time a substitution of this carboxyl group. the activities and selectivities for integrin subtypes avb , a b and aiibb have been explored and give interesting results. development of gnrh-iii-antracycline conjugates as multifunctional drug delivery systems for targeted chemotherapy targeted chemotherapy based on the cell specifi c or overexpressed receptors on tumors might be an effi cient therapeutic approach for the treatment of cancer. expression of gonadotropin-releasing hormone (gnrh) receptor was identifi ed on different types of tumors. it has been shown that gnrh-iii (glp-his-trp-ser-his-asp-trp-lys-pro-gly-nh ) isolated from see lamprey has antiproliferative activity on numerous tumor cells, and signifi cantly less potency on releasing gonadotropin hormones (lh, fsh); therefore, it is a more selective antitumor agent than the human gnrh derivatives. in our work, gnrh-iii was used as targeting moiety for the preparation of multifunctional drug delivery systems for targeted cancer chemotherapy. daunomycin (dau) and doxorubicin (dox) as antineoplastic agents were attached to the side chain of lys of gnrh-iii through amide, oxime, hydrazone or ester bonds, either directly or by insertion of an enzyme cleavable tetrapeptide spacer. stability studies of the conjugates were performed in human serum, as well as in the presence of chymotrypsin. the effect of chemical structure of the conjugates on in vitro antitumor activity was studied using different cancer cell lines (mcf- human breast, ht- human colon and c murine colon cancer cells). oxime bond linked dau-gnrh-iii conjugate was selected for in vivo experiments using c colon tumor bearing mice. the conjugate showed similar antitumor activity as the free drug, but less toxic side effect and longer survival of the animals was determined in the case of the application of the conjugate. guillon, g. ( ) . it was also shown to be highly selective for the human v b receptor ( ) . we now report the synthesis and some pharmacological properties of three fl uorescent hv br ligands (a,b,c), based on modifi cations of the lys residue in d[leu ,lys ]vp, with alexa (a), alexa (b) and antraniloyl (atn) (c). the fl uorescent peptides a, b and c exhibit the following affi nities for the hv br: (a) ki = . nm, (b) ki = nm and (c) ki = . nm. peptides a and c exhibit moderate affi nities for the hotr and very weak affi nities for the hv ar and for the hv r. on v b-transfected att cells, they activate plc coupling as evaluated by stimulation of ip levels. they also induced receptor internalization visualized by accumulation of fl uorescence in endosomial vesicles. one or more of these new fl uorescent hv br ligands promise to be useful tools for studying human v b receptor localization or traffi cking. distribution of prolyl oligopeptidase in the mouse wholebody sections and peripheral tissues prolyl oligopeptidase (pop) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than -mer, including many bioactive peptides. the distribution of pop in the brain has been studied but little is known about the distribution of peripheral pop. we used immunohistochemistry to localize pop in mouse whole-body sections and at the cellular level in peripheral tissues. furthermore, we used a pop activity assay to reveal the associations between pop protein and its enzymatic activity. the highest pop protein densities were found in brain, kidney, testis and thymus, but in the liver the amounts of pop protein were small. there were remarkable differences between the distribution of pop protein and activity. the highest pop activities were found in the liver and testis while kidney had the lowest activity. in peripheral tissues, pop was present in various cell types both in the cytoplasm and nucleus of the cells, in contrast to the brain where no nuclear localization was detected. these fi ndings support the proposed role of pop in cell proliferation in peripheral tissues. the dissociation of the distribution of pop protein and its enzymatic activity points to nonhydrolytic functions of pop and to strict endogenous regulation of pop activity. the rapid cytoplasmic entry of cationic cell-penetrating peptides in the past few years, our understanding of the cellular import of cellpenetrating peptides has evolved rapidly. the initial concept of cell entry by direct permeation of the plasma membrane, was followed by endocytosis as a major route of import at least for most cellpenetrating peptide-cargo conjugates. more recently, we and others observed a rapid cytoplasmic delivery of fl uorescein-labeled analogs of the cationic cpps nonaarginine and tat-peptide at subtoxic lower poster abstracts micromolar concentrations ( ). this import originates from spatially confi ned zones of the plasma membrane and depends on the presence of heparan sulfate proteoglycans. these molecules have been proposed to interact polyvalently with the guanidinium groups of the arginine residues. moreover, the threshold for the induction of this import could be lowered by inhibition of endocytic routes of entry. recently, it was reported that the absence of serum in the medium lowers the threshold for this uptake process ( ) . in summary, these observations suggest that the concentration of peptide associated with the plasma membrane is a decisive trigger for this import process. currently, it is not known, to which degree this cytoplasmic entry of labeled conjugates at higher concentrations is based on similar molecular mechanisms as the direct permeation of unlabeled conjugates that has also been observed at lower concentrations ( ). here we summarize the current knowledge on direct transport across the plasma membrane of cationic cpps and present new data on the molecular events involved in this uptake process. plasma membranes have numerous essential functions for maintaining cellular homeostasis. however, the membranes are also barriers to intracellular delivery of various therapeutic molecules. for improvement of their translocations, we developed a novel method using gala peptide/cationic lipid complexes. gala, a -residue amphipathic peptide with a repeat sequence of glutamic acid-alanine-leucine-alanine, was designed to mimic the function of viral fusion protein sequences that mediate escape of virus gene from acidic endosomes to cytosol ( ) . when attached with bioactive cargoes, the gala peptide may thus serve as intracellular vector bearing effi cient endosomal escape function. however, because of negative charges from glutamic acids ( -residues) in the gala sequences, access of the peptide on negatively charged cell surface would be not so effi cient. to overcome this problem, cationic lipid was employed as an gadhesive h for pasting the gala peptide onto cell surface to accomplish effi cient cellular uptake. we examined the ability of gala peptide as a delivery vector using fitc as a model of membrane-impermeable low-molecular weight drugs. when fitc-gala ( ƒÊm) was administrated to hela cells, co-addition of cationic lipid, lipofectamine (lf ), drastically increased the effi ciency in uptake of fitc-gala. in a time-dependent manner, the fitc-gala escaped from endosomes, and diffuse fl uorescent signals were observed in both cytosol and nucleus. also the gala/cationic lipid system was applied for the intracellular delivery of fitc-avidin protein ( kda). when fitc-avidin was mixed with biotinylated-gala/ lf complexes, fitc-avidin ( nm) was internalized into cells effectively. in the absence of these complexes, internalization of fitcavidin was poor. these results suggest the usefulness of our approach for intracellular delivery using gala peptide and cationic lipid. the membrane repair response masks membrane disturbances caused by cell penetrating peptide uptake even though cell-penetrating peptides are able to deliver cargos of different sizes into cells, their uptake mechanism is still not fully understood and needs to be elucidated in order to improve their delivery effi ciency. recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. however, the direct penetration of hydrophilic peptides through the hydrophobic plasma membrane is highly controversial. three proteins involved in target cell apoptosis -perforin, granulysin and granzymes share many features common in uptake of cell-penetrating peptides e.g. they bind proteoglycans on the plasma membrane. the uptake of perforin activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, due to extracellular calcium infl ux. upon activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane resealing it within seconds. in this study we present evidence that the membrane repair response is able to mask damages caused by cell-penetrating peptides when they internalize cells, thus preventing leakage of endogenous molecules out of the cell. the hypothalamic decapeptide gonadotropin-releasing hormone (gnrh-i; gnrh symmetric dimer derivatives ([ in vitro (cellular uptake and antiproliferative effect of the dimer derivatives) and in vivo (comparison of per os and intraperitoneal administration) antitumor effect of these symmetric dimers were investigated. the cellular uptake of dimer derivatives were studied by fl ow cytometry (bd lsr ii) on mcf- (human breast cancer) and ht- (human colon carcinoma) cell lines using carboxifl uorescein labeled symmetric dimer derivative. the cells were treated with different concentration of synthetic dimers and after the treatment were analysed by fl ow cytometry in order to investigate their cellular uptake. the antiproliferative effect of symmetric gnrh-iii dimers were studied on mcf- , ht- and t -d (human breast cancer) cell lines. ht- xenograft was applied for studying in vivo antitumor effect of gnrh-iii dimers in different administration routes. the cellular uptake and the antiproliferative effect are cell type dependent as it can be found in the literature. we found that symmetric dimer derivatives of gnrh-iii signifi cantly decreased the tumor volume in vivo ( %). iib receives intracellular signals (inside-out signaling) that allow cytoplasmic proteins to interact with the cytoplasmic domains of iib subunits, resulting in platelet aggregation. the aim of this work is to inhibit platelet thrombus formation by specifi cally disrupting the insideout signalling pathway using synthetic peptides based on the cytoplasmic region of subunit, residues - . peptide analogues derived from - region ( - , - (ptyr ), - , - and - ) were synthesized in their free, palmitoylated and/or tagged with the tat( - ) signaling sequence and carboxyfl uoresceinlabeled in order to investigate their membrane permeability, as well as their inhibition potency on the platelet aggregation. from the biological assays in prp and washed platelets we concluded that the modifi ed peptides that carry either palmitoyl-group or the tat( - ) signaling sequence penetrate platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. acknowledgements: the gsrt (epan yb/ ) and e.u. for the fi nancial support. ; aib = -aminoisobutyric acid) is a potent mitochondriotoxic analogue of mp that demonstrates a signifi cant intracellular co-localization with mitochondria. through co-operation with a protein of the mitochondrial permeability transition pore vdac, mitoparan specifi cally promotes apoptosis. in contrast, cytc - , a cryptic fragment of human cytochrome c, is a moderately potent apoptogenic cpp that demonstrates a strong propensity for colocalization within the endoplasmic reticulum. using a sychnologic modular design, we have synthesized and evaluated a broad range of chimeric constructs combining apoptogenic cpp (message) and peptidyl address motifs. the overriding objective of these studies was to enhance cytotoxicity and/or develop target-selective drug delivery vectors. address motifs that target both plasma membrane and nuclear envelope protein structures included i) the integrin-specifi c rgd sequence, ii) a fas ligand mimetic wewt, iii) heptagastrin (aygwmdf) that targets a novel membrane binding site on high grade astrocytoma and, iv) a mimetic of fg nucleoporins (nups). incorporation of peptidyl address motifs, by simple n-terminal acylation or via a fl exible aminohexanoic acid (ahx) linker, produced chimeric constructs with enhanced cytotoxic potencies. most signifi cantly, ld values readily achievable in vivo were demonstrated by the modular apoptogenic cpp z-gly-rgdf-mitoparan, (ld = . m) and ac-nfkfglss(ahx)cytc - (ld = . m). in conclusion, targetspecifi c modular design of apoptogenic cpp is a promising strategy for the study and therapeutic induction of apoptosis. phage display screening of geminin-binding peptide and validation of its therapeutic use in tumors yoshida, kenichi meiji university, japan dna replication is controlled by the stepwise assembly of a pre-replicative complex (pre-rc). geminin is a component of the pre-rc and plays a role in preventing incorporation of the minichromosome maintenance protein complex into the pre-rc via binding and inhibiting cdt function. it may be possible to design low-molecular-weight chemicals that would bind to geminin to suppress the aberrant cellular proliferation in tumor cells. a random -mer peptide phage library was used for the selections. phage was selected by panning on immunotubes coated with recombinant geminin. positive clones were identifi ed by a screening phage produced from single colonies for specifi c binding to gst-geminin in elisa. fluorescent-labeled peptide linked to the sv nuclear localizing signal was synthesized by solid-phase synthesis, using fmoc chemistry. by detecting fl uorescein fl uorescence to mark specifi c transfected cells, we examined the effects of the peptide transfection on the synthesis of new dna in hct cells. by screening a random peptide phage library, we identifi ed a certain peptide sequence bound to geminin. using a series of mutant geminin, elisa test revealed the amino acid residues - are responsible for the peptide binding. we found fewer brdu positive cells following transfection of the geminin-binding peptide than following that of the control peptide. this study suggests that the chemical peptidomimetics of this peptide might form the basis for the development of drugs that could be used to prevent tumor progression. acknowledgements: this study was supported by kakenhi. it has been previously demonstrated that n -( -metoxyfumaroyl)-l- , diaminopropanoic acid (fmdp) is a strong inhibitor of glucosamine- phosphate synthase, a potential target for antimicrobial chemotherapy. fmdp is transported into the cells when incorporated in a peptide chain and hydrolyzed by intracellular peptidase releasing free inhibitor as the "warhead" component, which can react with the target enzyme. the concept of utilizing of peptide transport system for delivery of toxic amino acids into the microbial cells is characterized by authors as "illicit transport". unfortunately, peptides are not stable in physiological fl uids, due to activity of peptidases. analogs with modifi ed amide backbone are more resistant towards enzymatic degradation. it has been shown that enzymatic hydrolysis of endothiopeptides is often signifi cantly slower than natural peptides. in our studies we synthesized nva[csnh]-fmdp, an analog of nva-fmdp, which is a strong antimicrobial peptide. because of the fact that the "warhead" component in this peptide contains an amide backbone, fi rstly we decided to synthesize free fmdp with thioamide backbone. the results of enzymatic kinetics studies showed that in this way modifi ed compound is much weaker inhibitor of glucosamine- -phosphate synthase than fmdp. it was the reason why we planned and carried out a multi-step synthesis in order to obtain a potential more resistant towards enzymatic degradation, antimicrobial peptide containing a thioamide backbone only between nva and fmdp. activity of nva[csnh]-fmdp nva-fmdp against selected microorganism in medium containing blood serum was determined. neuroprotective effects of cortexin and cortagen in rats with focal brain ischemia and brain trauma there is a growing body of evidences that natural peptides and their smaller synthetic analogues have good outlooks for medical use. three compounds were studied in this research -cortexin, cortagen and nîleylcortagen. cortexin is a balanced combination of oligo-peptides isolated from calf/porcine cortex. the number of non-clinical and clinical studies confi rmed cortexin effi ciency as nootropic and neuroprotective drug. in spite of benefi cial results the natural source of the drug implies some diffi culties for it general use. to overcome this issue a number of putative cortexin synthetic analogues were developed. among them is tetrapeptide cortagen. in order to improve the peptide transport to the brain and lipophility the hydrophobic oleyl radical was added to the peptide chain. the aim of research was to study the effects of these peptides on recovery of conditional refl ex in active avoidance paradigm after heavy brain trauma and neurologic defi cit dynamics induced by focal brain ischemia in rats. males albino rats were involved in experiment. closed craniocerebral trauma was modeled by weight-drop method. focal cerebral ischemia was induced by cortical phototrombosis. experimental therapy started in . hours after mechanical action and continued for days. synthetic peptides were administrated i.p. once-a-day in dose of - mcg/kg. cortexin was used at the same schedule in dose of mcg/kg. cortagen and cortexin administration in dose of and mcg/kg respectively resulted in pronounced neuroprotective effect in rats with focal cerebral ischemia. the observed effects were proved by limb-placing test. cortexin experimental therapy resulted in earlier restoration of conditional refl ex starting from the rd day after brain trauma. nîleylcortagen had weaker benefi cial effect with signifi cant improvement in conditioned response recovery by the th day. probiotics such as lactobacillus rhamnosus gg provide health benefi ts beyond their mere nutritive value, and are useful in the treatment and prevention of several diseases. isolation and use of the anti-apoptotic factor(s) would ultimately culminate in the development of novel therapeutic agents in enteropathy resulting from chemo-and radio-therapy in the treatment of cancer patients, which could in turn be administered in a consistent and pharmacologically sound manner. in present study, intestinal epithelial cells were isolated from wistrar rats (n= ), and were grown in dmem medium supplemented with % foetal bovine serum at °c in a humidifi ed % co atmosphere. after h, these cells were treated with bioactive peptides isolated from lgg ( - ìmol/ lit) for h, and were incubated with camptothecin ( ìmol/ lit) for and h, and cell viability was measured by mtt assay. caspase activity assay was carried out to determine caspase and caspase activity. moreover, cell lysates were also pretreated with caspase inhibitor ( ìm). dna fragmentation assay was carried out to determine the increase in dna fragmentation. bioactive peptides signifi cantly prevented camptothecin induced apoptosis in rat intestinal epithelial cells. the treatment of intestinal epithelial cells with camptothecin for h resulted in fi ve-fold increase in dna fragmentation, and resulted in three-fold increase in caspase activity, compared with controls. pre-treatment with bioactive peptides ( ìmol/ lit) signifi cantly attenuated the camptothecin-induced dna fragmentation by % (p< . ), and signifi cantly inhibited caspase and caspase activity by % and %, respectively, after h of camptothecin exposure (p< . ). hence, it was concluded that novel bioactive peptide from lgg were found to be potential anti-apoptotic agents and hence could be administered as therapeutic molecule for treatment of enteropathy resulting from chemo-and radiaton-therapy. cisplatin belongs to the most powerful and useful anticancer agents. it binds strongly to dna in regions containing several guanine units, forming pt-dna links within strands. through disrupting base-pairing guanine to cytosine cross-links lead to unwinding of the dna. as a result cisplatin works against both types of cells, destroying cancer and normal type ones. therefore more selective delivery system of platinum to cancer cells is still needed. based on the evidence of the presence of -and -opioid receptor types in carcinoma cells we proposed to use opioid peptides as selective carriers for delivering platinum ions to the cancer cells. we designed hybride molecules which combine two fragments. one part of the molecule contains the opioid pharmacophore and the other fragment is designed to form a complex with platinum ion. such molecule can serve not only as carrier for platinum, but also give a strong analgesic effect. as a result such hybride molecule should express analgesic properties and provide anticancer activity. we will present synthesis of a few opioid peptide-platinum(ii) complexes, the binding affi nity at the opioid receptors and effect on the proliferation of the human glioblastoma cells. peptides derived from the c-terminal heptad repeat region of the hiv fusogenic protein gp are potent inhibitors of viral infection, and one of them, t (enfuvirtide), is used for the treatment of therapy-experienced aids patients. the mechanism of action of these peptides is to interfere bioactive peptides with the fusion of the viral and target cell membranes, and it is known that hiv entry takes place in membrane microdomains ('lipid rafts') enriched in cholesterol and sphingolipids. therefore we explored the advantage of targeting a peptide fusion inhibitor to membranes by addition of a lipid moiety, specifi cally a cholesterol group. since derivatization with lipids is also known to improve the half-life of peptide therapeutics, we chose the antiviral peptide c , which is more potent than t in cell-based assays, but unlike t has a very short half-life in vivo. we show here that attachment of cholesterol to c (c -chol) dramatically increases its antiviral potency against a panel of hiv strains, including primary isolates: for strain hxb , ic = pm for c -chol, versus pm for c , and pm for enfuvirtide). consistent with the anticipated mechanism of action, c -chol accumulates at the site of action, since washing of the target cells after incubation with the peptide, but prior to triggering fusion, increases ic only -fold, relative to -fold for c . moreover, cholesterol must be strictly positioned at the c-terminus of c , in line with the need of an antiparallel orientation of the c-peptide relative to n-peptide trimeric coiled-coil, present in the fusion-active conformation of gp . in addition to boosting antiviral potency, derivatization with cholesterol has the expected benefi cial effect on the peptide pharmacokinetics. we believe that these fi ndings may be of general utility for viruses, which share with hiv the dependence on a type i fusogenic machinery. multiple sclerosis (ms) is an autoimmune demyelinating disease mediated primarily by cd + t cells of the th subset. the design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of ms. we designed and synthesized a number of peptide analogues by mutating the principal tcr contact residue based on mbp - epitope. the synthesis of the linear peptide agonist mbp - , as well as of the cyclic analogue was carried out by the fmoc/tbu methodology, utilizing the -chlorotrityl chloride resin. cyclization was achieved using obenzotriazol- -yl-n,n,n',n'-tetramethyluronium tetrafl uoroborate (tbtu) and -hydroxy- -azabenzotriazole, , , collidine allowing fast reaction and high yield cyclization product. the purifi cation was achieved using hplc reversed-phase chromatography and the peptide purity was assessed by analytical hplc and by mass spectrometry (esi-ms). agonist and antagonist (linear and cyclic) peptides were conjugated to reduced mannan. immune responses were diverted from th to th in sjl/j mice and generated antibodies which did not cross react with native mbp protein. ( ) . due to its high effi cacy and specifi city, hn represents a potential lead to new therapeutic approaches of ad. however, the molecular mechanism(s) of hn function(s) are not yet fully elucidated. wild-type hn and hn-derivatives were synthesized by spps and amino acid sequences and homogeneities of the rp-hplc purifi ed peptides ascertained by esi-and maldi-mass spectrometry (ms). the complex formation between hn and a ( - ) was studied by affi nity-chromatography and high resolution ms (fticr-ms), as well as by immunoanalytical (elisa) techniques. the binding sites between the two peptides were identifi ed by applying proteolytic epitope extraction/excision procedures [ , ] integramide a, an effi cient inhibitor of the coupled reaction of hiv- integrase, is a -mer linear peptide characterized by c -methylated -amino acids ( iva, isovaline, and aib, -aminoisobutyric acid, residues) that was isolated from fungal extracts of dendrodochium sp. the amino acid sequence was fully elucidated by the merck group a few years ago (s. b. singh et al., org. lett. , , - ) . on the other hand, the chiral sequence was only partially determined. in particular, the precise stereochemistry of the iva -iva dipeptide (known to contain one d-and one l-residue) near the c-terminus was not reported. to solve this unsettled issue and to assess integramide a primary structure-bioactivity relationship we performed by solution methods the total chemical independent syntheses of both l-d and d-l -mer diastereomers and compared their properties with those of the natural inhibitor. for an unambiguous, complete stereochemical assignment of integramide a we relied heavily on hplc and nmr techniques. our results clearly indicate that the chirality sequence of the iva -iva dipeptide of the natural product is l-d. the two integramide a diastereomers were also evaluated as inhibitors of hiv- integrase in the coupled reaction of proviral dna into the host cell dna. . a prototype peptide, r - , spanning rantes residues to , contains two hydrophobic clusters of fundamental biological importance connected by a nonessential hydrophilic linker. r - has been rationally modifi ed to improve its ccr binding and its antiviral potency. nmr studies on the modifi ed peptides revealed important similarities and differences with the three-dimensional organization of rantes. although these peptides are consistently more active as dimers, an increase in anti-hiv- activity of the monomeric forms was observed in parallel with their molecular evolution. in addition, no tertiary interactions could be detected by nmr, indicating an autonomous folding of the monomers also in the context of dimeric peptides. the most potent peptide designed so far, rmax, shows anti-hiv- activity in the low nanomolar range (vangelista et al, in preparation). strikingly, a similar potency was observed in the same assay with t , an hiv- gp -derived peptide currently licensed for use in aids therapy. these results provide a rationale for the use of rantes-derived peptides as new candidates for treatment and prevention of hiv- infection. % of the population older than years and % exceeding the age years are affected by alzheimer's disease (ad).( ) a factor in the pathogenesis of ad is the cerebral deposition of amyloid fi brils as senile plaque. bace initiates the pathogenic processing of app by cleaving at the n-terminus and the resulting c membrane bound c-terminal peptide can then be hydrolyzed by -secretase to form apeptide (a or a ). bace is not only a promising target cause it is a key player in formation of a but also because bace knock-out mice are viable and free of the gross phenotypic changes. our group was recently able to shown that the phosphino dipeptide (pdp) isostere is a suitable replacement of the hydroxyl ethylene isostere in om - ( ) resulting in pseudo peptidic inhibitors of about same potency. [ , ] in our search for conformationally restrained pdp isostere bace inhibitors we speculated that the p and p cyclization would lock the active conformation of the cyclic linear pseudo peptidic inhibitor in the n-terminal region. first the detail analysis of the crystal structure of om - bound to bace ( ), revealed that the ideal macrocyclus should consist of a -membered heterocycle. on this basis we developed a macrocyclic inhibitor with p and p cyclized side chains containing a pdp isostere with improved serum stability. specifi c tumor-targeting, via tumor-associated antigens (taa), selectively expressed or over expressed on tumor cells, is the goal of modern cancer therapy aimed at overcoming non-specifi c toxicity of most anticancer drugs. the expression of taa varies among different tumors and patients, resulting in highly variable response to tumor targeted therapies. therefore, diagnosis should provide information on the expression of the targeted antigen in each patient, thus allowing to predict possible effi ciency of a therapy mediated by targeting agents directed to the same tumor antigen. in this approach, the molecule used for tumor cell tracing should be as close as possible to that used for therapy. the use of peptides as tumor targeting agents was envisaged years ago with the fi nding that receptors for different endogenous regulatory peptides are over-expressed in several primary and metastatic human tumors, and can be used as tumor antigens (reubi jc. j nucl med ; : - ). in previous works, we demonstrated that peptides synthesized in a branched form, result in molecules that are resistant to proteolytic activity and can retain (or even increase, through multivalent binding) peptide biological activity. a branched peptide that targets neurotensin (nt) receptors, known to be over-expressed in a number of tumors, including colon, pancreas and prostate carcinoma (reubi jc. endocr rev ; : - ) and was conjugated to effector units and proved to be stable and active in vivo (falciani c, et al. mol cancer ther. ; : - ). here, we created new nt-based molecular tools conjugated to different fl uorophores and chemotherapeutics and demonstrated that branched peptides can be modulated either as tracers for measuring the presence of the specifi c target in primary tumor or metastasis on human surgical resections, or as specifi c drug-carriers, which use the same target to enter and eventually kill tumor cells in vitro and in vivo. aquaculture has become an increasingly important activity, as an immediate source of animal protein required for several countries growing population. vaccination of fi sh against bacterial and viral diseases is one of the best strategy for controlling certain infectious bioactive peptides diseases in aquaculture worldwide, thus decreasing the need for antibiotics, and increasing cost-effectiveness and net profi ts. infectious pancreatic necrosis virus (ipnv) is a pathogen of farm fi sh with a worldwide distribution. peptides derived from ipnv protein (vp ), a major structural protein, could be used for development of effective vaccine against ipnv, according to a search of vp antigenicity we selected two peptide sequences, k pyvrledetpqg (vp - - ) and n fslaeqpanetk (vp - - ), that are expected to be highly immunogenic. these peptide sequences were synthesized in their n-terminus iodoacetylated form and conjugated to ac-(lys-aib-cys) -nh , a cell penetrating sequential carrier (cpsc). conjugation of four copies to cys side chains of the carrier was realised by the chemoselective ligation method. the resulted constructs were purifi ed by hplc and characterized by esi-ms. immunizations are in progress in order to test their immunoprotective potency. acknowledgements to the gsrt (greece-egypt project -å) for the fi nancial support. for more then two decades, the rgd sequence is known to bind integrins, e.g., , v , and iib among many others. it is present in the natural ligands for these integrin receptors, as in fibronectin, vibronectin, or fibrinogen. recently, it was shown that fibronectin in which the rgd sequence has been mutated into an rge sequence (which is known not to be recognized by integrins) still has the ability to interact with its receptor. however, it forms fn fi brils only with a slightly different phenotype than wild-type fibronectin ( ). a hypothetical model for these unexpected results was proposed by curnis et al. ( ) . the ngr (asn-gly-arg) sequence, present at four positions in the fi bronectin molecule, is able to undergo a rearrangement to isodgr (isoasp-gly-arg), which shows activity on v and -with less potency -on . the mechanism of asn-deamination is already known for a long time and has widely been considered to be a process of degradation, acting as a biochemical clock that limits protein lifetimes in vivo ( ). curnis et al. were the fi rst to show that the deamination process increases protein function instead ( ). these fi ndings stimulated us to create a library of cyclic peptides containing the isodgr sequence. a screening of this library showed various new peptides with high activity and selectivity towards the integrin receptor . the correlation between the intensities of muramyl peptides adjuvant effect and nod activation. it is well known that essential activity of muramyl peptides -minimal structures of bacterial cell wall -are adjuvanticity. the comparison of the adjuvant effect of these compounds with the ability to activate nf-kb pathway through nod was examined. the adjuvant activity of di, tetrasaccharide peptides and stearoyl containing derivatives has at least two peaks in dose-response curves and greater of them correlates with respective dose-response data for nf-kb stimulation through nod . introduction of stearoyl moiety, with the aim of improving muramyl peptide interaction with the cell membrane and subsequent intracellular delivery, infl uenced the corresponding activities in vitro, but did not correlate with improved effects in vivo experiments. the comparison of the adjuvanticity in vivo and the nod activation in vitro revealed clear correlation between two responses. these fi ndings confi rm the view that nod pathway activation should account, at least in part, for the adjuvant effect of these compouds. the correlations of the nf-kb pathway induced by muramyl peptides with the aim of enhancing their adjuvant activity were investigated. the thymus gland plays an important role in overall immunomodulation. the studies in early s by several authors have established that thymus is necessary for the normal development of immune response. it is thought to be responsible for the development and regulation of tcell immunity, acting through endocrine mechanism ( ). it is known that thymus peptides play an important role in the development, maturation, differentiation, and activation of t-lymphocytes. investigation of the function and properties of this gland shows that the thymus contains pharmacologically active components with immunological properties. when the immune system is challenged, thymic peptides seem to regulate the expression of various cytokine and monokine receptors on t-cells and induce secretion of il- , interferon alpha, and interferon gamma. up to date several thymic extracts have been isolated using different biochemical methods of extraction, homogenization and purifi cation. these are, usually, semipurifi ed aqueous and lipid calf thymus extracts. the goal of this study was to determine the biological activity of peptide components, isolated from the calf thymus. extract of calf thymus was prepared and fractioned into lipid and nonlipid (peptides) fractions. the nonlipid fraction was isolated and characterised by biuret, ir , nmr and hplc methods. analyses of ir and nmr spectra indicated the presence of charasteristic bands and peaks for peptides. results estimated from hplc analyse showed that molecular masses of isolated peptides were below daltons. biological activity was accesed by using proliferation of thymocites and splenocites in vitro in the presence of mitogen, and obtained results showed signifi cant imunomodulatory effect. keywords: thymus, peptides, immunomodulators, thymocites, splenocytes, proliferation according to contemporary knowledge about proteins, every protein can play the role of a precursor of biologically active peptides. bioactive peptides isolated from food proteins display various activities, for example: antihypertensive, antithrombotic, immunomodulating, opioid and antibacterial. the database of protein and bioactive peptide sequences designed in chair of food biochemistry (www.uwm.edu.pl/biochemia) contains information on proteins which are precursors of bioactive peptides. the database, biopep, enables an evaluation of food proteins according to the following criteria: the frequency of the occurrence of fragments with the given activity in a protein chain, a potential activity of protein fragments, and profi les of a potential biological protein activity i. e. the type and location of a bioactive fragment in the protein chain. the biopep database was used to determine the profi les of potential biological activity of food proteins and classify them into families and subfamilies. results that we obtained indicate that the greatest number of bioactive peptides can be released from milk proteins. we also analysed structural properties of bioactive fragments encrypted in protein chains which are predicted to be accessible for endopeptidases. the application of biopep and msblast software enabled us selection of a fragments with high degrees of identity to the celiac-toxic peptides in food proteins sequences. based on the biopep, we are able to design the processes of the release of bioactive peptides from protein sequence and with the use of mass spectrometry -to identify released peptides. the detailed results of authors in silico food proteins analysis will be presented. the computational methods and the above-mentioned tools can be applied for designing food with the special designed and desired properties (functional food) as well as production of nutraceuticals i. e. food with therapeutic properties. neurotensin analogs with high affi nity and selectivity at human neurotensin receptor . neurotensin (nt), pglu -leu -tyr -glu -asn -lys -pro -arg -arg -pro -tyr -ile -leu -oh, exerts numerous physiological actions in the central nervous system and in the periphery. studies in animal models have suggested its involvement in the modulation of dopamine transmission, hypothermia, analgesia, locomotor activity, cardiovascular function, and others. at present, three receptors are known to bind nt; two of them are g-protein coupled-receptors (ntsr and ntsr ). the physiological effects of ntsr have been extensively studied but the functions associated with the nt binding to ntsr are less well defi ned. the c-terminal fragment of nt, arg -arg -pro -tyr -ile -leu -oh, has been long recognized as an essential and suffi cient segment for the effective interactions of nt with the receptors; a short peptide, arg -arg -pro -tyr -ile -leu -oh, designated nt( - ), displays binding affi nity similar to that of the full-length nt at both ntsr and ntsr . in this study, the role of arg in the interactions of nt( - ) with the human ntsr and ntsr was examined through ligand structure-function studies. the side chain of arg was determined to not be critical for binding to hntsr but essential for binding to hntsr . several analogs of nt( - ) are reported which are high affi nity hntsr ligands (ic = . to nm) and more than -fold selective versus hntsr . human umbilical vein endothelial cells (huvecs) form tubular networks when cultured on top of the matrigel basement membrane preparation, refl ecting the ability of the cells to form blood vessels. using this model, we have previously shown that psa (also known as klk ) inhibits endothelial cell tube formation( ), indicating reduced angiogenic potential. furthermore, we have shown that the antiangiogenic activity of psa is related to its enzymatic activity [ , ] . we have developed peptides that stimulate the enzymatic activity of psa towards a small chromogenic substrate. these peptides also enhance the anti-angiogenic activity of psa both in vitro and in vivo. this supports our hypothesis that enhanced psa-activity by our peptides could be used to reduce tumor angiogenesis and, thus, to reduce tumor growth. the most extensively studied tads are those that contain acidic tads and one extremely important protein is the human tumour suppressor protein p . given the presence of repetitive stretches of acidic amino acids tads are generally disordered in the free state and this has also been demonstrated for p tad. using a combination of nmr spectroscopy, isothermal titration calorimetry and site-directed mutagenesis studies we have recently characterized the interaction of the p tad with the pleckstrin homology (ph) domain of the tfb / p (yeast/human) subunit of tfiih. the nmr structure of the tfb / p tad complex demonstrates that p forms a short alpha-helix in complex with tfb ( ). this structure is an important step towards developing a sequence code for acidic tad binding to the ph domain of tfb /p . such detailed structural information is essential to design molecules that modulate transcription activators such as p . we will present the structure of the p /tfb complex, structural and biophysical characterization studies with peptides designed to mimic acidic tads and compete with p for binding to tfiih. integrins are a family of transmembrane cell surface receptors, which mediate cell-cell and cell-matrix adhesion. the binding of integrins to their natural ligands is the molecular basis of physiological processes such as cell adhesion, migration and signal transduction of cells, as well as of patho-physiological processes. thus, small molecules capable of interfering with this integrin-natural ligand binding process have pharmacological potential in the therapy of cancer and infl ammatory diseases. the amino acid sequence rgd, present on many of the natural ligands, is a prominent recognition motif of integrin ligands. synthetic rgd-containing peptides are an excellent starting point for the identifi cation, synthesis and development of selective integrin ligands. the affi nity and selectivity of the peptide ligands towards different integrins depend strongly on the secondary structure of the sequence and the overall three-dimensional shape. cyclization is frequently used as a method to reduce the accessible conformational space. additionally, the incorporation of non-natural conformationally constrained amino acids can greatly affect the secondary structure of the peptide, in such a way that the synthetic ligands prefer to adopt a particular conformation. the aim of this investigation are small cyclic peptides containing the rgd motif and constrained amino acids (such as -methylated amino acids, -amino acids or dehydroamino acids) that exhibit well-defi ned conformational properties. the present communication describes the synthesis of different cyclic rgd peptides with the general sequence c-(-arg-gly-asp-xaa-yaa-) and the evaluation of their activity as ligands for the v and integrins, present on human cells. acknowledgments: this work is supported by a marie curie intra-european fellowship from the th framework programme. in recent years there has been increasing interest in the design of chemical agents capable of inducing dna interstrand crosslinking (isc), which, shutting down the dna replication process, represents by far the most cytotoxic of all the alkylation events. quinone methides (qms) are interesting compounds that have been proposed as intermediates in a large number of chemical and biological processes. the asymmetry introduced by the presence of two electronically different substituents, carbonyl and methylidene, on the cyclohexadiene ring imparts a strong dipolar character to quinone methides not found in benzoquinones and quinodimethanes. qms have been successfully used to accomplish nucleoside alkylation and dna isc by photochemical and fl uorideinduced activation. apart from their involvement in biological processes, o-qms react with a variety of nucleophiles (michael addition), resulting in substituted hydroquinones. they also function as effi cient heterodienes in diels-alder reactions and undergo a variety of self dimerization reactions. here we present the synthesis of a new series of synthetic o-qms (bis-napthalene type) conjugated with rgd analogue peptides. the biological activity of these compounds is under investigation. aknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii) and particularly the program pythagoras ii for funding the above work. platelets aggregation causes clotting in the blood vessels during the blood circulation due to several reasons. factor viii (fviii), a blood coagulation glycoprotein, is a key component of the blood coagulation system. fviii in its activated form (fviiia) acts as a cofactor to the serine protease fixa in the conversion of the zymogen fx to the active enzyme (fxa). the role of fviiia is to increase the catalytic effi ciency of fixa in the activation of factor x (fx). the target of this research is the synthesis of biologically active peptides, which are expected to inhibit selectively the maximisation of thrombin production depended on factor ix (fix) and accordingly the additional activation of platelets. these peptides are based on the regions in which the fviii interacts with fix. glycoprotein fviii is composed of three distinct domain types in the arrangement, a -a -b-a -c -c . the sequence - of a subunit is the following: ser -val-asp-gln-arg-gly-asn-gln this work covers the synthesis and biological evaluation of linear and cyclic head to tail peptides, analogs of the loop sequence - of the a subunit, aiming at the inhibition of interaction of fviiia with fixa. substitutions have been taken place at asn , which are related to the pharmaceutical groups at the side-chain, like asp(r) . all the synthesized analogs are purifi ed (rp-hplc) and identifi ed (esi-ms). the synthesized peptides analogs were investigated for their inhibitory activity and tested for clotting defi ciency by measuring their activated partial thromboplastin time (aptt) in vitro. these results will be discussed in relation with their biological activity against the fviiia factor of blood coagulation. nerve growth factor (ngf) is an homodimeric protein that binds two different cellular receptors: ) the transmembrane tyrosine kinase receptor trka, a member of tyrosine kinase family; and ) p , a member of the tumor-necrosis-factor receptor superfamily. both receptors are involved in the activation of different intracellular signal transduction cascades. small peptides retaining the most essential elements of ngf may be useful either as agonist or competitive antagonist in the treatment of several neurodegenerative desease and nerve injuries. the n-terminal fragment of ngf was previously demonstrated to be an important determinant for affi nity and specifi city in the binding to trka. crystal structure of ngf-trka complex, site-directed mutagenesis studies and substitution of individual amino acids, contributed to identify within the ngf molecule the most relevant domains for its biological activity in the loop- ( - ), loop- ( - ) and in the nterminal region ( - ). we synthesized twenty peptides mimicking the loop and linked together with aminoacid-based spacers (n glycines) or with two unit of -ammino- , -dioxaottanoic acid with or without the n-terminal region. l l and l l homodimeric loops, moreover l and l were also synthesized, as single loop, and their biological properties were compared to l l and n-l l activity . the n-l l (hpifhrgefsvadsvsvwvgdctdikgkctgacdgkqc) and l l (ctdikgkctgacdgkqc) peptides showed a good ngf agonist activity at concentration as low as um. both were able to induce differentiation of chick dorsal root ganglia (drg) and to stimulate the tyrosine phosphorylation of trka but not of trkb receptor. in addition the l l peptide was able to induce the pc cells differentiation into sympathetic-like neurons. moreover the l l peptide was shown to reduce neuropathic behaviour and restore neuronal function in a rat model of peripherical neuropathic pain, thereby suggesting a potential therapeutic role for this peptide. short proline-rich peptides interact with sh domains, exhibiting little or no secondary structure before their binding to the cognate proteintargets. under these conditions the binding process of a proline-rich peptide with the sh domain shows unfavorable binding entropy, likely resulting from a loss of rotational freedom on the formation of the ppii helix. with the aim of stabilizing the ppii helix conformation in sh binding motifs, in the previous years, we replaced the proline residues of the hpk proline-rich decapeptide, ppplppkpkf (p ), either with -r-(fp) or with -s-(fp) fl uoro-l-proline at different i, i+ positions. the interactions of the fl uoroproline-peptides with the sh domain of cortactin protein were analyzed quantitatively by non-immobilized ligand interactions assay by circular dichroism (nilia-cd), whereas cd thermal transitions were measured to correlate their propensity to adopt ppii helix with their affi nity for sh . results show that although the introduction of the fp residue stabilizes the ppii helix conformation of peptides in a position-dependent manner, the induction of a stable peptide conformation does not increase the ligand affi nity towards the sh domain of cortactin. to explore the effect of electron-withdrawing substituent in the pro residue, the fp residues of p were replaced by the natural amino acid -r-hydroxy-proline (hyp). unexpectedly, nilia-cd and cd thermal transitions results showed that the hyp-containing peptides exhibit a stable conformation in aqueous buffer and k d values lower than the corresponding fp peptide-analogues. in particular, hyp peptide containing hyp residues at i, i+ and i+ positions adopts the greater percentage of ppii helix conformation over the entire studied temperature range and shows a k d value comparable to that of the parent p peptide. we are now searching for inhibitory peptides able to reduce survivin function in breast cancer cells. we take advantage of the observation that survivin forms homodimers in vivo and have derived short peptides representing the dimerization domain. to enable intracellular expression and visualization of these short peptides, they will be fused to a fl uorescent carrier protein. alternatively, the peptides comprising the dimerization domain will be inserted into a scaffold protein. this allows the presentation of the peptides in a constrained and stabilized conformation. we will analyze the effects of lentiviral expression of these fusion peptides in cancer cells expressing high levels of survivin. the peptides will be analyzed for their ability to inhibit proliferation, cell-cycle progression and/or to induce apoptosis in cancer cells. cd c/cd ( x ) is a member of the leukocyte integrin (cd or integrin) subfamily of adhesion molecules and it is expressed on monocytes, macrophages and granulocytes. its ligands include fi brinogen, ic b, lps, collagen type i and denatured proteins as well as intercellular adhesion molecules icam- and icam- . icam- is a red cell specifi c membrane glycoprotein that was fi rst described as anerythrocyte blood group antigen lw (landsteiner-wiener) and later it was found to belong to the family of intercellular adhesion molecules. the fi rst reported receptors of icam- were leukocyte integrins cd a/cd and cd b/cd . later it has been reported to bind to several other integrins as well. latest reported interaction is to cd c/cd . the binding of icam- to integrins expressed in macrophages might clarify some of the controversy concerning the recognition and uptake of senescent red blood cells in spleen. another function of icam- / macrophage integrin interactions could be in retaining the maturing red cells in bone marrow until they are ready to be released in the circulation. adhesion between icam- and integrins have also been found to be important in different pathological situations and inhibition of these adhesion events could be of important therapeutic value. we have studied the interaction between icam- and cd c/cd using peptides derived from both binding partners using pepspot method and synthetised peptides. the inhibitory or activating role of these peptides could beof clinical importance in pathological conditions where abnormal red cell adhesion is observed. the regulatory effects of relaxin in mammals realize via receptors of the serpentine type. in this work we studied the relaxin activating effect on the adenylyl cyclase (ac) activity in the rat tissues and muscle tissues of invertebrates using a peptide strategy. the strategy involved the synthesis of the peptides - and - , as well as the palmitatemodifi ed peptide - , all corresponding to the c-terminal region of the third intracellular loop (c-icl ) of the human type relaxin receptor lgr . the peptides - and - -lys(palm) had a dose-dependent activating effect on the ac activity and gtp-binding in myocardium, brain and, to a smaller extent, skeletal muscles of rat. the - -lys(palm) had a stronger ac effect than the longer peptide - , because of the possibility to be anchored in the membrane by the hydrophobic radical and, hence, to interact with the g protein more effectively. in mollusks and earthworm muscles, the stimulatory effects of both peptides were weak. competitive inhibition of the stimulating effects of relaxin by the lgr -derived peptides indicates that the relaxin signal in myocardium and brain is transmitted to ac via lgr receptor. in skeletal muscles of rat and muscle tissues of invertebrates, the inhibition of ac and gtp-binding stimulating effects of relaxin by peptides was almost indiscernible. it can be concluded that relaxin controls the ac of these tissues via the receptor different from the relaxin receptor lgr . the stimulating effects of lgr -derived peptides also decreased in brain and myocardium in the presence of Ñterminal peptide - of mammalian g s -subunit and after cholera toxin treatment. thus, relaxin stimulates ac via lgr receptor and g s protein in myocardium and brain, and the coupling between receptor and g s protein is mediated by the interaction of receptor Ñ-icl and Ñ-terminal segment of g s . acknowledgements: supported by rfbi (grant - - ) and «russian science support foundation». normal tissue function depends on adequate supply of oxygen through blood vessels. angiogenesis is a fundamental process by which new blood vessels are formed and which is highly regulated in healthy individuals. however, many diseases are driven by unregulated angiogenesis. excessive angiogenesis is associated with cancer, rheumatoid arthritis, psoriasis, while insuffi cient angiogenesis results in ischemia or artherosclerosis. many new angiogenic modulators have been developed in the last years, mostly to inhibit angiogenesis but only few peptide-based angiogenic stimulators have been reported. there are data in the literature suggesting roles of small peptides or basichexa peptides as angiogenic modulators like ringseis et al. reporting effects on endothelial cell function (such as ec proliferation) and fazekas et al. describing effects for the latter. endostatin, an endogenous inhibitor of angiogenesis, among its proteolitic fragments contains both, an inhibitor and a stimulator of angiogenesis. we considered it possible that fragments of basic heptapeptide d-phe-cys(-)-tyr-d-trp-lys-cys(-)-thr-nh (tt- ), a strong antitumor agent, could also act as angiogenic modulators. the heptapeptide was developed in our laboratory and has been recently in a clinical trial (phase ii). we synthesised, characterized and tested partially protected di-and tripeptide fragments of it such as boc-d-phe-cys(acm)-tyr-ome, d-phe-cys(acm)-tyr-ome, boc-tyr-d-trp-cyclohexilamide and h-tyr-d-trp- -adamanthylamide. the biological activity of the compounds was tested in vitro using an immortalized kaposi sarcoma cell line to determine their pro-anti-angiogenic character. to test the angiogenic potential of the best compound, the aorta ring assay was used, which by using intact vascular explants reproduces more accurately the environment in which angiogenesis occurs. in this study we report the synthesis and angiogenesis modulating effects of the peptides. somatostatin is a neuropeptide that regulates several functions of the endocrine and exocrine systems. it also affects cell proliferation and neurogenic infl ammation through a family of g protein-coupled receptors. neurogenic infl ammation plays signifi cant role in the pathogenesis of numerous infl ammatory diseases (such as asthma, arthritis, allergy and migraine). inhibitory effect of somatostatin on infl ammation is well known but the pharmaceutical use of the native peptide is limited due to its broad spectrum of anti-secretory effects and short plasma half-life time. tt- , a heptapeptide analogue of somatostatin (developed by our research group and it is in clinical phase ii trials) has selective antitumour and anti-infl ammatory effect without regulating other endocrine or exocrine processes. receptor-ligand binding experiments with various analogues of the somatostatin as well as tt- [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh ] verifi ed that the side chains of tyr, d-trp and lys are important pharmacophoric groups for somatostatin-like biological activity. linear, cyclic and branching derivatives were designed and synthesised using above amino acids to selectively inhibit infl ammatory actions. unnatural moieties also were applied to enhance their enzyme resistance. linear and cyclic peptides consist tyr and d-trp, while ring closed ones contain lys too. branching peptidomimetics have the same, fl exible core [tris( aminoethyl)amine] and three protected or unprotected amino acids situated in equal positions. the biological activity of the compounds was evaluated by in vitro assay of substance p release and in vivo assay of plasma protein extravasation. the most potent agents strongly inhibited substance p release by - % and one of them showed strong anti-infl ammatory activity when administered orally. the structure of the novel compounds and the relationship between their structure and biological activity will be discussed. urotensin ii (u-ii), a potent vasoconstrictor, is found in diverse species, including human. several biological studies indicate that u-ii is the most potent mammalian peptide vasoconstrictor reported to date, and it appears to be involved in the regulation of cardiovascular homeostasis and pathology. in order to elucidate the importance of trp residue for receptor interaction and biological activity recently we have designed, synthesized new analogues where trp was replaced with constrained analogues ltpi or dtpi ( , , , -tetrahydronorharman- -carboxylic acid). the tpi residue was replaced in both agonist p u and antagonist urantide sequences. on these new ligands we performed biological and nmr conformational studies. the new ligands will be used in further biological investigations of the ut receptor. gnrh analogs as carriers for targeted suicide gene delivery suicide gene therapy represents one of the promising approaches to the cancer treatment. an application of herpes simplex virus thymidine kinase (hsvtk)/ganciclovir system possesses additional advantage due to bystander effect on neighboring cancer cells. overexpression of gnrh receptors in the case of most adenocarcinomas creates the basis of gnrh analogs use as carriers for targeted suicide gene delivery. we investigated different manners of gnrh molecule modifi cation; their infl uence on peptide/dna complex formation and its penetration into cancer cells. analogs, containing nls from large antigen of sv were synthesized using combination of boc-and fmoc-chemistry. depending on peptide structure (agonist or antagonist) nls was attached via position or of the natural molecule. the competition experiments demonstrated that internalization of peptide/ dna complex into hepg cells is mediated by specifi c receptor binding. moreover, nls/dna complexes, lacking gnrh moiety were unable penetrate cellular membrane. subsequent studies permit to identify the infl uence of analog structure on in vitro effi ciency of suicide gene therapy, followed by acyclovir treatment. it was shown that application of reference peptide gene delivery system damaged about % of tumor cells. use of cationic peptide conjugated with rgdf sequence provides high effi ciency of treatment, however can not ensure selective action on cancer cells. gnrh analogs completely suppress tumor growth in vitro due to specifi c interaction of peptide/dna complex with correspondent receptor. the effi ciency of suicide gene therapy depends on peptide structure and is in favor of agonists as compared to antagonists. preliminary data of experiments in vivo on laboratory animals demonstrated practical utility of tested peptides in the course of intravenous administration. thus, it was shown that gnrh analogs containing nls moiety represent promising candidates for the delivery of hsvtk gene into the cancer cells. the structural-functional study of putative grape (vitis vinifera) uncharacterized protein sequences was performed. we developed a special method of computer analysis ( ) for this. this method has allowed to reveal new potentially active regulatory oligopeptide sequences yet not investigated experimentally. information on grape amino acid sequences of public databases [ , ] , computer database erop-moscow (endogenous regulatory oligopeptides) . containing the information on structure and functions of known natural oligopeptides and specially created computer programs were used for this. protein amino acid sequences were compared with all known oligopeptide sequences in this method. as a result several tens of grape oligopeptide sequences were elucidated. the similarity of their sequences with the known oligopeptide structures of other biological species was the basis for the prediction their potential functional properties. it has been shown that grape contain putative regulatory oligopeptides possessing functions of antibacterial and antifungal agents, enzyme inhibitors, calmodulin binding structures, rapid alkalinization factors, etc. the primary structure similarity of grape sequences was found not only with plant species but with bacteria, fungi, and animals also. cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. their stability derives from a headto-tail cyclised backbone coupled with a cystine knot arrangement of the three-disulfi de bonds. taking advantage of this stable framework we developed novel vegf-a antagonists by grafting a peptide epitope involved in vegf-a antagonism onto the stable cyclotide framework. antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. a grafted analogue showed biological activity in an in vitro vegf-a antagonism assay at low micromolar concentration and importantly the in vitro stability of the linear epitope was markedly increased using this approach. in general, the stabilization of bioactive peptide epitopes is a signifi cant problem in medicinal chemistry and in the current study we have shown the cyclotide framework is ideally suited for such stabilization. cystine rich scaffolds are emerging as valuable templates in drug design and in the current study we have shown that the cyclotide scaffold, with the advantageous features of a knotted disulfi de core and a cyclic backbone, has signifi cant potential in stabilizing a wide range of bioactive peptide epitopes. gonadotropin releasing hormone (pglu-his-trp-ser-tyr-gly-leu-arg-pro-gly-nh , gnrh) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. a large number of linear gnrh analogues has been synthesized and tested for several medical uses. leuprolide acetate (pglu-his-trp-ser-tyr-(d)leu-leu-arg-pro-nhet, lpa) is a potent gnrh agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of leuprolide are known. moreover, non crystallographic data is available for the receptor of gnrh (g protein-coupled receptor). the aim of this study was to characterize the conformation of leuprolide and its modifi ed linear analogue (pglu-his-trp-ser-tyr(ome)-(d)leu-leu-arg-aze-nhet) in dmso solution (which simulates better the receptor environment) using nuclear magnetic resonance (nmr) and molecular modeling techniques. by using both nmr and molecular modeling we have characterized the secondary structural preferences of these gnrh analogues. structural determinants of binding to the mu-opioid receptor -an important target in analgesia -attracts great scientifi c attention. many natural and synthetic peptides and peptidomimetics were shown previously to bind to the mu-opioid receptor selectively but there is no consensus about what structure is responsible for such biological activity. no high resolution structure of this receptor is available and the binding site of ligands is not exactly known despite numerous site-directed mutagenesis studies. this suggests that the determination of structural aspects of mu-opioid activity should focus on the ligands. mu-opioid ligands with similar affi nity and selectivity should possess at least one common structural feature in which they differ from other ligands of different affi nity and selectivity. comparative structural analysis of such ligands, considering adequate representation of binding conditions may reveal key features of bioactivity. in this study ten mu-opioid receptor ligands, damgo, tyr-w-mif- , morphiceptin, endomorphin- and and their analogues, possessing different affi nity and selectivity were examined using molecular dynamics. conformational preference of these molecules was determined in aqueous and dmso media which were meant to model different possible binding environments. no structural trend, correlating with previously measured bioactivities was observed in aqueous media. in dmso it was found, that a preference for trans orientation of the tyr side chain and gauche (-) orientation of the third aromatic side chain, the free rotation of the phe side chain and a high propensity of bent backbone structure is favorable for high affi nity binding to the mu-opioid receptor, while deviations from these criteria results in variable loss of bioactivity. constellation of these four key conformational parameters may be a guiding principle in the future for the design novel mu-opioid receptor ligands. in cui-catalyzed azide-alkyne , -dipolar huisgen's cycloaddition -prototypic "click reaction"-is a recently developed synthetic procedure to obtain cyclopeptides carrying, as a rigid linking unit, the lactam bioisoster, , -disubstituted [ , , ] triazolyl ring ( ). we have recently reported the synthesis and conformational analysis of the , -disubstituted- [ , , ] triazolyl containing cyclopeptide derived from the sequence of the potent i-to-i+ side chain-to-side chain lactam-containing antagonist of parathyroid hormone-related peptide (pthrp) .. the conformational properties of triazolyl-containg peptide were compared to those of the corresponding lactam analog. cd and nmr studies revealed that, despite a slight difference of the backbone arrangement, triazolyl containing cyclopeptide and lactam-containing cyclopeptide share a common orientation of the side chains ( ). here we present the structural study of a new library of disubstituted- [ , , ] triazolyl containing cyclopeptides designed to obtain different cycle dimensions and different triazolyl-ring positioning. cd and nmr analysis in different solvent systems shows that both, the dimension of the cycle and the specifi c positioning of [ , , ] triazolyl ring are critical to fully resemble the conformational properties of the potent lactamcontaining antagonist of parathyroid hormone-related peptide (pthrp). the somatostatin (srif) is a cyclic tetradecapeptide, which exerts inhibitory effects on the secretory processes in the endocrine and exocrine systems, and the cell proliferation through somatostatin receptors (sstr - ). sstrs are distributed throughout human body, not only in normal cells but also in tumor cells. most of the somatostatin analogues developed for clinical use, such as octreotide which act longer than somatostatin are being used in the diagnosis and treatment of endocrine tumors. the use of these analogues as antitumor agents has been limited because of their antisecretory effects and poor oral bioavailability. tt- [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh ], was reported by keri et al. to have potent antiproliferative activity without antisecretory action .. based on the above, we aimed to design and synthesize somatostatin analogues with more potent antiproliferative activity and high oral bioavailability. we synthesized pyrazinone ring containing cyclic peptides ( ) and linear peptides which are substituted at the c-terminus lys with hydrophobic and rigid groups. our focus was on the active sequence: tyr-d-trp-lys. we also examined their antiproliferative activity and found that boc/h-tyr-d-trp- -adamantylamide exhibited the most potent antiproliferative activity higher than that of tt- . furthermore, on the best analogues we studied dna fragmentation by facs analysis and cellular morphology. the results demonstrated that these somatostatin analogues induced cell death by apoptosis. kobe gakuin universit, japan background and aims: endomorphin- (em- : h-tyr-pro-phe-phe-nh ) has high affi nity and selectivity for the mu opiod receptor ( ). we focus on the pro residue, which imposes strong restraints on the conformation of the peptide chain or induces cis-trans isomerization of x-pro bonds. we substituted the pro with -azetidinecarboxylic acid (aze) or piperidinecarboxylic acids (pip) to increase or decrease the conformational fl exibility. in this paper, we deal with the synthesis of em- analogues containing aze and pip and the evaluation of the biological functions of the analogues on the opioid receptors. methods: the synthesis of peptides was achieved according to the procedure of okada y. et al. ( ) the fi nal products were identifi ed by maldi-tof mass spectrometry and elemental analyses. the receptor binding activity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from cos- cell membranes expressing each opioid receptors. for the evaluation of the biological function of peptides, the gpi and the mvd tests were performed. the new analogue of csf (glc) ., [pro ,asn (glc),thr ]csf is characterized by a type i -turn around the minimal epitope asn(glc), and it was demonstrated to show the highest antibody affi nity in competitive elisa in multiple sclerosis (ms) patients' sera and thus it appears as a promising tool for the detection in patients' sera of specifi c autoantibodies. in previous studies, we synthesized and tested different fragments of this new glucosylated peptide, [pro ,asn (glc), thr ]csf , identifying the shortest sequence [pro ,asn (glc),thr ] csf ( - ) able to detect antibodies by elisa in ms patients' sera. this heptapeptide is characterized by thr in position generating a characteristic n-glycosylation consensus sequence. according to the results obtained with the linear heptapeptide in ms, we applied the backbone cyclicization method to develop two backbone cyclic libraries of glycopeptides based on the sequence of the linear hepta active peptide. backbone cyclization is a method that allows obtaining cyclic peptides without changing the natural sequence or the chemical character of the amino acid residues, in order to enhance activity, stability to metabolic degradation, selectivity and bioavailability ( ) . the fi rst library contains a gly building unit at the c-terminus and is connected to the n-terminus by linker of various lengths (n= , , , ) . in the second library, his of the heptapeptide is replaced by gly building units with various alkyl chains (n= , , , ) [fmoc-n (n alloc(n-alkyl))gly-oh] that is connected to the n-terminus by linker of various lengths. twenty cycloglycopeptides were synthesized, and screened by competitive elisa s on ms patients' sera to select the most bioactive cyclic glycopeptide. peptide nucleic acids have become, arguably, one of the most interesting of dna mimics. owing to their high chemical stability and resistance towards nucleases and proteases, they are very attractive as antigene/ antisense agents, molecular biological tools and for genetic diagnosis. the lack of charge and polar groups in the backbone decrease their solubility in aqueous environment and their ability to cross cell membranes, reducing their performance in in vivo applications. in order to overcome these problems -to improve solubility, increase affi nity and specifi city of binding, a number of analogues were synthesized. this study describes the synthesis of pna-monomers on the base of non-protein amino acids analogues of basic amino acids lys and arg. nucleobases are the second residue we have selected. studies will include replacement for example of the uracil, with the fl uorinated analogue. growing evidences indicate that n-glycosylation is a co-translational modifi cation that, either native or aberrant, may play a fundamental role in a large number of biological events. in particular among postand co-traslational modifi cations glycosylation plays a crucial role in the immune system. in fact, almost most of all the key molecules involved in the immune response are glycoproteins. there are growing evidences of defects in glycosylation with diseases that assets to pathway oligosaccharides as code words. in previous studies, we demonstrated that the presence of a -dglucopyranosyl moiety on an asn residue at position of csf (glc) is fundamental for auto-ab recognition resulting the fi rst multiple antigenic synthetic probe (msap) able to detect autoantibodies in ms patients' sera. up to now we have investigated the carbohydrates infl uence and specifi city in ms antibody recognition introducing several glycosilated building blocks in the msap sequence (i.e. glc, man, glc glc, gal, glcnac on the side chain of ser, thr, asp, glu, hypro) .. due to microheterogeneity and the extremely high specifi city of carbohydrateprotein interaction we included in our library screening, the ribose. we report the synthesis of new asn-derivatives bearing on the side chain ribose rib and rib linked by an n-glycosidic bond and protected for spps.these building blocks introduced in the csf -turn scaffold lead to the new ribosylated peptides contributing to the library of glycopeptides to fi shing out families of autoantibodies specifi c for different autoimmune diseases. fraczyk, justyna; kujawska, nina; kaminski, zbigniew, j. we designed and prepared supramolecular structures formed from nlipidated oligopeptides immobilized in the regular pattern on the cellulose surface which are able to specifi c binding of ligand molecule. due to the conformational fl exibility of the fragments forming the supramolecular structure, the shape and prosperities the binding cavities are adjusted the most effectively to requirements of the guest molecules. the previous studies documented that process of binding guest molecules is highly selective, reversible and competitive. therefore, we supposed that under favorable circumstances the structures could operate as catalysts if suitable molecular fragment are included inside the binding pocket. in order to verify this hypothesis we prepared library of supramolecular hosts with catalytic triade: his asp(glu) ser, incorporated into the binding pocket .. for the fi rst generation library the rate of hydrolysis of p-nitophenyl esters of n-protected amino acids was measured by spectrophotometric determination of liberated p-nitrophenole in buffered, aqueous methanol and compared with appropriate data obtained in the absence of catalytic structures. the most active catalyst were selected from the library and their stability, selectivity and ability for re-use was studied. for the second generation of library the stereoselectivity of artifi cial esterase we present here a new technique for identifying very small quantity of peptide mixtures that are selected out from a peptide library in solution, by using multi-component fl uorescence labeling. the technique is basically a modifi cation of positional screening method associated with a : correspondence between the amino acid at the i-th position and the type of the fl uorescence lebel at the n-terminal. -dimensional fl uorescence spectroscopy was employed for identifying the fl uorescence labels in the mixture of peptides that bound to target cells or target proteins. the results of the new screening method will be presented for peptides that specifi cally bind to human cancer cells. show increased biological activity in a dimeric form . in recent years there have been signifi cant efforts to obtain minimized versions of naturally occurring proteins such as dimeric dna binding proteins which retain their function. the gcn basic region peptides were connected trough a disulfi de bond to give a dimer which specifi cally bound the ap -dna sequence. dimeric peptides and proteins were obtained also by non covalent interactions. in this work we propose a strategy for obtaining by expressed protein ligation (epl), one pot protein homodimers covalently connected at the c-terminus. the synthetic strategy was extended also to the synthesis of heterodimers. epl is a protein engineering tool for the chemo and region-selective modifi cation of proteins based on the use of intein containing constructs. in this work dimers were obtained by reacting a new bi-functional linker with carboxyl-activated polypeptides. we synthesized a linker containing two cysteines in a n-terminal-like position, separated by an ethylendiamine spacer, and obtained thioester proteins by intein mediated splicing reactions. this strategy affords chemically stable dimeric proteins. the linker can be easily modifi ed at need, changing the lenght and rigidity of the spacer between the cysteines. this strategy has potential in biochemical and bioorganic applications, for obtaining minimized and/ or modifi ed natural proteins and for joining two different proteins at the c-terminus position. this technique will be extended to the synthesis of dimeric proteins mimicking the transcription factor ap- . previous studies showed that csf (glc), , a designed glycopeptide characterized by a -d-glucopyranosyl moiety, can detect and isolate specifi c autoabs in sera of a signifi cant number of ms patients. this synthetic ag could be considered a mimetic of aberrantly glycosylated myelin proteins triggering autoimmunity in ms. myelin oligodendrocyte glycoprotein (mog) is considered a putative autoag in ms. our aim is to obtain mog properly glycosylated to characterize the molecular mechanisms of ab-mediated ms and to design new antigenic probes to detect autoabs as biomarkers. production of specifi c glycoproteins may benefi t from a chemical approach, such as expressed protein ligation (epl) a protein engineering strategy useful to introduce noncanonical amino acids and biological probes into proteins. epl allows synthetic and recombinant polypeptides to be chemoselectively and regioselectively joined together. the recombinant rmog ed ( - ), obtained as c-terminal thioesther by protein splicing, will be ligated to the peptide fragment [gly ,a sn (glc)]mog ed ( - ) bearing a cys residue at the n-terminus. an alternative strategy exploits the selective reaction between a glucosyl iodoacetamide derivative and the cys free thiol of a protein. a site directed mutation has been performed on rmog to introduce a cys residue at its native site of glycosylation. the semi-synthetic proteins will be tested by elisa using ms patients' sera. automation is an identifi ed goal in the peptides r&d at lonza. this approach would facilitate and accelerate peptide production. this is highly desired within peptides r&d, where the need exists for rapid synthesis of peptides to fulfi l iso and gmp projects requirements. automated peptide synthesisers are available on the market but many have limitations which make them inappropriate for lonza (e.g., low scale, coupling systems limitation, pre-activation procedure at low temperature). furthermore, there is no obvious standard instrument for scalable spps equipped with pat. ge healthcare provides scalable and complete solutions for automated solid-phase synthesis of oligonucleotides from small research amounts to full commercial production ( mol- mol) based on the Äkta, oligopilot and oligoprocess™ platforms. the unicorn™ software provides control and monitoring of the processes. ge healthcare synthesisers are designed around fl ow-through column reactors, giving faster kinetics and lower solvent and reagent peptide biotechnology and diagnostics consumption compared to batch synthesisers. based on experience with scale-up of oligonucleotides, ge healthcare and lonza are confi dent that the fl ow through-column technology can be scaled up for peptides to a cost effi cient process. this is a strong argument that the potential of the Äkta platform as a peptide synthesiser should be explored. ge healthcare was approached by lonza to develop a peptide synthesiser based on the Äkta platform instrument to compete with the state of the art in the peptide fi eld. the aim is to develop the fl uidics, programming and chemistry methods of the Äkta system to lonza's needs for routine production of . g to g of crude peptide, and later scale up to kg. this synthesiser, controlled by the unicorntm software, has the capability to perform synthesis using on-line mixing or pre-activation at different times and temperatures of amino acids, coupling reagents, solvents and additives. results from peptides will be presented. neuropeptides are produced from precursor proteins by selective cleavage at specifi c sites. classical biosynthetic cleavage occurs at basic residues due to the activity of a small number of well-known proteases. however, with the discovery and characterization of new neuropeptides, a new non-classical pathway has been described with cleavage occurring at tryptophane, leucine and other residues. neuropeptide-processing peptidases involved in this new non-classical pathway are completely unknown but essential for correct processing of certain neuropeptides. therefore, we are interested in identifying proteases involved in the non-classical pathway using activity-based proteomics. for this purpose, we fi rst designed and synthesized some peptides based on the sequence the mature neuropeptides described in the literature but with an active functional group able to bind the active site of the proteases of interest. bound peptides were labelled with biotin or rhodamine by click chemistry, and the brain and pituitary proteome was then characterized by in-gel analysis and multidimensional nlc-ms/ms. several proteases that may be involved in neuropeptide processing have been identifi ed in this experiments. further studies concerning cloning, protein expression and activity assays will confi rm their role in biological tissues. the incidence of autoimmune diseases continues to increase. although an enormous research effort has been directed in understanding this increment, etiology of human autoimmunity remains enigmatic. primary biliary cirrhosis (pbc) is a chronic cholestatic liver disease characterized by destruction of bile ducts and the presence in the serum of antimitochondrial antibodies (ama positive in - % of patients), directed against the e , which is one of the three component enzymes of the -oxo acid dehydrogenase multienzyme complex family chiefl y pyruvate dehydrogenase complex (pdc-e ). environmentally induced co-or post-translational modifi cations of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc .. in this context it is possible to take advantage of a unique technology that allows the monitoring of peptide epitope modifi cations that will lead to the identifi cation of altered autoantigens that the human host will recognize as foreign, similarly to what recently reported in multiple sclerosis ( ) . our approach will lead to new diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. herein we report the synthesis of lipoamide and glyco-peptides characterized by a -hairpin structure with the modifi cation on the tip of the -turn as peptidomimetic of native antigens involved in pbc. in order to identify the best synthetic antigens and to clarify the role of the -turn in exposing the modifi cation we compared the antibody recognition in pbc sera by elisa using the modifi ed peptides in comparison with the proposed autoepitope of pdc-e [pdh ( - ) ]. the synthetic antigens were able to detect by elisa autoantibodies in % of ama negative sera of pbc patients confi rming that ptm-peptides are useful diagnostic/prognostic tools. alzheimer fs disease is characterized by the abnormal accumulation of amyloid peptide (a ) into extracellular fi brillar deposits known as amyloid plaques. a can self-assemble into soluble oligomers, protofi brils, and amyloid fi brils, and all of these aggregated forms contain signifi cant -sheet structure. the core of a ( - residues) containing hydrophobic region is a key to promoting a aggregation. in the previous study, we constructed green fl uorescent protein (gfp) variants which have the core part of a sequence on the surface -sheets. it has been demonstrated that the gfp variants inhibit a aggregation .. in this study, we have utilized a small protein, insulin-like growth factor ‡u receptor domain (igf r-d ) as a scaffold, and a part of a sequence was incorporated into the -sheet surface of igf r-d . igf r-kk and igf r-ka were designed by substituting two a derived sequences for some amino acids in igf r-d as parallel and anti-parallel -sheet models, respectively, of the a aggregates. these insoluble proteins expressed by e coli. were solubilized by denaturing buffer, and the denatured proteins were refolded in conditions permitting formation of native proteins. after refolding, the proteins were purifi ed as monomers by size exclusion chromatography. we have investigated the interaction between a and the designed protein variants by surface plasmon resonance studies. as a result, igf r-kk bound tightly to a more than igf r-ka. inhibitory activities of a fi brillization in the presence of igf r-kk, igf r-ka, or wild type igf r-d were evaluated by thiofl avin t (tht) binding assay. it was demonstrated that igf r-kk inhibited a fi brillization effectively. because of their high structural fl exibility. on the other hand, a stranded -structure is presumed to be more stable than a single strand as a fi bril forming intermediate. therefore, the aggregation mechanism of prion protein remains to be further studied on their precursor structures. recently, we have established a novel strategy to determine the amino acid sequence for amyloid formation, based on their essential interactions. a number of fi bril forming peptides at positions from to in the amino acid sequence were obtained by our calculation method. in order to confi rm whether aggregates of the candidate peptides consist of the -stranded -structure, a series of peptides consisting of g residues-turn- residues h were prepared. several candidate peptides, of which regions are - , - , - , and - , showed the typical enhanced fl uorescence intensities in the thiofl avin t-binding assay, suggesting the amyloid formation. ir spectra of these peptides showed the typical bands corresponding to the -structure. in addition, the peptide ( - ) exhibited a shoulder band at cm - in ir spectrum, refl ecting a turn structure. these results revealed that the amyloids of the peptide ( - ) are constructed with the -stranded -structure, which may be formed by intra-molecular hydrogen bonds. in conclusion, the results obtained here indicate that the sequence from to could be a key region for the transition from a normal to an abnormal structural state. single-molecule force spectroscopy provides a powerful tool to investigate biomolecular interactions. a different approach measures forces required for breaking a bond in a differential format by comparison with a known reference bond of dsdna ( ). here we apply this molecular force balance to the integrin v , which is over expressed in ovmz- cells. this protein interacts with ligands containing an -arg-gly-asp-(rgd) motive. ligands containing the rgd sequence were made by peptide synthesis and linked to a gcn -peptide using a peg-spacer. for detection carboxyfl uoresceine was introduced to a lysine side chain between both peptides. the gcn -peptide can be recognized by specifi c anti-body fragments ( ) and can act as a reference bond. these force balances was linked to a pdms-surface, whereas the reference bond is attached to the surface and the rgd-peptide is accessible to the integrin. when the pdms-stamp gets in contact with the ovmz-cells, the interaction of ligand and receptor can occur. by applying a force at the stamp the force balance is stretched and the weaker bond brakes. investigation of the fl uorescence-level on stamp and cells enables the localization of the balance construct and thus an estimation of the bond force. first stamp-experiments on living cells showed that the gcn anti body interactions are too strong to act as a reference system. since the binding forces of dna are well investigated by afm and small dna-molecules were already used as force sensor, dna can act as a more sensitive reference. thus in a further approach the rgd-peptide was linked to a biotinylated peg. this enables the application of short double-stranded dna as a reference bond via a fl uorescence-labelled streptavidin. using this system the force balance could be transferred to the v integrines located in the cell membrane of ovmz-cells. nanogaps, which allow making electrical contact to structures on the nanoscale, are increasingly used for the preparation of biosensors. the positioning of the synthetic or biological species inside the nanogap must be controlled to reach optimal electrical or detection properties. ( ) ( ) ( ) in this context, the chemical properties of the layer between the electrodes is of prime importance, since they will impose the imbibition of the nanogap and permit the formation of chemical bonds between the molecules of interest and the substrate. thin fi lms can be characterized by a variety of physical or chemical methods. however, the characterization of the chemical properties of nanogaps is complicated because of the small size of the substrate delimited by the electrodes. in this context, novel experimental tools are needed for probing rapidly the chemical reactivity of nanogaps. we show here for the fi rst time that a specifi c functional group in a - nm nanogap can be detected by combining peptidecapped gold nanoparticles and electrical detection.( ) a semicarbazide layer and semicarbazone chemoselective ligation was used in this proofof-concept study, which thus required the preparation of stable peptidecapped gold nanoparticles modifi ed by aldehyde groups and control gnps derivatized by amide groups. the chemoselective insertion of gold colloids into the nanogaps led to current increases from to orders of magnitude, in accord with the number of gold nanoparticles in the nanogaps detected by scanning electron microscopy. we present the electrical detection of immunoglobulin g (iggs) from human serum using a nanogap-based biosensor. the detection method is based on the capture of iggs by a probe immobilized between gold nanoelectrodes of to nm spacing. the captured iggs are further reacted with secondary antibodies labelled with gold nanoparticles (gnps). insertion of gnps into the nanogap resulted in increasing the conductance through the nanogap. the use of a chip with ninety nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specifi c and differential capture of human antibodies by arrayed probes. we obtained a -fold higher quality factor with protein a compared to goat anti-murine antibodies. this method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples. in the past several decades, hundreds of peptides have been identifi ed which have specifi c biological activity and are highly potent in in vitro assays but lack the prerequisite pharmacokinetics to become effi cacious human therapeutics. we have developed a novel antibody-based platform technology that provides an improved pharmacokinetic profi le for biologically active peptides, resulting in a long duration of action. one such mimetibody™, cnto , is a novel erythropoietin (epo) receptor agonist. although cnto bears no sequence homology to erythropoietin, it is a potent erythropoietin receptor agonist, rescuing epo dependent cells from apoptosis in vitro and stimulating erythropoiesis in vivo. studies were done in normal rats to explore the pharmacodynamics and pharmacokinetics of cnto in normal rats and to demonstrate its effi cacy in rat models of anemia. in vitro, cnto was approximately fold less potent than rhepo in stimulating the growth of ut- epo cells. despite this lower in vitro potency, when compared to rhepo and darbepoietin in normal rats, a single subcutaneous dose of cnto resulted in a longer-lived reticulocytosis and longer-lived increase in hemoglobin. also, cnto caused only minor changes in red cell distribution width (rdw) or mean cell volume (mcv) and led to the release of mature reticulocytes. we have also shown that cnto was effi cacious in rat models of anemia and in a rat model of pure red cell aplasia. taken together, our data show that cnto is a novel stimulant of erythropoiesis in rats. this platform has been applied to other biologically active peptides as well and has proven to be a robust platform for enhancing the pharmacokinetics of peptides that would otherwise be rapidly cleared. cell penetrating peptides (cpps) have been recognized as promising tools for the delivery of different therapeutic molecules. previous studies in our laboratory have shown that the s ( )-pv peptide accumulates inside cells very effi ciently through a rapid, dose-dependent and nontoxic process. formulations based on the s ( )-pv cell penetrating peptide presented great potential for the delivery of plasmid dna, which may prove useful for gene-based therapies. in the present work, we aim to ) investigate the relevance of the dermaseptin-derived sequence and of the nuclear localization signal to the effi ciency of the overall process of plasmid dna delivery by the s ( )-pv and related peptides; ) compare the potential of the s ( )-pv peptide to mediate plasmid dna delivery with that of the extensively studied tat cpp. a comparative analysis of the transfection effi ciency mediated by the systems based on the s ( )-pv, reverse nls and scrambled peptides was performed in tsa and hela cells. in general, for both cell lines, the reverse nls peptide mediated transfection at effi ciencies comparable to those observed for the s ( )-pv peptide. however, transfection mediated by the scrambled peptide was signifi cantly less effi cient than that obtained for the s ( )-pv and reverse nls peptides. to compare the biological activity of the s ( )-pv with that of the tat peptide, we transfected tsa cells with various s ( )-pv-and tatbased formulations. our results have shown that both peptides enhanced the activity of cationic liposome-based systems. above a threshold peptide/cationic liposome/pdna charge ratio ( / / ), the enhancing effect was independent of the peptide used, although for the lowest charge ratios, this effect seemed to be more relevant in the case of the s ( )-pv peptide. higashi, nobuyuki; kawamura, yoko; koga, tomoyuki doshisha university, japan the aim of tissue engineering is to replace failed organs with new functional tissue and organs. to realize this, materials are needed, which can direct the growth of cells to generate new tissue. to control and direct cell behavior, a defi ned biomimetic environments is needed, which surrounds the cells and promotes specifi c cell interactions. the rgds sequence has been recognized as the cell attachment site of the natural extracellular matrix. the purpose of this study is to fabricate rgdscarrying nanoscaffords towards cell adhesion using a self-assembling technique. here we synthsize two types of rgds-based materials, one of which is an amphiphilic triblock peptide composed of leu and lys (rgds-l k l ) that has been revealed to self-assemble into ƒÀ-sheet nanofi ber under specifi c conditions ., and another one is a rgdsended polystyrene (pst-rgds) that is a typical artifi cial polymer. these peptide and peptidomimetic were prepared by solid phase synthesis using fmoc-chemistry and by coupling fmoc-chemistry and atom-transfer radical polymerization (atrp) method, respectively. rgds-l k l was found to self-assemble into ƒÀ-sheet-based nanofi ber at ph . , and by lowering ph the conformational change from ƒÀ-sheet to random coil structure was induced, giving no aggregation of the peptide. when mouse nih/ t cells were seeded on the rgds-l k l nanofi bercoated plate, successful cell adhesion and spreading were observed, but not for the rgds-l k l random coil-coated plate, indicating the importance of the rgds sequences densely and regularly located at the nanofi ber surface. the utility of pst-rgds nanofi lms will be discussed, in comparison with that of nanofi bers. the role of deiminated protein antigens in the diagnosis of rheumatoid arthritis autoantibodies directed against citrulline-containing peptide (acpa) have high specifi city of rheumatoid arthritis (ra). citrullinated proteins are formed by posttranslational modifi cation, namely by deimination of arginine residues in protein sequences by peptidylarginine deiminase enzymes (padi). autoantibodies to deiminated (citrullinated) proteins are the most specifi c serological markers of rheumatoid arthritis. rheumatoid arthritis symptoms develop gradually, and it is diffi cult to precisely date the beginning of the disease. these antibodies are detectable already years before the fi rst clinical symptoms of the disease. the aim of our study was to identify the epitopes of vimentin and fi laggrin derived-peptides targeted by ra specifi c antibodies to provide further information about the nature of the initial autoantigenic substance. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" (chiron mimotopes peptide system) non-cleavable kit. identifi cation of epitope structure of antigenic proteins represents one of the major applications of these technologies. the peptides were prepared in duplicates. citrullinated peptides and the nonmodifi ed counterparts containing arginine instead of citrulline residues were synthesized in order to compare their respective reactivities. in the "indirect" elisa experiments the presence of acpa was determined using serum samples of ra patients and healthy blood donors. this series of experiments effi ciently identifi es citrullinated epitopes of fi laggrin and vimentin as a potential antigenic target for ra specifi c antibodies. the determination of epitopes of these proteins could be important for the development of appropriate diagnostics in this most frequent human systemic autoimmune disease. at the present time the human lysosomal enzymes cathepsins b and l, cysteine proteinases of c family, attract great attention. they not only take part in protein degradation but also appear to play role in other important physiological processes, for example, antigen presentation, caspase-independent cell death and involved in different diseases such as osteosarcoma, acute pancreatitis, tumor invasion and metastasis. but selective detection of these enzymes is diffi cult because determination of their enzymatic activity is carried out using substrates also correspond to specifi city of trypsin-like enzymes. the chemo-enzymatic synthesis of selective substrates of cysteine proteinases of c family was developed. these compounds are chromogenic glp-phe-ala-pna (i), glp-val-ala-pna (ii) and fl uorogenic substrates abz-phe-ala-pna (iii), glp-phe-ala-amc (iv). peptides were obtained in preparative quantities and were characterized by the data of amino acids analysis, hplc, mass-spectrometry, spectrophotometry (i and ii) and fl uorimetry (iii and iv). the specifi c activities of cathepsins b, l and similar enzymes of plants papain, bromelain and fi cain were determined using synthesized substrates and commercial available substrates z-phe-arg-pna, z-arg-arg-pna and bzl-arg-pna. the specifi c activities of enzymes of other classes -serine (chymotrypsin, trypsin and subtilisin), aspartic (pepsin) and metalloproteinases (thermolysin) -were also defi ned with these substrates. it was shown, that glp-phe-ala-pna, glp-val-ala-pna and glp-phe-ala-amc are not detectable cleaved by enzymes of other classes. acknowledgements: this work has been supported by rfbr grant ¹ - - à. to implement a new electrochemical biosensor for autoantibody detection in ms we used csf (glc) analogues, properly modifi ed at n-terminus with ferrocenyl and ferrocenyl-thiophosphine derivatives, as "electrochemical probes" in cyclic voltammetry ( ) . the electrochemical properties of ferrocene, coupled to thiophosphine ability to build simple monolayers on gold surfaces, allow peptides to be anchored on the working electrode used for detection. in particular, -fcphp(s)abu organometallic amino acid was specifi cally designed to be used directly in solid phase peptide synthesis. the organometallic moiety introduced in the new glycopeptides did not affect autoantibody recognition as demonstrated both in sp-elisa and in inhibition experiments. an electrochemical monitoring was able to detect interactions of the modifi ed glycopeptides with isolated antibodies from ms patients' sera. we demonstrated a detection sensitivity comparable to elisa method. therefore, the new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology. troponin is a structural protein complex, located on the thin fi lament of the contractile apparatus. it is composed of three protein subunits: troponin i ( kda), troponin c ( kda), and troponin t ( kda) and exists as isoforms specifi c to the cardiac and skeletal muscle cells, respectively. cardiac troponins are released in the peripheral blood during irreversible cardiac muscle damage in a time-specifi c manner. rapid troponin elisa assays based on the production of specifi c antibodies against the whole complex or individual subunits have been shown to possess suffi cient sensitivity and specifi city for use in the emergency departments. however, their usefulness sometimes is limited by various factors, such as the selection of epitopes for antibodies production, derived from cardiac troponins representing high homology against the skeletal isoforms, interfering blood factors etc. aiming to contribute in the fi eld of developing highly sensitive and specifi c reagents for the detection and isolation of cardiac troponins in the sera of patients with cardiovascular diseases, we selected epitopes derived from the cardiac isoforms for production of antibodies, mainly based on their predicted immunogenicity, the minimum homology against the skeletal isoforms and the lack of interferences of the produced antibodies with various blood factors. the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions and the resulted conjugates were used as immunogens for releasing anti-troponin specifi c antibodies. the performed elisa experiments revealed the high affi nity and specifi city of the produced anti-troponin antibodies against the native protein. a chemical reverse approach for autoimmune diseases diagnosis alcaro an increasing number of individuals throughout the world is affected by autoimmune diseases, a large and diverse group of disorders that are categorized by tissue injury or pathology. although the incidence and prevalence of individual autoimmune diseases are not high, the population burdens of the disease are large and underestimated. thus, reliable diagnostic/prognostic tools are necessary for an early diagnosis and for monitoring disease activity. in this scenario, we proposed a 'chemical reverse approach' based on the use of patients' sera to screen synthetic modifi ed peptides. we showed that this approach could lead to the effective identifi cation of specifi c probes able to characterize highly specifi c autoantibodies as disease biomarkers of highly relevant autoimmune diseases such as multiple sclerosis and rheumatoid arthritis [ , ] . toscana biomarkers is a r&d company involved in the application of the "chemical reverse approach" to different autoimmune conditions for the development of innovative diagnostic/prognostic tests. as a number of autoimmune diseases have been associated with posttranslational modifi cations, which alter the function and immunogenicity of protein/peptide antigens, we are synthesizing and screening focused peptide libraries based on post-translational modifi ed amino acid, conformational and minimal epitope diversity. lead compounds can be thus used as antigenic probes for specifi c recognition of autoantibodies as biomarkers of diseases in the set up of diagnostic/prognostic assays ( ). autoimmune diseases are considered now as a plague. in fact some autoimmune diseases previously considered rare are actually increasing their frequency because of an earlier diagnosis (e.g. celiac disease). possibly also an environmental factor (bacterial and/or viral infection) should contribute to autoimmune diseases. the idea we have been investigating for years and more recently proposed by others, is that aberrant post-translational modifi cations, i.e. glycosylation, deimination etc., create neoantigens triggering autoantibodies. this could explain why proteins (both recombinant and isolated), components of target organs or tissues, are failing. anyway, it is evident that one single biomarker will never enable to reach successful diagnostic & prognostic tools. on the contrary, synthetic peptides specifi cally modifi ed (with sugars, citrulline, lipoyl moieties, etc.) are interesting tools to fi shing out of patients' sera these autoantibodies. we have recently reported that this can be effi ciently done following a "chemical reverse approach" .. we successfully applied this strategy in the development of the fi rst multiple sclerosis antigenic probe [msap] : an n-glucosylated peptide characterised by a -hairpin structure exposing at the best the minimal epitope asn( -glc) involved in antibody recognition ( ) . a wider application of our sap is obtained by its citrullinatation and/or galactosylation ( ) useful for rheumatoid arthritis or lipoylation for investigating primary biliary cirrhosis. in addition to being able to show the viral infection from a serum sample within a few minutes, at the point of care, the poc assays are inexpensive, easy-to-perform and do not require special equipment or laboratory. as antigen in the poc assay we used a -amino acid peptide in four branches of a lysine core, with the kyvtgin sequence in the middle. the peptide antigen was conjugated to gold particles and absorbed to a fabric ribbon and dried. in the test, a serum sample is added together with buffer, and the solution dissolves the conjugate. a positive result is obtained when the antibodies together with the gold conjugate bind to anti-human-igg on the nitrocellulose, and form a specifi c coloured line which can be detected in the test window. serum samples were collected from patients with acute b infection, and control samples were drawn many years after infection; additional control sera came from subjects devoid of b antibodies. the assay was shown to be stable in accelerated stability study. the conditions for industrial scale manufacturing were evaluated and the sensitivity and specifi city were addressed, whereby the assay proved to be highly specifi c for acute b infection. alzheimer's disease (ad) is a chronic neurodegenerative disease characterized by a progressive loss of memory and cognitive decline, for which the aggregation and plaque-formation by the -amyoild (a ) polypeptide has been identifi ed as a key event. recently, unpaired variable domains of llama single chain antibody (vhh) fragments against a have been found to exert considerable therapeutic potential for ad. vhh represents the smallest antigen-binding unit with a molecular size of ~ kda, compared to ig-heavy and light chain variable domains, fab fragments and complete igg antibodies. human cystatin c (hcc) is a cystein protease inhibitor present in all human body fl uids which has a propensity to co-associate with a -plaques/fi brils, and has plaque-inhibitory properties. using proteolytic extraction and excision of the llama-vhh-a ( - ) immune complex (e.g., trypsin, glu-c protease) in combination with electrospray ionization (esi)-and maldi-mass spectrometry, the a -binding epitope was identifi ed at the middle-carboxyterminal domain of a , a ( - ). an analogous mass spectrometric approach was employed for the identifi cation of the binding epitopes of the hcc-a -complex, using immobilized hcc and a -affi nity matrices. an almost identical minimal epitope to that of the vhh-anti-a -antibody ( a ( - ) ) was found, which binds to a specifi c c-terminal domain of hcc, hcc( - ) .. the identifi ed hcc epitope peptide was found to specifi cally inhibit a -oligomerization in vitro, in agreement with the a -epitope domain interefering with the a -aggregation. the identifi ed a and hcc epitopes represent new lead structures for designing neuroprotective inhibitors of the a -aggregation process, and for molecular ad diagnostics. using peptide arrays to reveal mechanisms of apoptosis aspp is a pro-apoptotic protein that stimulates the p -mediated apoptotic response. the c-terminus of aspp contains ankyrin repeats and an sh domain (aspp ank-sh ), which mediate its interactions with apoptosis-related proteins such as p and bcl- . we have used a combination of membrane-bound peptide arrays and biophysical methods to study the protein-protein interactions of aspp at the molecular level in order to reveal their possible role in apoptosis. using peptide arrays, we have mapped the binding interfaces of aspp with its partner proteins such as bcl- , nf-b and other proteins that are involved in apoptosis. we then applied the peptide array results to study profoundly the interactions of aspp with proteins from the anti-apoptotic bcl- family. we found that aspp ank-sh binds to bcl- , bcl-xl and bcl-w at two homologous sites in all three bcl proteins tested: (i) the conserved bh motif (ii) a binding site for pro-apoptotic regulators. quantitative biophysical analysis of the interaction with the free peptides revealed that the binding was selective, and the bh domain of bcl- binds tightest to aspp . we propose a mechanism in which aspp induces apoptosis by inhibiting functional sites of the anti apoptotic bcl- proteins. the array screening results also served as a basis for docking studies that resulted in binding model for the complex between the full length protein bcl- and aspp ank-sh . we conclude that the use of combinatorial methods such as peptide arrays, combined with quantitative biophysical techniques, can signifi cantly contribute to better understanding of biological pathways. micron-sized monodispersed polystyrene for synthesis, tagging and biological screening of small molecules peptide arrays are useful tools to characterize antibodies, to determine sequence specifi cities of enzymes (e.g. kinases) or to fi nd interaction partners to given peptide sequences. one popular format for such arrays is a cellulose sheet with hundreds of synthetic peptides bound to it. these spot-arrays have been used successfully in a broad range of applications since their invention years ago .. a drawback is the use of large reagent volumes and the limited throughput with only one copy of the library. celluspots™ represent a new method [ , ] that allows the production of hundreds identical peptide arrays from a single synthesis run on individual membrane disks. the peptides are synthesized on a modifi ed cellulose support which is dissolved in a cleavage-mixture after the synthesis. resulting solutions of peptide-cellulose-conjugates are then spotted onto coated microscope slides by conventional spotting techniques. the identical arrays are useful tools for large, parallel sera screening projects. there is a great importance in biological macromolecules integration within electronic devices. this is since these molecules are expected to be suitable both as the active components of electronic devices, or as guides for bottom-up assembly of hybrid structures. the goal of this research is to study the assembly and electronic properties of novel devices that exploit synthetic protein molecules. here we show the fabrication of de-novo protein based diode-like devices. we have chosen coiled coil protein structures, which can adopt two conformational states that differ in their internal molecular dipole. the proteins have been equipped with surface binding groups, typically cysteine thiols, on both ends. dithiol bridges at one side facilitated self assembly on gold surfaces. protected cys residues were placed on the other end that after deprotection will allow successive binding processes in order to complete the device assembly process. characterization of the correct folding in solution has been achieved by hplc and cd. the dependence of the dipole in the dimeric protein has been shown using large scale kelvin probe and high resolution kelvin force microscopy (kfm) measurements. we believe that this work will contribute to the understanding of electronic processes in proteins and may serve as foundation for their exploitation in device confi gurations by making use of the ability to trigger conformational changes in order to control device activity. synthesis of new tetracyclic rgd peptides for specifi c tumor cell targeting. the integrin family of adhesion molecules participate in important cell-cell and cell-extracellular matrix interactions in a diverse range of biological processes. the avb integrin is overexpressed in several types of cancer cells and play an important role in angiogenesis as well as in tumour cell migration by interacting with vitronectin on the extracellular matrix mainly through the recognition of the tripeptide sequence rgd. the search for highly selective ligands to target the endothelial cell integrin avb is currently the focus of many research groups as they may represent new therapeutics or valuable diagnosis in a number of areas such as metastasis, angiogenesis, arthritis and retinopathy. to date, the cyclic (-rgdfx) peptides and related n-methylated analogues developed by kessler's group are among the most active and selective compounds for the avb integrin receptors. for instance, the use of c(-rgdf(nme)v-) is actually evaluated in several clinical trials. therefore, we designed a new class of cyclic tetrapeptides. rgdk peptides were fi rst synthesized by solid phase synthesis then cyclized in solution through a urea bond using n,n'-carbonyldiimidazole. using skmel and a cells, expressing and non-expressing avb respectively, we demonstrate that one of our peptide showed a better internalization than the reference peptide crgdfe. our strategy allowed the coupling of the best cyclic recognition peptide through its extracyclic acidic group to any molecular moieties containing an amino group that makes him a great cadidate for easy multimerization. along this line, different applications are currently developped in our group. design in this study, we described a simple method for aminocylation of unprotected saccharides with mildly activated amino acids esters as potential ace inhibitory activity. as a model reaction, we investigated esterifi cation of sucrose -" royal carbohydrate" with cyanomethyl ester of benzyloxycarbonyl-phenylalanine.the difference of reactivity between the eight hydroxyl groups is a key factor in chemical exploration. in polar solvent as dmf or dmso the primary positions might react faster than secondary ones if the reaction is essentially sensitive to steric interactions. on the other hand, the reaction might be more sensitive to the activity of the alcohol functions. in this respects, g-oh has been shown to be the most reactive among eight hydroxyl groups of sucrose. after removal of benzyloxycarbonyl group, the products possess groups which can accommolated in the hydrophobic s and s subsites of angiotensin i converting enzyme. the free amino group in the amino acid-carbohydrate esters can also serve as good ligands for zn + in the ace active site. carbohydrates possess both hydrophobic and hydrophilic groups in their structure and could also bind with enzyme subsites. the measurements of ic value of newly amino acyl esters of carbohydrates are in progress. synthetic polyelectrolytes (pe) have been widely used to modify proteins via complex formation and covalent attachment, increasing (or reducing) the immunoreactivity and/or immunogenecity of originally antigenic proteins and improving their in vivo stability with prolonged clearance times. such conjugates seem to be great importance for medicine and immunobiotechnology in particular with respect to drug delivery and vaccine innovation. synthetic peptides are promising candidate vaccines for the control of viral diseases. previous studies with foot-and-mouth disease virus (fmdv) have identifi ed fragments of isolated vp protein and synthetic peptides from vpi which stimulate antibody production, albeit of poor neutralizing activity, or are recognized by antivirus antibodies. fmdv is an attractive model with which to study the potential of peptide-based synthetic vaccines. in this study, we sought to evaluate the immunogenecity of a candidate containing - synthetic peptide epitops of vp capsid protein of fmdv. the immunogenic properties of the conjugates were also investigated and the relationship between immunogeneticity and structure formation in the solutions is analyzed. a new high immunogenic protein-polymer complex and conjugates with antibody production, processing relatively prolonged times was obtained. it was obtained that a single immunization of mice with pepeptide bioconjugates without classical adjuvant increased the primary and secondary peptide-specifi c immune response to fmdv. polyacrylic acid (paa) is a well known bioactive polymer (bioadhesive nano-and microparticles, ph-sensitive paa grafted poly(vinylidene fl uoride) membrane bags, ultrafi ne cellulose fi ber surfaces grafted with paa for enzyme immobilization, paa-polyvinylpyrrolidone biopolymeric systems for the treatment of the dry eye, etc. paa, their alkyl-esters and non-toxic copolymers with vinyl pyrrolidones are strong adjuvants for primary and secondary responses and that they are promising alternatives to the mineral oil-based adjuvants presently used in various veterinary vaccines. in this study, the interaction between peptide epitops of vp protein of foot-and mouth disease (fmdv) and polyacrylic acid (paa) will be discussed on the basis of the experimental results obtained by the size-exclusion chromatography (sec) with online quadruple detection system: uv absorption (uv), refractive index (ri), right angle light scattering (ls) and viscosity (vis) detectors and the binding coeffi cient, i.e., the binding ratio of peptide to polyacrylic acid. human igg-specifi c binding peptides to distinguish normal and abnormal conformers: applications for igg purifi cation and detection in recent years, human immunoglobulin g (igg) attracts attention as protein of the main format of the antibody medicine. in the purifi cation of igg, protein a originated from bacteria is frequently used as a affi nity ligand, but several problems such as the contamination of endotoxin and the deterioration of protein a by the repeated use were pointed-out in the use of protein a. on this account, the development of low molecular ligands and mimic peptides which can be used instead of protein a has been performed. we have searched for the peptides which specifi cally bind to human igg using a t random peptide phage library and, as a result, succeeded in isolation of two kinds of peptides called type i and type ii. type i peptide recognize normal structure of human igg and can be used for the purifi cation and the detection of igg. however, it not only binds to human igg but also to mouse and rabbit igg with comparatively high affi nity. on the other hand, type ii peptide is extremely specifi c only for human igg. a more important thing is that type ii peptide does not bind to igg in serum, but get possible to bind to igg antibody purifi ed by a protein a column. from this result, we elucidated the abnormal structure of igg is generated by acid condition used in the elution from a protein a column and type ii peptide recognizes this specifi c structure. in this presentation, we report the results that the type i peptides functioning as an affi nity ligand instead of protein a can be applied for the purifi cation system of the antibody by improvement by amino acid substitutions and chemical conversion. furthermore, we also report the generation condition of an abnormal conformer of igg structure by ph and the temperature and the effective removal of the generated specifi c conformer by the type ii peptideimmobilized column. acknowledgements: this research was partially supported by a grant of practical application research from japan science and technology agency. the isolation and detection of low abundant enzymes and proteins is one of the most challenging tasks in bioanalytical and pharmacological fi elds, especially if information about their state of activity is required. for this purpose tailored solutions for addressing the members of a protein family are required. peptide chemistry provides established methods to assemble building blocks to construct such molecular probes. we chose matrix metalloproteinases (mmp) for validation of such an approach. this protein family processes and degrades various extracellular matrix proteins and possesses a highly conserved catalytic site with a zinc ion in its centre. most of the known and potent inhibitors are peptidomimetics containing zinc chelating hydroxamate groups (e. g. marimastat). although it binds reversibly, it is potent and active against a wide range of metalloproteinases. it was chosen as synthetically available binding group to target the protein in its active state. it was modifi ed by peptide chemistry in order to introduce multiple functions. depending on the purpose, different reporter groups, photoreactive or cleavage sites were chosen. the probes were tested positively to inhibit various human recombinant mmps using activity assays. mmp were isolated using streptavidin coated magnetic beads and a biotinylated probe. furthermore a photoreactive group was introduced to enhance the binding to the target protein. a covalent interaction was achieved by irradiation of a probe protein mixture, isolation with magnetic beads and cleavage from the beads. pyclock superior coupling reagent for biosensors construction based on peptides coupling onto solid phase polymer atias, danit; abu-rabeah, khalil; marks, robert s. electrochemically biosensors are valuable analytical tools for the monitoring of various biological/ chemical molecule levels, and tremendous research effort has been put into the development of such accurate analytical devices. one crucial aspect in the fabrication of a biosensor is the deposition of biological macromolecules in high amounts with retention of their specifi c activity. among the various deposition methods of biomolecules, such as direct adsorption onto the surface, cross-linking, covalent attachment by carbodiimide chemistry is one of the few methods that allows a stable deposition into a biocompatible environment. hence the urgent need for proteins coupling with high yield to solid surface in the aim of biosensors construction. however the use of carbodiimide as peptide coupling agents was found to be limited in various aspects. thus, during the activation of hindered carboxylic components, such as those involved in bulky protein coupling reactions to solid phase fi bers pyclock was found to be very effi cient for slow coupling reactions and it can be used in excess to assure a complete activation of the carboxylic function. in this study -chloro-benzotriazole- -yl-oxy-tris-pyrrolidino-phosphonium hexafl uorophosphate coupling reagent (pyclock) was shown to have better performance than -ethyl-[ -(dimethyl¬amino)propyl]- ethylcarbodiimide (edac) and hydroxysulfosuccinimide (nhss) in peptides large sequence coupling to solid phase fi bers. cantel, sonia; valmalle, charlene; subra, gilles; enjalbal, christine; martinez, jean general approaches used in protein identifi cation and characterization involve consecutive purifi cation steps. then the desired protein extract is submitted to mass spectrometry analysis (lc-ms/ms) after enzymatic digestion. technical diffi culties are involved in determining the pmf of a protein particularly in relative low abundance. a typical protein will give rise to at least twenty to thirty peptides after trypsin digestion. not all of these peptides will appear in maldi analysis. one factor that is believed to cause incomplete detection is competition for protonation during the ionisation process inducing ion discrimination. we have recently developed a new technology allowing specifi c labeling of lysine residues in proteins and easy maldi-ms detection and identifi cation of labeled peptides following protein hydrolysis ( ) . nhydroxysuccinimide ester of -cyano- -hydroxycinnamic acid (chca) was used as a labeling reagent to increase maldi signal of lysinecontaining peptides in cytochrome c proteolytic mixture. this original approach enables to discriminate labeled peptides of interest among other abundant peptides. herein, we report the optimization process to investigate the limits of this tool. a defi ned receptor-binding domain (rbd) on the viral spike protein (s) mediates the attachment of sars-coronavirus to its cellular receptor, angiotensin-converting enzyme (ace ). we have synthesized peptide libraries for identifi cation of rbd binding epitopes by surface plasmon resonance (spr) and saturation transfer difference (std) nmr spectroscopy. three dodecapeptides were identifi ed to have signifi cant affi nity to ace ; the best of them had a kd = m . further refi nement yielded a hexapeptide from y to l (ykyryl) of s that bound ace at kd= m. std nmr spectroscopy reveals close contacts of the aromatic tyrosine residues to the receptor. the peptide was also analyzed for antiviral activity using an in vitro assay that measures sars-cov infection of vero cells. at a concentration of mm virus replication was reduced about fold. no replication occurred at peptide concentrations above mm. the peptide blocks the binding site on ace that is necessary for the virus to infect the cells. it can be used to design peptidomimetic compounds as entry inhibitors for sars-cov. to exclude other effects that were due to unspecifi c inhibition of viral replication, the peptide was tested during an alpha virus infection of vero cells. no signifi cant inhibition was observed. however, for the human corona virus nl that causes severe colds in humans and that uses the same receptor a clear inhibition could be observed comparable to the results obtained for sars-cov. additionally, we have synthesized a hexapeptide library with amino acid substitutions of the chemical lead ykyryl and measured their binding affi nity to ace via spr to analyze the importance of the individual amino acids. spr studies indicate an important role of r . prostate cancer is one of the most common malignancies in men and is responsible for more deaths than any other cancer, except for lung cancer. in the last decade we have developed bradykinin antagonist peptides (b , b ), peptide dimer (b , suim-(d-arg-arg-pro-hyp-gly-igl-ser-d-igl-oic-arg) , suim: suberimidyl; hyp: trans- -hydroxyproline; igl: -( -indanyl)glycine; oic: s, as, asoctahydro- h-indole- -carboxylic acid) and four generations of our small molecule, bkm- which showed high inhibition against lung cancer in vitro and in vivo. some of the compounds also showed inhibition against prostate cancer. it is well known that prostate cancer metastasizes into bones. while prostate cancer itself has many treatment options, no drugs currently exist for the treatment of bone metastatic prostate cancer. we modifi ed our potent anti-cancer small molecules by incorporating an aminobisphosphonate group to target these compounds to bone. bkm- , f c-oc y-atmp (f c: , , , , pentafl uorocinnamoyl; oc y: (o- , -dichlorobenzyl)-tyrosine; atmp: -amino- , , , -tetramethylpiperidine) is the fi rst generation of our small molecules. this compound consists of three parts: a, an acyl group; b, a tyrosine amino acid residue and c, an amide group. we introduced aminobisphosphonate or aminobisphosphonate derivatives at position c, and the new n-(bis-phosphonatoalkyl)amide small molecules had anti-cancer activity against prostate cancer metastases in mice. one of these compounds has been shown to be effective against the growth of pre-established human prostate tumors in mouse skeleton through the induction of apoptosis and blockade of survivin expression. the synthesis of these aminobisphosphonate small molecules, the structure relationship studies and the biological activity of these compounds in cultured human prostate cancer cells and animal models will be presented. hayouka novel, potent and selective angiotensin iv short analogues the hexapeptide angiotensin iv: h-val-tyr-ile-his-pro-phe-oh (ang iv) mediates a wide range of physiological actions, including control of blood fl ow and cognitive enhancement. it exerts its effects by binding to at receptors, which are widely distributed across tissues. the at receptor has been identifi ed as the insulin-regulated aminopeptidase or irap. it has been proposed that ang iv exerts its action by inhibiting the catalytic activity of this enzyme. we have reported that the -homo amino acid containing analog h- hval-tyr-ile-his-pro- phe-oh (al- ) is a potent, selective and stable ang iv antagonist, in which the hval is responsible for stability and the hphe for selectivity. in this study we report the new ang iv short analogues. the incorporation of erythro-mephe or tic resulted in analogues which have great ability to inhibit the hydrolysis of leu-p-nitroanilide by irap or by ap-n. our data showed that the full ang iv sequence is not necessary for high potency. peptides with pro deletion containing tic or erythro-mephe were even more potent than full ang iv sequence. moreover a peptide design and synthesis of a non -peptide par thrombin receptor antagonist, using cyclohexane as template receptors that mediate thrombin action are attractive drug discovery targets because of their involvement in cardiovascular pathophysiology (dysregulation of platelet aggregation and endothelial cell function). the cellular actions of thrombin are, in large part, caused by the activation of proteinase-activated receptors (pars) , and (pharm. rev. : ). the serine proteinase thrombin cleaves and activates cellular par in many pathophysiological settings associated with hemostasis, tissue injury and the proliferation of vascular smooth muscle and tumor cells. in the present study, we synthesized a novel non-peptide par mimetic, based on a conformational analysis of the s fllr tethered ligand sequence of par in order to inhibit the cellular actions of thrombin. the rational design, based on nmr constraints and molecular dymamics, led to compound (fig. ) containing the spatially closed key pharmacophoric guanidyl and phenyl groups, attached to cyclohexane as a template. compound , inhibited both tfllr-amide ( m) and thrombin ( . and u/ml)-mediated calcium signaling in a cultured human hek cell assay (j pharm. exp ther. : ). aboye, teshome leta ; clark, richard j ; craik, david j. ; göransson, ulf biomedical centre, sweden; institute for molecular bioscience, australia the cyclic cystine knot motif, as defi ned by the cyclotide peptide family, is an attractive scaffold for protein engineering ( ). however, to date the utilization of this scaffold has been limited by the inability to synthesize members of the most diverse and biologically active subfamily, the bracelet cyclotides. here we describe the synthesis and fi rst direct oxidative folding of a bracelet cyclotide, cycloviolacin o , and thus provide an effi cient method of exploring the most potent cyclic cystine knot peptides ( ) . the linear chain of cycloviolacin o was assembled using fmoc solid phase peptide synthesis and cyclized by thioester-mediated native chemical ligation, and the inherent diffi culties of folding bracelet cyclotides were successfully overcome in a single step reaction. the folding pathway was characterized and included predominating fully oxidized intermediates that slowly converted to the native peptide structure. angiogenesis, the process of new blood vessel formation, is important in both physiologic and pathologic situations, and its inhibition can be useful in the fi ght against several diseases, such as tumor development, diabetic retinopathy, etc. one of the key factors in promoting angiogenesis is the vascular endothelial growth factor (vegf), which exerts its biological activity through interaction with specifi c receptors, vegfr- (flt- ), vegfr- (kdr, flk- ) and vegfr- (flt- ) . vegf is over-expressed in all examples of pathologic angiogenesis, and its effects on tumor growth are mainly mediated by kdr. our approach to novel anti-angiogenic drugs is centered on the search for inhibitors of vegf-kdr interaction. directed mutagenesis studies allowed the identifi cation of a surface of vegf recognized by kdr, with residues arg , ile , lys , his and glu , located in a -hairpin of loop , identifi ed as essential for the interaction. most residues in this region are also located in the interface of interaction between vegf and some none-humanized phage-displayed antibodies with potent antiangiogenic activity, suggesting a binding to vegf similar to that of vegf receptors. starting from vegf - fragment (mrikphqgqhi), we have designed different hydrocarbon-bridged analogues to preserve the -hairpin native structure. this communication will describe the solid-phase synthesis of olefi n-bridged (c=c) peptides and their saturated (c-c) analogues. considering that the -turn of native vegf - is slightly distorted, due to the presence of an extra amino acid residue, in addition to the bridged undecapeptides, two series of decapeptide analogues have also been prepared, just by removing the residues glu or gly . the conformational behavior of linear and bridged-peptides has been analyzed by nmr (noe, h and c chemical shifts). the ability of these peptides to adopt the native vegf -hairpin structure will be compared with their anti-angiogenic activities. integrins constitute a family of heterodimeric, transmembrane cell adhesion receptors which connect cells to the scaffolding proteins of the extracellular matrix. the pioneering observation that integrins -especially v and -are hallmarks of metastatic cancer and seriously involved in the process of tumor angiogenesis turned them into attractive targets for cancer therapy. out of that, the inhibition of integrin function is a major challenge in medicinal chemistry. potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (amd). especially the subtype has recently been drawn into the focus of research due to its genuine role in angiogenesis.( ) in here, we describe the rational design and the synthesis of high affi nity binders and the optimization of their activity and selectivity against v by means of extensive sar-studies and docking experiments.( ) starting from a tyrosine scaffold( ) we succeeded in getting compounds with affi nities in the low and even sub-nanomolar range and selectivities of fold against v . the insights about the structure-activity-relationship gained from the tyrosine based ligands could then be successfully transferred to ligands bearing an aza-glycine( ) scaffold to yield ligands with affi nities of even sub-nanomolar range and selektivites exceeding . fold. modeling studies and biological activities of a nonpeptide at receptor angiotensin ii antagonist the octapeptide angiotensin ii (h-asp-arg-val-tyr-ile-his-pro-phe-oh) is the major factor of the renin-angiotensin system (ras) and plays a signifi cant role in the regulation of arterial blood pressure. in the present study, we have modeled a losartan analogue, the non-peptide angiotensin ii at antagonist, -butyl- -hydroxymethyl- -{[ '-( htetrazol- -yl)biphenyl- -yl]methyl} imidazole (v ). structure activity relationship (sar) and molecular modeling studies indicate close proximity of hydroxymethyl and tetrazole pharmacophoric groups of antagonist v and losartan. conformational analysis was performed using a grid scan search in order to derive all the possible conformations from which six energy local minima (syn and anti) were extracted after a cluster analysis. furthermore, these different conformations were superimposed with losartan, where a spatial correlation among the pharmacophoric groups is observed. antagonist v showed similar potency with losartan in our in vivo model with anesthetized rabbits and in vitro binding studies to at receptor. at specifi c concentrations compound v showed higher affi nity compared to losartan indicating that reorientation of butyl and hydroxymethyl groups on imidazole template allows a better binding to at receptor. h-benzotriazolium -[bis(dimethylamino)methylene]- -chloro-,hexafl uorophosphate ( -), -oxide (hctu) is a non-toxic, non-irritating and non-corrosive coupling reagent [ ] [ ] . seven biologically active peptides (ghrp- , - acp, oxytocin, g-lhrh, c-peptide, hamylin - , and -amyloid - ) were synthesized with reaction times reduced to deprotection times of minutes or less and coupling times of minutes or less using hctu as the coupling reagent. no expensive coupling reagents or special techniques were used. total peptide synthesis times were dramatically reduced as much as . hr ( . days) without reducing the crude peptide purities. it was shown that hctu can be used as an affordable, effi cient coupling reagent for fast fmoc solidphase peptide synthesis. human sdf- contains sixty-eight amino acids and is a member of the chemokine family of peptides. this long peptide was synthesized step-wise using our quality control conditions in hours. the reaction times were then reduced to deprotection times of x min and coupling times of x . min, resulting in a total synthesis time of hours. the effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities were compared. a small portion of crude peptide was purifi ed using an rp-hplc column and the mass of the fi nal product was confi rmed with maldi-tof mass spectrometry. references: . steven l. kunkel and nuria godessart, chemokines in autoimmunity: from pathology to therapeutics, autoimmunity reviews, , - ( ). pedersen, søren l.; sørensen, kasper k.; jensen, knud j. despite the development of new coupling reagents and solid supports, spps is still often faced with diffi culties in the assembly of long or "diffi cult" sequences, e.g. due to aggregation and steric hindrance giving rise to incomplete reactions. the use of convenient and precise heating with microwaves for spps has gained in popularity as it for many syntheses has provided signifi cant improvement in terms of speed, purity, and yields, maybe especially in the synthesis of long and "diffi cult" peptides. thus, precise microwave heating has emerged as one new parameter for spps, in addition to coupling reagents, resins, solvents etc. we have previously reported on microwave heating to promote a range of solid-phase reactions in spps. here we present a new, fl exible semi-automated instrument for the application of precise microwave heating in solid-phase synthesis. it combines a slightly modifi ed biotage initiator microwave instrument, which is available in many laboratories, with a modifi ed semi-automated peptide synthesizer from multisyntech. a custom-made reaction vessel is placed permanently in the microwave oven, thus the reactor does not have to be moved between steps. mixing is achieved by nitrogen bubling. washing steps are automated, however the activated amino acid derivatives have to be added manually. first, we developed optimized protocols for short cycle times in semi-automated spps with general fmoc chemistry. we utilized a microwave-compatible temperature probe for exact temperature measurements during microwave heating. then we developed protocols for on-resin reductive amination for anchoring of the fi rst amino acids to a bal handle. finally, we used the new instrument and the optimized protocols to assist in the synthesis of a range of diffi cult and long sequences. we believe that these successful syntheses demonstrate that this semi-automated instrument and the methods developed for it, can be an effi cient starting point for spps with microwave heating. investigation of polyelectrolyte-antigenic peptide conjugates by fl uorescence spectroscopy in proteins or peptides, it is possible to localize the interaction between protein and pe at certain protein domains. we investigate covalent binding mechanism of synthetic peptides which include tryptophan residue in the peptide sequence with copolymers of acrylic acid and n-vinylpyrolidone (vp\aa) depending upon the weight concentration ratio of components by fl uorescence method. antigenic peptides are synthesized by microwave assisted solid phase peptide synthesis method. characterization of these peptides are performed with lc-ms. subsequently these crude peptides are purifi ed by preparative hplc system. peptide-polymer covalent conjugation are performed in organic and pbs media. covalent conjugation are carried out by carbodiimide method. carbodiimide is used for the activation of carbonile groups of synthetic polymer which is the binding area of peptides that includes free amino groups. from the analysis of peptide-pe conjugates, it is possible to discuss the mechanism of the conjugate formation and structure of forming particles. according to the fl uorescence analysis results, free peptide which containing trp residue, gives fl uorescence spectrum. however, after the conjugation reaction it is observed that products max decreased and it is characterized as blue shift of max. this indicates that when conjugates formed, trp is completely isolated from aqueous solution. antiangiogenic thrombospondin type repeat analogs: synthesis, structure, and biological activity the thrombospondin type repeat (tsr) has been shown to inhibit angiogenesis and tumor growth in a number of in vivo models of cancer, but the details of this activity, including structure-function relationships, are not well understood. we explore these tsr elements in further detail via structural and biological evaluation of a series of tsr analogs. the tsr domain has a characteristic fold consisting of a two-stranded -sheet and a third 'rippled' strand. three tryptophans on the 'rippled' strand form cation-stacking interactions with two arginines on the adjacent sheet, forming an extended cation-network. in addition its structural importance, it has been postulated that the surface formed by this interaction is a key site for protein-protein recognition. a synthetic approach to the generation of novel tsr analogs, utilizing spps and native chemical ligation, has allowed us to rationally introduce unnatural amino acids in an effort to probe the structural and functional landscape of this clinically relevant protein domain. we have synthesized analogs of the second tsr domain of human thrombospondin- (tsr ) designed to probe the importance of the cation-stack. the arginine residues have been replaced by ornithine and citrulline, and thermal denaturation experiments by circular dichroism have been used to estimate stability differences between the mutant tsr and the native. taken as a set, these thermodynamic measurements provide insight into the stability afforded by a cationinteraction in the context of a native protein fold. synthetic access to the tsr domain also provides the opportunity for the introduction of various modifi cations, including the introduction of aminooxy groups as chemical handles, pegylation for in vivo stability, and biotinylation to create an affi nity caputure reagent for identifi cation of protein-protein interaction partners. the tetradecapeptide bombesin (bbs) has a high affi nity for the gastrin-releasing peptide (grp) receptor. these receptors are overexpressed in human tumors such as breast and prostate cancers. therefore they can serve as targets for in vivo imaging and therapy of these tumors with m tc-and re-radiolabeled bbs analogues, respectively. for the radiolabeling, a chelator is attached to the n-terminus of the bbs analogues. our group developed the (n his)ac chelator, which is easy to synthesize and has a very high affi nity for the m tc-and retricarbonyl complexes. however, an important part of the injected radiolabeled bbs analogues accumulated in healthy organs, such as liver and kidneys. to improve the pharmacokinetic properties, a bbs analogue was glycated via the lys side chain of the spacer using the maillard reaction. unfortunately, during glycation, overalkylation on the secundary amine of the chelator was observed. therefore, two alternatives for this glycation were examined. the fi rst one was based on the chemoselective reaction between a hydroxylamine (incorporated into the spacer) and an aldehyde (glucose). the second alternative was the cu(i) catalyzed cycloaddition between an alkyne (incorporated into the spacer) and an azide (azido-glucose). these approaches for carbohydration circumvented the problem encountered during glycation via the maillard reaction. glycation by the cu(i) catalyzed cycloaddition was, by far, the easiest way to incorporate the glucose moiety. moreover, after m tc labeling, this analogue showed the best biodistribution and the best diagnostic properties of all glycated analogues. an alternate approach to improve the pharmacokinetics consisted of including different polar spacers such as ³hglu, ³hasp, ³hlys, ³hser and -nh(ch ch o) co-between the (n his)ac chelator and the bombesin sequence. in particular the analogues with a negative charge ( ³hglu, ³hasp) in the spacer showed a favorable effect on the pharmacokinetics. determination of binding ratio of hydrofobic peptidepolymer conjugates by using fl uorescamine assay budama battal, yasemin; derman, serap; mansuroðlu, banu; mustafaeva, zeynep yildiz technical university, bioengineering department, turkey non-fl uorescent -phenylspiro-[furan- ( h), -phthalan]- , '-dione (fl uorescamine) reacts readily with primary amines in amino acids, peptides and proteins to form stable, highly fl uorescent compounds (fl uorophors). fluorescamine has been used to detect free amino groups on peptides or completion of coupling reactions in solid phase peptide synthesis and not only in aqueous solution but also in organic solvents and on solids. in this study, peptide epitops of vp capsid protein of foot-and-mouth-diseases virus - amino acid residues (val-lys-ile-asn-asn-thr-ser-pro-the-his-val-ile-asp-leu-met-gln-thr-his-gln-his-gly) were synthesized by sigma. we synthesized the covalent conjugate of - amino acid residues with copolymers of acrylic acids and nvinylpyrolidone (vp\aa) at different ratio of components (npeptide/ npolymer) and investigated the mechanism of condensation reaction by using different physicochemical analyses as hplc, fluorescence spectroscopy and fluorescamine assay. for determination of binding ratio of hidrofobic peptide-polymer congutages, fl uorescent measurements were performed in the presence of fl uorescamine at the wavelengts of excitation nm and emission nm. when conjugation reaction completed, the amount of free amino groups had decreased and observed that fluorescence intensity had decreased and the estimated degree of primary amino group after conjugation, calculated from fl uorescence spectrums. ( -phenylspiro-[furan- ( h), -phthalan]- , '-dione) which heterocyclic dione is a reagent for the detection of primary amines on peptides or completion of coupling reactions in the picomole range. its reaction with amines is almost instantaneous at room temperature in aqueous media, organic solvents ect . fluorescamine reacts with primary amines (in peptide, protein ect.) to form highly fl uorescent product (fl uorophors) whereas the reagent and its degradation products are nonfl uorescent ( ). this is the basis of a fl uorescent protein assay [ , ] . fluorescamine is used in many sensitive detection methods, e,g., characterization of poly-l-lysine (pll)/dna complexes post-modifi ed with a multivalent hydrophilic polymer ( ), or synyhetic peptide-polymer conjugates. in this study, foot-and-mouth-diseases virus vp capsid protein's synthetic peptide epitope which containing tryptophane (trp), - (p ) amino acid residues (try-ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) were synthesized by using the solid-phase methods. we synthesized the covalent conjugate of copolymers of acrylic acids and n-vinylpyrolidone (vp\aa) with - amino acid residues at different npeptide/npolymer ratio and investigated the mechanism of condensation reaction by using fluorescence spectroscopy and fluorescamine assay. in the fl uorescamine assay the conjugate solution was measured on a pti qm- steady state fluorescence spectrometer with an excitation wavelength of nm and emission at nm. after the conjugation reaction, the amount of free amino groups had decreased because of binding carboxyl group and observed that fluorescence intensity had decreased. determination of free amino group degree after conjugation, calculated from fl uorescence spectrums. newly developed high strength and chemically stable silica gel based preparative reversed phase packing materials kuriyama, naohiro; shoji, noriko; morishita, kiyoshi; omote, masakatsu ymc co., ltd., japan a new high strength silica gel and a bonding technology based on preparative bulk packing materials for hplc have been developed to provide improved recovery, selectivity, and longer life time for the preparative peptide separations. the novel preparative silica particle was successfully prepared by the new generation process, which allows the higher gel density than typical silica gel and the particle size distribution would be practically mono-dispersed character. for the effective reversed phase peptide separations, pore size and pore volume of these new particle were optimized depending on the molecular weight of peptides. to enhance chemical stability and selectivity under the typical peptide purifi cation conditions, the combination of chemical bonding method and functional group density was optimized for maximum performance. by repeated packing and unpacking of this synthesized gel with large dynamic axial compression column, it was demonstrated that no fi ne has appeared and no back pressure increasing has occurred comparing to commercially available packing materials. also cost effective peptide purifi cation with high loadability, productivity, and recovery was achieved with signifi cant small and large peptides. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide produced by tissue-specifi c alternative mrna splicing of the calcitonin gene. the cgrp peptide signals through a seven transmembrane g protein-coupled receptor (gpcr) belonging to the secretin receptor family. co-expression of the calcitonin-like receptor (clr) with receptor activity modifying proteins- (ramp ) forms a mature cgrp receptor on the cell membrane surface. cgrp is widely distributed in the peripheral and central nervous systems. in the later, cgrp is expressed in trigeminal ganglia nerves and when it is released has potent dilator effects on cerebal and dural vessels. through this mechanism, cgrp is involved in the regulation of blood fl ow to the brain and painsensitive meninges. the pathology of migraine has been associated with the vasodilation effects of cgrp on cerebal circulation. it has been reported that the cgrp peptide consist of an alpha-helical n-terminal region, a fl exible center region, and two putative c-terminal beta-turns. truncation of the fi rst seven residues in cgrp results in antagonists of the cgrp receptor (ic nm), however, cgrp( - ) is rapidly degraded in plasma. here, we report the iterative design and synthesis of new high affi nity cgrp antagonists with signifi cantly increased plasma stability, and higher affi nity for the human cgrp receptor. cordopatis, paul; pappa, eleni; zompra, aikaterini; magafa, vassiliki; diamantopoulou, zoi; lamari, fotini; katsoris, panagiotis university, greece mammalian gonadotropin releasing hormone (gnrh-i) and the sea lamprey gonadotropin releasing hormone type iii (gnrh-iii), belong to the class of conserved gonadotropin releasing hormone peptides. in addition to the classic hypophysiotropic action of gnrh-i, it has been shown that many malignant cells, such as prostate cancer cells, secrete gnrh-i and express the gnrh-i receptor/s. gnrh-iii has no endocrine activity in mammals even at high doses, but has been shown to suppress directly the growth of breast and prostatic cancer cells in vitro. in a continuation of our previous work and in order to study the effect of modifi cations in positions and of leuprolide, an agonist of gnrh-i, on prostate cancer cell proliferation, we synthesized ten new conformationally restricted analogues. d-leu of leuprolide was substituted by d-lys, d-or l-glu and ser by n-me-ser. we also synthesized fi ve new analogues of gnrh-iii and studied their effect on prostate cancer cell proliferation. asp in position of gnrh-iii was substituted by asn and glu, pro was substituted by á-aminoisobutyric acid (aib) and trp and/or trp by d-trp. peptides were synthesized by the solid phase methodology and fmoc/but chemistry in high yields. results show that the inhibitory effect of gnrh-i analogues on the proliferation of human prostate cancer cells depends on the nature of the substituted amino acid in position . incorporation of d-trp in positions and of gnrh-iii preserved its antiproliferative activity, whereas all other modifi cations reduced its activity. mastoparan (mp) is an antimicrobial cationic tetradecapeptide with following primary structure inlkalaalakkil. this amphiphilic -helical peptide was originally isolated from the venom of wasp paravespula lewisii. however some mp analogues (tetradecapeptides with different amino acids sequence) have been also isolated from the venom of hornets, yellow jackets and paper or solitary wasps. they are rich in hydrophobic amino acids such as leu, ile, ala and commonly posses lys residues. mp shows a variety of biological activities such as inhibition of the growth of gram-positive bacteria, activation of mast cell degradation and histamine release, activation of phospholipase a and c or erythrocyte lysis. mp is also turned out to enhance the permeability of artifi cial and biological membranes and activate gtp-binding regulatory proteins by a mechanism analogous to that of g protein-coupled receptors. nowadays many mastoparan analogues have been synthesized. some of them contains in primary structure the mastoparan sequence but they loss some of the properties of mp, such as transportan (a cell penetrating peptide, consisting n-terminal fragment - of galanin and mastoparan at the c-terminus linked with lys residue) or its truncated analogue, transportan- . in present study we have designed and synthesized several new chimeric mastoparan analogues composed of mp and other biologically active peptides (e.g. galanin, rnaiii inhibiting peptide) and containing natural or unnatural structures. next we examined each of these hybrid constructs as well as natural peptides for they antimicrobial activities. acknowledgement: this work was supported by the university of gdansk grand bw - - - cinnamic acid derivatives as esters, amides and glycosides are known to have antibacterial, antiviral, antiinfl ammatory, antiproliferative, immunostimulatory etc. properties. some of them (amide and ester analogues of caffeic acid with natural amino acid esters) exibit stronger antioxidative activity. in particular thiazole, oxazole and imidazole amino acids that may play a key role in biological activities of unusual peptides are also important intermediates for natural product synthesis and peptidomimetics. here we report the synthesis of novel cinnamic acid amides with oxazole containing glycyl-methyl ester as potential antioxidants. the amides were synthesized from sinapic, coumaric, ferulic acids and the corresponding glycyl-methyl ester hydrochloride form using n-ethyl-n-( -dimethylaminopropyl) carbodiimide hydrochloride (edc) and -n,n-(dimethylamino)-pyridine (dmap). their antioxidant activity was evaluated by , -diphenyl- -picrylhydrazyl dpph ( , diphenyl- -pycrylhydrazyl) test. the venoms of marine cone snails contain a complex mixture of peptide neurotoxins known as conotoxins ( ) . they are a diverse class of biomolecules that exhibit exquisite selectivity and potency for a wide range of pharmacological targets in the central nervous system, making them valuable tools for studying the mechanisms of neurotransmission and pain. despite their diversity, conotoxins have evolved from a relatively small number of rigid disulfi de bonded frameworks that give rise to a series of intervening loops of amino acids that project outwards from the framework and interact with the receptor binding site. receptor targets are generally determined by the shape of the framework, with the amino acids within the framework infl uencing receptor subtype specifi city. this work aims to use positional scan libraries ( ) based on / -conotoxin frameworks to study the interactions of amino acids with the binding site of neuronal nicotinic acetylcholine receptors, and to discover new ligands that possess greater potency and selectivity for different subtypes these receptors. the disulfi de bond frameworks were formed by oxidizing each mixture in dilute aqueous buffer and their correct formation monitored by cd spectroscopy. an á nachr functional assay was used to screen each mixture to determine the activity of each mixture and the results of this assay were used to design a series of individual candidates for use in further structure activity relationship studies. melittin effect on the sensory neurons prelevated from double transgenic vs. normal mice melittin is a aa peptide. it is the active compound of bee venom and a powerful stimulator of phospholipase a . its antimicrobial effect was extensively studied in the literature, but little is known about its neuroactive properties. melittin increases the cell membrane permeability in excitable and non-excitable tissues, as a result of its interaction with the negatively charged phospholipids. our interest was focused on the algesic properties of melittin and on its potency against the electrophysiological parameters of sensory neurons from dorsal root ganglia. in our study, we have performed patch-clamp studies, by the whole-cell confi guration. primary cell cultures were obtained from double transgenic mice tcr-ha+/ins-ha+ with type i diabetes. this animal model is well-suited for the study of neuropathic pain and it enables us to test the analgesic effects of melittin ( . - m). our recordings indicate that the active-peptide modifi es the algesic profi le of the sensory neurons, in particular by acting against the capsaicinactivated ionic currents. in addition, we have monitored the action of melittin on the i h currents, na + voltage -dependent currents, delayed rectifi er k + currents. a particular interest was focused on the recordings of ca + voltage-dependent currents. the maximal effect was obtained at a dose of m melittin, and at higher doses the active-peptide strongly disturbs the lipid membrane order. in conclusion, the algesic profi le of the sensory neurons from double transgenic mice is signifi cantly modifi ed by the neuroactive melittin in comparison with the profi le of neurons from balb/c mice. these electrophysiological data are important, and are very well corroborated with the increased thermal and mechanical hypersensitivity induced by melittin itself and the bee venom extract, that have been proved their effi ciency in behaviour tests . the toxic effects of amps tested on mammalian cell cultures the amps (antimicrobial peptides) is a class of molecules that belongs to the innate immunity. the ability of some of these peptides to penetrate the microorganism's external membrane and to induce their death suggests that this class of molecules may represent a new generation of antibiotic pharmaceuticals. in our work, we focused on the amps side effects on mammalian cells. the target was to characterize the toxicity of several amps evaluated on in vitro cellular cultures. the following amps were used in our experiments: melittin, magainin i and ii, cecropin a and mastoparan. the viability of cells in cultures was assessed by mts test on chinese hamster lung fi broblasts v and on human lymphocytes primary cultures. concentrations in the range of . - m were used. the ic value for each type of amp was evaluated from the viability versus concentration curves. haemolysis assays were also performed for these active-peptides. based on ic values, melittin has a stronger citotoxicity than magainin i and magainin ii. on the other hand, magainins appears to posses higher haemolytic properties than melittin. the less cytotoxic peptide seems to be mastoparan. these types of results are very useful to characterize the ability of amps to induce toxic effects in human body during the treatment. acknowledgements: financial support by the cnmp research grant pnii number - / . the melanocyte-inhibiting factor (mif- ), which is the tripeptide pro-leu-gly-nh , was isolated in the conventional way from bovine hypothalamus tissue. mif- represents a class of naturally occurring opiate antagonists with varying activities in independent situations. the purpose of the present study was to investigate the analgesic activity of mif- and its analogues modifi ed at position with unnatural amino acids cav, slys, sleu, sile and snle. the experiments were carried out on male wistar rats ( - g). the changes in the mechanical nociceptive threshold were measured by the paw-pressure test using an analgesiameter (ugo basile). mif- and analogues (all in dose mg/ kg) were administered intraperitoneally (i.p.). mif- exhibits a weak analgesic effect and selective affi nity for the -opioid receptor. mifanalogues -mif-cav, mif-sleu, mif-ile and mif-nle were found to have a naloxone-reversible analgesic effects. pacap (pituitary adenylate cyclase-activating polypeptide) exists in two biological isoforms, i.e a -amino acid peptide and a shorter form of residues corresponding to the n-terminal part of pacap . previous structure-activity relationships studies revealed that the n-terminal segment is essential for the activation of the specifi c and selective type i pacap receptor (pac ). in order to clarify the molecular requirements for pac activation, we initiated structure-activity investigations of the n-terminal part of both pacap isoforms. in particular, an important library of analogs focusing on the fi rst seven residues (his -ser -asp -gly -ile -phe -thr ) was rationally developed and pharmacologically evaluated for their capacity to bind the pac receptor and to induce ca + mobilization in chinese hamster ovary (cho) cells stably expressing the human pac receptor. ala scan demonstrated that the carboxylic acid function of residue asp and the benzyl group of phe are essential feature for proper pac receptor activation. the inversion of chirality of residues ile , phe and thr caused a loss of affi nity whereas the incorporation of d-amino acids in positions , or did not affect the binding properties of pacap. moreover, using a n-methyl scan, we showed that the n-terminal domain of pacap is not tolerant to back-bone modifi cations. however, replacement of ser by pro did not decrease signifi cantly the potency of the peptide while substitution of this same residue by d-pro totally inhibited the biological activity of pacap. furthermore, other structural constraints reduced the potency of pacap, suggesting that the n-terminal domain needs fl exibility to bind and activate the pac receptor. finally, residues - of c-terminally extended peptides played a favorable role for the binding affi nity towards the pac receptor as pacap analogs demonstrated highest binding affi nity compared to their pacap counterparts. neutrophil elastase-dependent synthetic host defence pro-peptides for the treatment of bacterial infections in cystic fi brosis patients chronic infection and infl ammation play a major role in the pathophysiology of lung disease in cystic fi brosis (cf). besides pseudomonas aeruginosa, there is increasing evidence that other multidrugresistant organisms, such as methicillin-resistant staphylococcus aureus (mrsa) are implicated in the activation of the infl ammatory response in cf patients. cationic host defence peptides, multifunctional mediators of the innate immune system, have been recognised as promising candidates for the development of novel antimicrobial agents ( ). most of these macromolecules are produced as inactive prepropeptides and are proteolytically activated to release a c-terminal cationic peptide with antimicrobial activity. we thought to mimic this natural mechanism to target cf pathogens with synthetic host defence propeptides. saltresistant, all-d cationic antimicrobial peptides ( ) were conjugated at their n-termini to a polyglutamic acid sequence to compensate the net positive charge of the parent peptide, through a linker selectively degraded by a disease-associated enzyme (neutrophil elastase). in vitro effects of the all-d p peptide candidate on planktonic and biofi lm forms of pseudomonas aeruginosa and staphylococcus aureus clinical isolates were assessed. reactivation studies of the propeptide were performed in presence of purifi ed neutrophil elastase and in physiologically relevant conditions, using bronchoalveolar lavage fl uids from cf patients. the nmr structures of the propeptide and active p sequences were also compared. these studies confi rmed that the propeptide remains inactive in the absence of neutrophil elastase and that the latter enzyme can potentiate its antimicrobial activity. synthesis and biological activity of a series of aza pseudopeptides related to rfa, the endogenous ligand of gpr rfa, a novel neuropeptide of the rfamide family, is the natural ligand of the previously orphan receptor gpr . both rfa and gpr mrnas are highly expressed in hypothalamic nuclei of rodents, and icv injection of rfa induces a potent orexigenic effect in mice. recently, it has been shown that gpr -defi cient mice suffer from osteopenia. analysis of the rfa precursor reveals that it may generate several additionnal rfa-peptides including an n-terminally extended form ( rfa) and a truncated form ( rfa ( - ) ). rfa and rfa increase dose-dependently [ca + ]i in gpr -transfected cells while rfa( - ) is about times less potent than rfa. molecular modeling under nmr constraints of rfa shows that the n-terminal region encompasses an -helix and the c-terminal region adopts a -turn in dpc micelles. the c-terminal peptide rfa( - ) exhibits major distortions of this turn that may be responsible for its weak potency. the aim of this work was to introduce an aza residue in rfa( - ) in order to stabilize the -turn. sequential aza -counterpart substitution of amino acids at positions and enhanced by and folds the potency of rfa( - ), respectively. the aza -phe analog was times less potent than rfa( - ). replacement of the native ser by the aza surrogate of (ho)homothr (aza -hth) to favor hydrogen bonding, generated an analog that was folds more potent than rfa( - ). in contrast, substitution of residues to led to analogs totally devoid of effect on calcium mobilization. as the -turn is centered on the ser of rfa, we can assume that the aza -hth moiety partially mimics the -turn in the rfa( - ) sequence. these data constitute the fi rst step towards the development of new gpr analogs that could prove useful for the treatment of feeding disorders and/or osteoporosis. acknowledgements: supported by inserm (u and pnr-re) and the région haute-normandie. olm is recipient of a fellowship from mesr. it is well known that microtubule targeting agents (mtas) are used in clinical treatments as anticancer agents. recently, it has become clear that some mtas also function as gvascular disrupting agents (vdas), which induce tumor-selective vascular collapse. as one of such candidates, a natural cyclicdipeptide, phenylahistin (plh) exhibiting colchicine-like anti-microtubule activity, has been our focus. we have succeeded in synthesizing plh, and performed the structure activity relationship study of its derivatives. from the biological evaluations, according to these results, a highly potent derivative npi- (ic = nm, ht- ) was selected, which is now in phase i clinical trial as an anticancer drug in the us. furthermore, we have established the synthetic route of npi- and its derivatives. about analogs were prepared so far and screened by ht- citotoxicity assay. as a result, several highly potent derivatives ware developed. although npi- and its derivatives are believed to be recognized around the colchicine binding site on tubulin, the three-dimensional structure of npi- could not be superimposed over that of colchicine. in order to understand the precise binding mode of npi- , we developed a highly potent cytotoxic derivative, kpu- (ic = . nm, ht- cells) with a benzophenone structure. because benzophenone is recognized as a photo-reactive group, we synthesized biotin-tagged kpu- derivative as a photoaffi nity probe. since this probe has biological activity enough to function to tubulin, photoaffi nity labeling was performed to analyze the binding site. as a result, irradiation-time-dependent labeling towards tubulin was observed. the labeling was also dose-dependently inhibited by colchicines addition, suggesting that the probe specifi cally recognizes around the colchicine binding site on tubulin. chemical biology study for determining the precise binding site is now in progress. conformational analysis of the new temporin analogues: gln ta and pro tl. temporins are antimicrobial peptides (amp €™s) isolated from the skin of red european frog rana temporaria. they are active particularly against gram-positive bacteria, candida species, fungi. they have the ability to bind and permeate both artifi cial and biological membranes. we have recently investigated by spectroscopic means two members of this amp family temporin l (fvqwfskflgril-nh ) and temporin a (flpligrvlsgil-nh ) ( ). based on the nmr results, we developed two new analogues of these peptides named pro tl (fvpwfskflgril-nh ) and gln ta (flqligrvlsgil-nh ). biological data indicate that pro tl has a higher antimicrobial activity and a lower hemolytic activity than the native peptide tl. in contrast, gln ta more hemolytic than the parent peptide ta. the conformational behavior of the new analogues was investigated in membrane mimetic environment (sds and dpc micelles) by spectroscopic and computational methods. diagnostic nmr parameters observed for glu ta and pro tl indicate a conformational propensity toward helical structure. for glu ta, bturn structures are also observed along the n-terminal fragment of the peptide. we are currently studying the conformational behavior of the four peptides also by molecular dynamics simulation in explicit solvated dodecylphosphocholine and sodium dodecylsulfate systems. these simulations would provide a realistic picture of the interactions between the peptide and models of bacterial and mammalian membranes. where xaa was selected form a collection of cyclic and acyclic nonaromatic amino acids. the peptides were prepared by standard spps methods using either the fmoc or boc strategy and tested in vitro for their agonistic potency and effi cacy at the v a and the related receptors. unlike avp, which has aromatic amino acids in positions and , these analogues contain only non aromatic residues, but are still potent v a agonists. compounds with cyclic aliphatic residues (e.g. cha) in position were found to be generally more potent v a agonists than the ones containing acyclic residues. the ala analogue ([ala ,ile ,dab ]vp) was totally inactive (no signifi cant v ar agonism up to nm) as pharmacology and medical peptide chemistry reported previously for [ala ]avp ( ). the cha containing peptides are slightly less potent as v ar agonists than their phe counterparts, but the overall in vitro pharmacological profi le of the two classes of compounds is similar. selected new analogues were also tested in vivo in a rat pressor model. the compounds were found to be effective in raising arterial blood pressure and appeared to be longer acting in vivo when compared to avp and related compounds, as demonstrated by slower disappearance of their pressor effect at equieffective doses. detailed experimental procedures, the results of biological testing, and sar discussion will be presented. the impact of lithium cations on the peptide bond lithium ions play an important role in some biological processes, e.g. in the treatment of neuronal dysfunctions and some metabolic pathways. however, the mechanisms for these actions are not known up to now. recently it has been shown that lithium cations infl uence the isomerisation state of peptide bonds which is essentially pronounced in the case of the amino acid proline. such an isomerisation goes hand in hand with conformational changes possibly resulting in altered folding and structuring. thus, lithium cations might essentially infl uence the biological function of peptides and proteins. to shed light on the underlying mechanism we carried out detailed studies on several model peptides. thus, the isomerisation as well as the kinetic properties of the peptides have been determined employing various techniques of nmr spectroscopy. besides, quantum chemical studies on li + -peptide complexes were performed to get insight into the structural aspects of the cation-peptide interaction. enhanced screening and understanding of hypolipidemic peptides assisted by informatics hypolipidemic peptides targeting the bile acid to inhibit cholesterol absorption in intestine has been indicated by several naturally obtained short peptides. these bile acid-binding peptides are known to disrupt the micellar formation of bile acid for capturing intestinal cholesterol, and lead the aggregates pass the intestine without absorbance. such effect also disrupts the hepatic circulation of bile acids, which accelerates the conversion of stored cholesterol into lacking bile acids. however, traditional extraction procedure of peptides form natural products is time-consuming. therefore, to enhance the screening of such functional short peptides, we have combined the array-based screening strategy with bioinformatics strategy, to design effective experiments by prediction of physicochemical peptide structures. as a result, we resulted in signifi cantly higher screening effi ciency and high bile acid affi nity compared to random screening. we hare report the introduction of one of supervised learning algorithms, fuzzy neural network, and unsupervised algorithm, hierarchical clustering scheme for such functional peptide screening. analogues of the kinin b receptor antagonist r- bearing n-terminal lipid moieties. adrenomedullin (am) is a -amino acid peptide that shares structural similarities with regulatory peptides belonging to the calcitonin family (calcitonin, cgrp and amylin). am is known to produce a potent vasodilation via the coupling to the calcitonin receptor-like receptor (crlr) associated with a chaperone protein, the receptor-activity modifying-protein (ramp). binding studies performed in mammalian tissues revealed that the largest distribution of am binding sites was found in the heart and lungs. in fact, the pulmonary blood vessels display a vast abundance of am binding sites that act as clearance receptors. pulmonary arterial hypertension (pah) is a condition associated with obliteration of pulmonary arterioles which carries a very poor prognosis, in part because of delayed diagnosis due to the lack of specifi c noninvasive diagnostic tools. likewise, there currently exists no molecular imaging agent to diagnose pulmonary embolism, a pathological condition that occurs when a blood clot blocks a lung artery. therefore, due to the high density and incidence of am receptors in the cardiorespiratory system, am could represent a key-target for diagnosis with radiolabeled am-related drugs. in this study, we looked at structureactivity relationships and synthesized various am fragments exhibiting high binding specifi city for am receptors but reduced biological activity. moreover, we introduced different chelating moieties into these am peptides to evaluate the possibility of labelling those molecules for imaging purposes. using cell binding assays, we observed that a cyclic moiety combined with the am( - ) sequence is able to maintain a good affi nity for am receptors. furthermore, we showed that the incorporation of a -amino acid sequence into the peptide chain was the best chelating moiety that allows high effi ciency labelling with mtc. hence, our study strongly suggests that am derivatives are promising leads for lung specifi c imaging. m tc-labeled analogs with pansomatostatin properties may lead to clinically useful radiotracers and further search for analogs displaying improved biological profi le is warranted. and for the rat (r) sst by competition binding assays in ar - j cell membranes with [ i-tyr ]octreotide as radioligand. after m tc-labeling, radiopeptide internalization was studied in ar - j cells. biodistribution of [ m tc]demotates was studied in healthy swiss albino mice and for [ m tc]demotate , , and in ar - j tumor bearing mice. results: demotate showed the highest affi nity binding for the hsst and the rsst , while introduction of one asp linker(s) at the n-terminus diminished the affi nity for the hsst and the rsst and led to lower internalization rates. substitution of thr by asp in demotate resulted in good affi nity for the rsst and lower affi nity for the hsst . single (d)phe -substitution/ or thr substitution by asp combined with asp introduction at the nterminus/ led to inferior binding affi nity, poor internalization capacity and poor uptake of the respective radiopeptides in target-organs and, in the case of [ m tc]demotate , , and , in ar - j tumors in mice. conclusion: introduction of asp residues in the original [ m tc]demotate chain exerted a negative effect on receptor affi nity, internalization capacity and targeting of somatostatin binding sites in mice precluding their use as hydrophilic pharmacokinetic modifi ers. the family of secreted aspartic proteinases (saps), which are encoded by ten distinct genes, is an important virulence factor of the human pathogen candida albicans.( ) due to an increase in the number of candida strains that are resistant to the drugs currently used in therapy, these proteinases are highly promising new drug target candidates. based on the knowledge of sap -substrate specifi cities( )and x-ray structural studies of sap in complex with inhibitors,( ) we designed and synthesized a series of sap inhibitors by modifying the structure of the pentapeptide pepstatin a, which is a potent yet non-selective aspartic proteinase inhibitor. these inhibitors were synthesized manually via a spps methodology using a -cl-tritylchloride resin and fmoc-protected amino acids. in addition, the key residue of the designed peptide inhibitors, the -amino acid statine (sta), which was prepared according to a slightly modifi ed literature procedure, . was further protected at its hydroxyl group as a silyl ether for the purpose of avoiding competitive side reactions during amino acid couplings. we prepared new inhibitors by varying the pepstatin a structure at the p , p , or p ' position. all variants showed effi cient inhibition when screened against the isoenzymes sap , sap , and sap , and some of them exhibit ic values similar to or even lower than that observed for pepstatin a. peptide library is a general technique for screening specifi c peptides that bind to a target protein. but, in a standard screening method, peptide libraries need to be fi xed on large carriers like phage, beads and so on. the large carriers would affect the binding between peptides and the target protein. in this work, we develop a new method for screening peptide libraries without carriers. we attached a variety of fl uorescent amino acids to peptide libraries and the peptides that bind to target proteins were identifi ed through -dimensional fl uorescence spectroscopy in aqueous solution. in this presentation, -different fl uorescent amino acids were synthesized or purchased. of those amino acids, we newly synthesized fl uorescent amino acids. -d fl uorescence spectra of those amino acids were measured. they were different in fl uorescence excitation/emission wavelengths. excluding those of weak fl uorescence intensities, fl uorescent amino acids are selected. those fl uorescent amino acids are mixed in methanol/water (ph . ) (= / (v/v)) and the -d fl uorescence spectrum was measured. then, concentrations of the component amino acids were evaluated by a linear least-squares method. the results suggested that all fl uorescent amino acids can be quantifi ed. the set of fl uorescent amino acids combined with the -d fl uorescence spectroscopy technique will be a powerful tool for screening peptides and other drug compounds without using any carriers. n-methyl phenylalanine-rich peptides as potential blood brain barrier shuttles malakoutikhah, morteza; teixidó, meritxell; giralt, ernest institute for research in biomedicine, barcelona, spain several peptide families containing n-methylated amino acids were designed and synthesized using solid-phase peptide synthesis (spps). the permeability of these compounds was studied by parallel artifi cial membrane permeability assay (pampa) and immobilized artifi cial membrane chromatography (iamc) so as to select the best peptides in terms of length, terminal groups and amino acid replacement to be used as carriers that pass through a model of blood-brain barrier (bbb) by passive diffusion for non-permeating agents. furthermore, their enzymatic stability in human serum and their cell viability were tested by mtt assay. these peptide families showed great stability and nontoxicity. the three best peptides were coupled to levodopa and assessed. these peptides transferred levodopa through an artifi cial membrane and transformed it from a non-passive permeating drug into a compound able to cross the membrane by passive diffusion. the structure-activity study of insulin-like peptide (insl ) requires the design and synthesis of various analogues. in order to test these for their receptor binding affi nity, a high-throughput receptor binding assay is needed. we have therefore developed an effi cient solid phase synthesis protocol to prepare specifi cally mono-labeled human insulin-like peptide (insl ) for the study of its interaction with its g-protein-coupled receptor, rxfp . a commercially available chelator, diethylene triamine pentaacetic acid (dtpa), was coupled to the n-terminus of solid-phase bound insl a-chain and then a coordination complex between eu + and dtpa chelator was formed. after combination of the purifi ed aand b-chains together with sequential formation of the three insulinlike disulfi de bonds, the labeled peptide was purifi ed at high yield using high-performance liquid chromatography with high ph buffer to prevent the liberation of europium from chelator. saturation binding assays were undertaken to determine the binding affi nity (pk d ) of labeled insl for rxfp in hek stable cell line expressing rxfp . the binding affi nity of dtpa-labeled insl ( . ± . ) was comparable to that of i-labelled insl ( . ± . ). the effi cient solid-phase synthesis has provided a novel lanthanide-coordinated, dtpa-labeled insl with excellent sensitivity, stability and high specifi c activity and which is superior to the traditional i-insl . this labeled peptide can be used in a high-throughput screening of insl analogues in structure-activity studies. multimodal tumor imaging using mri and pet/spect provides comprehensive diagnostic information as it combines anatomical information (by mri) and functional information (by pet or spect). development of dota-derived imaging probes is advantageous for multimodal imaging as it accommodates many metal ions employed in different imaging modalities including gd(iii) for mri. however, high relaxivity and enhanced specifi c up-take at the targeted site is important for the gd-based mri contrast agents. the dota-based prochelators required for the multivalent vectorization of targeting ligands were synthesized by functionalizing cyclen or do a-tertbutyl ester with modifi ed glutamic acid. multivalent conjugation of bombesin peptides, which specifi cally target tumors expressing gastrinreleasing peptide (grp) receptors, yielded the corresponding mono-, di-, and tetravalent bombesin analogues. the conjugates showed excellent chelating properties with gd(iii) (for mri applications) and also with in, lu and ga (for radiopharmaceutical applications). the in and lu labeled divalent conjugates showed rapid internalization and slower externalization rate compared to their corresponding monovalent analogues as studied with prostate tumor cell lines. the gadolinium complexes of monovalent and divalent conjugates showed signifi cant relaxivities at mhz ranging from . to . mm- s- at °c. the values represent the to fold enhancement of relaxivity compare to clinically employed dotarem® (gd-dota). further, the relaxivities increased signifi cantly from monovalent to divalent conjugates. this is highly promising as we would expect much higher relaxivities in case of tetravalent conjugates, which are currently under investigation. in conclusion, the work demonstrates the development of high relaxivity multivalent bombesin analogues, which could be employed for the multimodal imaging such as pet/mri or spect/mri with improved tumor targeting capabilities. a new highly potent dota-conjugated bombesin antagonist for grpr-positive tumor targeted imaging peptide receptors are very promising targets for tumor imaging. the somatostatin receptors were successfully targeted with peptides labeled to -emitters ( in, ga and m tc) and positron emitters ( f, ga, y) for diagnostic imaging. for tumor targeting, radiolabeled agonists were developed as they usually trigger the internalization of the peptidereceptor complex. we have recently shown that somatostatin-based radiolabeled antagonists may not only have a higher tumor uptake than equipotent agonists but also a longer lasting tumor uptake. among the most promising receptors to be targeted, the bombesin receptors are of great interest as they are overexpressed in major human tumors such as prostate and breast. the aim of this study was to develop a radiolabeled bombesin-based antagonist conjugated to the macrocyclic chelator dota which allows effi cient and stable labeling with a variety of two-and three-plus charged radiometals for imaging (pet, spect). we compared its in vitro pharmacologic properties and biodistribution in the pc- mouse model side-by-side with the potent agonist [ nat, in]-do a-ch co-g- -aminobenzoyl-q-w-a-v-g-h-l-m-nh (amba). in(iii)-amba was shown to be a potent agonist by ca + fl ux and immunofl uorescence studies and in(iii)-rm was effi ciently antagonizing the activity of in(iii)-amba. the pharmacokinetics showed a distinct superiority of the radioantagonist with regard to the high tumor uptake as well as to all tumor to normal tissue ratios. [ ga]-rm showed similar pharmacokinetic than [ in]-rm with a lower initial kidney uptake and a faster wash out from the kidney. the pet/ ct scintigraphic studies of [ ga]-rm in the animal model confi rm the signifi cant high tumor uptake and tumor to background ratio. as we found for somatostatin receptor-targeting radiopeptides,bombesin-based radioantagonists also appear to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of grpr-positive tumors cell penetrating peptides (cpp) are a special class of peptides that possess the property to traverse the formidable barrier of the plasma membrane and deliver cargos into cells. using cpp as vectors and dna, mrna or proteins/enzymes as potential intracellular targets, a new generation of intracellular contrast agents (cas) can be developed. these agents have prospective use for molecular imaging (both optical and magnetic resonance imaging) by targeted labeling of cells. aiming to image the presence of specifi c mrnas or enzymes, two mrna targeting (contains a pna sequence antisense or non-sense to the target mrna of dsred) and one enzyme targeted (contains a unit cleavable by -galactosidase) cas were tested for their activity in the presence and absence of respective targets. the antisense targeting ca, their nonsense derivative and the enzyme targeted ca were taken up effi ciently into cells by an exclusively endosomal mechanism as observed by fl uorescence microscopy. cell free binding assays proved a specifi c interaction with a synthetic target for the antisense but not for non-sense ca. magnetic resonance studies showed a higher uptake in transgenic dsred expressing cells than the parent cells. however, no difference was observable for antisense versus non-sense ca in dsred cells, due to the vesicular entrapment which is preventing the specifi c interaction between ca and cytosolic target. since a comparable cellular distribution was visible for the enzyme targeted agent, a specifi c accumulation in -galactosidase containing cells is also unlikely. the results show that even though the designed cas were effi ciently taken up into cells, they can interact specifi cally with the target only if colocalization is achieved. however, a lack of specifi city is caused by the endosomal entrapment. further modifi cations are required to achieve the release from endosomes or a direct uptake into the cytosol. the infl uence of pegylation on the tumor accumulation of frop- , a tumor specifi c peptide identifi ed by phage display the pool of natural peptides suitable for the tumor targeting is limited and therefore novel lead structures identifi ed by screening of phagedisplayed libraries are highly warranted. however, transfer of the novel peptide sequences to clinical application is often diffi cult. we had shown that the coupling of dota to frop- , a peptide identifi ed in phage display libraries resulted in a fundamentally improved in vitro binding capacity. however, a biodistribution study revealed that the slow binding kinetics frop-dota (h-glu-asn-tyr-glu-leu-met-asp-leu-leu-ala-tyr-leu-lys(dota)-cys-nh ) allowed the excretion to forestall a signifi cant tumor accumulation. the aim of this study was to investigate whether the conjugation of peg to frop-dota results in a derivative with a prolonged residence time in the blood. frop- bearing a c-terminal dota residue to allow labeling with in- and a cysteine to attach a maleimido-modifi ed da peg oligomer was obtained by solid phase synthesis. the conjugate was purifi ed by size exclusion chromatography. several attempts to couple different peg derivatives on the solid support did not proceed satisfactorily and therefore the peg residue was attached in solution. the breast cancer cell line mcf- showed a relative low accumulation of the pegylated peptide in the cells. in contrast, biodistribution studies of the labeled conjugate in mice bearing human fro - showed a time dependent increasing uptake of the pegylated peptide with a high retention (at h p.i. % of the maximal activity concentration persisted in the tumor). the highest uptake values were determined at min. p.i. reaching . %id/g tumor as compared to . % %id/g observed for the non-pegylated derivative at min p.i. apparently pegylation provides a substantially improved stabilization in the circulation which allows a stable tumor accumulation. in vivo spect/ct imaging of intracranial human glioblastoma xenografts with indium-labeled homing peptide. huhtala, tuulia* ; enbäck, julia* ; rautsi, outi ; weisell, janne ; laakkonen, pirjo* ; närvänen, ale* *equal contribution. university of kuopio, finland; university of helsinki, finland phage display libraries provide a powerful tool to identify new biologically active peptides that bind specifi cally to their target molecules. different tumors express a distinct range of molecular markers on their vasculature providing a possibility to use peptides for targeting. tumor-homing peptides offer an opportunity to target, image and destruct the target tissue. glioblastomas represent the most aggressive form of brain tumors. we have identifi ed a novel peptide, coop, using an ex vivo / in vivo phage display screen. this peptide homes specifi cally to the early stage astrocytoma model. in addition, the peptide homes to the u human glioblastoma xenografts (enbäck and laakkonen, unpublished data). we have studied the biodistribution and tumor homing properties of indiumlabelled peptide in mice bearing intracranial human u glioblastoma tumors using spect/ct imaging. coop peptide was conjugated with the dtpa (diethylenetriamine pentaacetic acid) via amino terminus and labeled with indium. imaging was performed in four different time points ( minutes, hour, hours & hours), and the organ and tissue specifi c radioactivity was measured after the scarifi cation of the animals. intravenously injected in-labeled coop peptide accumulated in the u braintumors. the amount of the peptide increased with time and a clear radioactive accumulation was seen in / animals at hour and in / animals at hours after injection. due to the small size of the dtpa-peptide conjugate the majority of the molecules were secreted via the kidneys during the fi rst minutes. at h timepoint the tumorto-brain tissue ratio was , . we report here that the coop peptide, which specifi cally homes to the brain tumor vasculature and tumor cells, strongly accumulated in vivo to the xenografted human glioblastoma tumors in mice. this is the fi rst time when a synthetic peptide has been used in spect imaging of brain tumors in animal mode. failure of the development of a novel peptide tracer for molecular imaging of cancer therapy response - ( ) have shown that fl uorescently labeled derivatives of the hvggssv motif accumulated in tumors undergoing therapy. this peptide motif had been identifi ed by the in vivo screening of phage-displayed peptide libraries. the ability of the peptide to differentiate between responding and nonresponding cancers after treatment could be utilized for the development of promising tracers to monitor cancer therapy. the goal of this study was to investigate whether radiolabeled derivatives of the hvggssv motif can be used for the noninvasive determination of therapy response in vivo. therefore three different conjugates were synthesized by solid phase synthesis using fmoc chemistry. the fi rst peptide was n-terminally modifi ed with the chelator dota (dota-ggghvggssv-conh ) to allow radiolabeling with metallic radioisotopes such as in or ga. a glycine linker was placed at the n-terminus to separate the peptide motif from the chelator. in order to obtain derivatives accessible to radioiodination a tyrosine was introduced at the n-terminus (h n-yggghvggssv-conh ) of the second peptide. the third peptide was biotinylated at the n-terminal amino group and bound to radioiodinated streptavidin (sa-biotin-ggghvggssv-conh ). the biodistribution was monitored by scintigraphy in mice bearing a -tumors treated with a vegf receptor-specifi c inhibitor (su ). this study revealed that the in labeled hydrophilic dota conjugate shows a rapid renal clearance, the iodinated peptide accumulates mainly in the liver and kidneys. all of the peptide derivatives do not specifi cally accumulate in treated tumors after i.v. injection. in contrast to the promising results shown by han z. et al. our study shows that the hvggssv motif can not be easily adapted to radiolabeling approaches with clinical relevance. in conclusion, the peptide motif is not attractive for further clinical evaluation as new diagnostics for the imaging of cancer response. a novel approach to improve cellular delivery of -aminolaevulinic acid: new ala-containing peptide prodrugs for photodynamic therapy. photodynamic therapy (pdt) is a binary therapeutic modality which is currently under investigation for the treatment of several kinds of malignancies. it relies on the interaction of two individually harmless components: a photosensitiser and an external radiation. the interaction of the photosensitiser with light of the appropriate wavelength and molecular oxygen results in the generation of cytotoxic species, namely singlet oxygen and/or radicals, and localized destruction of tumours or infected tissue. in -aminolaevulinic acid photodynamic therapy (ala-pdt), exogenous administration of ala is employed to generate elevated intracellular levels of the natural photosensitiser protoporphyrin ix (ppix), via metabolism through the haem biosynthetic pathway. however this approach suffers from several drawbacks associated with ala's lack of stability at physiological ph and its highly hydrophilic nature, which prevent it from crossing biological membranes and hence limit tissue penetration. ala delivery with synthetic peptide prodrugs is a promising way to address these problems. in this work we report the synthesis and characterisation of a series of peptide prodrugs of general structure ac-xaa-ala-or, where xaa is an alpha amino acid, chosen to provide a prodrug with appropriate lipophilicity and water solubility. the uptake of the compounds and metabolism to ppix in pam keratinocytes, relative to ala is evaluated by fl uorescence spectroscopy, and further quantifi ed by recovery and chemical derivatisation of intact/ partially metabolised prodrugs. in a parallel study, we have also explored the possibility of coupling of ala and ala prodrugs to cell penetrating peptides (cpp). the conjugation of one or more molecules of ala to a cpp sequence represents an interesting approach to enhanced topical delivery of ala. preliminary results of these studies are reported herein. acknowledgements: thanks are due to biotechnology and biological sciences research council (grant bbd ) bioshuttle as a carrier for temozolomide transport into prostate cancer cells if metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. temozolomide (tmz) as an oral applicable chemotherapeutic substance has been proven to be effective and well tolerated with toxicity especially for brain tumors. unfortunately tmz was ineffi cient in the treatment of symptomatic progressive hormone-refractory prostate cancer. this may have different reasons like the short plasma half-life of tmz, a non adapted application schema and as a result, an insuffi cient bioavailability. to improve the specifi city, we built our so called tmz-bioshuttle-construct with cathepsin b (ctsb) mrna specifi city. this complex combines, a transmembrane transporter molecule connected via a disulfi de bridge to an antisensepeptide nucleic acid (pna) against a docking site in exon of the ctsb mrna. furthermore this part is connected via a ctsb cleavable peptide substrate to a nuclear localization sequence coupled with tmz. inside the target-cell, the pna recognizes the cytoplasmic ctsb mrna and after annealing (the pna/rna hybride is not a substrate for rnase h) results in a cell-specifi c retention of the tmz in the cytosol especially of ctsb-expressing cells. then, after the cathepsin b-mediated cleavage in the cytoplasm, the nls-sequence is separated and activated for an ran/importin-mediated transport of the tmz-cargo into the nucleus of the target cells. this bioshuttle-mediated tmz transfer could be a step forward to a successful therapy with higher specifi city, avoiding adverse reactions and circumventing the previous therapy limiting situation. the intracellular delivery of protein segments and other bioactive molecules using membrane -permeable peptides has been investigated in multiple aspects. most of the currently recognized cell penetrating peptides (cpp) are of cationic nature and derived from viral, insect or mammalian proteins endowed with membrane translocation properties. these peptides enter the cell by a receptor independent mechanism, which is poorly understood and carry only one epitope. our target was to achieve a cell-penetrating carrier able to transport more than one bioactive molecule. to this aim we designed a cell penetrating sequential carrier (cpsc) formed by the repetitive -lys-aib-cysmoiety, which incorporates the cysteine residue for anchoring the bioactive molecules through a thioether bond. the lysine free side chain possesses the cationic nature of the construct while the á-amino isobutyric moiety could induce a helicoid peptide backbone with amphipathic characteristics. to test the ability of the cpsc to penetrate the cell membrane we synthesized the carboxyfl uorescein -labelled cpsc, cf-[lys-aib-cys(-ch conh )] -nh , while the cf-tat-cys(-ch conh )-nh analogue, which is a small basic peptide that has been shown to deliver a large variety of cargoes into the cells, was used as a positive control. the results indicated that cpsc had a homogeneous distribution into the cytoplasm suggesting that cpsc may provide a new powerful biological tool for drug transportation. acknowledgements: the gsrt and eu (pened ÅÄ ) for the fi nancial support. amphipathic pro-rich peptides as intracellular drug delivery systems pujals, silvia; fernandez-carneado, jimena; giralt, ernest institute for research in biomedicine, spain shared among many of the known cpps. in our laboratory described a novel group of cpps: amphipathic proline-rich peptides. the principal advantages of these compounds are non-cytotoxicity, nonviral origin and high solubility in aqueous media. the best candidate for cpp among the amphipathic pro-rich peptides was sap (sweet arrow peptide), (vrlppp) .( ) derivatives with hydrophobic moieties, such as fatty acids or silaproline, have shown highly improved internalisation effi ciency; an all d-amino acid version of the cpp sap was shown to be completely protease resistant and was evaluated in a preliminary in vivo study. cd and tem studies regarding the self-assembly properties of this family of peptides highlight the possible role of aggregated species in the internalisation process. finally, these cpps have shown to be internalised via caveolae or lipid-rafts mediated endocytosis, which circumvents the lysosomal route of degradation. in order to test a challenging cargo for sap, gold nanoparticles were conjugated to sap. thereafter, the transport of au np by sap in hela cells has been studied by tem. while np alone are not internalised, np-c-(vrlppp) are clearly uptaken. studies with qds (quantum dots) and egfp as sap cargo are currently being done in our laboratory. . pujals, sílvia; giralt, ernest. proline-rich, amphipathic cell-penetratingpeptides. addr ( ), , - . novel cysteine-rich cell penetrating peptide: effi cient uptake and cytosolic localization introduction: crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. cell penetrating peptides are actively used as the delivery tool for intracellular delivery of various cargos. however, confi nement of biomolecules into endosomes limits their use for intracellular targeting. therefore, there is a need for vectors capable of transferring cargo molecules directly into the cytoplasm. herein, we focus on the development of a novel cpp (derived from polypeptide crotamine .) which shows an effi cient uptake at low concentrations ( . m) and cytosolic distribution along with vesicular uptake. methods: series of peptides were synthesized by fmoc strategy, introducing mutations in cro ( - ) (proposed cpp sequence in crotamine). all were n-terminally labeled with fl uorescein isothiocyanate for optical imaging. structure activity relationship (sar) studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fl uorescence spectroscopy and microscopy. results: amongst synthesized peptides, one of shorter length showed the best intracellular delivery and cytosolic distribution at lower concentration ( . m) when compared to other cpp. replacing or deleting cysteines had negative impact on internalization. results also displayed the involvement of tryptophans in cellular uptake indicating, along with cationic amino acids, the importance of each residue in this optimized sequence. conclusions: sar studies identifi ed a novel cell penetrating peptide showing, besides of endosomal uptake, also an effi cient delivery into the cytoplasm at low concentrations. thus, this peptide might prove useful for effi cient transmembrane delivery of agents directed to cytosolic targets. peptide nucleic acid (pna) is a dna mimic consisting of the four common bases of dna on a pseudopeptide backbone that makes it extremely stable in biological fl uids. antisense pna is targeted against mrna in cytoplasm in a sequence specifi c manner. however, the main hindrance to the effective use of pnas has been their relatively poor uptake by cells. endosomal release or direct uptake into cytosol of agents is mandatory for attaining mrna based targeting. there are reports on the cell penetrating peptide (cpp) based delivery system. it has also been reported that conjugates of cholesterol and sirnas facilitate cellular import ( ) . the aim of this study was to synthesize different sequences of cholesterol coupled antisense pna and to compare its uptake characteristics with a cpp-pna conjugate. the synthesis of pna (anti-dsred pna (agcgcctgtacc), specifi cally targeted to mrna of dsred, a red fl uorescent protein) conjugated to cpp (d-tat) or cholesterol was performed in fully automated synthesizer (prelude, protein technologies, inc.) using continuous solid phase chemistry. to increase the solubility in water, linkers (aeea) and additional charged amino acids were coupled or the sequence of peptide, pna and cholesterol was changed. all compounds were labelled with fitc to confi rm the cellular uptake by fl uorescence microscopy and spectroscopy. cell uptake studies showed that the cpp bound pna was located predominantly in vesicles indicating an endosomal uptake mechanism and subsequent entrapment in vesicles. cholesterol bound pna was also effi ciently internalized. however, it was also located inside vesicles without detectable cytosolic distribution. pna-cholesterol has fewer synthetic steps than pna-cpp. however, it was also located inside vesicles restricting its applicability for mrna targeting. the effi cient uptake might make it a promising cellular delivery agent after further improvements. opioids are gold standards in pain treatments. unfortunately, fast development of tolerance and dependence creates limitations in application of these drugs in chronic pain treatment. over twenty years ago we have proposed development of multitarget medicines as a new avenue of drug discovery. identifi cation of numerous endogenous components that participate in the formation, transmission, modulation and perception of pain signals offers various strategies for the development of new analgesics. neurotensin is an endogenous neuropeptide that play important regulatory role of pain transmission. therefore, it was interesting to develop chimeric analogues that hybridize opioid and neurotensin active components. in such chimeric compounds the possible structural interference may signifi cantly infl uence on interaction of both components with target receptors and/or biological barriers transport systems. in this communication we present synthesis, pharmacological profi le and structural analysis of new potent chimeric compounds. acknowledgements: presented studies have been supported in part with eu grant normolife (lshc-ct- - ) artifi cial ribonucleases based on short peptides. the synthesis of molecules that are capable of nonrandom rna cleavage has found a variety of important applications in molecular biology. for instance, such molecules are used as structural probes for nucleic acids in solution, or in rational design of novel anti-infectives, since rna is the genetic material of many pathogenic viruses. here we represent design and synthesis of peptide-like molecules mimicking the catalytic site of natural ribonucleases (a and t ): series f aa -aa -aa -phe -oalkyl; aa -glu/lys, aa -thr/ser/lys, aa -glu/ lys/thr/arg; series r glu -x -arg -gly -oalkyl; series k glu -x -lys -gly -oalkyl; -gly, -ala, -aminobutyric acid, aminohexanoic acid, p-aminobenzoic acid; series l aa -aa -aa -l (l )-aa -aa -aa , l - , -dioxa- , -dodecanediamine, l - , -diaminododecane; aa -aa -glu, lys, ser, arg. ability of artifi cial rnases to rna cleavage was shown in experiments with mer rna hiv- . the effi cacy of rna cleavage depends on artrnases structure (for example, on location of negative and positive charged amino acids (glu, arg lys) in peptide). the effi cacy of the most active compounds was - % at h incubation. anti-infl uenza activity in vitro of such artrnases was investigated by inhibition of reproduction virus a/hong kong/ / (h n ) in tissue cultures of chorioallantoic membranes (ccm) of - days age chick embryos. acknowledgements: this work was supported by rfbr ( - - a, - - -bel_a), pharmamed ruxo- -n - , the grant of novosibirsk region government for the best young scientists. (ziagen)-( s,cis)- -[ -amino- -(cyclopropylamino)- hpurin- -yl]- -cyclopentene - -methanol is a synthetic carbocylcilic nucleoside analogue with inhibitory activity against hiv. serious and sometimes fatal hypersensitivity reactions have been associated with abacavir. a possible way to increase the side effects is by modifying the known antiviral drugs with various amino acids. the aim of this study was to design and to synthesize of new amino acids and peptide (gly, gly -gly) and thiazole containing (gly, gly -gly) esters prodrugs of abacavir and to explore their activity on the hiv. chemical stability of some purine analogues in the search of new prodrugs effective against herpes simplex virus, series of acyclovir- -[( -hydroxyethoxy)methyl]guanine (acv) esters with peptidomimetics, as well as abacavir-( s,cis)- -[ -amino- -(cyclopropylamino)- h-purin- -yl]- -cyclopentene- -methanol derivatives have been synthesized and tested for antiviral activity. the chemical stability of some of them is studied at ph and . and temperature of °c. a high-performance liquid-chromatografi c (hplc) method was developed for quantifi cation of the unchanged ester concentration. the promelittin represents a readymade cancer pro-toxin; the prodomain inhibits cytolytic activity and the sequence of the prodomain could be manipulated into a substrate for activating proteases. here we present the development of a novel promelittin pro-toxin that can be activated by the serine protease fi broblast activation protein (fap). fap is over expressed on the surface of reactive stromal fi broblasts present in the stroma of human epithelial tumors. fap is not expressed by tumor epithelial cells or by fi broblasts in other tissues, thus making fap a pantumor target for pro-toxin development. peptides containing truncated pro-domain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. once the length was optimized, modifi ed promelittin peptides were generated that contained previously identifi ed fap substrate sequences in the prodomain. these peptides were digested with fap and tested in vitro against fap+ and fap-cell lines in order to determine their fap cleavage potential and toxicity. our lead promelittin peptide was found to be effi ciently activated by fap and selectively toxic to fap+ cell lines with an ic value in the low micromolar range that is similar to that of melittin. pharmacology and medical peptide chemistry region of highly hydrophobic and the antigenic part of hbsag by using microwave assisted solid phase peptide synthesis (spps). tryptophan at the n terminus of the sequence was added by our research group for fluorescence dedection analysis. this peptide was conjugated with synthetic polyanions. different conjugates comparised each other. the synthesized bioconjugates analysized by choromatographic and fl uorometric methods. the fi nal product peptide was characterized by lc-ms and purifi ed by rp-hplc. the bioconjugates were synthesized using different activation mechanisms and the different initial molar ratios (npeptide/npolymer = , , , , ) of the polyanions and the peptide. also physicochemical properties of the bioconjugates were investigated. the structure and characterization of the synthesized conjugates was analyzed by various choromatographic and fl uorometric methods such as hplc, gpc and fluorescence spectrometer. the reaction of high temperature solid-state catalytic isotope exchange (hscie)( ) between insulin and spillover tritium was studied. hscie reaction with the solid mixture containing mg recombinant human insulin was performed for min at o c using % pd/baso catalyst. the product of the reaction was purifi ed by hplc on waters delta-pak c a . x mm column. tritium labeled insulin with specifi c radioactivity ci/mmol and radiochemical purity of more than % was obtained. performic acid oxidation of cysteine residues and subsequent acid hydrolysis were used to analyze the distribution of tritium. it was demonstrated that specifi c radioactivity of À and b chains amounted to . and . ci/mmol respectively. all the amino acids contained tritium and its content in his residues of the b chain was equal to %. experiments for the evaluation of the biological activity of tritium labeled insulin were performed on cd- - -week-old awake male mice. activity of labeled insulin was compared with the activity of standard insulin. insulin was injected subcutaneously at a dose of . u/kg. twofold statistically signifi cant lowering of glucose level was observed min after injection ( , + , and , + , mmol/l for standard and tritium labeled insulin, respectively). lowering of the glucose level in animals obtaining labeled insulin was the same, as in animals obtaining standard insulin. thus, hscie reaction of insulin with tritium doesn't change its hypoglycemic activity. cortexin is polypeptide (up to kda) brain extract accepted for clinical use in several countries including russia due to its positive effects on memory, attention, and cortical processes. a synthetic peptide analog of cortexin, cortagen (ala-glu-asp-pro), was recently developed. the heptapeptide semax (met-glu-his-phe-pro-gly-pro) is an analogue of the acth( - ) fragment, which is completely devoid of any hormonal activity associated with the full-length acth molecule. both cortexin and semax are currently effectively used for treatment of brain hypoxia and ischemia with comparable clinical benefi ts. however, the cellular and molecular mechanisms underlying the action in the brain are mainly unknown. in present work we studied effects of cortagen, cortexin, semax, its analogue pro-glu-pro (pgp) on glutamate-induced neuronal death and intracellular ca + homeostasis deterioration in cultured cerebellar granule cells. peptide drugs gain increased interest as a new supra-group of therapeutics between the classic-organic, small-molecule drugs and the large biotechnology-derived bio-drugs. they are used in different therapeutic areas like allergy, anti-infection, oncology, obesity, etc… due to their particular structure and biochemical origin, pharmaceutical development of a peptide drug poses special challenges which will be discussed here, exemplifi ed by own research as well as literature data. currently, there are about a dozen classical peptides described in pharmacopoeia, excluding the derived peptidomimetics like ace-inhibitors. these pharmacopoeial peptide-drugs, together with the approved marketing authorisations of new peptidic entities, are a good starting point to look at the desired characteristics. last, the regulatory developmental guidelines are in a general way seldom taken peptide drugs into account, leaving an interpretational gap between the two current main supragroups of therapeutics. after the initial active pharmaceutical ingredient (api) synthesis, analytical characterisation is aimed at integrity and purity evaluation of the api, which is also required for biomedical experiments. in this analytical characterisation, sample treatment issues like solubility and adsorption are considered as well. the chemical and plasma-metabolic stability, a critical parameter for peptides, is to be assessed to obtain kinetic and mechanistic information. functionality is tested in vitro using cell-and organ-based protocols, including ligand binding studies, as well as in vivo encompassing adme and target-organ confi rmation like brain. the pharmaceutical drugability information thus obtained allows further development decisions including required api modifi cations and proof-of-principle drug delivery formulations. lysine dendrimers and starburst copolymers as new carriers of anticancer drugs based on the complexes of platinum and gold. though the complexes of metals such as platinum and gold are very promising compounds for the treatment of different types of cancer, the low solubility complicates their application as medicines. in this work a series of lysine dendrimers of different structure and starburst copolymers of lysine and glutamic acid was studied as solubilizing carriers of anticancer drugs based on the complexes of platinum (cisplatin) and gold. cd analysis showed the small changes in the structure of the polymers upon metal binding. the esem study revealed that the presence of the amino groups on the surface of the carriers is crucial for the metal ligation. the highest content of metal bound was found in the samples with high amount of lysine residues. while the content of gold was rather low in all the samples (about %), the copolymer of lysine and glutamic acid with the ratio : as well as lysine dendrimer of fourth generation had to % of platinum linked to the carrier. the absence of the bromine in the samples in the case of gold complex allowed to assume the covalent bond between metal and polymer. in the same time all the conjugates were highly soluble in water. the peculiarities of the synthesis and the anticancer activity in different cell lines will be discussed. acknowledgements: the work was supported by the "russian science support foundation". prospects for the development of synthetic peptide vaccines against hepatitis c no vaccinoprophylaxis of this disease or cardinal treatment means has been developed till now because of the problems produced by high genetic variability and heterogeneity of hepatitis c virus (hcv) isolates, lack of both hcv infection models and effi cient expression systems for the virus and its components. the problem of anti-hcv vaccine development can be solved via a principally new approach, the creation of artifi cial synthetic immunogenic constructs with the help of bioinformatics and high-throughput screening technologies. two approaches exist towards the development of anti-hcv peptide-based vaccines: one is to develop vaccines that stimulate cytotoxic t-cell response (peptides loading dendritic cells; the so-called "cell vaccines") and the second is to construct immunogenic peptides raising protective antibodies. however, the fi rst approach may lead to the massive hepatocyte death and the development of the heavy hepatic injury during hcv infection. in order to develop peptide immunogenic constructs able to raise antibodies against whole native hcv envelope proteins analysis of amino acid sequences of these proteins belonging to different hcv genetic variants has been performed. this analysis has allowed to choose the most conserved and presumably functionally important protein fragments. immunogenicity of these fragments in the whole protein molecules has been determined and interaction sites for one of the putative hcv receptor, heparansulfate, have been revealed with the help of peptide scanning. despite of the low immunogenicity of the most fragments inside the whole protein, antipeptide antibodies against them have been obtained via immunizing by conjugates of the peptides with certain carriers. two synthetic peptide immunogenic constructs have been developed that have raised antibodies against whole hcv envelope proteins. synthetic immunoactive fragments of endogenous proteins: selection and application for diagnostics and immunotherapy we have selected and synthesized the immunoactive fragments of four endogenous proteins: nucleophosmin, survivin, prion and alpha- subunit of acetylcholine receptor (achr). nucleophosmin and survivin are overexpressed in tumor cells and exhibit an antiapoptotic activity. their detection in tumor cells could be used for tumor differential diagnostics and selection of optimal chemotherapy scheme. accumulation of pathogenic isoform of prion protein in brain causes the neurodegenerative prion diseases and antiprion antibodies as known could be used for immunotherapy of prion disorders. alzheimer's disease (ad) development is accompanied by an accumulation ofamyloid peptides ( a) in brain and destruction of neurons . one of the ad development hypotheses assumed that a when binding to the alpha- achr forms a complex that penetrates into the cells, resulting in the neurons death. we have proposed that alpha- achr could be a new target in the ad therapy and antibodies against alpha- subunit could prevent its binding to a. selected synthetic fragments of these four proteins were able in a free nonconjugated to a protein carriers state induce antibody response in experimental animals. obtained rabbit antibodies against fragments of nucleophosmin and survivin were affi nity purifi ed. antibodies against nucleophosmin detected monoand oligomeric forms of this protein in immunoblotting of hela cells lysates. antibodies against survivin were able to detect this protein in immunohistochemical test of the breast cancer tumor samples. rabbit antiprion antibodies interfered with patigenic prion isoform in scn a neuroblastoma cell culture. it was shown that synthetic alpha- achr fragments induced the antibodies formation in mice with symptoms of ad which penetrated the blood brain barrier, regenerated the spatial memory and normalized the beta-amyloid level in animal's brain. acknowledgements: supported by ras program mcb and rfbr grants - - , - - . investigation peptide-based cyclin a inhibitors: new tools to understand and to regulate the cell cyle-apoptosis signalling pathway the knowledge of the aetiology of cancer has increased considerably in the last two decades. the objectives have moved from unspecifi c cytotoxic chemotherapeutics to the identifi cation of small molecules (and antibodies) with a well defi ned molecular target. proliferation of eukaryotic cells is under control of a series of concerted molecular mechanisms defi ned as the cell division cycle whose progression is tightly governed by members of the cyclin-dependent kinase family. the protein-protein complexes formed between different cyclins and cdks (cdks) are central to cell cycle regulation. these complexes have been object of extensive research in cancer programs. considerable effort has been focused on the development of atp-competitive small molecule inhibitors of cdks. however, in general, these compounds have several alternative protein targets that compromise their demanded selectivity. cdk- was recently suggested to be dispensable for cell proliferation, although this does not appear to be the case for cyclin a, which therefore is defi ned as a more appropriate target for drug design. we have identifi ed an hexapeptide (nbi ) that inhibits the kinase activity of the cdk -cyclin a complex through selective binding to cyclin a ( ). the characterization of the inhibitory mechanism revealed that the hexapeptide does bind neither to the atp site nor to the cyclin recruitment site. a cell permeable derivative of the nbi peptide induces apoptosis and inhibits proliferation of tumor cell lines. we believe that the current structural and mechanisms of action studies on nbi will be useful for characterizing and controlling the important relation between cell cycle and apoptosis signalling pathways. the average size and size distributions of protein, polymer and peptideprotein or polymer-protein mixtures (complexes) and conjugates was investigated using photon correlation spectroscopy with a zetasizer nano zs instrument. the zeta potential measurements of this complex and conjugates were carried out in the folded capillary cell of the zetasizer nano zs instrument the same as used for dynamic light scattering measurements. in this study we investigate that size and zeta potential of synthetic peptide-carrier protein covalent conjugates. synthetic peptides are small antigenic molecules and not strong immunogens because of their small size for this reason, synthetic peptide must be coupling to a suitable carrier proteins or polymers (bsa, klh, ova) for increasing their immunogenity. most of the vaccine development studies are focused on the preparation of a good effective adjuvant. in this study we prepare carrier-protein (bovine serum albumin protein)-synthetic peptide conjugates whit carbodiimide method by using -ethyl - -( dimethylaminopropyl) carbodiimide hydrochloride (edc). for one protein molecule we studied with different ratios of peptides (npeptid/ nbsa) and synthesized the conjugates at these ratios. conjugates are purifi ed by using different column systems. purifi ed conjugates are characterized and the mechanism of the binding process was investigated comparatively with synthetic peptide, bsa and bsa-synthetic peptide physical mixture by using zeta-sizer nano zs depent on the time. an analysis of deletion mutants of the pld d domain defi nes short regions within the pld interacting with ped/pea : implications for the development of peptides-specifi c antagonist. ( ) . several studies have revealed that it regulates cellular functions by binding components of intracellular transduction pathways. recent reports also evidenced that it binds to phospholipase d (pld ) and enhances its stability, resulting in increased intracellular levels of diacylglycerol( ), deregulating protein kinase c signalling and generating resistance to insulin action on glucose transport ( ) . thus, disrupting the interaction between these proteins by a cell-penetrating compound represents a novel strategy for improving insulin sensitivity in target cells. the expression of d domain, (the shortest ped-interacting region with pld ) in l skeletal muscle cells stably overexpressing ped, reduces the interaction of this protein with pld ( ), suggesting that this region could bind ped preventing its interaction with the whole pld and restoring insulin action. aim of this work is the identifi cation of d crucial residues for ped interaction and the development of specifi c antagonists. we expressed three soluble truncated d domains, determining whether binds to ped like the d wild type, by carrying out dose-response elisa. only one of these regions exhibited an effi cacy similar to d -wt and functional cellular data support this evidence. to further investigate the d residues involved in ped binding, we prepared a set of overlapping peptides covering the d -region identifi ed. our results suggest that the n-terminus region of d encompassing residues - is involved in ped/pea recognition and, currently, cellular experiments on peptides are underway. ew-peptide family. one family, two drugs. two dipeptide analogs -l-glu-l-trp ( ) and -d-glu-d-trp ( ) -had been registered in russia as immunomodulator thymogen ( ) and immunosuppressor thymodepressin ( ). these two drugs possessed reciprocal activities in vivo [ , ] . the individual peptides were initially separated by preparative hplc from the crude thymus homogenate and sequenced. dipeptide l-glu-l-trp was the most active in the majority of in vitro and in vivo tests. in the process of sar studies, the "signal" role of l-glu-l-trp in the immune response was discovered, as well as the critical role of manifestation of in vitro effects as well as the infl uence of precise chemical and optical structures on biological activity of different ewpeptide analogs will be discussed. the experiments were conducted on blood neutrophiles, monocytes, thymic epithelial cells, lymphocytes, thymocytes, endothelial cells of human blood vessels and newborn blood cord. we evaluated the peptide effects on the phagocyte activity of cells, on the expression of functionally important membrane molecules and cell activation markers, and on the capacity of cells for mutual adhesion. the dose dependence data of and on such activities as cytokine production, the processes of stimulation and suppression of apoptosis and proliferation of immunocompetent cells will be presented. ion exchange chromatography (iec) is widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro, specially designed for separation of proteins, peptides and nucleic acids. ymc-biopro iec columns are based on micron @porous and non-porous hydrophilic polymer beads with low nonspecifi c adsorption, and they show higher binding capacity and higher recovery of biomolecules compared to conventional iec columns. the completely spherical and monodispersed beads, with optimal packing technology, provide high theoretical plate number and symmetrical peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp ion exchangers. in this poster, we will show benefi ts of ymc-biopro iec columns and some example cases of superior separation of important biomolecules, such as monoclonal antibody and dna. the analysis of proteins and peptides by rp-hplc is important in the development of well-characterized biotechnology pharmaceuticals. since polypeptides interact only slightly with the stationary phase after desorption, short columns are suited for fast separations. also, by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short hplc columns packed with . um, large pore c silica for ultra-fast biomolecule analysis. due to the short column format, high fl ow rates and optimal separations are possible, with moderate backpressures (< psi), using a conventional high-pressure gradient, binary pump system. narrow-bore lc and lc-ms analyses were performed on a binary or a quaternary hplc system, with detection by uv or ms (ab/mds sciex q trap). the solvent system comprised of acn/water or n-propanol/water plus one or two ion pairing agents (formic acid, tfa, hfba) at . to . %. a fl ow rate of . ml/min ( cm/h), x higher than typical for a . mm id column, allowed for ultra-fast one minute separations of proteins with broad physicochemical characteristics. closely related insulin variants (bovine, sheep, human) may be separated in under . minutes. while the typical hplc run time for the separation of hemoglobin chains on conventional columns ranges from to minutes, runs under two minutes may be realized with the large pore, sub-two micron column. accordingly, fi ve different hemoglobin samples (from different animal species) may be compared after a total of only minutes (includes run and equilibration time). highly reproducible runs of a synthetic peptide mixture can be completed in < minutes, including equilibration with column volumes between runs. intact igg in sheep serum may be separated from albumin in < min. using a n-propanol/water solvent system containing . % tfa. new peptides and triterpene derivatives as dimerization inhibitors of hiv- protease protein-oligopeptide fragmentomics zamyatnin the substantiation and defi nition of the term "fragmentomics" is given. within the framework of this scientifi c direction the theoretical structural-functional analysis of all possible fragments of protein molecule can be performed with the purpose of determination of its sites which could be potential sources of the regulatory oligopeptides. the data on the primary structure of proteins from public protein databases, information from the erop-moscow database ( ) that contains data on the structures and functions of the natural oligopeptides, and special computer program complex were used for it. as a result the natural regulatory oligopeptides both representing exact structures of protein fragments and containing protein fragments were found. the method has allowed also to reveal new potentially active sites of protein amino acid sequence yet not investigated experimentally. it was shown that different fragments of the food protein molecules are involved in amino acid sequences of many natural antimicrobial oligopeptides, toxins, neuropeptides, and hormones. these results confi rmed deep relationship between the basic regulatory systems. in connection with the obtained data, the process of oligopeptide biogenesis, the possibility of natural formation of regulatory oligopeptides from different protein molecules, formation of exogenous oligopeptides pool, and correspondence of the obtained results with the conception of oligopeptide continuum are discussed. a possible practical importance of active fragments of proteins in regulatory processes of a living organism is noted. the binding of peptides containing tyrosine residue to -cyclodextrin cyclodextrins form inclusion complexes with various organic molecules. aromatic amino acid residues bind to -cyclodextrin with deep penetration of the cyclodextrin cavity. in case of oligopeptides binding depends on the peptide conformation. the formation ofcyclodextrin inclusion complexes with the tyrosine residues within three peptides was investigated using steady-state fl uorescence spectroscopy and molecular dynamic simulations. the free energy along the reaction pathway delineating the inclusion of tyrosine's aromatic ring into -cyclodextrin was computed using umbrella sampling molecular dynamic simulation. the association constant and the corresponding association free energy were derived by integrating the potential of mean force over a representative ordering parameter. the three peptides studied consist of eighteen amino acids and the tyrosine residues are located at the position , or . selected sequences are fragments of notch receptors. (notch =ykieavqsetveppppaq, notch =tlsyplvsvvsesltper, notch =pyplrdvrgepleppeps). out of three peptides only notch binds strongly to -cyclodextrin with binding constant similar to that of actyrnhme. the binding of cyclodextrins with phenolic compounds involves nonspecifi c van der waals and hydrophobic interactions and depends on accessibility of tyrosine sidechain. role of carboxyl groups in the secondary structure and function of sturgeon gonadotropin free negatively charged carboxyl groups were selectively modifi ed (neutralized) in sturgeon (acipenser güldenstädti br.) gonadotropic hormone (gth) and subunits. carboxyl groups, in and in subunit, were neutralized by the reaction with glycine ethyl ester. investigation of re-associated -dimers (recombinants) comprising one or both modifi ed subunits showed that specifi c hormonal activity was completely lost while immunoreactivity was lowered in comparison with that of the standard -dimer. cd-spectroscopy of the modifi ed subunits did not indicate considerable changes in their spatial structure. conclusion was made that free cooh-groups of gth are important as bearers of the negative charge necessary for the hormone activity on the level of the hormone-specifi c membrane receptors. wagner, nathaniel; dadon, zehavit; yishay, eliya; ashkenasy, gonen ben gurion university of the negev, israel living cells can process rapidly and simultaneously multiple extracellular input signals through the complex networks of evolutionary selected biomolecular interactions and chemical transformations. recent approaches to molecular computation have sought to mimic or exploit various aspects of biology. a number of studies have adapted nucleic acids and proteins to the design of molecular logic gates and computational systems, while other works have affected computation in living cells via biochemical pathway engineering. we described recently the graph structure and experimental analysis of a self-organized synthetic peptide network ( - ). the system was designed rationally to operate in neutral aqueous solutions based on sequence selective auto-and cross-catalytic template-directed coiled-coil peptide fragment condensation reactions. here we show that such de novo designed synthetic networks can also mimic some of the basic logic functions of the more complex biological networks. consequently, we describe experimental and simulation studies that highlight the possibility of segments of the networks to express all sixteen two-input boolean logic functions ( , ) . many studies deal with the folding of double helices in nucleic acids. in contrast, much lesser attention is given to double helices in peptides. nevertheless, the investigation of peptides with alternating l-and d--amino acids, like gramicidin a, shows various double helix patterns with antiparallel and parallel arrangements of the strands. in this study, we want to give a systematic overview on the possibilities of double helix formation in -peptides on the basis of ab initio mo theory. according to general principles, double helices with characteristic intermolecular hydrogen bonding patterns were generated and verifi ed as minimum conformations at the hartree-fock level employing the - g* basis set. the calculations show several types of antiparallel and parallel double helices with different stability, which is strongly infl uenced by backbone substituents. the dengue virus causes a spectrum of clinical symptoms, ranging from mild dengue fever to the severe forms of dengue hemorrhagic fever and dengue shock syndrome. there are four dengue virus serotypes (den - ). the dengue virus ns protease is an attractive target for development therapeutics against dengue. the aim of the study was to investigate the prime side specifi city of dengue virus ns protease substrates using proteochemometrics, a new technology for drug target interaction analysis. a set of internally quenched peptides were designed using statistical molecular design (smd), synthesized and assayed with proteases of four subtypes of dengue virus for michaelis (k m ) and cleavage rate (k cat ) constants. the obtained data were subjected to proteochemometrics analysis, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. the interpretation of the models suggested that considerably differing physico-chemical properties of amino acids (aa) contribute to k m and k cat activities. it was found that for k cat activity only p ' and p ' prime side residues played important role, while for k m all four prime side residues, p '-p ', were important. high cleavage rate (characterized by k cat ) was obtained for peptides having small aa (ser, gly, ala) at the p ' position and small or fl exible aa at the p ' position. for high affi nity (low k m ), at the p ' position aa should be small and moderately hydrophilic, while acidic residue is highly unfavorable. for the p ' position, the most favorable was trp; however, this position allowed a broader diversity of aa. for the p ' position, cys was more benefi cial than the aa present in native cleavage sites of the dengue polyproteins. for the p ' position, the best affi nity would be obtained with ala, gly or cys. the models may be used to modify each p' substrate position to optimize separately substrate affi nity and cleavage rate for den - proteases. identifi cation of novel bioactive peptide sequences from human proteins for the development of potential therapeutics many protein-protein interactions are facilitated by the binding of peptides recognition modules to short linear motifs within the target proteins's primary sequence. therefore, synthetic peptides based on parent sequences of human proteins containing functional motifs are useful tools to uncover protein signaling and interactions. to date, peptide studies typically derive sequences from a single identifi ed protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. the objective of the novel bioinformatic approach presented here was to determine whether rational design of oligopeptides specifi cally targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function ( ) . the aim of this project was to identify peptides that span residues strongly conserved in the corresponding proteins in other species (orthologues), but that differ from those in related human proteins (paralogues). synthetic selection rules to avoid unfavorable amino acid combinations and excessively hydrophobic peptides were devised to reduce the risk of by-products during synthesis, postsynthetic degradation and solubility problems. high-throughput in vitro platelet function assays of fatty acid-modifi ed cell-permeable peptides corresponding to these regions identifi ed many agonists and antagonists of platelet function. the combined bioinformatic and experimental screens of human protein subsequences can therefore be used to validate functions of candidate proteins and provide templates for the development of potential therapeutics. denessiouk, konstantin; denesyuk, alexander; johnson, mark s. Åbo akademi university, finland many small molecule ligands (or parts thereof) with important roles in the biochemistry of the cell are recognized by different proteins with different cellular functions. these proteins do not necessarily share a common fold since their three-dimensional structures can differ completely such that they are considered to be unrelated proteins. surprisingly, we see on numerous occasions that similar ligands are often recognized in a very similar way by means of a common structural motif formed from main-chain elements of several consecutive amino acids within the ligand-binding site of the proteins. these short polypeptide segments can be found in many protein folds, and variations in their structure can often explain different elements of protein function. in particular, we have characterized the presence of a nucleotide binding motif present in more than protein folds that function in part to recognize the adenine ring system in many essential biological ligands (e.g. atp, nad(p), fad, fmn, coa, etc.). furthermore, a study of unrelated folds among pyridoxal phosphate dependent enzymes led to the characterization of a c nn structural motif for protein recognition of phosphate ions common to fold-representative protein structures that belong to different folds. interestingly, amino acid side chains play little or no role in ligand recognition, explaining the evolutionary independence of such binding mechanisms (i.e. the motifs are found in unrelated folds), while possibly refl ecting the types of interactions that were characteristic of the earliest protein-ligand interactions during early stages of molecular evolution. bioinformatics meets medicine: structure-based peptide design as a basis for drug development here, we present different structure-based approaches on the design of peptides mimicking the whole surface, a specifi c region of a protein or even tumor markers. the potential of such methods stretches from the development of peptide-derived drug candidates to immunological screenings. mimicry of binding sites: we have developed superficial, a program that proposes peptide libraries representing the entire surface or just regions of proteins. these peptides can be synthesised, e.g. using the spot-synthesis technique, and tested for their ability to mimic linear as well as non-linear binding sites. as a proof of principle, a library of peptides representing the surface of lysozyme was generated starting from a crystal structure of a lysozyme-hyhel- complex. adjacent sequence segments were linked by spacers to conserve local conformations. disulfi de bonds were generated to tether the peptides. binding assays against the hyhel- antibody identifi ed several peptides representing the non-linear recognition site of the lysozyme. translation of a tumor marker into peptides: the tumor-associated carbohydrate antigen gd is an established target for immunotherapy in neuroblastoma. we proposed peptidic mimics of this ganglioside for active immunisation against the tumor marker gd using molecular modelling. the ability of these peptides to mimic the carbohydrate moiety was verifi ed with in silico docking experiments. in a next step they were successfully tested as mimotopes in a mouse model. design of switchable peptides: photo-switchable compounds are becoming increasingly popular for a series of biological applications. goal is the reversible photo-control of structure and function of biomolecules. a required death domain for the binding of proapoptotic proteins (e.g. bak) to the hydrophobic groove of anti-apoptotic proteins is the bh helix. inserting the photo-reactive compound hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. the identifi cation of peptides that bind to antibodies is an important step in characterizing antibody specifi city in order to study molecular recognition. numerous approaches have been developed to select peptides that bind with desired specifi city and affi nity to antibodies of interest. in our study peptide arrays produced by spot technology were used to develop and optimize binders to antibody derived by mutations from the anti-p (hiv- ) single chain fv antibody, cb - . three mutations were introduced in the complementarity determining region of the light chain being in close proximity with epitope-homologous peptide. this mutated antibody had completely lost its affi nity for the epitope-homologous peptide. a substitutional analysis of epitopehomologous peptide was performed on cellulose, in which all positions of the peptide sequence were substituted by each of the naturally occurring amino acids. the peptides representing the binding activity in spot membranes were synthesized using solid phase synthesis and their binding activity was confi rmed by fl uorescent polarization method. a substitutional analysis indicated that binding to mutated antibody could be restored and improved by combining favourable substitutions at the n-and c-terminal residues of the epitope-homologous peptide. this approach has allowed us to generate binders from non-binders to mutated antibody in high micromolar range. it is well known that physiological regulatory peptides that act as hormones and neurotransmitters are produced by the specifi c cleavage of their precursor proteins that per se have no biological functions. during these processes, many fragmented peptides are also produced from the same precursor proteins. it is expected that they may have various unexpected biological activities, but their biological functions have not been investigated in depth. the neutrophil-activating peptides we recently purifi ed turned out to be the peptides that are cleaved from mitochondrial proteins by proteolysis, suggesting that fragmented peptides produced by maturation and degradation of functional proteins may also have various biological functions [ , ] . therefore, we named such functional cryptic peptides hidden in protein sequences cryptides, and those cryptides that are derived from mitochondrial proteins "mitocryptides" in particular ( ). we then identifi ed many mitocryptides by the combined investigation with "dry" and "wet" experiments, i.e., the sequences of mitocryptides that activate g proteins were predicted based on the distribution of charged and hydrophobic residues and their activities were examined on granulocytic cells. receptors for these peptides were also characterized by the direct cross-linking experiments between peptides and their targeted proteins. moreover, it is demonstrated the presence of a novel signaling mechanism involving mitocryptides. the comprehensive identifi cation of cryptides is expected to lead to the elucidation of various cryptic signaling mechanisms. the -benzoylphenylalanine (bpa) residue is widely used as a photoaffi nity label for the study of intermolecular (peptide) ligand-protein (receptor) interactions, where it is thought to function by hydrogenabstraction from met residues followed by covalent c-c bond formation of the resulting radical pair. we are carrying out detailed studies of this reaction in a series of fi ve, structurally rigid, hexapeptides of general sequences boc-u x bu y mu z -ome and boc-u x mu y bmu z -ome, where b = l-bpa, u = aib, m = l-met, and u x +u y +u z = , with a view to determining the effects of spacer length (u y = - ) on the rate of the intramolecular excited state reaction and the chemical and d-structures of the resulting products. the triplet state lifetimes of the bpa residues in the compounds, determined in a deoxygenated, dilute, acetonitrile solution by laser fl ash photolysis, vary in the following order: ubu mu, = ns; umu bu, = ns; ubumu , = ns; ubmu , = ns; and ubu m, = ns. in addition to the information these data provide on the structural requirements for intramolecular excited state quenching in the molecules, they also serve to defi ne the conditions necessary for optimal intramolecular reaction in preparative photolysis experiments. accordingly, the products resulting from ubu mu and ubumu have been prepared in high yields, isolated, and structurally characterized by hplc, mass spectrometry, nmr, and cd techniques. for each of the two photoinduced macrocyclizations, two diastereomers, arising exclusively from the bpa diradical regioselective attack on the met s-methyl group, were found. gattin, zrinka; van gunsteren, wilfred eth, switzerland during the past decades, the dependence of the secondary structure of peptides on their amino-acid sequence has been the focus of numerous investigations. in addition to naturally occurring peptides based onamino acids, peptides based on -amino acids, have raised particular interest because of their potential for pharmaceutical use. these peptides often have high folding propensities and it has been found that peptides with as few as four residue may fold into a stable secondary structure. -peptides offer the possibility to investigate the folding-unfolding equilibrium in the context of very small systems, therefore they have also been the subject of a number of investigations based on atomistic molecular dynamics (md) simulations which revealed that the foldingunfolding equilibrium typically occurs on time scale of nanosecond to tens of nanoseconds. how the backbone bound heteroatoms (oh, nh ,f) infl uence the structure of a peptide is little known by now, both experimentally as well as theoretically. we performed several md studies on the conformational behavior of centrally placed hala( -met) fl uoro and hydroxy analogues, hala( -f) and hala( -oh), as function of chirality and level of substitution to investigate the infl uence of these factors on secondary structure formation and stability. all -peptides were simulated in methanol solution at temperature of k and a pressure of atm using the gromos biomolecular simulation software and the gromos a force fi eld. the ensembles of trajectory structures were analyzed in terms of conformational space sampled by the peptide, folding behavior, structural properties such as hydrogen-bonding and in terms of the level of agreement with the available experimental nmr data, noe's and j-coupling constants. the nitroxide spin-labelled , -cyclic amino acid poac was synthesized, resolved and its absolute confi guration assigned (k . wright et al., tetrahedron, , , - ) in order to be used as a spin-probe to evaluate the -helicalpeptide secondary structure. a series of -hexapeptides, b o c -a c p c -p o a c -p o a c -a c p c -a c p c -a c p c -o m e , b o c -a c p c -p o a c -a c p c -p o a c -a c p c -a c p c -o m e , boc-acpc-poac-acpc-acpc-poac-acpc-ome, and b o c -a c p c -p o a c -a c p c -a c p c -a c p c -p o a c -o m e , based on the ( r, r)-poac enantiomer, combined with ( s, s)-acpc for confi gurational homogeneity of the amino acid components, was designed. in these hexapeptides two poac residues are incorporated at positions i, i+n (n = - ) to observe conformation-related spin-spin interactions. the peptides were synthesized by n-to -c chain elongation of n -boc protected peptide segments in solution. their conformational analyses, performed by cd, ft-ir absorption and esr spectroscopic techniques, will be also described. computational design has been successful in creating new proteins. we focus on the design of a -sandwich protein which is challenged by general problems as h-bonds between neighboring strands being dependend on a tightly packed hydrophobic core and aggregation. an improved version of our program propac was tested to repack the backbone of several natural protein structures with high reproducibility. starting with backbone coordinates we found amino acid sequences by packing amino acid side chains with defi ned conformations (rotamers) into the core of -sandwich proteins. in a fi rst approach we have assembled four synthetic -hairpins on a cyclic peptide template (tasp) to form a -sandwich with two identical four-stranded antiparallelsheets. improvement of surface residues reduced the aggregation of the proteins. spectroscopic analysis shows a fold with a free energy near - kj/mol and confi rms the -structure by cd and ftir. we improved the design to a partial asymmetric -sandwich assembled from three different -hairpins. the synthesized molecule was the fi rst -sandwich molecule showing a defi ned fold in d-nmr. this data and results from the symmetric betabellins ( ) suggest that symmetrical sheets do not fold into defi ned structures. to synthesize completely asymmetric sheets we simplifi ed the synthesis of -sandwiches by directed coupling of two four-stranded antiparallel -sheets. the computed proteins were selected for tight packing of the hydrophobic core and analyzed for a stable fold during dynamic simulations. one of our goals is to fi nd a correlation between the experimentally determined protein stability and the parameters used for the selection of the residues in the hydrophobic core and at the surface. biocatalyst-catalyzed peptide synthesis using inverse substrates as acyl donor sekizaki previously we reported that the p-amidinophenyl esters behave as specifi c substrates for trypsin and trypsin-like enzymes. in these esters the site-specifi c group (charged amidinium) for the enzyme is included in the leaving group portion instead of being in the acyl moiety. such a substrate is termed an inverse substrate ( ) . inverse substrates allow the specifi c introduction of an acyl group carrying a non-specifi c residue into the trypsin active site without recourse to a cationic acyl moiety characteristic of conventional substrates. we also showed in an earlier study that inverse substrates were applicable to enzymatic peptide synthesis. therefore, a general method for the preparation of a variety of inverse substrates would be valuable. we designed two series of new type inverse substrates, p-(amidinomethyl)phenyl and m-(amidinomethyl)phenyl esters derived from n-(tert-butyloxycarbonyl)aminoisobutylic acid, were prepared. we also analyzed the kinetic behavior of trypsin towards these synthetic esters ( ) . they were found to be readily coupled with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide. the optimum condition for the coupling reaction was studied by changing the organic solvent, ph, and acyl acceptor concentration. the optimum standard condition was selected as follow: acyl donor (inverse substrate), mm; acyl acceptor (l-alanine p-nitroanilide), mm; enzyme, m; % dmso-mops ( mm, ph . , containing mm cacl ); Ž. an -aminobutyric acid containing dipeptide was obtained in high yield. streptomyces griseus trypsin was a more effi cient catalyst than the bovine trypsin. it was found that the enzymatic hydrolysis of the resulting product was negligible. peptides derived from nk- selective for phosphatidylserine as novel cancer agents chemotherapeutic agents, commonly used as anti-cancer drugs, have severe side effects, also affecting healthy human cells. natural antimicrobial peptides, and their derivatives, have gained interest as potential anti-cancer agents. under normal conditions, due to the asymmetric distribution of plasma membrane lipids across the bilayer, mammalian cells comprise phosphatidylserine (ps) only in the inner leafl et. in the case of malignant transformation inner leafl et ps can move to the outer leafl et and act as a surface marker. the surface exposure of negatively charged ps on various tumour cells makes these cells susceptible to killing by cationic membranolytic peptides such as nk- ( ).the aim of this study is to develop short peptide sequences still acting selectively towards ps exposed on cancer cells without damaging healthy cells. in order to use this new strategy with ps-specifi c peptides it is necessary to analyze the lipid composition of mammalian cancer and non cancer cell membranes. further as a basis for peptide activity studies the biophysical characteristics of cancer cell membranes and healthy counterparts with respect to lipid composition were determined by investigation of liposomal mimics composed of phosphatidylcholine and/or phosphatidylserine by dsc and x-ray. these model systems were also used to screen the activity of a series of peptides derived from nk- . fluorescence spectroscopy was applied to test the release of fl uorescence marker molecules from liposomes composed of solely ps, pc or pc/ps mixtures in the presence of various concentrations of peptides. data revealed that some nk- derived peptides have a high affi nity towards ps causing signifi cant leakage of liposomal content, whereas healthy mammalian cell mimicking pc liposomes were not affected. optimized peptides resulting from these experiments will be used for in-vitro studies on prostate cancer cell lines. in most moths, the sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (pban), a - amino acid neuropeptide that is released from the subesophageal ganglion into the hemolymph in response to physiological and environmental cues. pban exerts its pheromonotropic effects by binding to pbanr, a member of the rhodopsin-like family of g protein-coupled receptors (gpcr) that is predominantly expressed in the pheromone-producing cells of the female pheromone gland. the structure-activity relationship studies for bombyx mori pban have revealed that the shortest peptide with pheromonotropic activity is the c-terminal pentapeptide-amide, pban( - )-nh (fsprl-nh ), and that the c-terminal amide group is required for the pheromonotropic activity of pban. in this study, we have analyzed the solution structures of the c-terminal decapeptides derived from bombyx mori pban with an amidated and a free c-termini by two-dimensional nmr. the pheromonotropically active decapeptide-amide, pban( - )-nh (srtryfsprl-nh ), and its inactive counterpart, pban( - )-oh (srtryfsprl-oh), were dissolved in three kinds of solvents: ( ) mm sodium phosphate buffer (ph . )/ mm nacl/ . % nan in %(v/v) h o/ %(v/ v) d o (buffer a), ( ) mm dodecyl phosphocholine (dpc)-d in buffer a, and ( ) %(v/v) , , -trifl uoroethanol (tfe)-d in buffer a. the nmr data indicated that these decapeptides did not adopt specifi c conformations in buffer a, but they adopted specifi c conformations in the presence of dpc micelles or tfe. these peptides exhibited different chemical shifts for some protons in all the solvents used, and they took similar but different conformations in the presence of dpc micelles. the conformational difference between these peptides may refl ect their difference in pheromonotropic activity. structuring and membrane interactions of the human antimicrobial cathelicidin ll cathelicidins are a family of vertebrate host defence peptides with both a direct capacity to inactivate microbes and to modulate components of the innate and adaptive immune systems. they are characterized by a conserved pro-region carrying individual, highly variable antimicrobial sequences, that become active only after proteolytic release, and with quite different structural and aggregational features that lead to differential biological effects on prokaryotic or eukaryotic cells. ll- is the only human cathelicidin, and displays a broad-spectrum, medium sensitive antimicrobial activity in vitro that is accompanied by some cytotoxicty towards eukariotic cells at antimicrobial concentrations, and a strong capacity to modulate host immune and healing processes at lower concentrations. these activities likely all involve interaction with biological membranes at some point, and are related to its amphipathic, helical structure and strong tendency to aggregate in specifi c conditions. the latter is an evolved feature absent in some primate orthologues, whose activities appear more limited to a direct antimicrobial action. the structural and aggregational behaviours of ll and selected primate orthologues were systematically investigated by biophysical and biochemical methods. these included cd spectroscopy in different buffers, sds micelles or anionic or zwitterionic luvs, transmission ftir spectroscopy, dye release from liposomes, and atr-ftir spectroscopy on supported lipid monolayers, followed by atomic force microscopy to probe morphological effects on lipid order. data was also collected from antimicrobial, cell lysis and cytofl uorimetric assays, giving a more complete picture of the possible mode of action of these helical peptides, and highlighting the importance of biochemical parameters such as structuring and dimerization/oligomerization processes in the selective interaction with biological membranes, leading to cytotoxic or immunostimulatory effects. oligomeric structure of fowlicidin- , an antimicrobial/ anti-endotoxic peptide from the family of cathelicidin, in lipopolysaccharide bilayer a recurring theme in the structure-function correlation studies of the cationic antimicrobial peptides (amps) is that the formation of oligomeric structures in lipid environments by amps. self-assembly of amps may result disruption of the membrane (outer/inner) structures of the microorganisms by forming pores or ion channels. however, high-resolution oligomeric structures of amps in lipid environments are diffi cult to obtain. to-date, oligomeric structure at atomic resolution of any antimicrobial peptide has not been reported. secondary structures of amps are usually obtained in perdeuterated sds or dpc micelles, as a mimic to the inner cytoplasmic membrane, by nmr spectroscopy. cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. these peptides are synthesized as a part of large precursor proteins containing a well conserved cathelin domain and an extremely variable, in terms of length and amino acid compositions, cterminal region. the c-terminal part of the cathelicidins is bestowed with antimicrobial and lps neutralizing activities. here, we report a tetrameric structure of a -residue active fragment of fowlicidin- or vk , one of the fi ve cathelicidins found in chicken, in lipopolysaccharide by nmr spectroscopy. the tetrameric structure of the vk determined from trnoe is highly helical. a large number of trnoe cross-peaks were observed connecting residues far apart in the sequence. calculated structures reveal an anti-parallel arrangement of four helices. the interface of the tetramer appears to be rich in polar or charged residues delineating a plausible pore or channel. most of the non-polar residues are found to be exposed indicating plausible interactions with non-polar fatty acyl chains of lps or with membrane in general. the oligomeric structure of vk peptide may be useful to understand structure/activity relationship, in particular pore formation, by the helical antimicrobial peptides. the interaction of the antimicrobial peptide nk- with different lipid systems antimicrobial peptides are important components of the natural defense system of most living organisms against invading pathogens. these are relatively small (below kda), cationic and amphipathic peptides of variable length, sequence and structure. in this study, the antimicrobial peptide nk- , which is a amino-acid residue derivative of the cationic core region of nk-lysin, has been used. the used membrane mimetic model systems have different dimensionality: two-dimensional (monolayers at the air-liquid interface) as well as three-dimensional (vesicles, micelles) systems of different phospholipids. the aim of this study was to investigate the infl uence of peptide-lipid interactions on the lipid structure and vice versa on the secondary structure of the peptide. the peptide secondary structure was measured by cd (circular dichroism spectroscopy) in bulk and irras (infrared refl ection-absorption spectroscopy) at the air-liquid interface. the structure of lipid langmuir monolayers has been examined by numerous techniques such as pressure-area isotherm measurements, fl uorescence and brewster angle microscopy and x-ray techniques. charged as well as zwitterionic phospholipids have been used to study the adsorption of nk- . the peptide adsorbs at the air-buffer interface due to its amphiphilic character. it also inserts into uncompressed phospholipid monolayers. the peptide reorients from random coil in bulk to -helix lying fl at at the interface. the incorporation of nk- into a dppg monolayer leads to a different orientation (either -helix with an oblique orientation or random coil) and to the fl uidization of the aliphatic chains. nk- infl uences also the structure of ordered dppg domains. to assess the location of the peptide nk- in the lipid matrix, specular x-ray refl ectivity studies were carried out. wadhwani, parvesh; reichert, johannes; buerck, jochen; ulrich, anne s. karlsruhe institute of technology, germany antimicrobial peptides (amps) constitute an essential part of innate immunity and act against an external microbial invasion where as cell-penetrating peptides (cpps) can carry a biologically functional molecule through the cell membrane and are relevant in drug delivery developments. fusogenic peptides (fps) are active on membraneinterfaces and are instrumental in fusion of membranes which is vital for various fertilization and viral processes. membrane fusion properties of short amps and cpps have been seldom tested and have therefore eluded the attention of most investigators, instead only their cytotoxicity is investigated. there is general lack of understanding if these peptides are fusogenically active. we have investigated the ability of amps and cpps to trigger membrane fusion. as an example, hiv- -fusion peptide fp is used as benchmark to compare fusion activity of various amps and cpps. most fps are believed to be unstructured or conformationally fl exible ( -helix or -sheet); therefore the secondary structure of the peptides before and after the fusion reaction is investigated and correlated to their respective ability to execute membrane fusion. our results show that various amps and cpps which are capable to switch their secondary structure are also able to promote fusion to an extent that is even higher than the known hiv- fusion peptide fp . these results will be presented in the poster. interaction of the minimal active peptide sequence of human growth hormone releasing factor with negatively charged liposomes zschörnig, olaf; thomas, lars; weigelt, heiko; köhler, guido university of leipzig, germany the most bioactive peptides such as hormones and neurotransmitters do not exist in an ordered structure in aqueous solution. the conformation of the peptide changes from this fl exible unordered structure into an inherent one, only when it reaches a biomembrane. that's why it is important to investigate the such peptides like growth hormonereleasing factor (ghrf) in the presence of lipid bilayers. human ghrf is an amidated peptide consisting of amino acids residues. a lot of studies has show that the residues n-terminal part are the active core of the peptide. we studied the interaction of human ghrf ( - ) with phospholipid membranes. the interaction of ghrf ( - ) with the liposomes was investigated fl uorescence spectroscopy. to detect a fl uorescence signal the peptide were labelled at fi rst, th, th and th position with dansyl. the fl uorescence intensity of ghrf ( - ) at different lipid-peptide-ratios were analysed using a model, which includes the hydrophobic and the electrostatic interaction between the peptide and the phospholipid membrane. the hydrophobic binding constant of ghrf( - ) and its effective charge were determined using this model. further the peptides position in the membrane were investigated using spin labelled phospholipids, which are able to quench fl uorescence over large wavelength arrays. the quenching effi ciency will decrease by increasing the distance between fl ourophore and spin probe. the use of mol% tempo-pc, -doxyl-pc, -doxyl-pc and -doxyl-pc in the phospholipid composition of the liposomes allows the determination of the peptides membrane penetration depth. all results indicate, that the binding of ghrf (aa - ) to phospholipid membranes is increased strongly by increase of the surface charge density of the liposomes. the peptide is arranged parallel to the membrane surface and is localized in membrane-water-interface of the liposomes. folding propensity and biological activity of selected antimicrobial peptides bozzi, argante ; di giulio, antonio ; aschi, massimiliano ; rinaldi, andrea c. university of l'aquila, italy; university of cagliari, italy the innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (amps) to resist microbial invasion. crafted by evolution into an extremely diversifi ed array of sequences and folds, amps do share a common amphiphilic -d arrangement. this feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. it is generally agreed that amps are essentially unstructured in the aqueous phase and fold upon contact with the membrane, adopting an amphiphilic fold. this favours absorption of peptides onto lipid bilayer and their subsequent integration into the membrane with expansion of the outer leafl et, which in turn leads to membrane thinning and permeabilization. however, recent observation suggest that things might be more complicated than previously believed. indeed, md simulations coupled to cd spectroscopy, studies with model membranes, and antimicrobial assays, have shown that, at least for some peptides, a signifi cant correlation exists between the conformation adopted by the peptide in solution, i.e. before the interaction with membranes, and its antimicrobial activity. the linear peptides we have studied following this approach include two members of the frog skin-derived temporin family, namely temporin a and temporin l, and two members of amphibian bombinins h, i.e. bombinins h and h . we observed that the presence of a partially folded structure in water solution may facilitate, both thermodynamically and kinetically, the peptide folding in the microbial membrane, and thus favour biological activity. looking for such built-in conformational characteristics could well help to rationalize the different spectrum and level of activity recorded for cationic alpha-helical amps on membraneenveloped targets, and assist the design of improved analogs and biomimetic synthetic peptides with antibiotic properties. investigation and computational modelling of antimicrobial peptides linser the uprising resistance of pathogenic bacteria against treatments with conventional antibiotics emerged an acute search for alternatives. one class of promising alternatives are naturally occurring antimicrobial peptides. we present a comparative study of computational modelling of peptide properties with structural characterization of the interaction and antibacterial or haemolytic activity of three peptides (nk-cs, nkcs-[lp] and nkcs-[aa]). we compared computational interaction models with measurements of antibacterial and haemolytic activity, small angle x-ray and surface plasmon resonance data, and structure predictions by circular dichroism (cd). all peptides were active against escherichia coli (gram negative) and staphylococcus carnosus (gram positive) bacterial cultures, but the haemolytic properties against human red blood cells were found to be poor and indicated the peptides' selectivity. cd studies of the peptide secondary structure confi rmed the prediction of peptide helicity. the antibacterial activity can be correlated with a change of the hexagonal phase transition temperature of -palmitoyl- -oleoyl-snglycero- -phosphoethanolamine (pope) as determined by small angle x-ray scattering (saxs). the calculated peptide membrane affi nity is not related in linear way with the antibacterial activity. the reason for this might be aggregation as shown by surface plasmon resonance. the inverse hexagonal phase transition temperature was increased by the peptides and this promotes a positive curvature of the membranes. we assume that this curvature fi nally leads to the disruption of the model membranes. in summary an over all helical structure, electrostatic and hydrophobic parameters as well as strong amphipaticity are good measures to describe antibacterial peptide interaction. kier, brandon; andersen, niels university, united states over the last decade, -hairpins have emerged as excellent model systems for probing the intrinsic stabilities of portions of -sheet structure and for studying the sequence dependence of the folding dynamics of -strand association and alignment. for many decades, it has been established that peptide helices can be substantially stabilized ( - kj/mol) by starting the sequence with an n-cap (acetyl, asp or sede). no comparable strategy has appeared for reducing the end-fraying of -hairpins. fully folded models of hairpins have typically been constructed by conversion to a cyclic system: inserting another turn favoring sequence (e.g. d-pro-gly, pg) at the end of the hairpin strands. we now report a set of favorable through-space interactions that can serve to cap -hairpins. the discovery followed from nmr studies of ac-w-ixgk-wtg (x = p, n). these studies indicated a favorable face-to-edge (fte) w /w interaction which also allows for a g -hn to w indole ring h-bond and the sequestration of the thr hydroxyl by h-bonding to the acetyl. these interactions produce spectroscopic diagnostics: a cd exciton couplet together with extreme upfi eld shifts for he of the c-terminal trp ( . - . ) and hn of the c-terminal gly ( . - . ppm). these feature disappear when x = l-pro and the gly-hn shift returns to its coil value (as in ac-wtg) upon removal of the n-terminal ac. in its most favorable application, ch ch co-wipglwtgps, we were able to establish that dgu at k was . kj/mol ( . % folded) by backbone hn h/ d exchange protection measures. we now report that these interactions are retained in longer hairpins. peptides of the general formula, ac-w-(zz)ningk(zz)n-wtg (n = , , or ) display enhanced fold stability. in each case, the ac is essential to prevent end-fraying and to elicit the full set of chemical shift diagnostics of the " -cap". the unique structural features of polyproline peptides to adopt an extended, relatively rigid polyproline ii (ppii) backbone conformation in aqueous solution, has led to their widespread application as a "molecular ruler" in biological and biophysical investigations. in the left handed ppii helix the peptide bonds show the all-trans conformation resulting in an interterminal distance, which increases by . to . Å per proline residue. the same backbone conformation has been identifi ed in important proline rich protein-protein recognition motifs involved in the regulation of physiological processes like transcription, cell growth, cytoskeleton rearrangement and postsynaptic signal transduction via modular sh , ww or evh domains. to investigate the structural features of polyproline and proline rich sequences by fl uorescence resonance energy transfer (fret) and other spectroscopic methods, these peptides were labelled n-and c-terminally with different fl uorescent dyes. for synthesis of homopolymeric polyproline peptides with a sequence length of more than amino acids a fragment condensation strategy has been applied to prevent the occurrence of shorter side products which can perturb distance measurement by fret. spectroscopic analysis of these peptides indicated that even for short polyproline sequences (< residues) there is a remarkable deviation from the expected ppii conformation. fret measurements revealed that polyproline peptides show an increase of the mean interterminal distance of about . Å per proline residue. single molecule fret measurements of polyproline peptides with a length of amino acids point to a heterogeneous ensemble of different conformations caused by randomly distributed cis conformations resulting in diverse species with different interterminal distances. similarly the structural consequences of the integration of non-proline amino acids in a polyproline sequence have been investigated by fret. the unusual helix stability of a vegf mimetic peptide understanding helix stability and formation is a prerogative to elucidate mechanism of protein folding and design helix peptide with specifi c activity. peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of non natural amino acids or introducing constraints. in this communication, we report the stability characterization, via cd, nmr and md studies, of a designed, -helical, -mer peptide, composed only of natural amino acids, which activates the vegf-dependent angiogenic response ( ). this peptide shows an unusual thermal stability whose structural determinants have been determined. two factors, the n-terminal region and an hydrophobic interaction i, i+ , are found as playing a mayor role of this remarkable stability ( ) . these results could have implication in the fi eld of protein folding and in the design of helical structured scaffolds for the realization of peptides to be applied in chemical biology. adiponectin, a cytokine secreted by adipose tissue, which has been shown to affect lipid and glucose metabolism, attracts interest because it is a target for therapeutics in the metabolic syndrome. the molecule has a tendency to associate to form various multimers. although this multimer formation could modulate its biological function, the details of this mechanism are still unclear. thus studies on this system from the aspect of molecular assembly are interesting. the molecule consists of three domains, i.e. a variable (v), a collagen-like (c) and a globular (g) domain. the structure of the g domain determined by x-ray crystallography showed that it exists as a trimer but the remaining c and v domains have not been well characterized. we synthesized various parts of the molecule, c, g, vc, cg, and full length vcg in e. coli. the cd spectra of cg and vcg showed a positive peak around nm which is the hallmark of collagen structure. this may be attributed to the repeats of typical collagen type triplet, x-y-gly. the temperature dependency of these cd spectra showed the three states conformational changes of these peptides. the lower transition, where the positive peak disappeared corresponds to the melting of collagen like structure and the higher one corresponds to that of globular structure of the g domain. the apparent molecular weights determined by ultra-centrifugal analysis showed that these peptides exist in trimeric form at the intermediate state. however, neither c nor vc showed such transitions. these results showed that, contrary to our expectations, the contribution of the triplet of x-y-gly is not the dominant one for stabilizing the trimeric state. in order to explore the stabilizing factor, we are carrying out various experiments, such as mutation analysis, titration of ca ++ , redox reaction of disulfi de bonds and so on. one result is that ca ++ was shown to be a factor for increasing the thermal stability of the trimer. are v mutants of amyloidogenic protein -human cystatin c more or less resistant to denaturation conditions? jankowska, elzbieta; orlikowska, marta; radulska, adrianna; szymanska, aneta university of gdansk / faculty of chemistry, poland cystatins are natural inhibitors of cysteine proteases -enzymes widely distributed in animals, plants and microorganisms. human cystatin c (hcc) has been also recognized as an amyloidogenic protein directly involved in formation of pathological fi brillar aggregates which deposit in the brain arteries of elderly persons causing cerebral amyloid angiopathy ( ). our studies were performed to explore possibilities of preventing this lethal disease. the overall d architecture of monomeric human cystatin c is not known but its fold could be anticipated from the structure of the dimer which has been already determined ( ) . the dimeric cystatin c is created through the exchange of 'subdomains' between two molecules ( d domain swapping) and consists of two identical subunits in great extent reconstituting the fold of the monomeric chicken analogue of hcc. it is possible that similar mechanism is involved in cystatin c oligomerization and fi brilization process. the most signifi cant structural changes during dimerization process are observed in the l loop ( - , qivag). this loop occurred to be a hinge region in the d domain swapping event. with the aim to check implications of greater or decreased stability of this loop for dimerization and aggregation propensity of human cystatin c, we designed and construct hcc l mutants with val residue replaced by asp, asn or pro, respectively. the structural studies of these mutants and their thermal denaturation process have been performed by means of cd and ftir spectroscopies. results of these studies will be presented. azobenzene-mediated photomodulation of a collagen triple helix monitored by ir spectroscopy ( ), our most recent efforts were addressed to the design and synthesis of a photoswitchable collagen triple helix. by replacing in suitable positions of the ac-(gly-pro-hyp) -nh a pro and hyp residue with ( s)mercaptoproline, respectively, a side chain-to-side chain crosslinking of the collagen peptide was afforded with a purposely designed azobenzene derivative. as expected from modeling studies, self-association of the modifi ed collagen model peptide into a stable triple helix was observed with the trans-azobenzene clamp, while its photoisomerization to the cis isomeric state leads to unfolding processes as well assessed by nmr structural analysis ( ) . unfolding pathways can be studied by comparing ftir difference spectra induced by temperature or light. we found the photomodulation of the triple helix to be reversible and the effi ciency of the photoisomerization increased with temperature reaching a maximum value shortly below the melting point. ir spectroscopy was thus used to identify the optimal temperatures required for structure destabilization at suffi cient extents to enable unfolding by the weak driving force of the azobenzene clamp. the results confi rmed the correctness of the design and the ability of the azobenzene switch to photocontrol this complex tertiary structure, thus allowing for time-resolved monitoring of the triple-helix unfolding process. studies on various collagen model peptides (x-y-gly) where x and y are often imino acids, pro or hyp r ( (r)-hydroxyproline), have shown that hyp r at the y position plays an important role in stabilizing the collagen triple helix. however substitution of non-natural (s)-hyp (hyp s ) at both positions decreases the thermal stability of triple helix as (pro-hyp r -gly) exists in a stable triple helical state, whereas (hyp s -pro-gly) and (pro-hyp s -gly) are in a single coil state. similar effects on the stabilities of triple helices are provided by the substitution of fl uoroproline. however there is an exception that (fpro s -pro-gly) takes a triple helix at c whereas the counterpart, (hyp s -pro-gly) , is in a single coil state. although the difference could be explained by the steric hindrance of hydroxyl group in s confi guration, it is still controversial how much this hindrance could obstruct the triple helix formation. therefore, even though apparently the tripeptide with the hyp s -pro-gly sequence is not capable of triple helix formation, we could expected that (hyp s -pro-gly) n forms a triple helix as n increases. the transition temperature of (pro-pro-gly) n was shown to have the chain length dependency. here, we synthesized (pro-pro-gly) n and (hyp s -pro-gly) n (n= , ) and investigated their thermal stabilities. the cd spectra of (hyp s -pro-gly) showed the conformational transition from collagenlike triple helix to random coil with the melting temperature at c. the apparent molecular weight, determined by the sedimentation equilibrium method, showed that (hyp s -pro-gly) exists as trimer at c and as monomer at c. the melting profi le was also clearly detected by dsc. @thus, it is concluded that the existence of hyp s at the x position is not essential to interfere the triple helix formation. we are on course to investigate the stabilizing mechanism of the collagen structure. the infl uence of o-glycosylation on the folding and stability of -helical coiled coil peptides glycosylated proteins have been shown to play a key role in many biological events like cell-cell communication, immune response, cell adhesion, intracellular targeting, protease resistance, and many other processes. recently, there is a growing interest in the effect of glycosylation on the secondary structure of proteins, because of the association with the so called conformational diseases that arise from the dysfunctional aggregation of proteins in not native conformations. in this study we used -helical coiled coil based peptides as model systems to investigate the effects of serine-linked -galactose on both the secondary structure and amyloid formation tendency. thehelical coiled coil structural motif consists of two to seven -helices which are wrapped around each other with a slight superhelical twist. its simplicity and regularity have made it an attractive system to explore fundamentals of protein folding.( ) the importance of the coiled coil motif is obvious with the amount of % of all amino acid residues in the protein data bank being part of a coiled coil motif. at fi rst we examined to which extent and at which positions a glycosylation is possible without destroying the coiled coil structure. therefore, we systematically incorporated one to six serine-linkedgalactose units into several solvent exposed positions of a amino acid long coiled coil peptide. the hydrophobic dimerization face was not modifi ed. the preformed l-ser(ac --d-gal)-oh building blocks were introduced by convenient solid-phase synthesis following the fmocstrategy. folding and stability of the glycopeptides were monitored by cd spectroscopy. the amyloid formation tendency was investigated by a tht fl uorescence assay. aging, associated with decreasing protein homeostasis (proteostasis) capacity and increasing oxidative stress, is a prominent risk factor for amyloid diseases. alzheimer fs disease (ad), which is one of the most common amyloid diseases, involves intra-and extracellular amyloid formation by the amyloid bata peptide (abeta). however, how abeta can aggregate in vivo even though its physiological concentration (pc) is much lower than its critical concentration (cc) for aggregation is a mystery. we have proposed that covalent modifi cation of abeta by small molecule oxidation products can explain how abeta can form amyloid at pc. the aldehyde-bearing cholesterol oxidation product ( ), which can modify abeta by schiif-base formation, is an example of the products that could affect ad onset. however, signifi cant questions about the modifi cation of abeta by ( ) persist, including: does modifi cation by ( ) lower the cc of abeta aggregation into the pc range? and, is abeta modifi ed by ( ) able to aggregate at low concentrations on a biologically relevant time scale? in this study, these questions are answered by studying chemically synthesized analogs of abeta that are site-specifi cally modifi ed by ( ) at asp , lys , or lys . modifi cation at the different sites has a similar effect on the thermodynamic propensity for aggregation. in contrast, the effect of metabolite modifi cation on aggregation kinetics depended strongly on the modifi cation site. abeta modifi ed at lys formed amorphous aggregates fastest and at the lowest concentrations. in contrast, the appearance of thiofl avin-t positive aggregates at higher concentration was fastest for abeta modifi ed at asp( ). the infl uence of modifi cation site on the nature of the aggregates suggests that amorphous aggregation and fi brillization place different conformational demands on abeta. furthermore, these studies may partially explain how abeta can aggregate at nm pcs when the cc of unmodifi ed abeta is in the ƒÊm range. code, christian; domanov, yegor; kinnunen, paavo k.j. antimicrobial peptides are ubiquitous in nature and consist of several families of cationic amphiphilic peptides. they partition into lipid membranes and promote the segregation of anionic phospholipids ( , ) . we have shown several antimicrobial peptides, e.g. temporin b and l, indolicidin, and magainin to activate secretory phospholipase a (pla ) ( , ) and we concluded that this could represent synergistic action of these peptides/proteins in defense against microbes. the fact that the sequences of these amps are very different suggest rather non-specifi c mechanism to be involved. to pursue the latter in more detail we used förster-type resonance energy transfer (fret) between labeled pla and temporin b. interestingly, fret coincides with concentration dependant activation of pla hydrolysis of dipalmitoylphosphatidylcholine (dppc) liposomes. accordingly, temporin b and pla interact forming a supermolecular complex terminating into amyloid-like fi brils ( ). homo-oligomeric fi bers are formed on a slower time scale when pla interacts alone on dppc liposomes ( ) . a general mechanism of peptide induced pla activation forming heterooligomeric cofi brils is suggested. human islet amyloid polypeptide forms lipid-encased amyloid fi brils on supported lipid membranes domanov, yegor; kinnunen, paavo university of helsinki, finland islet amyloid polypeptide (iapp) forms fi brillar amyloid deposits in the pancreatic islets of langerhans of the patients with type diabetes mellitus and its misfolding and aggregation are thought to contribute to -cell death. increasing evidence suggests that iapp fi brillization is strongly infl uenced by lipid membranes and, vice versa, the membrane architecture and integrity is severely affected by amyloid growth. we performed direct fl uorescence microscopic observations of the morphological transformations accompanying iapp fi brillization on the surface of supported lipid membranes. within minutes of application in submicromolar concentrations, iapp caused extensive remodelling of the membrane including formation of defects, vesiculation, and tubulation. the effects of iapp concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. growth of amyloid fi brils was visualized using fl uorescently labelled iapp or thiofl avin t staining. two-colour imaging of the peptide and membranes revealed that the fi brils were initially composed of the peptide only, and vesiculation occurred in the points where growing fi bres touched the lipid membrane. interestingly, after - hours of incubation iapp fi bres became "wrapped" by lipid membranes derived from the supported membrane. progressive increase in molecular level association between amyloid and membranes in the maturing fi bres was confi rmed by förster resonance energy transfer (fret) spectroscopy. the possible role of lipid wrapping in stabilization of iapp amyloid in vivo is discussed. fibrinogen-derived peptides that mediate cell adhesion: structure and activity studies interaction of human islet amyloid poly peptide with phospholipid membrane vesicles dannehl, claudia; zschörnig, olaf university of leipzig, germany amylin, also known as human islet amyloid polypeptide (hiapp), is a -residue peptide, suspected to play a major role in the malfunction of insulin secretion in diabetes mellitus type ii. co-secreted with insulin in the beta-cells, hiapp, in higher rates destroys the barrier function of the beta-cells, leading to a failure in insulin production. because of its amyloidogenity, aggregates of fi brils can be observed in the islands of langerhans to indicate its overexpression. we studied the physico chemical properties of hiapp by observing changes in its structure depending on time and the surrounding media using maldi-tof-ms, atr ft-ir-spectroscopy. to understand the process of penetration and toxicity to cells, we performed leakage measurements of carboxyfl uoresceine containing phospholipid large unilamellar vesicles by means of fl uorescence spectroscopy. moreover we determined membrane binding of dansyl-labeled hiapp. at physiological ph value, hiapp is positively charged and thus negative charges at the phospholipid membrane surface accelerate the process of peptide folding. being random coil as initial state, a mixture of anti-parallel betasheet and alpha-helices emerges in time. in the presence of negatively charged phospholipids, hiapp aggregates can be seen within a few minutes after titration. also in absence of any free charges, as seen in water, fi brils grow and after an incubation for hours at °c, some alpha-helices are twisted and after two weeks, no random coil is detected anymore. titration of hiapp to carboxyfl uoresceine fi lled liposomes, showed different results concerning equilibrium time and maximal extent depending on age and preparation of the peptide. in particular the composition of the vesicles seems to determine their stability in the presence of hiapp. nanoparticle induced folding and fi bril formation of a coiled coil peptide nanoparticles present large surface areas and are capable to catalyze fi bril formation of peptides. ( ) . one assumes that the nature of surface controls which of the peptides will interact with the nanoparticles and that an enhanced local peptide concentration reduces the lag-time for aggregation. in previous studies, we reported the design of a coiled coil peptide that can adopt a random coil, -helical structure, and -sheet folding in dependence of ph and peptide concentration. ( ) here, we expose this peptide to au nanoparticles that are either negatively or positively charged. the interaction of peptide and nanoparticle was investigated by cd and uv/vis spectroscopy, gel electrophoresis, and transmission electron microscopy. we found that electrostatic interactions with au nanoparticles can affect the peptide folding resulting in more than one secondary structure, namely, a competition between the -helical and -sheet structures. the latter folding is very likely a consequence of the high local peptide concentration on the surface of nanoparticle that facilitates a -sheet fi brillation of peptide. moreover, several factors such as ph, peptide concentration, and size of the nanoparticle have a strong infl uence on the nanoparticle-mediated folding. these results provide valuable information on pharmaceutical applications of nanoparticles and will help to reduce possible adverse effects. antimicrobial peptides are synthesized by all living organisms as a part of their innate immune system. those molecules are endowed with a broad spectrum of activity against pathogens. they can be divided into two classes differing in the mechanism of killing: membrane disruptive antibiotics cause a dysfunction of the membrane and subsequent cell lysis; non-membrane disruptive peptides are focused on the intracellular targets. in both situations the initial stage consists in the interactions with the cytoplasmic membrane, in most cases without the exploitation of any receptors. a non-receptor type of interactions decreases the possibility of development of microbial resistance and makes peptides a very potent alternative to conventional antibiotics. in the face of the reduced effi ciency of traditional drugs there is an urgent need for the design of new therapeutic agents with the optimized activity. monte carlo simulations of peptide-membrane interactions can be one of the strategies. that method takes in the account biophysical properties of a membrane as well as structural and physicochemical nature of peptides. in our work we are focused on the development of new analogues of nkcs, a derivative of potent antibiotic peptide nk- . in the present study the outcome of monte carlo simulations is compared to experimental results of antibacterial tests performed against escherichia coli. the insight into the molecular mechanism of peptides activity is obtained in vitro using saxs method and artifi cial systems mimicking a bacterial cytoplasmic membrane. the results indicate that monte carlo modelling is a good tool to predict the peptide -membrane interactions and can be very useful for the design of novel antibiotics. a - peptide: structural features in membrane mimicking systems amyloid (a ) peptides, the hallmark of alzheimer disease, depending upon conditions, undergo conformational transition from soluble monomers to the highly toxic -sheet oligomers which form the mature fi brils. conformational and biological analyses were carried out on several different a fragments, to understand the role of the single residues in the fi brillation process. a - , is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. we recently reported the conformational analysis of the synthetic a - under several solution conditions, and analyzed the modulation of the conformational behaviour of a - peptide, by interaction with nicotine-like molecules. a - is the -mer a peptide, composed of the a - fragment, and additional n-terminal residues, known to be endowed with fi bril disaggregating activity. a - encompasses part of hydrophobic ( - ) and hydrophilic ( - ) a -regions. due to the amphipathic character of this fragment, common to the full a - and a - , a tossicological mechanism involving a -peptide-membrane interaction may be hypothesized. on these bases we decided to perform the structural investigation of the fragment a - in membrane mimicking systems including micelle and vesicles aggregates, differing for composition and structural complexity. high resolution three dimensional structure was solved by nmr spectroscopy in negatively charged sds and in dpc/ sds mixed micelles. fluorescence and epr spectroscopies were used to monitor the peptide lipid interaction. the data agree on the critical role played by the membrane composition on the stabilization of different conformers, potentially driving to different oligomeric and/or polimeric toxic species. machan, radek; jurkiewicz, piotr; benda, ales; hof, martin j.heyrovsky institute of physical chemistry, czech republic charge and hydrophobicity are important determinants of membrane activity of antimicrobial peptides. the model peptide lah whose charge can be easily controlled by ph of the medium ( ) is a very convenient tool to study the relationship between physical properties of peptide molecules and their membrane activity. it was shown that the changes in the charge of lah molecules result in changes in their orientation with respect to phospholipid bilayers ( ). in the present study, effects of lah on the properties of phospholipid bilayers at different ph values are studied by several methods. its infl uence on the lateral mobility of lipids within an spb and on the stability of suspensions of large unilamellar vesicles were characterized by fl uorescence correlation spectroscopy, showing evidence of peptide induced aggregation of lipids at basic ph. changes of hydration and local viscosity within the bilayer following changes in orientation of peptide molecules were measured by solvent relaxation technique. the effect of phospholipid charge was also taken into account in each case. several bioactive peptides, such as antimicrobial, cell-penetrating or fusogenic peptides, exert their biological function by interacting with cellular membranes. therefore, structural data on the location of these molecules inside lipid bilayers are very important for a detailed understanding of their mechanism of action. fluorescence spectroscopic methods are particularly suited to the study of peptide-membrane association, but give only low-resolution information on peptide position in the lipid bilayer. molecular dynamics simulations, on the other hand, can provide a very detailed picture of the peptide-membrane interaction, but need to be validated by quantitative comparison with experimental data. we applied several fl uorescence approaches, together with md simulations, to the investigation of two antimicrobial peptides: the lipopeptaibol trichogin ga iv, and pmap- , a member of the cathelicidin family. to perform the spectroscopic studies, a variety of peptide analogues containing a single fl uorophore were synthesized. fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the two peptides inside lipid bilayers, in particular as a function of peptide/lipid ratio. molecular dynamics simulations were performed by a "minimum bias" approach, starting from a random mixture of water, lipid and peptide, and following the spontaneous self-assembling of the lipid bilayer. the fi nal membrane-bound d-structure is in quantitative agreement with the position of the fl uorescent labels determined by depth-dependent quenching experiments. for both peptides investigated, the atomic details of md simulations provide new insights on the mechanism of membrane destabilization. acknowledgements: with the support of the ministry of education, university and research, and of foreign affairs of italy. cavaco-paulo, artur university of minho, portugal surfactant proteins (sp-) are found in lungs of mammalians. most surfactant proteins have a carbohydrate binding domain (crd) and neck domain. crd have defense function in mammalian mucosa's, by eliminating microbes, due to strong binding to sugars in the cell walls. neck domains are found to order phospholipids allowing the exchange of oxygen between the alveoli and blood. x-ray structures indicate that neck domains are alpha-helixes, but circular dicroism indicates that in the presence of phospholipids, those peptides present a disordered structure. neck domains with residues from natural sequences of sp-a, sp-b, sp-c, sp-d have been tested to be delivered in lipophilic media. only disordered structures would have the ability to cross the lipid barriers. the results obtained here indicated that neck domains possibly can be used as carriers to deliver molecules across skin and hair. fraternal twins ! -peptides and oligoureas are isosteric, isostructural foldamers endowed with yet distinct biomolecular recognition properties. in the fi eld of peptidomimetics, there has been a sustained interest towards the design of non-natural oligomeric backbones with new folding patterns. over the past years, the amide linkage has become the quintessential motif to elaborate folding oligomers. aliphatic and aromatic oligoamides (peptoids, -, -peptides) have provided numerous helical-folded structures, many of which have shown interesting biological activities ( ). interestingly, substituting urea for the ch -co-nh units in the -peptide backbone represent a spectacular case of isosteric and iso-structural replacement. detailed nmr studies of the resulting oligoureas revealed a helical fold very similar to that reported for the cognate -peptides. defi nitive confi rmation of this isostructural relationship came with the recent x-ray structure determination of the canonical . -helix of oligoureas. how such isosteric and isostructural oligoamide and oligourea backbones compare in biomolecular recognition events is an interesting question that we attempted to address in the present work. notably the two systems were compared for their antimicrobial activity, membrane interaction and disruption properties. both -peptides and oligoureas designed to mimic globally amphiphilic alpha-helical host-defense peptides have been synthesized and tested. the results showed a surprising dichotomy in bactericidal activity between the two isostructural systems and enlightened the unique antibacterial of amphiphilic oligourea helices. to question whether this functional difference results from differential membrane disruption activities, we have undertaken detailed physicochemical investigations using negatively charged phospholipid membranes as model systems. understanding of the cellular uptake of an amphipathic cell penetrating peptides complexed with sirna konate, karidia; divita, gilles; heitz, frédéric crbm umr -cnrs, france the effi ciency of delivery of macromolecules into living cells is very important for therapeutic purposes. the discovery of a class of peptides, known as cell-penetrating-peptides (cpps), with the ability to mediate translocation of various cargoes both in vitro and in vivo has provided new perspectives in the fi eld of delivery. we have designed a new secondary amphipathic cpp, caddy, which can transfect sirna. many studies have tried to elucidate cpp internalization mechanisms and there is currently still much controversy between endocytosis phenomena and direct interaction with membranes. however, elucidating the interactions between peptides and lipid membranes is essential to understand how caddy delivers cargoes in cells. to highlight these interactions, we have applied biophysical method to phospholipids monolayer and bilayer as model plasmic membranes. regarding the increase of the phospholipids monolayer surface pressure in presence of caddy, it is obvious that this peptide have high affi nity with lipids. while cholesterol presence does not induce anything in peptide insertion on a phospholipids monolayer, the cargo and a widely studied partner of the internalization: heparan sulfate of the gag' s family, do not induce the same behaviour. the intrinsic probe of caddy, tryptophan residues, and the fitc probe bound to the cargo allowed to follow and identify interactions between these two entities by fl uorescence spectroscopy. adding a bilayer vesicular solution unsettle these interactions, tryptophan residues interact with phospholipids while fitc probe are less embarrassed by peptides. caddy alone in solution is not structured but cd measurements show a typical spectrum of helical conformation in presence of phospholipids vesicles. and when the peptide complex its cargo, cd spectrum show a contribution of the sirna relevant of a conformational change inside both partners interacting together. high membrane coverage as the basis of antimicrobial peptide activity the interaction of the antimicrobial peptides (amps) omiganan (h-ilrwpwwpwrrk-nh ) and bp (h-kklfkkilkyl-nh ) with model bilayers was characterized. the activity and selectivity of these peptides could be attributed to a strong preference towards anionic membrane model systems, which mimetize bacterial membranes. regarding the interactions with bacterial membrane models, there were marked differences in the interaction patterns, as well as in functional properties of the peptides at high peptide:lipid ratios. these differences occurred for both peptides, despite their being unrelated in sequence and in occurrence in nature. such events at high membrane coverage could represent the equivalent at the molecular scale of the conditions at which the antimicrobial activity of the peptides is triggered. although the lipid: peptide ratios at these transitions are lower than phospholipids per peptide molecule, the plausibility of this hypothesis was demonstrated taking into account an estimate of the amount of lipid per bacterium, and the bacterial concentration in minimum inhibitory concentration (mic) assays. according to the partition constants obtained towards bacterial membrane models, these peptides are expected to reach, at the mic, precisely those high concentrations in the membrane. in addition, surface charge neutralization was shown to occur in these conditions. activity at high membrane coverage is thus likely not only for these peptides but also for any peptide displaying high membrane affi nity and micromolar mics, which is common amongst amps. biosensors based on artifi cial membrane system that permits the functional reconstitution of transmembrane receptors have been studied for the past decade. immobilized liposome encapsulates intracellular chemicals in the internal water cavity and provides a potential advantage over supported planar lipid membrane. to prepare a stable liposome adlayer, we have proposed an immobilizing method based on small-peptide modifi cation of solid surface. in the present study, we investigated kinetics of liposome adsorption on the surface-bound synthetic peptides which have several alanine, lysine and tryptophan residues as amphiphilic segment with a cysteine at terminus. the quartz crystal microbalance studies showed that the peptide sequences can be divided into two groups according to liposome-size dependence of langmuir adsorption constant. while the initial adsorption processes could be satisfactorily described by simple langmuir adsorption kinetics, the amounts of liposome adsorbed irreversibly were increased as time advances. the results from afm reveal that most of the liposomes are bound to peptidic surfaces as single fl attened particles without fusion together for several hours. we next performed the detection of ganglioside gm -lectin interaction on the liposome surface. all the binding constants and binding amounts, as observed by qcm, were fairly consistent with each other and and showed the effectiveness of our approach. herce, henry rensselaer polytechnic institute, united states recently we have proposed theoretically an energy independent pathway for the uptake of cell penetrating peptides (cpps) that challenges fundamental concepts associated with protein membrane interactions, h. d. herce and a. e. garcia, pnas, , ( ) . this mechanism involves strong interactions between cpps peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of cpps peptides by diffusing on the pore surface. this mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifi cally the arginine amino acids, contribute to the uptake. we will describe the details of this mechanism and present novel experimental results that directly validate the model. a comparative studies on lipid affi nity of cell penetrating peptides in presence or absence of cargo a growing number of natural and /or synthetic peptides with cell membrane penetrating capability have been identifi ed and described in the past years. these molecules have been considered as targeting structures for the delivery of bioactive compounds into various cell types. although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. in this work we try to connect the capability to interact with model lipid membrane of cpp and their structural and chemical characteristics in order to obtain a biophysical classifi cation that predicts the behavior of cpp-cargo molecule in cell system. indeed, the interaction with cell membrane is one of the primary step in the interaction of cpp with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how cpps may translocate a membrane without destroying it and how this interactions come into play in shuttling cpps via different routes with different effi ciency. we analyzed by fl uorescence spectroscopy the binding properties of six different cpps (kfgf, antp and tat derived peptides, and oligoarginine peptides containing , or residues) in absence or presence of the same cargo peptide (the [ - ]ptyr fragment of hs protein). the binding properties were correlated to the conformational and chemical characteristic of peptides, as well as to the cell penetrating properties of the cpp-cargo conjugate. results show that even if certain physico-chemical properties (conformation, positive charge) govern cpp capability to interact with the model membrane, these cannot fully explain cell-permeability properties. great deals of data show that alzheimer's pathology lead to the loss of neuronal functionality and synaptic plasticity. these effects seem to be the consequence of a toxic effect of a peptides related to their ability to modify cell membrane homeostasis. in particular, a peptides, as full length or in fragments, being in oligomeric or polymeric form, could alter membrane fl uidity and compromise its functionality. we have previously demonstrated that the - fragment of a peptide a ( - ) is able to penetrate into the outer leafl ets of the membrane. furthermore, it is able to alter membrane fl uidity changing membrane response to cholesterol, and thus affecting its ability to form low fl uid membrane regions named lipid rafts. a ( - ) is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. flavonoids are a group of naturally occurring, benzo--pyrone derivatives, ubiquitous in plants. they are endowed with tumor prevent activity and they act as antioxidants through a membrane mediated molecular mechanism involving the membrane ion transport. on the basis of the common ability of fl avonoids and a peptide of altering membrane fl uidity, we carried out an epr spectroscopic analysis of several fl avonoids -specifi cally quercetin, naringenin, rutin and naringin -in , -dioleoyl-sn-glycero- -phosphocholine (dopc) vesicles. the modifi cations of the dopc physio-chemical environment were analyzed in presence of fl avonoids and beta-amyloid fragment a ( - ). our results indicate that the addition of fl avonoids induces a decrease in membrane fl uidity. this effect is retained in presence of a ( - ). thus fl avonoid compounds could be able to antagonize the effect induced by amyloid peptide on membrane fl uidity and in this respect they could be therapeutically useful as neuroprotective agents. analyse lipid peptide interactions of p and lipo-p with membrane mimicking represented by micelles and vesicles. nmr structures of p and lipo-p in mixed sds/dpc micelles are reported; the positioning of p and lipo-p peptides with respect the lipidic surface is analyzed using -doxyl stearic and -doxyl stearic acid. epr analysis is carried out in the zwitterionic dimyristoyl phosphatidylcholine and the anionic dimyristoyl phosphatidylglycerol vesicles using several different lipid spin labels. both peptides bind to lipid bilayers and trp residues and lipidic chain of lipo-p show a important role in the process of peptide adsorption onto the membrane, to exert its antiviral activity. studies on human cell membranes were necessary to further establish the role of membranes in these peptides mode of action. this interaction was assessed by evaluating the effects that these peptides have on the membrane dipole potential of human erythrocytes, using the fl uorescence probe di- -anepps. in the presence of enfuvirtide or t- , a decrease in the di- -anepps fl uorescence excitation ratio dependent of peptide concentration was observed. these results show that t- has ten times more affi nity to the erythrocyte membrane than enfuvirtide, a factor that can be associated with the adsorption of t- on cholesterol rich membranes observed on the studies with membrane model systems. moreover, as a fraction of hiv associates with erythrocytes in vivo, these cells can have a role in delivering these peptides to the viral surface. the improved clinical effi ciency of t- relative to enfuvirtide may be related to its higher partition coeffi cient and ability to adsorb to rigid lipid areas on the cell surface, where most receptors are located upon membrane fusion. moreover, adsorption to the sterol-rich viral membrane helps to increase the local concentration of the inhibitor peptide at the fusion site. the platelet receptor iib plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affi nity for fi brinogen, which forms bridges between adjacent platelets and assembles them into an aggregate. the iib activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, that of talin with the cytoplasmic tail is the most important. it has been recently suggested that talinmediated iib activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) regions with talin f domain and that the n ply motif of , which can be phosphorylated at y , plays a critical role in this process. to evaluate the interaction of talin with the tail of integrin we designed and synthesized two peptides corresponding to the md and mp parts of in their carboxyfl uorescein-labeled form (md: cf-r akwdtannplyke and mp: cf-k llitihdrke ). emission and anisotropy fl uorescence spectroscopy was used to quantitatively assess the affi nities of these peptides for talin. furthermore, to challenge the role of the y phosphorylation in talin-iib interaction we also studied the binding of talin to the modifi ed analogue of md, cf-r akwdtannpl(ptyr) ke . our experiments revealed that the md and mp parts of bind tightly to talin and that y phosphorylation has an inhibitory effect on this binding. finally, circular dichroism studies of all peptides in aqueous solutions and mixtures with tfe were also performed in order to characterize structure-affi nity relationships. acknowledgements: gsrt and eu (epan yb/ ) for fi nancial support. the infection of human cells with hiv requires two initial steps: binding of the viral glycoprotein gp to the receptor cd followed by the interaction between the v loop of gp and a seven helix transmembrane coreceptor (ccr on macrophages or cxcr on t cells). the n-type glycosylation at asn of the v loop is assumed to play a crucial role in this process. in order to understand the interaction in detail, it is important to analyze the binding of v -glycopeptides to the membrane integrated coreceptors on an atomic scale. here, we present binding studies between multiple v -peptides and -glycopeptides and the coreceptor ccr by stdd nmr and surface plasmon resonance (spr). spr experiments were carried out by immobilizing the (glyco)peptides on an spr chip by amide coupling. several concentrations of ccr overexpressing human osteosarcoma (hos-r ) cells were passed over the sensor surface. the role of individual amino acids in the binding process to ccr can be analyzed by using different v -peptides and -glycopeptides. additionally, the observation of interactions between the glycopeptide ligands and the receptor proteins embedded in liposomes is possible by using the saturation transfer double difference (stdd) nmr technology. stdd nmr allows removal of all unwanted signals resulting from native binding processes in cells. ( ) . we used ccr liposomes derived from hos-r cells. substraction of an std spectrum of ccr liposomes from an std spectrum of the same concentration of liposomes incubated with a ligand allows recording of clean stdd nmr spectra presenting only signals of protons of the ligand interacting with the transmembrane receptor.( ) k d values of the ligand with respect to ccr have been determined in series of experiments with varying ligand concentrations. we could show that the binding epitope between hiv and ccr is formed by the carbohydrate at asn as well as the peptide. this knowledge is important for the design of new inhibitors. temperature dependant methionine proximity assays highlights conformational variations occurring through the mechanism of peptidergic gpcr activation arsenault, jason; clément, martin; leduc, richard; guillemette, gaétan; lavigne, pierre; escher, emanuel university of sherbrooke, canada g protein coupled receptors are invaluable for cell signal transduction mediated by external stimulus. pharmacological efforts towards these targets are of primordial importance albeit few efforts have resulted in structural characterisation, although rational drug design necessitates such information. recent advances in crystallisation of the -adrenergic receptor have been highly insightful and such a structure corroborates our results on the human angiotensin ii type receptor (hat ) using the methionin proximity assay (mpa) in identifying ligand receptor contact points. unfortunately, physical methods such as crystallography are still far from routine procedures and are limited to a static picture of a given receptor. in the present contribution we propose a more accessible method that permits analysis conformational variations of different receptor states through an energy landscape, spanning from low energy conformations to conformations at physiological temperatures. photolabelling, mpa mutants constructed on the wt receptor, the constitutively active receptor (cam) and a corresponding non-activable mutant across the temperature range reveal activation-status dependent labelling patterns. as an example, position becomes signifi cantly less accessible in the cam receptor compared to the wt receptor. ligand accessibility can be classifi ed from easily accessible (low temperature photolabeling) to less accessible residues (higher temperature photolabeling). this labelling pattern can be associated to the activation status of the receptor and may allow quantifying and identifying structural changes occurring during receptor activation. such structural understanding is crucial for future endeavours in rational drug design. capillary electrophoresis for diffi cult characterization of hardly soluble polypeptides recently, we have designed and synthesized polypeptides able to stimulate the natural plant defenses. these homopeptides were obtained by ring opening polymerization of n-carboxyanhydride (nca) with several initiators. ratio nca/initiator is determinant for the length of the polymers. on the bases of elicitor activity, we identifi ed a leader, called lapp , which results from l-ala-nca polymerization initiated by alaninol. the mixture of poly(alanine) with different lengths is diffi cult to analyze considering important solubility problems. malditof confi rmed polymerization despite limitation due to molecular discrimination during ionization. nmr in deuterated tfa was possible but only afforded the number-average degree of polymerization (dp). to obtain more detailed characterizations, we developed separation by capillary electrophoresis in new solvents mixtures based on hexafl uoroisopropanol (hfip) and water. this technique allowed us to separate the oligomers with baseline resolution. different parameters infl uencing electrophoretic separation were investigated and optimized conditions were established. this technique enabled a full characterization of the polymer distribution, with determination of number-average dp, weight-average dp and polydispersity index. the pertinence and the reliability of capillary electrophoresis for characterization of non-water soluble polypeptides have been confi rmed, with analysis of short and long peptide chains. weakly polar interactions support polypeptide structures although the sequence of a polypeptide appears to determine the secondary and tertiary structure, weak inter-residue interactions such as between aromatic side chains and other aromatic side chains, aliphatic side chains and the peptide backbone may contribute signifi cantly to the stability of the fi nal folded structure. recent advances in structural bioiformatics and computational chemistry made it possible to study precise strurctural features and energetics of these interactions in model peptides and miniproteins such as tc b, app and c-vhp. the interaction energies of the weakly polar interactions are of the same order as of the hydrogen bonds which occur in biopolymers ( - kj/ mol). furthermore, these interactions not only stabilize local secondary structures but also entire tertiary folds of miniproteins. acknowledgements: this work was supported by the nih-inbre grant (p rr ) and the carpenter endowed chair in biochemistry, creighton university trusova, valeriya; gorbenko, galyna v.n. karazin kharkov national university, ukraine a number of so-called conformational diseases including neurological disorders (parkinson's, alzheimer's and huntington's diseases), type ii diabetes, spongiform encephalopathies, systemic amyloidosis, etc., are associated with the deposition in tissue of highly ordered aggregates of specifi c peptides and proteins. despite the main structural elements of amyolid fi brils (particularly, cross--structure) are well-characterized, the mechanisms of fi brillogenesis remains poorly understood. accumulating evidence substantiates the idea that formation of fi brillar structures can be initiated and modulated by peptide/protein-lipid interactions. membrane-related determinants of fi brillization are thought to involve conformational changes of the peptide/protein, increase of its local concentration at lipid-water interface, specifi c orientation of aggregating species, neutralization of the peptide/protein surface charges by anionic lipid headgroups, particular arrangement of the inserted and solvent exposed segments of the peptide/protein molecule, etc. the present study was undertaken to explore the formation of lysozyme (lz) amyloidlike fi brils in the model lipid-protein systems. lipid component of the model system was represented by liposomes prepared from zwitterionic phosphatidylcholine (pc) and anionic phosphatidylglycerol (pg) lipids in the molar ratio : . fluorescence microscopy studies performed with fl uorescein-labeled and rhodamine-labeled lz showed the presence of long lz fi bers (length > ìm). the to elucidate the nature of the events preceding lz self-assembly into amyloid-like structures, the protein adsorption onto pc:pg vesicles was examined by monitoring fl uorescence changes of fl uorescein-labeled lysozyme. the observed sigmoidal shape of the adsorption isotherm is strongly suggestive of oligomerization of membrane-bound protein. it seems highly probable that such oligomers serve as nuclei in the membrane-assisted lysozyme fi brillogenesis structure and dynamics of photosystem ii lightharvesting complex revealed by high-resolution fticr mass spectrometric proteome analysis structure and dynamics of membrane-bound light-harvesting pigmentprotein complexes (lhcs), that collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identifi ed and characterized using high-resolution fticr mass spectrometry. lhcs from photosystem ii (lhcii) were isolated from the thylakoid membrane of arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel fi ltration into lhcii subcomplexes. tsekova, daniela university of chemical technology and metallurgy, bulgaria formation of fi brous supramolecular complexes from l-val derivatives and their arrangement in spherulites was studied applying spectrophotometric approach and electron-microscopic observations. experiments show that boiling one and the same compound with different initial concentrations in water to completely dissolving and posterior cooling to room temperature lead to formation of stable supramolecular complexes with different sizes and properties. they behave like polymer molecules which specifi c numbers of monomers depend on the initial concentration of the boiling solution, moreover at higher supersaturations they crystallize in sperulites which is typical for crystallization of polymer molecules. metal -organic gels based on the self-assembly of peptidomimetics and cu (ii) ions peptidomimetics constructed from l-val and nicotinic/isonicotinic acid are good low molecular weight gelators and their supersaturated solutions in a number of solvents turn into gels. they make complexes with some metal ions. mixing of pure solutions of the peptidomimetic and some cu (ii) salts lead to immediate gel formation in some solvents. this kind of compounds, named metal-organic frameworks (mofs), are new class of nanoporous materials and are very promising ones for applications in catalysis, pharmaceutical industry, etc. the stability and molecular structure of these complexes in some solvents is in the process of investigation. peptide metalloconstructs often possess particular conformations and hence display increased activities and metabolic stabilities. we investigated new rgd peptide analogs cyclized through oxorhenium / oxotechnetium coordination. the rgd sequence is known to bind specifi cally to of the known integrins, a family of integral proteins that plays an important role in tumor neoangiogenesis, development and proliferation. several cyclic rgd pentapeptides bearing an exocyclic tc- m oxotechnetium core have been proposed for molecular imaging, however few of them have displayed attractive selectivities and metabolic stabilities. structure, biological activity and metabolic stabilities of metallated peptides cannot be predicted. therefore, we preferred a combinatorial approach to generate a panel of tracers that may be evaluated by tumor imaging in mice. tracers were constructed from a rgd model of general formula : ns -x -x -x -rs where x , , are respectively arginine, glycine and aspartic acid analogs and r is a series of linkers that feature various lengths and geometries (ns is a n-bis(ethylthio) moiety). first attempts for synthesizing these peptides by the versatile ugi multicomponent reaction did not yield the peptides with suffi cient purities. a representative library of peptides was obtained by standard parallel peptide synthesis and purifi cation of all members. peptides were metallated either with rhenium or technetium using standard procedures. their resistance towards mice serum, glutathione and tumor extracts was evaluated and showed that compounds containing an aminoethanethiol linker displayed higher stabilities. infl uence of the chemical environment on isomers ratios of the metallated peptides was also investigated. finally, oxorhenium peptide coordinates were assayed as specifi c ligands of integrins. their oxotechnetium equivalents were evaluated for tumor imaging in mice. degradation products of desmopressin in phosphate/ citrate buffer it shows, that introduction of unsaturated residue to the peptide chain could be useful tool to design bioactive compounds with desirable structure [ ] [ ] . therefore full knowledge about relation between presence of dehydroamino acid and peptide's conformation is necessary to predict biological proper and to design newdrug. for that reason we have undertook conformational investigations of numerous peptides containing two dehydroamino acid residues ( z phe, e phe, ala) in peptide chain, in different position. the investigations were based on nmr measurements (standard d techniques and d experiments, typical for detection of hydrogen bonding) and theoretical calculations. conformational preferences of investigated systems were obtained on base of roesy and noesy experiments and calculations by use of x-plor and quantum chemical calculation is concentration overload or volume overload the best strategy for synthetic peptide purifi cation? as the complexity of synthetic peptides increases so the demands on the synthesis and purifi cation increase. whilst improvements in synthesis resins and techniques enables higher purity peptides to be produced, % purity is not achieved. for many applications post-synthesis purifi cation is still required. methods for the purifi cation at the mg level can be relatively straightforward but where there is a need to develop methods which may be used for larger scale production there are a number of additionly considerations. the method developed must be scaleable and the economics of the process must be compatible with the fi nal product costs. when looking to develop a purifi cation method the loading will be critical to the throughput and fi nal production costs. the selection of the purifi cation media and the purifi cation conditions are two of the major infl uences on loading as these determine capacity and resolution. however, it is also important to consider the the physical properties of the pepitide -especially its solubility. as part of the purifi cation method development a loading study must be performed -increasing the amount of peptide loaded onto the column. the most common way of doing this is to increase the concentration of the sample and keep the injection volume constant, concentration overload. but this does require that the peptide is readily soluble in a solvent compatible with the hplc purifi cation conditions. alternatively volume overload can be used where the concentration of the peptide solution is kept constant and the volume purifi ed increases. the most commonly used method is concentration overload. the work presented in this poster compares the two overload strategies, concentration overload and volume overload, for the purifi cation of synthetic peptides. the suitability of the two strategies for large scale manufacture will be explored. expansion of human stem cells by passive transmembrane transfer of homeoproteins . these results clarify the effect of hoxb in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein to the intrinsic lympho-myeloid differentiation potential of defi ned lymphoid progenitor subsets. finally, this supports the potential use of hoxb for hsc and hpc expansion in a therapeutic setting, by means of direct transmembrane protein transfer. tumor selective delivery by cell-penetrating peptides systemic delivery of therapeutic agents used for cancer chemotherapy today lead to undesired side effects and toxicity. increasing the selectivity of the anti-cancer agent will not only facilitate lower dosage for equal therapeutic effect, but also decrease the frequency of drug resistance. we have studied two different targeting approaches both based on cellpenetrating peptides. the fi rst approach is a chimera between cancer homing peptides and a cpp the other is a enzyme activated prodrug design. the cyclic peptide ccpgpegagc (pega) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. pega peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pvec, the conjugate is taken up by different breast cancer cells in vitro. additionally, the homing capacity of the pega-pvec is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. matrix metalloproteinases (mmps) are over-expressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. to exploit these characteristics, we designed a tumor cell-selective prodrug, by constructing modifi ed version of the already shown to be functional mtx-yta conjugate. it is an inactive pro form of a cell-penetrating peptide (nope) conjugated to the cytostatic agent metothrexate (mtx), selectively cleavable and thereby activated by mmps. characterization of hantavirus envelope structure hepojoki, jussi; strandin, tomas; vaheri, antti; lankinen, hilkka university of helsinki / haartman institute, finland hantaviruses are zoonotic viruses carried by different rodent species and if transmitted to man cause two severe diseases, the hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome to which there is no specifi c therapy. the hantavirus envelope consists of spike structures formed by surface glycoproteins gn and gc which anchorage to the viral lipid bilayer. the quaternary complexes formed by glycoproteins have not been solved. to begin with, it is important to understand the structure of virus particle in order to treat infection for example by prevention of membrane fusion. using pepspot technique the interaction site between gn and gc proteins was mapped to peptide level. to interpret interaction mapping results three dimensional ( d) models of gc protein were created. the structure of hantavirus was also investigated in this study by cryo-electronmicroscopy (cryo-em). according to our results, hantavirus spike consists of either four or eight glycoprotein units and the spike would consist of equimolar associations of both glycoproteins. overall it seems likely that the glycoproteins of hantavirus form a heterodimeric base unit analogous to semliki forest virus e -e glycoprotein complex, where the interaction between e -e proteins hides the fusion peptide in e protein and thus prevents premature fusion. isolation of a novel kda protein from ginger rhizomes having anti-fungal and anti-proliferative activity gill medicinal properties of ginger have been known for long. ginger has been used in traditional indian and chinese medicine and is effective on a wide range of ailments including diarrhea, nausea, respiratory disorders, infl ammatory diseases, arthritis etc. a number of constituents and active ingredients are present in ginger. recent studies have shown the role of ginger extract in the modulation of biochemical pathways involved in chronic infl ammation and thus providing evidences for the anti-infl ammatory role of ginger. mainly the medicinal properties and anti-proliferative activity of the ginger is because of the presence of certain pungent vallinoids, viz. .-gingerol and .-paradol, as well as some other constituents like shogaols, zingerone etc. we have identifi ed and purifi ed a novel anti-fungal and anti-proliferative protein with a molecular mass of kda from the crude extract of ginger rhizobium (zingiber offi cinales), belonging to the zingiberaceae family. the isolation procedure involved ion exchange chromatography using deae-cellulose and affi nity chromatography using affi -gel blue gel. crude extract was loaded on the deae-cellulose column equilibrated with mm tris-hcl buffer (ph . ). the unadsorbed protein fraction from deae-cellulose was further loaded on affi -gel blue gel column equilibrated with mm tris-hcl buffer (ph . ), from which the elution of adsorbed protein was done with the same buffer. the purifi ed protein of kda exhibited a potent anti-fungal activity against the mycelial growth in different fungal species, for example aspergillus fumigatus. in addition, the antifungal activity is also seen against important fungi, viz, fusarium and candida species. further, the purifi ed protein also showed % inhibition of cell proliferation at m concentration. the anti-proliferative activity was checked on human oral cancer (kb cells). search for native interacting partners of fl uorinated amino acids using phage display the introduction of fl uorine has proven to be a successful concept for improving the biological and pharmaceutical properties of drug candidates. the unique properties of fl uorine as well as its absence from the pool of canonical amino acids make fl uorinated amino acids a promising tool in the development of peptide based drugs.( ) however, the application of fl uorinated amino acids for rational protein design requires a comprehensive knowledge of their properties within a native protein environment. our research focuses on the effects caused by single substitutions by fl uorinated amino acids within a polypeptide environment.( ) based on a parallel, heterodimeric -helical coiled coil peptide we applied phage display technology ( ) to screen for preferred interaction partners of fl uorinated building blocks within the pool of the twenty canonical amino acids. three fl uorinated amino acids were introduced either at an a-or a d-position into one of the coiled coil monomers. the second coiled coil monomer was randomized at the four positions that represent the direct interaction partners of the fl uorinated amino acid within the dimerization domain. coiled coil pairing selectivity was used to determine the best binding partner out of the library. using the acylation method, fatty acyl chains are covalently linked to the free amino residues forming a stable amide bond. in this study, a novel method for acylation of proteins was developed using in situ prepared fatty acyl chloride dispersion in aqueous acetonitrile solution, which allows protein modifi cation under very mild conditions. chicken cystatin, a reversible kda inhibitor of papain-like cysteine proteases, was selected as a model protein due to its high potential to inhibit intracellular cathepsins. the protein was modifi ed using fatty acyl chlorides with , , , , , , and carbon atoms. based on the cell culture assays, we examined the transport properties of fatty acylated cystatin, the effectiveness of its internalization and effi ciency to inhibit intracellular enzymes, which was measured indirectly by cathepsin b inhibition. the experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin b. in contrast, non-acylated cystatin didn't cause inhibition as it was unable to enter the cell. the permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with carbon atoms long stearoyl chloride. additionally, chemical modifi cation did not infl uence the protein's immunogenicity. the results of our study provide clear evidence that fatty acylation greatly improves membrane permeabilization properties of proteins. daisogel wing takes your process separation to a higher level. custom made solution for your process peptide purifi cation problem can be easily delivered using the daisogel wing polymer grafted silica based platform. "silica or polymer?"-it used to be an evergreen debate when it came to choose the adequate stationary phase for process scale separation or purifi cation. silica based stationary phases feature much higher mechanical strength and stability, better controlled porosity and as a result higher separation effi ciency, while polymers boast wider ph range. the attempts to bring the good aspects of these opposite methods together failed so far, or failed to provide fl exible solutions. the new daisogel wing platform for silica surface polymer grafting preceding chemical modifi cation is presented. the revolutionary new technique offers high versatility: most variations of the base silica (different pore sizes or particle sizes of choice) can be combined with your choice of familiar chemical surface modifi cations to tailor make the perfect stationary phase for your given chromatography challenge. the polymer graft on the silica surface provides extreme shielding effect, the produced stationary phase displays outstanding ph stability and durability. any of your preferred and trusted regular silica surface modifi cations can be done on the top of the grafted polymer layer. you may enjoy the usual high performance separation, excellent effi ciency, high plate numbers, mechanical strength of silica based stationary phases with a dramatically extended ph range you expected so far only from polymer resins. you have the full range of choices of particle and pore sizes with the full choice of desired bonding chemistries. finally daisogel wing is here to deliver you the most versatile polymer grafted silica hybrid platform to provide the best solution for your demanding process scale peptide separations. przybylski, józef; rogala, piotr; siemaszko-przybylska, krystyna melanin, a natural compound formed in the tyrosine metabolic process. according to occurrence, melanin is divided into that present in true skin and in internal organs. the melanin synthesis and secretion process takes place in melanocytes under the control of two neurohormons: msh -melanocyte stimulating hormone and mch -melanocyte concentrating hormone. research has shown the signifi cant role of melanin in the process of breeding and storing, in regulating metabolism of fat tissue, and in carbohydrates regulation. melanin biopolymer fi nds application in it technology as neurotransmitters for non cellular intelligence. biologically active collagen constitutes the architectonics of all tissues and organs. due to its piezoelectric and dielectric properties it also provides storage and relay functions. for the needs of medical practice, transplants, pharmacy, cosmetology and information technology the key issue is to obtain a compound of melanin with biologically active collagen. this compound we named melanocollagen type i. melanocollagen type i is formed in result of protonating collagen amino acids with melanin, which yields coloured gel ranging: grey, graphite and black. /melanin (h+) + collagen melanocollagen type i/. the therapeutical capacity of biologically active collagen type i with melanin is an ideal formulation inhibiting aging and mitigating related neurovegetative and dementia symptoms, and in vivo transplantology. freeze dried melanocollagen type i retains the features of both collagen type i and melanin. it can be a formulation on its own or a supplement for in vivo and in vitro tissue engineering, biotechnology, information technology, pharmaceutical industry and cosmetology. a patent application for melanocollagen was placed with the polish patent offi ce on . . as p- , entitled: melanocollagen type i -method of obtaining. [ , ] . , -dhp lipid structure was calculated with ab initio quantum mechanics to obtain the charges for molecular dynamics with amber . force fi eld. dhp-lipid molecules were subjected to molecular dynamics from the initial structure of a periodic lipid bilayerwater box, with a small amount of excessive water on the lipid edges to ensure the mobility of lipid molecules. after ns of md simulation the lipid molecules with the fatty acid tails started to squeeze from one bilayer layer to another one. after ns few lipid molecules turned with their charged heads to the side of the lipid bilayer and after ns a profound tubular micelle structure began to form. the tubular micellae structure becomes more perfect during the course of simulation of ns. conclusion is that one of the gene transfection agent , -dhp lipid structures is a tubular micellae, and we could expect that such the micellaes are capable to form lipoplex for the dna transfection or peptide delivery. introduction a newly developed hydrophilic polymer-based ion exchange chromatography column for biomolecules. ion exchange chromatography (iec) is a widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro series, specially designed for separation of proteins, peptides and nucleic acids. the completely spherical and monodispersed porous / nonporous beads ( m), optimally packed with advanced technology, provide high theoretical plate number and symmetric peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp chemistries. in this paper, we will show features and benefi ts of ymc-biopro series. james p. tam, school of biological science, nanyang technological university, nanyang drive, singapore . viral entry requires fusion of the viral and cellular membranes. in all class- envelope viruses including hiv- , fusion is mediated by envelope glycoprotein which has a homotrimeric quaternary structure which forms a hairpin-like assembly of six-helix bundle ( hb) during the fusion event. the hb employs a -on- locking mechanism in which hrc (heptad repeating-carboxyl region) chains crosslink hrn (heptad repeating-aminal region) chains to enable membrane fusion. this locking mechanism confers avidity due to multi-chain interactions and a high genetic barrier to mutations in the viral entry strategy despite the high mutation rate of their envelope protein. this mechanism also provides inspiration to our approach in designing mutation-resistant entry inhibitors of hiv- . t /enfuvirtide, a synthetic hrc-peptide monomer designed to interrupt membrane fusion, is the fi rst and only fda-approved entry inhibitor against hiv- . however, t becomes ineffective in % of aids patients due to acquired resistance to t- resistant during the treatment course. our inhibitor design is based on a novel quaternary protein mimetic approach. key elements include a covalent-link parallel-chain construct mimicking the quaternary structure of gp in its pre-fusion state and an inhibition mechanism mimicking the multimeric interactions found in the highly conserved hb formation. our hypothesis is that covalent-linked, -chain quaternary mimetics (called mimetics) of hrc peptides can confer multi-chain binding to the hrn region in a multi-chain locking mechanism that may lead to mutation-resistant hiv fusion inhibitors. we also extend our design to -chain hrc mimetics (called mimetics) which may bind to the hrn as a -on- locking mechanism. in contrast, t and other single-chain peptide inhibitors with a -on- binding mechanism lacks the advantages offered by the quaternary mimetics and is susceptible to gp mutations. this report will describe all three types of inhibitors as a model to further our understanding of gp mutations and viral fusion mechanism in developing mutation-resistant entry inhibitors. references: . lain et al. cancer cell costentin, beauvillain, vaudry, proc. natl. acad. sci pnas , , . . references: . a.a.zamyatnin. fragmentimics of proteins and natural oligopeptides. biofi zika the fi rst genome sequence of an elite grapevine cultivar (pinot noir vitis vinifera l.): coping with a highly heterozygous genome the grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla the erop-moscow oligopeptide database press references: . alcaro galactosylated peptides, their preparation and use in autoimmune disease diagnosis new coupling reagents: development and industrial aspects assessment of new -cl-hobt based coupling reagents for peptide synthesis. part : coupling effi ciency study fast conventional synthesis of chemokine sdf- ( - ) on the symphony® references: . pardridge, w. m. drug discov.today alberto ; rondina, maria ; rosato maura ; quintieri synthesis and biological activity of new linear and cyclic shp- n-sh -ligands the sequence specifi city for most sh domains is dictated by the amino acids surrounding the py-residue (position ) and the c-terminal positions relative to py ( ). in contrast, the sh domains of shp- in addition to py+ and py+ depend on position py- ( ). it was further demonstrated that beyond this minimal consensus sequence the positions py+ and py+ also signifi cantly affect the binding affi nity and specifi city of the shp- sh domains ( ). for the investigation of shp- mediated signaling pathways, inhibitors of this phosphatase are of great interest. therefore, we focused our research on linear and cyclic peptide ligands based on the previous studies [ - ]. the new ligands were c-terminally prolongated according to the recognition determinants for py+ and py+ . except, an additional motif designed to occupy a basic gap on the surface of the ptp-domain was introduced. the latter was predicted to impair the n-sh -ptp dissociation process design of a directed molecular network restructuring artifi cial peptide networks by external triggering the road to non-enzymatic molecular networks boolean logic functions of a synthetic peptide network systems chemistry: logic gates, arithmetic units and network motifs in small networks peptides: the wave of the future references: . d'andrea l.d.; iaccarino g.; fattorusso r.; sorriento d.; carannante c.; capasso d.; trimarco b.; pedone c proc. natl. acad. sci agnieszka ; gofman nir ; willumeit, regine gkss research centre, germany; hasylab/desy, germany urtti novel cationic amphiphilic , -dihydropyridine derivatives for dna delivery dioleoyl phosphatidylethanolamine and peg-lipid conjugates modify dna delivery mediated by , -dihydropyridine amphiphiles masako ; kuriyama, naohiro poster abstracts lacking his-pro residues and stabilized by introduction of hval resulted in an analog h- hval-tyr-ile-tic-oh (al- ) that was very selective for irap versus ap-n and at( ) . the targeting properties of cell penetrating peptides cell penetrating peptides (cpps) offer the ability to penetrate across the plasma membrane of mammalian cells and to carry cargoes such as proteins, oligonucleotides and liposomes. due to this, cpps have gained high attention to improve the cellular uptake of the delivery of 'biologicals'. however, most of the research that has been performed with cpps restricted to in vivo studies. preclinical investigations are rare and the clinical validation of potential of cpps is required. a series of cpps were synthesized by solid phase peptide synthesis (spps) on an abi a synthesizer. the stability of these peptides in human serum was determined. subsequently, the uptake was studied in the following cell lines: sw , pc- , mh wt, hno in summary, the in vitro uptake studies did not reveal great differences that might have revealed a tumor type specifi city. in vivo studies revealed neither a distinct tissue uptake or tumor specifi city. design and synthesis of a tripartate paclitaxel prodrug for melanoma therapy therapy of melanoma continues to be a challenge since, regardless of the treatment used, long-term survival is quite uncommon. in an attempt to improve the effectiveness and decrease the toxicity of anticancer chemotherapy, one useful approach might be to administer a prodrug that specifi cally releases the active cytotoxic drug at the tumor site. in this work, we describe the synthesis of a peptide conjugate of paclitaxel potentially useful in the treatment of human melanoma, and characterized by the simultaneous presence of three functional domains: a "targeting domain", an "activation sequence", and the antitumor drug paclitaxel. the "targeting domain" of the prodrug is represented by an rgd-containing cyclic peptide, able to bind selectively to alpha-v beta- integrin, which is known to be highly over-expressed by both metastatic human melanoma cells, and endothelial cells of tumor vessels. the "activation sequence", responsible for the selective release of the drug, is a short peptide which is cleaved specifi cally by cathepsin b, a protease highly up-regulated in malignant tumors. the results of nmr conformational studies, as well as those of biological experiments aimed at evaluating the plasma stability of the prodrug and its ability to inhibit alpha-v beta- -mediated tumor cell adhesion to vitronectin, will be also presented. angiogenesis is a remodeling process characterized by the sprouting of new blood vessels from pre-existing ones. it occurs during embryogenesis and to a limited extent in the adult. vegf is a homodimeric protein and has been characterized as a prime regulator of angiogenesis and vasculogenesis; when cells lose the ability to control the synthesis of vegf, angiogenic disease ensues ( ) . in vitro studies show that vegf is a potent and specifi c angiogenic factor involved in the development of the vascular system and in the differentiation of endothelial cells ( ) . vegf biological function is mediated through binding to two receptor tyrosine kinases: the kinase domain receptor (kdr) and the fms-like tyrosine kinase (flt- ), which are localized on the cell surface of various endothelial cell types. this binding activates signal transduction and can regulate both physiological and pathological angiogenesis ( ) . vegf and its receptors are overexpressed in pathological angiogenesis, making this system a potential target for therapeutic and diagnostic applications ( ) . the extracellular portion of vegf receptors is comprised of immunoglobulin-like domains; deletion studies have shown that the ligand binding function resides within the fi rst three domains of flt- and in domains and of kdr. actually, no structural data are known on the extracellular portion of these receptors except for the second domain of flt- .. so, our aim is the cloning and the expression of part of extracellular domains of both vegf receptors for structural characterization and to be used in interaction studies with peptide ligands or small organic molecule. we are interested in an activation mechanism of receptor tyrosine kinases (rtks), especially, how the transmembrane (tm) and the intracellular juxtamembrane (jm) region couple ligand binding to tyrosine phopsphorylation. one of the rtks that we are working on is neu. neu (erbb ) is one of the four members of erbb receptor family. this rtk with a mutation in the transmembrane region (v e) has been recognized as a potent transforming oncogene product. structure of the tm region dimer and its structural difference between the wild type and v e mutant have been reported by smith et.al. ( ) , providing structural constraints for modeling the tm dimer. recently, we have performed a structural study on a tm-jm region of egfr (erbb ) showing that the tm helix breaks at the membrane interface and the unfolded jm region binds electrostatically to the membrane. we also have shown that ca + complex with calmodulin (ca +/cam) binds to the positively charged jm region and pulls this portion off the membrane. these results are consistent with an electrostatic engine model, postulated by mclaughlin and coworkers ( ) , for the autoinhibition and activation of the egfr. in this research, we are revisiting the structure of tm-jm region of neu to see if we can apply the electrostatic engine model to neu and to see if there is a structural difference between the wild type and the mutant tm-jm sequence. here we describe structural studies on the tm-jm sequence, neu( - ), reconstituted into bilayer vesicles. a combination of solid state nmr, infrared and fl uorescence measurements are used to draw conclusions that the unfolded jm binds electrostatically to the membrane and binding of ca + -calmodulin to the positively charged jm sequence can, under some conditions, reverse its charge and release it from the membrane. @the collagen triple-helix consists of three polypeptide chains, each of which takes the poly-l-proline ii form in a left-handed helix and undergoes a transition to a single coil state as the temperature increases. this characteristic structure is ascribed to the unique amino acid sequence x-y-gly, where x and y are commonly pro or (r)-hydroxyproline (hyp r ). @so far, various nmr studies have been done to explain the mechanism of folding and the thermal stability of the triple helical structure by using model peptides rich in imino acids. however, the complexity of nmr spectra imposed severe limitations on detailed analysis. the spectrum is complicated because of a large number of conformational isomers due to cis/trans isomerization around each imino acid causing various chemical shifts with small differences in h-nmr spectra. it has been demonstrated that the site directed n enrichment allows real time nmr monitoring of the folding of residues at specifi c locations of collagen model peptides ( ) . @to approach this problem, we have applied f-nmr study on various collagen model peptides containing (r)-fl uoroproline (fpro r ). compared to h and c, f chemical-shifts are extremely sensitive to small environmental changes around the nuclei and show a wide dispersion of chemical shifts. as a result, we not only distinguished the signals from the triple helix and unfolded states, but also differentiated the signals at intermediates states in d f-nmr spectrum of (pro-fpro r -gly) . @here, in this study, a combination of d nmr experiments including exsy, hoesy, and tocsy has been used to investigate detailed equilibrium properties of collagen model peptides. especially, in the d f-exsy spectra, we successfully observed many exchange cross peaks at high temperature. the f-nmr method shown here provides a clue to analyze the conformational transition, dynamics and stability of collagen triple-helix. by contrast to well-folded natural peptides, oligomers consisting of nonnatural building blocks, so-called foldamers, are highly stable towards proteolytic degradation which is a key feature in the development of peptide-based drugs. aside from the fact that foldamers consisting ofand -amino acid building blocks are able to display different secondary structures, the combination of different homologous building blocks to hybrid peptides has recently shown some promising results. [ , ] the ab initio mo theoretical studies have shown that / -hybrid peptides composed of alternating -and amino acid building blocks might be very well suited to mimic helical conformations. ( ) here we report the fi rst examples of -helical coiled coil formation with synthetic foldamers. based on the computational studies, we synthesized heterogeneous peptides by automated solid phase peptide synthesis (spps). purifi cation was carried out by hplc, and products were confi rmed by esi-tof-ms. furthermore, temperature-dependent cd spectroscopy was applied to investigate the stability of hetero-oligomers formed between, either / -or / / -hybrid peptides with a naturalhelical coiled coil peptide. in addition, the interaction potential with their natural -helical counterparts in aqueous solution were investigated by cd, and förster resonance energy transfer (fret). using reversed phase high performance liquid chromatography and two-dimensional gel electrophoresis, the lhcii proteins, lhcb - and fi brillins were effi ciently separated, and identifi ed by fticr-ms proteome analysis. some of the lhcii subcomplexes were shown to migrate from photosystem ii to photosystem i as a result of short term adaptation to changes in light intensity. in the mobile lhcii subcomplexes, decreased levels of fi brillins and a modifi ed composition of lhcii protein isoforms were identifi ed compared to the tightlybound lhcii subcomplexes. in addition, fticr-mass spectrometric analysis revealed several oxidative modifi cations of lhcii proteins ( ) . a number of protein spots in d-gels were found to contain a mixture of proteins, illustrating the feasibility of high resolution mass spectrometry to identify proteins that remain unseparated in d-gels even upon extended ph gradients. phosphinic analogue of the homophenylalanyl-phenylalanine dipeptide was demonstrated to be a potent, competitive inhibitor of cytosolic leucine aminopeptidase (lap, e.c. . . . ), mimicking the transition state of the reaction catalysed by the enzyme. exhibiting ki value at low nanomolar range it was ranked among the most potent inactivators of lap reported so far ( ) . recently, the compound has been also successfully employed to inhibit leucyl aminopeptidase of p. falciparum, a potential target protease for the development of new antimalarials ( ) . thus, its structure represented an attractive lead for the design and construction of next generations of modifi ed analogues. they were targeted towards both lap as well as a related metalloprotease -microsomal leucine aminopeptidase (apm, e.c. . . . ). these achievements, including recent results of studies on synthesis and activity, will be summarized here. we recently described the synthesis of some cyclic tetrapeptides bearing imidazole side chains and we analyzed, in detail, their copper(ii) binding properties in aqueous solution. the copper(ii) species obtained are of interest in relation to copper-protein active-site biomimetics. in particular, a -membered ring cyclic tetrapeptide c(lys-dhis-ala-his) (dk ) was synthesized by the solid-phase peptide synthesis method and its copper(ii) coordination properties were studied. surprisingly, all collected data strongly support the presence, at alkaline ph, of a stable peptide/copper(iii) complex that is formed in solution by atmospheric dioxygen oxidation. in order to clarify the mechanism of the copper oxidation through the average cu-n bond distance and to get information on the local geometry around copper, depending on the peptide sequence, we have collected experimental xanes and exafs spectra. the investigated cyclopeptide/copper complex shows pre-edge peak energy position and integrated intensity consistent with those of cu + model compounds. also, edge energy is consistent with the presence of trivalent copper. then, calculations performed on the exafs measures should give information on the local geometry, confi rming the square planar geometry proposed by us for this dk /cu(iii) complex. improved expressed protein ligation method for consecutive coupling of polypeptide fragments in the post-genomic era, to support the structural and functional studies of proteins, there is a growing demand for novel methods with which a wider range of selective protein modifi cations achievable. expressed protein ligation (epl) can be the method of choice for the preparation of unique protein derivatives not available by recombinant methods when the protein fragments extend the size accessible by solid phase peptide synthesis, and when extra chemical information (i.e. a special covalent modifi cation) is introduced into a well-defi ned part of the sequence. however, this method is not generally applicable, especially in the case when the target protein derivative is assembled from three or more polypeptide fragments. we demonstrate here an improved epl method that facilitates the effective ligation of large fragments in a consecutive way. the method employs a novel protection-deprotection scheme. supported by otka f and jános bolyai fellowship (cs.t.). protein ligation using protein trans-splicing , in order to facilitate protein ligation using synthetic peptides. highly effi cient fl uorescent labeling of proteins has been demonstrated in a site-specifi c manner by protein ligation using the newly designed intein. this approach could be applied for sitespecifi c modifi cation of proteins in vivo because protein trans-splicing is an autocatalytic process requiring no additional cofactor. moreover, protein trans-splicing approach could be extended to dual site-specifi c modifi cation using an additional intein. la doppel (dpl) is a glycosylphosphatidylinositol-anchored protein that exhibits % sequence homology with prion protein but lacks the octarepeat region. the role of prion is not yet completely known but prpc seems to be involved in cu (ii) traffi cking from synaptic clefts and in preventing neuronal oxidative damage. doppel is expressed in heart and testis and has been shown a regulator of male fertility. therefore, despite a high sequence homology and a similar three-dimensional fold, it's possible to hypothesize that the function of the two protein is not correlated. in the literature is reported that both prpc and of dpl are able to bind cu (ii) but the characterization of the complex species is well defi ned only for prpc. several binding sites of dpl are involved in the complexation with cu (ii). [ , ] in the present work we have focused our attention to the third alpha-helix of the dpl which is the preferential binding site for the metal ion. we have synthesized two peptide fragments relative to the - sequence of the dpl. the fi rst peptide has the native sequence whereas in the second fragment the asp was replaced by a asn residue. this substitution was performed to understand the role played by the carboxylate group of the asp residue in the complexation of cu (ii). cd ad nmr spectra were carried out on the two peptides in absence and presence of cu (ii) to investigate conformational features upon cu (ii) binding. finally, the complex species formed were completely characterized by using spectroscopic (nmr, cd, uv-vis, epr) and thermodynamic measurements (potentiometric titrations). three-dimensional ( d) domain swapping of proteins is a phenomenon associated with formation of oligomers and fi brils of several amyloidogenic proteins ( ) . the propensity of a particular protein to undergo domain swapping depends on factors like changes in environment or presence of specifi c mutations, but also on some topological determinants. it was proposed that domain swapping-prone proteins have some common "hot spots" in their structures that facilitate the process. one of these motives are so called "hinge loops" -turns showing enhanced propensity for unfolding due to conformational constraints ( ). cystatin c (hcc), main inhibitor of cysteine proteases in human body was shown to dimerize ( ) and oligomerize ( ) through domain swapping. hcc contains highly conserved throughout the cystatin family loop l , connecting two beta strands which, together with the only helix in the protein fold create a structural unit that undergoes the d process. in order to confi rm important role of distortions in l loop, which are centered around valine residue in position in the dimerization/ oligomerization process of hcc, we designed point mutations which should inhibit or promote aforementioned processes. as it was expected, substitution of val with either asparagine or aspartic acid residues abolished dimerization process almost completely. in contrast, v p mutant shows enhanced dimerization propensity. above observations are in good agreement with theoretical studies, performed on entire hcc and its fragment encompassing the hairpin structure centered on loop l . the expansion of the cag trinucleotide that produces polyglutamine (polyq) segments in several proteins is responsible for at least eight neurodegenerative diseases. a possible therapeutic approach would be to inhibit the polyq self-assembly process using disrupting agents. although, screening studies have identifi ed several polyq aggregation inhibitors, their mechanism of action remains unknown. this is due to the poor aqueous solubility and fast aggregation of uncharged polyq peptides, which complicate their study. the most common strategy to resolve these problems is to produce polyq stretches fl anked with charged residues. however, this strategy was found to modify the aggregation behaviour of polyqs, their aggregate structure and their affi nity for disrupting molecules. to circumvent these problems, polyq peptides containing morpholine moieties, whose charge is ph-dependent, were produced using fmoc-based chemistry on spps. following an acid treatment, the designed peptides were soluble in acidic aqueous solutions and their aggregation rate could be controlled by ph variations and followed by dynamic light scattering. furthermore, aggregation initiated at physiological ph provided uncharged fi brils allowing the evaluation of the effects of charged disrupting agents. the actions of known polyq aggregation inhibitors such as polyq-binding peptide (qbp ), trehalose and congo red were studied. peptide-protein and protein-protein interactions play key roles in most biological processes, and therefore represent valuable targets for drug discovery. an approach in the search of new chemical entities able to interfere with these interactions is the use of small molecules able to mimic or to induce precise aspects of specifi c peptide secondary structures. among the secondary structure elements, reverse turns have been shown relevant in many biomolecular recognition events. thus, different efforts have been directed to the design of turn mimetics or inducers. in this sense, -substituted azetidine- -carboxylates, synthesized by our group through a versatile procedure from amino acids, possess the dihedral angle restricted to approximately º or - º, depending on the absolute confi guration at the asymmetric carbon. these values are similar to those reported for the central residues of main types of -and -turns, and in fact, recent studies have shown the ability of the , -disubstituted azetidines to induce -turns when incorporated at the i+ position of model peptides. to know if this conformational behavior is distinctive of these restricted amino acids, we now investigate the infl uence of the incorporation of these amino acids at the i+ position of model peptides. with this aim, we have synthesized a series of simplifi ed tetrapeptide models, rco-l-ala-azx-nhme, in which the rco and nhme groups are simplifi cations of the i and i+ residues of the turn, respectively. for comparative purposes, dipeptide derivatives incorporating azetidine- -carboxylate, pro and -mepro have also been prepared. the turn inducing capacities of all these model tetrapeptides have been analyzed by molecular modelling, ¹h rmn, ft-ir and x-ray methodologies. the results have shown the importance of the , -disubstitution at the heterocyclic amino acid for stabilizing reverse turns, and the infl uence of the ring size on the induced turn type infl uence of position of dehydroamino acid in peptide chain on conformation of dehydropeptides containing two dehydroamino acid residues it is known that presence of c -c double bound in dehydroamino acid infl uences on dramatic limitation of conformational space, not only side-chain but also main-chain [ ] [ ] . according to the peptide's length and neighboring amino acid residues, dehydroamino acid exerts s-shaped, -turn or helical conformation in peptides [ ] [ ] [ ] [ ] [ ] . angiotensin-i converting enzyme (ace) somatic form bears two catalytic domains with two zn metal ions, while the testis form bears remarkable similarity to the aminoacid sequence of ace c-catalytic domain and only one zn metal ion. however, both exhibit their hydrolytic activity to vasoactive peptides such as angiotensin i (angi) and bradykinin (bk), though with different effi cacy. in order to study experimentally the possible interaction and binding events between the ace active site(s) and known ace substrates or inhibitors, we constructed peptide-based catalytic site maquettes (csm). -residue peptides were synthesized through solid-phase peptide synthesis having the aminoacid sequence that correspond to the ace val -ala n-domain segment and to the val -ala c-domain segment and enzyme's active sites were reconstructed through addition of zinc metal ion in solution. the resulting complexes have the metal ion bound in a native-like mode, as manifested by previously reported nmr data. the reconstituted peptides were titrated separately by captopril and angiotensin-i with a molar ratio ace:captopril/angi : and : , respectively. titration was followed by h nmr spectroscopy and spectral changes (chemical shifts, line broadening, etc.) were recorded and analyzed. after the end of angi titration the solution of interacting peptides was titrated with captopril and the titration was followed by h nmr spectroscopy. at the end of each titration d homonuclear h- h tocsy and noesy spectra were recorded. analysis of high-resolution nmr data probes the conformational variation of ace csm and peptide substrates upon interaction, and in the presence of captopril in ace-angi solutions. contemporary development in nanotechnology is fabrication of nanostructured materials using the peptide-based self-assembly. importance of peptide as monomeric components is their capacity to be engineered to mediate spontaneous supramolecular self-assembly and their inherent chemical nature that facilitates chemical and biological recognition process. tubular self-assembled peptide nanostructures are of special interest since these can serve in various applications including 'bottom-up' fabrication of molecular scaffolds, nanoelectromechanical systems and nanomachines. an important discovery by gazit group revealed that the simple dipeptide molecule of phenylalanine which is core recognition motif of the alzheimer's -amyloid polypeptide effi ciently self-assembles into well-ordered nanotubular structure ( ) which could be controlled by relatively simple chemical modifi cations at its termini. based on these observations and our interest in the synthesis of softmaterials for nanotechnology, we have successfully synthesized novel monomeric units of di-peptides with ureido functional group at nterminal of various hydrophobic l-amino acids. these derivatives have been prepared by using solid phase peptide synthesis protocol with cost effective process retaining the chirality of molecule. these ureido dipeptide formed nanostructures with high yield under mild and controlled condition in aqueous media. to fully understand the molecular and supramolecular mechanism guiding the formation of nanostructures we have selected a multidisciplinary approach by combining spectroscopic studies with advanced electron microscopic techniques. the morphology of nanostructures can be controlled by using variety of l-amino acids. these novel ureido di-peptides nanotubes can be used in fabrication of biocompatible peptide-based nanostructures and they will undoubtedly have nanotechnological applications. biomolecules are excellent in hierarchically organizing a characteristic structure by self-assembly. the spontaneous assembly of biomolecules has advanced a bottom-up approach for fabrication of nanostructured materials. we have developed fabrication of controlled nanofi bers through self-assembly of simple and short de novo designedsheet peptides with unique sequences [ , ] . the single straight nanofi ber with high regularity constructed by -sheet fi peptide (sequence: pkfkiiefep) was functionalized with proteins by biotinylated peptides ( ) and using functional anchors that have binding and functional groups. conjugation of self-assembled peptide nanofi bers with biomolecules such as peptides and proteins will expand their potentialities of the nanofi bers for various applications. to identify peptides binding to the self-assembled fi peptide nanofi ber, a phage display combinatorial screening using a random peptide library was performed. after fi ve rounds of biopanning, phage pools with highly specifi c affi nities to fi nanofi bers were screened. as a result of dna sequencing after phage cloning, phage clones were identifi ed. binding affi nities of these clones were quantitatively investigated by an enzyme-linked immunosorbent assay. these clones showed specifi c affi nities to fi nanofi bers, as compared with ff and vi nanofi bers having slightly differences of amino acid sequences. affi nities and specifi cities were dependent on the sequence of each peptide, indicating that different amino acid sequences contributed to peptide interactions with nanofi bers. infrared spectroscopy studies by using chemically-synthesized peptides showed that the peptide binds specifi cally to the fi nanofi ber with structural transitions. owing to their diversity in size and shape, easy access, and biocompatibility, peptides represent versatile units for the construction of h-bonded tubular assemblies and other biomimetic materials with potentially useful applications. so-called peptide nanotubes (pnts) have been obtained through multiple and complementary approaches ( ). originally designed from -peptides made of d-and l-amino acids ( ) , fl at macrocyclic systems forming cylindricalsheet like assemblies have diversifi ed to include oligoamides made from higher amino acid homologs as well as peptide hybrids (e.g. ,peptides). tubular sheet-like assemblies however are not restricted to oligoamides. the urea group which shares a number of features with the amide linkage, i.e. rigidity, planarity, polarity, and hydrogen bonding capacity is an interesting surrogate. we and other have demonstrated that macrocyclic biotic and abiotic n,n'-linked oligoureas have a unique propensity to self-organize into polar h-bonded nanotubes ( ). partial peptide backbone n-methylation has been introduced as a general strategy to generate truncated stacks (i.e. h-bonded dimers) useful to gain access to the thermodynamics of nanotube formation ( ). herein, we describe biotic macrocyclic amide/urea hybrids with partially nalkylated backbones as new candidates for the formation of h-bonded dimers. a cysteine branched pga (polyglutamic acid) hydrogel is being investigated for therapeutic peptide and protein controlled delivery. entrapment of hgh as a model protein in this hydrogel has been carried out in order to check its later release in different aqueous buffered media and stability. different physico-chemical analysis (by maldi-tof, sec-mals-ri, h-nmr, cd, …) of the released hgh showed no effect on destabilization of the protein. controlled release systems of proteins involve important challenges such as maintaining the protein integrity. researchers need an exhaustive understanding of protein stability issue in drug delivery formulations. common degradative reactions in proteins involve: oxidation, deamidation, stability of disulfi de bonds and aggregation, which may affect secondary and tertiary structure of proteins and thus, their biological activity. the high water content which imbibes the polymer network of hydrogels enables a good accommodation for proteins. polyamino acids have been already studied in some biotechnological applications, however, in spite of their potential, they have barely been taken into account for the development of controlled release microparticles. we show that proteins could be easily loaded into cys cross-linked pga hydrogels. the protein hgh has been loaded and released preserving its physico-chemical integrity. thus, these hydrogels are promising for their use as therapeutic protein delivery systems. totally synthetic collagen-like gels by intermolecular folding of designed peptides our goal is to develop artifi cial collagens that can use as safer and functional biomaterials. here, we report the development of collagenlike gels by means of the intermolecular folding of chemically synthesized peptides. the peptides are disulfi de-linked trimers of collagenous gly-x-y triplet repeats with self-complementary shapes. the self-complementary peptides are able to form elongated triple-helix through spontaneous intermolecular folding. upon cooling of the peptide solutions, hydrogels of peptide supramolecules formed. the gel-sol transition was appeared to be reversible, and the transition temperatures were found to be tunable by the design of the peptides. our strategy for the totally synthetic collagen will offer possibilities for the development of innovative biomaterials. recent advance in biotechnology enables us to fi nd the peptides with affi nity for nonbiological materials and with function of mineralizing inorganic materials. the use of the functional peptides is attracting a growing interest for bottom-up fabrication approaches of nanoscale devise. zinc oxide (zno), a semiconductor with a wide direct band gap, possess unique optical, acoustic, and electronic properties, so that it is one of most widely studied metal oxides for solar cells, ultra violet nanolaser, blue light-emitting diode and so on. this wide variety of applications requires various fabrications of morphologically and functionally distinct zno nanostructures. here, the peptides which are selected from the phage-displayed peptide library with a -mer on the surface, can bind zno particle but not other metal oxides particle, and further, the zno-binding peptides play an important role on the crystal growth of zno in its synthesis. the anti-zno peptide can assist the synthesis of zno nanoparticles from a zn(oh) solution even at ºc, and further, the peptide leads to the self-assembly of synthesized zno particles to fl ower-type morphologies. we describe the biomimetic zno synthesis using the artifi cial peptide with affi nity for zno. the design of nanoparticles suitably functionalized for applications in biology or medicine is an ongoing challenge for both chemists and biologists. nanoparticles such as quantum dots, gold nanoparticles or carbon based-materials offer, in addition to their remarkable physical properties, the possibility to be functionalized by biomolecules, making them suitable for sensing, detection, diagnostic, and/or therapeutic applications. recently, quantum dots have been designed for biological imaging owing to their unique size-dependent fl uorescence properties. however, their plausible toxicity still remains a major concern for in vitro and in vivo applications. nanodiamonds, (nds) are also candidates for biomedical applications considering their intrinsic or induced fl uorescence and biocompatibility. the work will describe: i) the functionalisation and the characterization of nm non-fl uorescent cnds and ii) their use in toxicity and transport studies in living cells after the grafting of a fl uorescent model peptide. we chose to introduce a nonpermeant fl uorescent peptide for the tracking of the nanoparticles on or inside the cells. nanodiamonds (cnds, nm) coated by silanisation or with polyelectrolyte layers have been grafted with a fl uorescent thiolated peptide via a maleimido function, leading to aqueous colloidal suspensions stable for months. for each step of the preparation, the diameter of the nds measured by dynamic light scattering (dls), the zeta potential, the amount of amino groups grafted onto the surface, as well as the stability of the different nds suspensions no cytotoxicity was observed up to hours incubation with cho cells with any of the prepared ( g/ml). their capacity to enter mammalian cells, and their localisation inside cho cells, have been ascertained by confocal microscopy, refl ected light and fl uorescence. hepatitis b virus is one of the most common problem and potential danger of all over the world and also in our country. it is known that the use of proteins and peptide antigens as vaccines has several potential advantages over whole viral or bacterial preparations. to elicit the maximum immunogenic response from synthetic peptide antigens ,it is generally necessary to bind the peptide to carrier protein. moreover, to realize the full potential of synthetic peptide antigens protein-peptide conjugate will have to be used with an adjuvant. we have developed new approches for obtaining highly immunogenic peptide conjugatessynthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. this concept drive us to create new immunogenic bioconjugates of hepatitis b surface antigenic polypeptides (hbsag) with synthetic pe. in this study we used the region - of the s gene. the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem), by using f-moc chemistry. copolymers of acrylic acid with different monomers were used as a pe for the conjugation with polypeptides. pe-peptide conjugates was synthesized by microwave assisted method. composition and structure of bioconjugates werw characterized by hplc, spectrofl uorometry and different spectrophotometric methods. protein-protein and protein-ligand interactions are among the few essential cell processes whose understanding provide us insights into fundamental events of the life cycle. aside from in silico methods, natively folded proteins are an absolute prerequisite in all current biotechnological tools used for the study of these interactions. however, the recently developed ianus peptide array has a potential to evolve in to a protein-free method for detection of protein-protein interaction sites. using this assay, protein-protein interactions could be represented by and investigated as peptide-peptide interactions using peptide pairs immobilized on a solid support.( ) . two main obstacles had to be overcome for successful implementation of this array: (i) the library size and (ii) identifi cation of interacting peptide pairs. if, for example, interactions between two small proteins of only approx. amino acids each should be analyzed, a library of ca. peptide pairs would be needed to cover all possible combinations of overlapping peptides derived from these proteins. although libraries of several thousand peptides were already successfully synthesized, the library size could be reduced drastically if the binding pocket of one protein is already known or if the protein-ligand interactions are studied. up to date, two methods for the identifi cation of interacting peptides in a library have been developed in our group. both methods will be demonstrated in studies of protein-protein and protein-ligand interactions. the role of the non-helical tailpiece in myosin ii assembly the hebrew university of jerusalem, israel self-assembly of macromolecules into large complexes is often mediated by folding of disordered regions. we used peptides to investigate the role of the non-helical tailpiece from non-muscle myosin iic (nm-iic) in its self-assembly process. nm-iic is a motor protein composed of a globular motor domain in the n-terminal region and a coiled-coil rod domain in the c-terminal region, which terminates with a non-helical -residue tailpiece (residues - ) . nm-iic molecules undergo self-assembly into fi laments that are necessary for its contractile activity. the tailpiece has an unfolded character and participates in the assembly process of nm-iic. the n-terminal region of the tailpiece (residues - ) has a net positive charge of + while its c-terminal region (residues - ) has a net negative charge of - . we designed peptides that correspond to the entire tailpiece and to its negative and positive regions. these peptides were used to study interactions with the rod, their structures in the free and bound states, and structural changes they undergo upon binding to residues - of nm-iic rod. fluorescence anisotropy binding studies showed that a peptide corresponding to the positive region of the tailpiece (residues - ) bound the nm-iic rod (residues - ) with an affi nity of m. circular dichroism studies showed that the positively charged peptide became folded upon binding the rod, indicated by a shift of the spectral minimum from nm to nm upon binding. a peptide corresponding to the negative region of the tailpiece (residues - ) did not bind nm-iic (residues - ). based on our results, we suggest that the positive region of the tailpiece is required for ordering and aligning neighboring molecules by electrostatic attraction, leading to fi lament formation. our results provide molecular insight into the role of the structurally disordered tailpiece of nm-iic in the selfassembly process. stefanidakis, michael; karjalainen, katja; pasqualini, renata; arap, wadih; koivunen, erkki md anderson cancer center, united states acute myelogenous leukemias (aml) are characterized with non-random patterns of medullary and extramedullary invasion. we hypothesized that a supramolecular complex, the leukemia cell invadosome, which contains certain integrins, matrix metalloproteinases (mmps) and other as yet unidentifi ed proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. here we show that the specifi c binding of mmp- to leukocyte surface ám integrin is required for pericellular proteolysis and migration of amlderived cells. an effi cient antileukemia effect was obtained by peptide inhibitors that prevented prommp- cell surface binding, transmigration through an endothelial cell layer, and extracellular matrix degradation. notably, the functional protein anchorage between ám integrin and prommp- described in this study does not involve the enzymatic active sites targeted by any of the existing mmp inhibitors. taken together, our results provide a biochemical working defi nition for the human leukemia invadosome. disruption of specifi c protein complexes within this supramolecular target complex may yield a new class of anti-aml drugs with anti-invasion (rather than cytotoxic) attributes. the molecular mechanisms of action of the polycationic lysine homoand heterodendrimers on functional activity of adenylyl cyclase signaling system (ac system) in the myocardium and the brain of rats were studied. the lysine homodendrimers of the third (i), the fourth (ii) and the fi fth (iii) generations, as well as the lysine heterodendrimers of the fi fth generation -[(nh ) (lys-glu) (lys-glu) (lys-glu) (lys-glu) (lys-glu) lys-ala-ala-lys(clac)-ala-nh ] (iv), [(nh ) (lys-ala) (lys-ala) (lys-ala) (lys-ala) (lys-ala) lys-ala-lys(clac)-ala-ala-nh ] (v) and [(nh ) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) lys-gly-gly-lys(clac)-ala-ala-nh ] (vi) stimulated by receptor-independent mechanism the activity of heterotrimeric g proteins, preferably of inhibitory type, and interacted with c-terminal regions of their -subunits. the homodendrimers ii and iii and heterodendrimer v were more effective g protein activators. the treatment of the membranes with pertussis toxin (inactivating g i protein), but not with cholera toxin, led to a decrease of g protein activation effect of the dendrimers. the lysine dendrimers disturbed the functional coupling of the receptors of biogenic amines and peptides hormones with g i proteins and, to a smaller extent, with g s proteins. it was illustrated by the decrease of regulatory effects of the hormones on ac activity and g protein gtp binding and by the decrease of receptor affi nity to agonists in the presence of the lysine dendrimers, as result of receptor-g protein complex dissociation. it was shown that the molecular mechanisms of the action and the g protein selectivity of the polylysine dendrimers are similar to those of mastoparan and melittin, natural toxins of insect venom, which also preferably activate g i/o proteins and inhibit g i/o -coupled signaling. acknowledgements: supported by rfbi (grant - - ) and «russian science support foundation». genetics, bielefeld university, germany dna-protein interactions are a key element in the regulation of cellular processes. transcription factors are able to recognize their cognate dna sequences and regulate the expression of proteins. as a model system, the specifi c dna binding of the transcription factor phob from e. coli is investigated in single molecule experiments. structurally, phob belongs to the family of winged helix-turn-helix proteins. it is composed of a transactivation domain (amino acids - ) and a dna binding domain (amino acids - ). after phosphorylation of the transactivation domain, the protein binds to specifi c dna sequences containing a tgtca consensus sequence. different c-terminally modifi ed protein epitopes representing parts of the dna binding domain of phob were chemically synthesized using microwave assisted solid phase peptide synthesis. the binding contributions of these molecules are compared to the complete dna binding domain ( - ). this protein was purifi ed using intein mediated protein splicing, an additional cysteine was ligated to the protein by intein mediated ligation. performing single molecule force spectroscopy experiments, kinetic off-rates were obtained, and sequence specifi c dna-binding of both peptide and protein was proven in competition experiments. alanine scans of strategic residues revealed the contributions of single amino acid residues for peptides and proteins. structural investigations of the peptides, the proteins and dna/protein complexes were performed using circular dichroism measurements. this method revealed structural differences of the peptides, proteins and dna upon complex formation. furthermore, the protein/dna or peptide/dna interaction was determined by surface plasmon resonance experiments and electrophoretic mobility shift assays. acknowledgment: this project was supported by dfg (sfb ). reversed-phase chromatography is important in the development of well-characterized peptide pharmaceuticals. several factors infl uence sensitivity, resolution, and retention for lc and lc-ms applications: bonding chemistry, pore size, particle size, column confi guration, and solvent composition. by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short -mm length hplc columns packed with . m, Å c silica for ultrafast, one-minute peptide analysis with moderate backpressures (< psi) using a conventional hplc system. for more complex peptide samples, such as protein digests, on conventional hplc, a novel column confi guration packed with . m, Å c is described for -to- min. separations that traditionally take to minutes. we also discuss fast two-minute separations on an ultra-high pressure system using a variety of . m, small pore columns with unique selectivity. "unknown-genome" proteomics-based identifi cation of a new nadp-epimerase/dehydratase from desulf. phosphitoxidans by inverted-pcr, edman-sequencing and high resolution mass spectrometry protein identifi cation in proteomics is generally based on the availability of genomic data. using amenable databases, identifi cation in "bottom-up" proteomics is often straightforward but is highly complex in the absence of genome data, which typically requires "de novo"-identifi cation approaches. we present here a new approach for identifi cation of proteins from a bacterial strain, desulfotignum phosphitoxidans, with unknown genomic background, using a combination of (i), inverted pcr of degenerate primers derived from n-terminal edman sequencing, and (ii) high resolution maldi-fticr mass spectrometric peptide mass fi ngerprint-proteomics of expressed proteins. desulfotignum phosphitoxidans is an anaerobic sulfatereducing bacterium from marine sediment in which phosphite oxidation was found crucial for energy metabolism. culturing under different growth conditions provided specifi cally expressed proteins with molecular masses of ca. kda in the presence of phosphate, which were subjected to d-gel electrophoretical separation for soluble and membrane fractions using pdquest comparative analysis. n-terminal sequences of the proteins, determined by edman analysis were used for inverted pcr of degenerate primers, and provided a series of orf candidates, one of which coded for a putative nad-dependent dehydratase. in a complementary approach, protein spots from the d-gels were excised, digested with lys-c protease, and digestion mixtures analysed by maldi-fticr-ms. the accurate peptide masses unequivocally matched to identify a new nad-dependant epimerase/ dehydratase. the detailed functional characterization of the new protein, and further development and application of this approach to proteome analysis with unknown genomic background, are currently in progress. based on specially treated large pore silica and enhanced with a proprietary bonding process, vydac ms reversed-phase (rp) hplc columns offer superior performance for peptides and proteins. the deamidation of human growth hormone (hgh) has been monitored for many years by rp using vydac columns. the vydac ms c column provides the best overall performance characteristics (recovery, resolution, and peak symmetry) for the common important assay of hgh and desamido hgh. although hydrophobic membrane proteins are particularly diffi cult to separate, the vydac ms c column provides better separation and recovery (up to % higher vs. other leading columns) for a reptilian reovirus p protein and myristolyated form, a component of a potentially new vaccine delivery system. separation of the trypsin digest of fetuin, a glycoprotein, exhibits improved selectivity for peptide mapping on a vydac ms c column compared to other c columns, revealing some peaks otherwise not seen. the improved selectivity for peptides on the vydac ms columns results in better primary structure defi nition and easier identifi cation of degradation products and other protein characteristics. hemoglobin peptides in mammalian tissues: facts or artifacts?yatskin, oleg; karelin, andrei; ivanov, vadim shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, russian federation comparative rp-hplc and ms analysis of peptide composition of rat brain, heart, lung and spleen acidic extracts was performed. the extracts were prepared from snap-frozen organs both in the presence and in absence of protease inhibitors ( - m pepstatin a, - m pmsf, mm edta) which stabilize the peptide composition of tissue acidic homogenates. the absence of inhibitors led to appearance of novel peptides and did not affect the concentration of predominant peptide components detected in samples prepared with protease inhibitors. therefore, the latter were considered as present in the tissues prior to extraction, i.e. as endogenous. the majority of predominant peptide components were found common in the studied tissues. we believe that the presence of predominant (> pmol/g) peptide components in the protease-inactivated tissue extracts can be reliably extrapolated to their in vivo presence in the tissues. the endogeneity of minor (< pmol/g) components requires a separate consideration. generally, the results of peptidomic studies strongly depend on sample preparation procedures involved, making diffi cult the straightforward comparison of the results obtained by different research groups. urine is a promising source of endogenous peptide biomarkers, because the protein concentration is -fold lower than in plasma but the peptide concentration is equal. urine contains small metabolites that interfere with peptide analysis and urine fl ow varies between subjects and within a day. these variations pose a challenge to urine sample preparation, which, in case of serum, has been shown to be critical for reproducibility. we aim to optimize the enrichment method for urine peptides, to characterize these and to fi nd potential biomarkers. all methods resulted in good peptide recovery if the peptide concentration was normal and metabolite content low. slightly different peptides were obtained with different methods. the df preferentially recovered hydrophilic peptides, while sec-spe recovered high molecular weight peptides better than the df-method. sec-spe provided the best peptide enrichment especially for samples with high metabolite concentration. normalization was critical especially with very dilute samples. without normalization the number of peptides detected was highly dependent on urine concentration. normalization against specifi c gravity proved the most reproducible results. we have identifi ed several peptides found in all samples and some differences between cancer and control samples. validation of these differences is currently under way. jalili, pegah ; ball, haydn sigma-aldrich, united states; in order to improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotintag), possessing three functional domains; a biotin group for binding to avidin, a base-labile -carboxy fl uorenyl methoxy-carbonyl ( -carboxy fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base, liberates a covalently bound gly-cys analog of the peptide(s) of interest, exhibiting improved rp-hplc retention and ms ionization properties compared to the precursor phosphopeptide sequence. the results obtained for a model peptide akt- and ovalbumin protein digest, demonstrated that the method is highly specifi c and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles/ l. in search of physiological substrates of brain prolyl oligopeptidase. prolyl oligopeptidase (pop) is a serine protease, which cleaves small peptides at the carboxyl side of an internal proline residue. substance p, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. pop has been implicated in a variety of brain processes including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension and mood disorders. however, there is no defi nite information about the physiological substrates of pop in brain. we have determined previously that some pop inhibitors, when administrated orally or intraperitoneal, are able to cross the blood-brain barrier and inhibit pop ex vivo. in this work also inhibition duration according to the doses were determined. using this system we studied the natural occurring peptide profi le in brain tissues from inhibited animals and compare it with the profi le in control tissue. we were able to design a method in which the background peptide level was importantly decreased which allowed us to identify, with high degree of confi dence, the peptides which were changed in rat hypothalamus upon pop inhibitor treatment. conclusions about the physiological substrates of pop are discussed. [ , ] . in the present work, we further studied the dependence of peptide production by the cells on their functional state. using rat hepatocytes as a model, we compared peptide sets generated by: ( ) primary hepatocytes in a differentiated state; ( ) dedifferentiated primary hepatocytes with fi broblast-like phenotype; ( ) rat hepatoma cells. peptides were extracted from the cellular lysates and supernatants by solid-phase extraction and then separated by rp-hplc. peaks were analyzed with maldi-tof and/or maldi-tof/tof. acknowledgements: this work was supported by ras presidium grant "molecular and cellular biology"; russian president grant "scientifi c schools" # . . ; rfbr grant # - - -a. triostin a is a naturally occurring quinoxaline depsipeptide antibiotic originally isolated from streptomyces s- - . with its two quinoxaline moieties it bis-intercalates into dna in a gc selective manner. triostin a contains n-methylated l-valine and l-cystein. the unmethylated analog tandem binds at selectively due to a different hydrogen bonding pattern. substitution of the disulfi de bridge of tandem by an azobenzene moiety leads to photoswitchable analogs. their photoswitchability was investigated and quantifi ed using uv, cd and nmr spectroscopy as well as hplc. in order to determine the three-dimensional structure, conformational analysis by nmr spectroscopy in combination with molecular dynamics calculations was carried out. in this process, distance restraints were obtained from nmr spectra measured in dmso-d and applied in distance geometry/simulated annealing followed by molecular dynamics calculations. as it is possible to distinguish between cis-and transisomer in nmr spectra due to the difference in the chemical shifts, structures of both isomers can be investigated. dna binding studies were carried out using an optical tweezers setup with -dna. furthermore, uv melting curves were recorded for the tandem derivatives in complex with dna. cd spectroscopy was applied to observe changes in dna structure upon formation of the complex. in addition to structural investigations, the antibiotic activity of the tandem derivatives was investigated in minimal inhibition concentration assays against gram-positive bacillus subtilis. acknowledgment: this work was supported by the dfg (sfb ). k. gaus gratefully acknowledges the fi nancial support (phd grant) by the international nrw graduate school of bioinformatics and genome research. cyclic peptide nanotubes are formed by self-assembly of cyclic peptides with alternating l-and d-amino acid. however, peptide nanotube selfassembly phenomenon had not yet been made cleared. intermolecular electrostatic interaction is one of the important driving forces to assemble cyclic peptides to peptide nanotubes. here in, a series of cycilc hexapeptides, cyclo(-l-lys-gly-) , cyclo(-l-glu-gly-) , cyclo(-l-lys-d-ala-) , cyclo(-d-glu-l-ala-) , cyclo(-l-trp-d-lys-) , and cyclo(-l-trp-d-glu-) , were designed and synthesized by solid-phase peptide synthesis using fmoc strategy. turbidity study revealed that the abilities of self-assembly formations of the : mixtures of cyclo(-l-trp-d-lys-) and cyclo(-l-trp-d-glu-) in aqueous solution are extremely higher than the abilities of each individual positively or negatively charged cyclic peptides. transmission electron microscope (tem) experiments showed that mixture of cyclo(-lys-xxx-) and cyclo(-glu-xxx-) formed fi brous structure. tem images indicated that, the diameters of the nanotubes were approximately - nm. diameter of similar cyclic peptides was found to be nm approx. these results also supported that the bundle formation of peptide nanotube. conformations of monomer cyclic hexapeptides were calculated based on nmr observation. estimated formation of peptide nanotube is consisted with the bundle structure. koèevar, nina; obermajer, nataša; kreft, samo faculty of pharmacy, slovenia therapeutic proteins offer a promising potential as effective drug compounds. however, proteins are generally poorly transported across biological membranes due to hydrophilicity. fatty acylation with reactive fatty acid derivatives represents one of the basic methods for increasing the proteins' hydrophobicity and improving membrane permeability. key: cord- -lrsqrc u authors: yañez-guerra, luis alfonso; zhong, xingxing; moghul, ismail; butts, thomas; zampronio, cleidiane g; jones, alexandra m; mirabeau, olivier; elphick, maurice r title: echinoderms provide missing link in the evolution of prrp/snpf-type neuropeptide signalling date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: lrsqrc u neuropeptide signalling systems comprising peptide ligands and cognate receptors are evolutionarily ancient regulators of physiology and behaviour. however, there are challenges associated with determination of orthology between neuropeptides in different taxa. orthologs of vertebrate neuropeptide-y (npy) known as neuropeptide-f (npf) have been identified in protostome invertebrates, whilst prolactin-releasing peptide (prrp) and short neuropeptide-f (snpf) have been identified as paralogs of npy/npf in vertebrates and protostomes, respectively. here we investigated the occurrence of npy/npf/prrp/snpf-related signalling systems in a deuterostome invertebrate phylum – the echinodermata. analysis of transcriptome/genome sequence data revealed loss of npy/npf-type signalling, but orthologs of prrp-type neuropeptides and snpf/prrp-type receptors were identified in echinoderms. furthermore, experimental studies revealed that the prrp-type neuropeptide pqdrskamqaertgqlrrlnprf-nh( ) is a potent ligand for a snpf/prrp-type receptor in the starfish asterias rubens. our findings indicate that prrp-type and snpf-type signalling systems are orthologous and originated as a paralog of npy/npf-type signalling in urbilateria. neuropeptides are neuronally secreted signalling molecules that regulate many physiological processes and behaviours in animals, including feeding, digestion, reproduction and social behaviour. they typically exert effects by binding to cognate g-protein coupled receptors (gpcrs) on target cells, which leads to changes in the activity of downstream effectors (e.g. ion channels, enzymes) (jékely et al., ) . investigation of the evolution of neuropeptide signalling has revealed that many of the neuropeptide systems found in vertebrates have orthologs in invertebrate deuterostomes (urochordates, cephalochordates, hemichordates, echinoderms) and protostomes (e.g. arthropods, nematodes, molluscs, annelids, platyhelminthes). thus, the evolutionary origin of over thirty neuropeptide signalling systems has been traced back to the common ancestor of the bilateria (urbilateria) (jékely, ; mirabeau and joly, ; elphick et al., ) . one of the neuropeptide systems that originated in urbilateria is neuropeptide y (npy)-type signalling. npy is a -residue peptide that was first isolated from the porcine hypothalamus tatemoto, ) but which is also expressed by neurons in many other regions of the nervous system (adrian et al., ; morris, ) and in peripheral organs such as the gut and cardiovascular system (holzer et al., ; farzi et al., ) . accordingly, npy is pleiotropic (pedrazzini et al., ) , although it is most widely known as a potent stimulant of food snpf-type signalling may be restricted to protostomes (mirabeau and joly, ) . subsequently, snpf-type peptides and a cognate receptor have been characterised in the bivalve mollusc crassostrea gigas, confirming the occurrence of this signalling system in the lophotrochozoan branch of the protostomes (bigot et al., ) . furthermore, the physiological roles of snpf-type neuropeptides have been characterised in c. gigas and in other molluscs (hoek et al., ; zatylny-gaudin et al., ; bigot et al., ) . important insights into neuropeptide evolution have been obtained recently by pharmacological characterisation of g-protein coupled neuropeptide receptors in invertebrate deuterostomes (kawada et al., ; roch et al., ; bauknecht and jékely, ; semmens et al., ; tian et al., ; yañez-guerra et al., ) . however, currently little is known about the occurrence and characteristics of npy/npf/prrp/snpf-related signalling systems in invertebrate deuterostomes. phylogenetic analysis of bilaterian g-protein coupled neuropeptide receptors has demonstrated the occurrence of npy/npf receptor-related proteins in ambulacrarians -the echinoderm strongylocentrotus purpuratus and the hemichordate saccoglossus kowalevskii (mirabeau and joly, ) . furthermore, the precursor of a putative npy/npf-type peptide was identified in s. kowalevskii (mirabeau and joly, ; elphick and mirabeau, ) . a candidate npy/npf-type precursor has also been identified in the cephalochordate branchiostoma floridae, but an npy/npf-type receptor has yet to be identified in this species (mirabeau and joly, ; elphick and mirabeau, ) . a more recent finding was the discovery of a family neuropeptide precursor-type proteins in echinoderms that contain a peptide that shares sequence similarity with npy/npf-type peptides (zandawala et al., ) . however, it is not known if these proteins are orthologs of vertebrate npy-type precursors and protostome npftype precursors. to address this issue, detailed analysis of the sequences of the echinoderm npy/npf-like peptides and precursors and the genes encoding these peptides/proteins is needed. furthermore, the receptors for echinoderm npy/npf-like peptides need to be identified. accordingly, here we show that npy/npf-type signalling has in fact been lost in echinoderms and report the discovery and pharmacological characterisation of a prrp/snpf-type signalling system in an echinoderm -the starfish asterias rubens. these findings provide important new insights into the evolution of neuropeptide signalling in the bilateria. the sequence of a transcript (contig ; genbank accession number mk . ) encoding the precursor of an npy-like neuropeptide has been reported previously based on analysis of neural transcriptome sequence data from the starfish a. rubens (zandawala et al., ) . here, a cdna encoding this precursor was cloned and sequenced, revealing that the open reading frame encodes a -residue protein comprising a predicted -residue signal peptide, a -residue npy-like peptide sequence with an n-terminal glutamine residue and a c-terminal glycine residue, followed by a putative monobasic cleavage site (figure -figure supplement a) . analysis of radial nerve cord extracts using mass spectrometry (lc-ms-ms) revealed the presence of a peptide with the structure pqdrskamqaertgqlrrlnprf-nh , showing that the n-terminal glutamine and c-terminal glycine in the precursor peptide are post-translationally converted to a pyroglutamate residue and an amide group, respectively ( figure -figure supplement b) . alignment of the sequences of the a. rubens neuropeptide and orthologs from other echinoderms with related peptides in other taxa revealed that they share sequence similarity with both prrp-type neuropeptides ( figure a ) and with npy/npf-type neuropeptides ( figure b) . however, the echinoderm peptides comprise - residues and are similar in length to vertebrate prrps, which are - residues as full-length peptides and in some species can occur as n-terminally truncated peptides due the presence of a monobasic cleavage site (hinuma et al., ; tachibana and sakamoto, ) . this contrasts with npy/npf-type neuropeptides, which are longer peptides ranging in length from to residues (fadda et al., ) . furthermore, by analysing sequence data from the hemichordate s. kowalevskii and the cephalochordate b. floridae, here we identified novel neuropeptides that share sequence similarity with the echinoderm figure . comparison of the sequences of echinoderm npy/npf/prrp-like peptides with related peptides in other taxa. (a) comparison with prrp-type neuropeptides. conserved residues are highlighted in black (identical) or grey (conservative substitutions) (b) comparison with npy/npf-type neuropeptides. conserved residues are highlighted in black (identical) or grey (conservative substitutions). the arrowheads indicate residues that have been shown to be important for the three-dimensional structure of the npy/npf-type peptides but which are not present in the echinoderm peptides. the colour coding of phyla is as follows: dark blue (echinodermata), light blue (hemichordata), purple (chordata), orange (platyhelminthes), red (lophotrochozoa), yellow (priapulida), green (arthropoda), grey (nematoda). the full names of the species and the accession numbers of the sequences are listed in figure -source data . figure continued on next page neuropeptides and with vertebrate prrps ( figure a) . thus, sequence alignment reveals that, in addition to a shared characteristic of a c-terminal rfamide or a ryamide (y and f being conservative substitutions), there are other residues in the echinoderm peptides that are identical or structurally similar to equivalently positioned residues in chordate prrps ( figure a) . contrastingly, the echinoderm peptides lack two proline (p) residues that are a conserved feature of the n-terminal region of many npy/npf-type peptides, with the exception of some peptides that have only one of these proline residues and a peptide in the cephalochordate branchiostoma floridae that has neither ( figure b) . furthermore, there are four other residues that are highly conserved in bilaterian npy/npf-type peptides -tyrosine (y), leucine (l), tyrosine (y), and isoleucine (i) residues, which are marked with arrowheads in figure b . these residues have been shown to be important for the formation of the three-dimensional structure in vertebrate npytype peptides (blundell et al., ; glover et al., ; glover et al., ; allen et al., ) , so these residues may likewise be important for npf receptor activation and bioactivity. importantly, none of these residues are present in the echinoderm peptides. it is noteworthy, however, that all but one of the aforementioned six conserved residues in npy/ npf-type peptides are present in a peptide from a species belonging to a sister phylum of the echinoderms -the hemichordate saccoglossus kowalevskii ( figure b ; mirabeau and joly, ; elphick and mirabeau, ) . collectively these findings indicate that the echinoderm neuropeptides originally described as npy-type peptides (zandawala et al., ) are not orthologs of npy/ npf-type peptides but are orthologs of chordate prrp-type peptides. therefore, henceforth we will refer to the a. rubens neuropeptide pqdrskamqaertgqlrrlnprf-nh as arprrp and we will refer to orthologs in other echinoderms equivalently. echinoderm prrp-like peptide genes have the same exon-intron structure as chordate prrp genes to investigate further the proposition that arprrp and other echinoderm prrp-like neuropeptides are orthologs of chordate prrps, we compared the exon-intron structure of genes encoding these peptides ( figure ). this revealed that a common characteristic is the presence of an intron that interrupts the coding sequence at a position corresponding to the n-terminal or central region of the echinoderm prrp-like peptides and vertebrate prrps. furthermore, in echinoderm prrp-like peptide genes and vertebrate prrp genes the intron interrupts the coding sequence in the same frame, at a position between the first and second nucleotide of the interrupted codon (a phase one intron), which is denoted by + in figure . genes encoding novel precursors of prrp-like peptides in s. kowalevskii and b. floridae also have a phase one intron. furthermore, in the b. floridae gene and in one of the s. kowalevskii genes (skow ) the intron is located in the region of the gene encoding the n-terminal part of the neuropeptide, whereas in the other s. kowalevskii gene (skow ) the intron is located in a region encoding the c-terminal part of the neuropeptide. the presence of a conserved intron in the same frame in echinoderm prrp-like peptide genes, the two s. kowalevskii prrp-like peptide genes and chordate prrp-type genes supports our hypothesis that the echinoderm and hemichordate prrp-like peptides are orthologs of chordate prrp-type neuropeptides. by way of comparison, echinoderm prrp-like peptide genes have a different exon-intron structure to npy/npf genes. previous studies have reported that a conserved feature of npy/npf genes is an intron that interrupts the coding sequence for npy/npf-type peptides, with the intron located between the second and third nucleotide of the codon for the arginine residue of the c-terminal rf or ry dipeptide (mair et al., ) . here we show this conserved feature in npy/npf genes in source data . accession numbers of the precursor sequences used for the peptide alignments in figure . species from several animal phyla, including a hemichordate (sister phylum to the echinoderms), chordates, molluscs, an annelid, a priapulid, an arthropod and a nematode (figure -figure supplement ). in echinoderm prrp-like peptide genes, the exon encoding the neuropeptide is likewise interrupted by an intron but it is located in a different position to the intron that interrupts the coding sequence for npy/npf-type peptides. thus, it does not interrupt the codon for the arginine of the c-terminal rf or ry motif, but instead it is located between the first and second nucleotide of the codon for a residue located in the n-terminal or central regions of echinoderm prrp-like peptides ( figure -figure supplement ) . another difference is that typically in npy/npf genes there is another intron that interrupts the coding sequence in the c-terminal region of the precursor protein, whereas in the echinoderm prrp-like peptide precursor genes the coding sequence for the c-terminal region of the precursor protein is not interrupted by an intron (figure -figure supplement ) . collectively, these findings provide further evidence that echinoderm prrp-like peptides are not orthologs of npy/npf-type neuropeptides. . the protein-coding exons are colour-coded to show regions that encode the n-terminal signal peptide (blue), the neuropeptide (red), monobasic or dibasic cleavage sites (green) and other regions of the precursor protein (grey). note that a common characteristic is that an intron interrupts the coding sequence in the n-terminal or central region of the neuropeptide, with the intron consistently located between the first and second nucleotides (phase one intron represented by + ) of the codon for the amino acid shown after intron. taxa are highlighted in phylum-specific colours: dark blue (echinodermata), light blue (hemichordata), purple (chordata). the full names of the species and the accession numbers of the sequences are listed in figure -source data . the online version of this article includes the following source data and figure supplement(s) for figure : source data . accession numbers of the sequences used for the gene structure analysis in figure and discovery of orthologs of snpf/prrp-type receptors in a. rubens and other echinoderms having obtained evidence that echinoderm npy/prrp-like peptides are not orthologs of npy/npftype neuropeptides but are orthologs of prrp-type peptides, we then investigated the occurrence in a. rubens and other echinoderms of proteins related to gpcrs that mediate effects of npy/npftype peptides, prrp-type peptides and snpf-type peptides in other bilaterians. using receptor sequences of h. sapiens npy-type, d. melanogaster npf-type, h. sapiens prrp-type and d. melanogaster snpf-type receptors as queries for similarity-based analysis of a. rubens neural figure . blosum cluster map of npy/npf/prpr/snpf-type receptors and closely related tachykinin-type receptors (tkr) and luqin/ryamide-type receptors (lq/ryar). nodes are labelled with phylum-specific colours, as shown in the key, and connections represent blast relationships with a p value > e- . note that the echinoderm receptors (boxed) have more connections with prrp/snpf-type receptors than with npy/npf-type receptors. the sequences of the receptors included in this figure are listed in figure -source data . the online version of this article includes the following source data and figure supplement(s) for figure : source data . accession numbers of the receptor sequences used for the clans analysis in figure . transcriptome sequence data, a transcript (contig ) encoding a -residue protein was identified as the best hit (figure -figure supplement ). furthermore, homologs of the a. rubens protein encoded by contig were also identified in other echinoderms for which genome sequences have been obtained, including the starfish a. planci, the sea urchin s. purpuratus and the sea cucumber a. japonicus, and importantly no other npy/npf/prrp/snpf-type receptors were identified in these species. to investigate relationships of the novel echinoderm receptors with other bilaterian neuropeptide receptors, we generated a sequence database including bilaterian npy/ npf/prrp/snpf-type receptors and other closely related receptors (tachykinin-type, luqin-type receptors) as outgroups. these receptor sequences were then analysed using two different methodologies. firstly, we performed a cluster-based analysis of the receptor sequences using clans ( figure ). this analysis revealed three main clusters: . a cluster comprising the outgroup receptors (tachykinin/luqin), . a cluster comprising npy/npf-type receptors and . a cluster comprising snpf-type receptors and prrp-type receptors. interestingly, the echinoderm receptors showed stronger connections with the snpf/prrp receptor cluster ( figure , black square) than with the npy/npf receptor cluster. these findings indicate that snpf-type receptors and prrp-type receptors are orthologous, as has been proposed previously based on cluster-based analysis of receptor sequences (jékely, ). furthermore, these findings indicate that npy/npf/prrp/snpf-type receptors in echinoderms are not orthologs of npy/npf-type receptors but are orthologs of snpf/prrp-type receptors. however, it is noteworthy that the lines linking the echinoderm receptors and nematode snpf-type receptors with other snpf/prrp-type receptors in clans are quite long (figure ) , which is indicative of sequence divergence. secondly, we performed a phylogenetic analysis of the receptor sequences using the maximum likelihood method. for this analysis, in addition to bilaterian npy/npf-type receptors, deuterostome prrp-type receptors and protostome snpf-type receptors, we included tachykinintype, luqin-type and gpr -type receptors as outgroups. this revealed that the echinoderm receptors are positioned within a branch of the phylogenetic tree that comprises npy/npf-type, prrp-type and snpf-type receptors, with the other receptor types included in the analysis occupying an outgroup position ( figure ). more specifically, the echinoderm receptors are positioned in a clade comprising snpf-type receptors, with bootstrap support of > %, indicating that the echinoderm receptors are orthologs of protostome snpf-type receptors. however, it is noteworthy that snpf-type receptors and prrp-type receptors do not form a monophyletic clade as would be expected for orthologous receptors. this may be a consequence of sequence divergence in the echinoderm and nematode snpf/prrp-type receptors that is reflected in the long branches leading to these receptors. because the phylogenetic analysis revealed that the echinoderm receptors are positioned in a clade comprising protostome snpf-type receptors (figure ), we also compared the sequences of echinoderm prrp-type peptides and protostome snpf-type peptides (figure -figure supplement ) and the structures of the genes encoding these neuropeptides (figure -figure supplement ). this revealed that sequence identity is restricted to a few residues in the c-terminal regions of the peptides and, furthermore, the echinoderm prrp-type peptides are much longer than protostome snpf-type peptides (figure -figure supplement ). this contrasts with the much higher levels of sequence similarity shared between echinoderm prrp-type neuropeptides and chordate prrp-type neuropeptides, as shown in figure a . another difference is that protostome snpf-type neuropeptide precursors typically give rise to multiple snpf-type peptides, whereas echinoderm prrp-type precursors are similar to chordate prrp-type precursors in containing a single prrp-type neuropeptide that is located adjacent to the signal peptide (figure -figure supplement ). accordingly, comparison of the exon/intron structure of the genes encoding prrp-type precursors in echinoderms and snpf-type precursors in protostomes also revealed limited similarity (figure -figure supplement ). collectively, our analysis of sequence data indicates that npy/npf/prrp/snpf-type receptors in echinoderms are not orthologs of npy/npf-type receptors but are orthologs of snpf/prrp-type receptors. therefore, henceforth we refer to these echinoderm receptors as snpf/prrp-type receptors and specifically refer to the snpf/prrp-type receptor in the starfish a. rubens as ar-snpf/ prrpr. furthermore, having identified ar-snpf/prrpr we proceeded to investigate if arprrp acts as a ligand for this receptor. . phylogenetic tree showing that a candidate receptor for the a. rubens neuropeptide arprrp is an ortholog of protostome snpf-type receptors. the tree includes npy/npf-type receptors, chordate prrp-type receptors and protostome snpf-type receptors, with gpr -type, luqin-type, and tachykinin-type receptors as outgroups to root the tree. interestingly, the candidate receptor for the a. rubens neuropeptide arprrp (red arrow) and orthologs from other echinoderms are positioned in a clade comprising protostome snpf-type receptors, whereas candidate receptors for prrptype peptides in the hemichordate s. kowalevskii are positioned in a clade containing chordate prrp-type receptors. note that npy/npf-type receptors form a distinct clade that includes an npy/npf-type receptor from the hemichordate s. kowalevskii, but no echinoderm receptors are present in this clade. the tree was generated in w-iq-tree . using the maximum likelihood method. the stars represent bootstrap support ( replicates, see legend) and the coloured backgrounds represent different taxonomic groups, as shown in the key. the names with text in blue represent the receptors for which ligands have been experimentally confirmed. the asterisks highlight receptors where the reported ligand is atypical when compared with ligands for receptors in the same clade. species names are as follows: aaeg (aedes aegypti), acal (aplysia californica), ajap (apostichopus japonicus), amis (alligator mississippiensis), apla (acanthaster planci), arub (asterias rubens), bbel (branchiostoma belcheri), bdor (bactrocera dorsalis), bflo a cdna encoding ar-snpf/prrpr was cloned and sequenced ( figure -figure supplement ) and its sequence has been deposited in genbank under accession number mh . . analysis of the sequence of ar-snpf/prrpr using protter revealed seven predicted transmembrane domains, as expected for a gpcr ( figure -figure supplement ) . the cloned receptor was then co-expressed with ga in cho-k cells expressing apoaequorin to produce the cell system cho-ar-snpf/ prrpr. synthetic arprrp (pqdrskamqaertgqlrrlnprf-nh ) was tested as a candidate ligand for ar-snpf/prrpr at concentrations ranging from À m to À m, comparing with cells incubated in assay media without the addition of the peptide. this revealed that arprrp at a concentration of À m triggers luminescence responses (defined as %) in cho-ar-snpf/prrpr cells that were approximately five times the background luminescence detected with the assay media used to dissolve the peptide ( figure a) , demonstrating that arprrp acts as a ligand for the receptor. furthermore, arprrp induced dose-dependent luminescence in cho-ar-snpf/prrpr cells with a halfmaximal response concentration (ec ) of .  À m ( figure b ). importantly, no response to arprrp was observed in cho-k cells transfected with the vector alone, demonstrating that the signal observed in cho-ar-snpf/prrpr cells exposed to arprrp can be attributed to activation of the transfected receptor ( figure -figure supplement ) . because arprrp contains a potential dibasic cleavage site (see underlined arginine residues in its sequence: pqdrskamqaertgqlrrlnprf-nh ), we hypothesised that the c-terminal pentapeptide of arprrp (lnprfamide) may also be generated from arprrpp in vivo. therefore, we also tested synthetic lnprfamide as a candidate ligand for ar-snpf/prrpr. however, this peptide did not induce luminescence responses in cho-ar-snpf/ prrpr cells ( figure b) . therefore, we conclude that the -residue amidated peptide arprrp is the natural ligand for ar-snpf/prrpr in a. rubens. the a. rubens luqin-type neuropeptide arlq also did not induce luminescence responses in cho-ar-snpf/prrpr cells, demonstrating the selectivity of ar-snpf/prrpr for arprrp as a ligand ( figure b ). the discovery of an npy-like neuropeptide, named npf, in a platyhelminth provided the first definitive molecular evidence that npy-type neuropeptides originated in a common ancestor of the bilateria (maule et al., ) . subsequently, analysis of transcriptomic/genomic sequence data has enabled identification of npy/npf-type neuropeptides and their cognate receptors in a variety of invertebrate taxa, revealing a high level of conservation of this signalling system in bilaterian phyla (zatylny-gaudin and favrel, ; fadda et al., ) . here we report the first detailed analysis npy/npf-related signalling systems in echinoderms -invertebrate deuterostomes that have provided key insights into the evolution of other neuropeptide signalling systems (semmens et al., ; tian et al., ; elphick et al., ; yañez-guerra et al., ) . recently, we reported the discovery of echinoderm proteins comprising putative neuropeptides that share sequence similarity with npy/npf-type peptides (zandawala et al., ) . however, here our detailed analysis of the sequences of these peptides and the genes encoding them has revealed that they are not orthologs of the npy/npf-type neuropeptides. consistent with this finding, orthologs of npy/npf-type receptors were also not found in echinoderms. therefore, we conclude that npy/npf-type neuropeptide signalling has been lost in the phylum echinodermata ( figure ). this is a noteworthy because, to the best of our knowledge, the only other taxon in which loss of npy/npf-type signalling has been reported are the urochordates, a sub-phylum of the phylum chordata (mirabeau and joly, ; figure ). the evolutionary and functional significance of loss of npy/npf-type signalling in echinoderms and urochordates is rubens prrp/snpf-type receptor ar-snpf/prrpr, the promiscuous g-protein g a and the calcium-sensitive luminescent gfp-apoaequorin fusion protein g a. for comparison, the background luminescence of cells that were not exposed to arprrp is shown (basal media; grey bar). mean values (± s.e.m) were determined from three independent experiments performed in triplicate (b). graph showing the selectivity of arprrp as a ligand for ar-snpf/prrpr. arprrp causes dose-dependent luminescence in cho-k cells expressing ar-snpf/prrpr, with an ec of . nm. ar-snpf/prrpr is not activated by a c-terminal pentapeptide fragment of arprrp (lnprfamide) or by the a. rubens luqin-type peptide arlq. each point represents mean values (± s.e. figure continued on next page unknown. however, insights into this issue may emerge from functional characterisation of npy/ npf-type signalling in other invertebrates. the nematode c. elegans is a powerful model system for functional characterisation of neuropeptide signalling systems (frooninckx et al., ) . however, npy/npf-type signalling has thus far only been partially characterised in this species. here, our phylogenetic analysis (figure ) indicates that there are two c. elegans receptors that are orthologs of npy/npf-type receptors: npr- , which is an orphan receptor, and npr- , which has been shown to be activated by the peptide mdanafrmsfamide (chalasani et al., ) . however, this peptide shares little sequence similarity with npy/npf-type peptides from other bilaterians. furthermore, receptor assays only showed activation at peptide concentrations of and mm (chalasani et al., ) , which are high when compared to other npy/npf-type receptors that are typically activated by ligands in the nanomolar range (bard et al., ; lundell et al., ; garczynski et al., ; saberi et al., ) . recently, based on similarity-based sequence alignments, it has been suggested that the mature peptide derived from the c. elegans protein flp- may be an ortholog of npy/npf-type peptides (fadda et al., ) . here, our analysis of the structure of the gene encoding the flp- precursor has revealed that it has the characteristic structure of npy/npf-type genes, with an intron interrupting the codon for the c-terminal arginine of the npf-type peptide sequence (figure -figure supplement ). thus, based on our analysis of c. elegans sequence data, we conclude that the npy/ npf-type peptide derived from the flp- precursor protein is likely to act as a ligand for the npr- and/or npr- receptors. this finding provides a basis for functional characterisation of npy/ npf-type signalling in c. elegans. if the echinoderm npy-like peptides are not orthologs of npy/npf-type neuropeptides, then what are they? here we show that these peptides share sequence similarity with vertebrate prrp-type neuropeptides ( figure a) . furthermore, analysis of the structure of the genes encoding the echinoderm neuropeptides revealed that the coding sequence for the neuropeptides is interrupted by an intron in the phase one frame, a feature that is also a characteristic of genes encoding vertebrate prrp-type neuropeptides (figure ) . these findings indicate that the echinoderm neuropeptides are orthologs of vertebrate prrp-type neuropeptides. to further address this issue we analysed echinoderm genome/transcriptome sequence data to identify candidate cognate receptors for the echinoderm prrp-like peptides. a cluster-based analysis of receptor sequence data using clans revealed the presence in echinoderms of receptor proteins that show strong connections with a receptor cluster comprising vertebrate prrp-type receptors and protostome snpf-type receptors (figure ) . accordingly, a previous cluster-based analysis of receptor sequence data has reported that vertebrate prrp-type receptors cluster with protostome snpf-type receptors, indicating that these receptors may be orthologous (jékely, ). a novelty of our analysis is the inclusion of several echinoderm receptor sequences. it is noteworthy, however, that whilst strong connections between the echinoderm receptors and prrp/snpf-type receptors in other taxa can be seen using clans, the lines linking to the echinoderm receptors are quite long (figure ). this suggests that the echinoderm receptors are orthologs of prrp/snpf-type receptors but have undergone sequence divergence. interestingly, a group of snpf-type receptors in the nematode c. elegans appears to be similarly divergent with respect to other snpf/prrp-type receptors (figure ) . . phylogenetic diagram showing the occurrence of npy/npf-type, snpf-type and prrp-type neuropeptide signalling in the bilateria. the tree shows the phylogenetic relationships of selected bilaterian phyla. a gene duplication event giving rise to the paralogous npy/npf-type (green) and prrp/snpf (purple) signalling systems is shown at a position in the tree corresponding to the common ancestor of the bilateria. phyla in which npy/ npf-type peptides/precursors and npy/npf-type receptors have been identified are labelled with green-filled squares. phyla in which prrp-type peptides/precursors and prrp-type receptors have been identified are labelled with blue-filled squares. phyla in which snpf-type peptides/precursors and snpf-type receptors have been identified are labelled with red-filled squares. the inclusion of an asterisk in filled squares indicates that activation of a receptor by a peptide ligand has been demonstrated experimentally. note that in the starfish asterias rubens (this study) a prrp-type peptide (blue triangle) is the ligand for receptor that has been found to be an ortholog snpf/prrp-type receptors (figure ) or an ortholog of snpf-type receptors ( figure ) ; hence this receptor is represented here as a red triangle. note also the mutually exclusive patterns in the phylogenetic distribution of snpftype signalling and prrp-type signalling, with the former found in protostomes and the latter found in vertebrates, cephalochordates and hemichordates, which is supportive of the hypothesis that these signalling systems are orthologous. our discovery of a prrp/snpf-type signalling system in echinoderms provides a missing link in the evolution of this neuropeptide signalling system. npy/npf-type signalling occurs in most phyla, but it has been lost in echinoderms and urochordates. the inclusion of a question mark for the putative npy/npf-type peptide identified in the cephalochordate b. floridae (mirabeau and joly, ; elphick and mirabeau, ) signifies that it is atypical of npy/npf-type peptides, which may explain why npy/npf-type receptors have yet to be identified in cephalochordates. the inclusion of a question mark in the c. elegans green square figure continued on next page to further investigate the relationship of the echinoderm receptors with snpf/prrp-type receptors, we performed a phylogenetic analysis of sequence data using the maximum likelihood method (figure ) . in this analysis, the echinoderm receptors are positioned in a clade comprising protostome snpf-type receptors. however, snpf-type receptors and prrp-type receptors do not form a monophyletic clade in the tree. interestingly, this finding has been reported previously as part of a wider analysis of neuropeptide receptor relationships in the bilateria (mirabeau and joly, ) . thus, there is inconsistency in the findings from cluster-based analysis (clans) (jékely, ; figure ) and phylogenetic tree-based analysis (mirabeau and joly, ; figure ) of receptor relationships. one possible explanation for this inconsistency would be that gene duplication in a common ancestor of the bilateria gave rise to two snpf/prrp-type signalling systems, which were then differentially lost/retained in bilaterian lineages, but in such a scenario gene loss in several lineages would have to be invoked. alternatively, the inconsistency may, at least in part, be a consequence of sequence divergence in echinoderm and nematode snpf/prrp-type receptors with respect snpf/prrp-type receptors in other taxa, which is reflected in their position peripheral to the main cluster of snpf/prrp-type receptors in the clans. accordingly, it is noteworthy that in the phylogenetic tree (figure ) there is a long branch leading to the echinoderm receptor clade and likewise nematode snpf-type receptors also have long branches (figure ) . nevertheless, collectively our sequence analysis indicates that the echinoderm receptors are orthologs of snpf/prrptype receptors. therefore, it was of interest to determine if echinoderm prrp-type neuropeptides act as ligands for snpf/prrp-type receptors in this phylum. here we show that the a. rubens prrp-type neuropeptide arprrp (pqdrskamqaertg qlrrlnprf-nh ) is a potent ligand for the a. rubens snpf/prrp-type receptor ar-snpf/prrpr ( figure ). these findings demonstrate for the first time the existence and molecular identity of a prrp-type signalling system in an echinoderm. furthermore, our identification of orthologs of arprrp and ar-snpf/prrpr in other echinoderms, including for example the sea urchin s. purpuratus, demonstrates the conservation of this signalling system in this phylum. in addition, our comparative analysis of sequence data has also enabled identification of genes/transcripts encoding prrp-type neuropeptides in the hemichordate s. kowalevskii and the cephalochordate b. floridae (figure ). previous studies have concluded that snpf-type signalling is paralogous to npy/npf-type signalling in protostomes (nässel and wegener, ) and that prrp-type signalling is paralogous to npy/ npf-type signalling in vertebrates (lagerströ m et al., ) . evidence that the prrp-type and snpf-type signalling systems may be orthologous has also been reported previously (jékely, ), but this hypothesis has not been tested experimentally. our discovery of a starfish prrp-type neuropeptide that acts as a ligand for a starfish ortholog of snpf-type receptors is important because it provides a missing link for reconstruction of the evolutionary history of prrp/snpf-type neuropeptide signalling (figure ) . comparison of the sequences of vertebrate prrp-type neuropeptides and protostome snpf-type neuropeptides reveals low levels of sequence similarity, which no doubt in part explains why prrptype and snpf-type neuropeptides have not been recognised as orthologs. in figure -figure indicates that the peptide identified as a ligand for the c. elegans npy/npf-type receptor (chalasani et al., ) does not have the typical features of an npy/npf-type peptide. the grey square for snpf in m. expansa, for which only transcriptome sequence data are available, indicates that snpftype peptides and snpf-type receptor(s) are likely to be present in this species because snpf-type peptides and snpf-type receptors have been identified in another platyhelminth species, s. mediterranea, for which a genome sequence is available. species names are as follows: h. sapiens (homo sapiens), c. intestinalis (ciona intestinalis), b. floridae (branchiostoma floridae), s. kowalevskii (saccoglossus kowalevskii), a. rubens (asterias rubens), p. dumerilii (platynereis dumerilii), l. stagnalis (lymnaea stagnalis), m. expansa (moniezia expansa), s. mediterranea (schmidtea mediterranea), c. gigas (crassostrea gigas), d. melanogaster (drosophila melanogaster), c. elegans (caenorhabditis elegans). silhouettes of representative animals from each phylum are from www.openclipart.com and they are free from copyright. supplement we illustrate this in an alignment of the echinoderm prrp-type neuropeptides and protostome snpf-type neuropeptides, with sequence identity restricted to a few residues in the c-terminal regions of these peptides. this contrasts with the higher levels of sequence similarity shared between echinoderm prrp-type neuropeptides and vertebrate prrp-type neuropeptides, as shown in figure a . furthermore, echinoderm prrp-type precursors are similar to chordate prrptype precursors in containing a single long neuropeptide, whereas protostome snpf-type precursors typically contain multiple smaller neuropeptides. thus, there is little evidence of orthology from comparison of echinoderm prrp-type and protostome snpf-type neuropeptide, precursor and gene sequences. consequently, our conclusion that the echinoderm prrp-type peptides are orthologs of protostome snpf-type peptides is principally based on the orthology of their receptors (figure ) and our experimental demonstration that a prrp-like peptide (arprrp) acts as a ligand for a snpf/ prrp-type receptor (ar-snpf/prrpr) in the starfish a. rubens ( figure ) . it is important to note, however, that this is not unprecedented in investigations of the evolution of neuropeptide signalling. thus, whilst the sequences of some neuropeptides and neuropeptide precursors are highly conserved throughout the bilateria, others are so divergent that they can be unrecognisable as orthologs. an example of the former are vasopressin/oxytocin (vp/ot)-type neuropeptides and precursors. an example of the latter are neuropeptide-s (nps)/crustacean cardioactive peptide (ccap)-type neuropeptides and precursors, which are paralogs of vp/ot-type neuropeptides and precursors (semmens et al., ) . thus, by way of comparison, npy/npf-type neuropeptides are similar to vp/ot-type neuropeptides in exhibiting a high level of sequence conservation throughout the bilateria. conversely, prrp/snpf-type neuropeptides are similar to nps/ccap-type neuropeptides in being highly divergent, with neuropeptides in protostomes and deuterostomes exhibiting modest sequence similarity. the discovery of prrp/snpf-type signalling in echinoderms has provided a unique opportunity to speculate on the ancestral characteristics of this signalling system in urbilateria. it is noteworthy that, by comparison with the protostome snpf-type peptides, the echinoderm prrp-type peptides have more features in common with the paralogous npy/npf-type peptides. prrp-type peptides are not as long as npy/npf-type peptides but they are nevertheless much longer than protostome snpf-type peptides. furthermore, it was the sequence similarity that echinoderm prrp-type peptides share with npy/npf-type peptides that originally facilitated their discovery (zandawala et al., ) . additionally, the structure of the prrp-type precursors is similar to npy/npf-type precursors because the neuropeptide is located immediately after the signal peptide, whereas this is not a feature of protostome snpf-type precursors. based on these observations, we propose that prrp-type peptides and precursors may more closely resemble the ancestral characteristics of the prrp/snpf type signalling system in urbilateria. furthermore, we speculate that the common ancestor of the paralogous npy/npf-type and prrp/snpf-type neuropeptide precursors may have been similar to npy/npf-type precursors with respect peptide, precursor and gene structure. then, following gene duplication, these ancestral characteristics were retained in the paralog that gave rise to the bilaterian npy/npf-type peptides/precursors. in contrast, the paralog that gave rise to prrp/snpf-type signalling diverged from the ancestral condition. however, the extent of divergence varies in the deuterostome and protostome lineages. in deuterostomes, the prrp-type peptides/precursors have many npy/npf-type characteristics and we conclude that this reflects less divergence from the proposed ancestral condition. conversely, in the protostomes, the snpf-type peptides/precursors exhibit little similarity with npy/npf-type peptides/precursors and we conclude that this reflects more divergence from the proposed ancestral condition. in conclusion, our discovery of a prrp/snpf-type signalling system in echinoderms has provided a missing link that unites prrp-type peptides in vertebrates and snpf-type peptides in protostomes as members of a bilaterian family of neuropeptides, as illustrated in figure . this represents an important advance in our knowledge of neuropeptide signalling systems in the bilateria and illustrates the value of insights from echinoderms in enabling reconstruction of the evolutionary history of neuropeptides. animals starfish (asterias rubens) were obtained from a fisherman based at whitstable (kent, uk). they were then maintained in a circulating seawater aquarium at~ ˚c in the school of biological and chemical sciences at queen mary university of london and were fed on mussels (mytilus edulis) collected near margate (kent, uk). cloning and sequencing of a cdna encoding the precursor of an a. rubens npy/npf/prrp-like peptide a transcript encoding the a. rubens precursor of an npy/npf-like peptide was reported previously (genbank: mk ) (zandawala et al., ) . however, in this paper we show that the npy/npflike peptide derived from this precursor shares more sequence similarity with prrp-type peptides. a cdna containing the complete open reading frame of the precursor was amplified by pcr using a. rubens radial nerve cord cdna, the forward primer aagtcaaaaggcgagcaaga, the reverse primer aaagggatgtggtgttggtg and q polymerase (neb; cat. no. m s). the pcr products were ligated into the pbluescript ii ks (+) vector (invitrogen; cat. no. k ) that had been cut previously with the restriction enzyme ecorv by performing blunt-end ligation with t dna ligase (neb; cat. no. m s). the cloning was confirmed by restriction enzyme digestion and sequencing (tubeseq service; eurofins genomics). structural characterisation of the a. rubens npy/npf/prrp-like peptide using mass spectrometry after confirming the nucleotide sequence of the a. rubens precursor of a npy/npf/prrp-like peptide by cloning and sequencing, mass spectrometry was used to determine the mature structure of the peptide. the methods employed, including extraction of peptides from a. rubens radial nerve cords, treatment of samples, equilibration of columns, reverse phase chromatography for the initial separation and injection into a orbitrap-fusion (thermoscientific) for tandem mass spectrometry (ms/ms), were performed using a previously reported protocol for the identification of the starfish neuropeptides (lin et al., ) . the methods employed for data analysis are described below. mass spectra were searched using sequest proteome discoverer (thermo fisher scientific, v. . ) against a database comprising forty-three different precursor proteins identified by analysis of a. rubens neural transcriptome data, including the a. rubens arprrp precursor and all proteins in gen-bank from species belonging to the asteriidae family and the common repository of adventitious proteins database (http://www.thegpm.org/crap/index.html). theoretical peptides were generated allowing up to two missed cleavages and variable modifications, including amidation (À . ) of c-terminal glycines and pyroglutamate (À . ) of n-terminal glutamines, and oxidation of methionine (+ . ). precursor mass tolerance was ppm and fragment ions were searched at . da tolerances. results from discoverer were collated and annotated in scaffold version . . (proteome software). sequence alignment of echinoderm npy/npf/prrp-like peptides with npy/npf-type peptides, prrp-type peptides, and snpf-type peptides from other taxa the amino acid sequences of echinoderm npy/npf/prrp-like peptides were aligned with the sequences of npy/npf-type peptides, prrp-type peptides and snpf-type peptides from a variety of bilaterian species (see figure -source data and figure -figure supplement -source data for lists of the sequences). to identify candidate ligands for prrp-type receptors in the cephalochordate b. floridae and the hemichordate s. kowalevskii, we analysed transcriptomic and genomic sequence data for these species (putnam et al., ; simakov et al., ) . the data analysed also included a list of predicted s. kowalevskii proteins kindly provided to o. mirabeau by dr. r.m. freeman (harvard medical school, usa). the methods employed to identify candidate neuropeptide precursors have been reported previously (mirabeau and joly, ) but here we had the more specific objective of identifying proteins with an n-terminal signal peptide followed by a neuropeptide with a predicted c-terminal rfamide or ryamide motif. this resulted in discovery of one candidate prrp-type precursor in the cephalochordate b. floridae and two candidate prrp-type precursors in the hemichordate s. kowalevskii. alignments were performed using mafft version ( iterations, substitution matrix; blosum ) and then manually curated. highlighting of the conserved residues was done using boxshade (www.ch.embnet.org/software/box_form.html) with % conservation as the minimum for highlighting. finally, the sequences were highlighted in phylum-specific or superphylum-specific colours: dark blue (echinodermata), light blue (hemichordata), purple (chordata), orange (platyhelminthes), red (lophotrochozoa), yellow (priapulida), green (arthropoda), grey (nematoda). comparison of the exon/intron structure of genes encoding npy/npf/ prrp-like peptides in echinoderms and genes encoding npy/npf-type peptides, prrp-type peptides and snpf-type peptides in other taxa the sequences of transcripts and genes encoding precursors of echinoderm precursors of npy/npf/ prrp-like peptides and precursors of npy/npf-type, prrp-type and snpf-type peptides from other taxa were obtained from genbank. the sequence of a predicted transcript encoding a second s. kowalevskii precursor (skow ) of a prrp-like peptide was determined based on a genscan prediction (burge and karlin, ; burge and karlin, ) from scaffold (genbank accession number nw_ . ). see figure -source data and figure -figure supplement source data for a list of the transcript and gene sequences analysed. the online tool splign (kapustin et al., ) (https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi) was employed to determine the exon/intron structure of genes and schematic figures showing gene structure were generated using ibs . (liu et al., ) . identification of a candidate receptor for the npy/npf/prrp-like peptide in a. rubens and analysis of its relationship with npy/npf/ prrp/snpf-type receptors in other taxa to identify a candidate receptor for the a. rubens npy/npf/prrp-like peptide, a. rubens neural transcriptome sequence data were analysed using the blast server sequenceserver (priyam et al., ) , submitting npy-type receptors from h. sapiens (genbank np_ . , np_ . , np_ . ), an npf-type receptor from d. melanogaster (genbank aaf . ), a prrp-type receptor from h. sapiens (np_ . ) and snpf-type receptors from d. melanogaster (genbank; np_ . ) and c.gigas (genbank xp_ . ) as query sequences. a transcript (contig ) encoding a -residue protein (http://web.expasy.org/translate/) was identified as the top hit in all blast searches and this has been deposited in genbank under the accession number mh . the protein sequence was also analysed using protter v . (omasits et al., ) . using blast, homologs of the a. rubens protein were identified in other echinoderms for which genome sequences are available, including the starfish acanthaster planci (xp_ . ), the sea urchin strongylocentrotus purpuratus (xp_ . ) and the sea cucumber apostichopus japonicus (pik . ). furthermore, no other npy/npf/prrp/snpf-type receptors were identified in these species. to investigate the relationship of the echinoderm receptors with neuropeptide receptors from other bilaterians, a database of receptor sequences was generated that included npy/npf-type, prrp-type, snpf-type, tachykinin-type, luqin-type and gpcr -type receptors (the latter three receptor types being included as outgroups), including representative species from the phyla chordata, hemichordata, echinodermata, mollusca, annelida, platyhelminthes, nematoda, priapulida, and arthropoda (see figure -source data for a list of the sequences used). a cluster-based analysis of the receptor sequences was performed using clans (frickey and lupas, ). an allagainst-all blast was performed using the scoring matrix blosum and linkage clustering was performed with an e-value of e- to identify coherent clusters. the clustering was first performed in d and then the map was collapsed to d to enable generation of the diagram shown in figure (see figure -source data for a list of sequences used). using the same receptor sequences, a phylogenetic tree was generated using the maximum-likelihood method. receptor sequences were aligned using muscle in the online tool ngphylogeny (iterative, iterations, upgmb as clustering method) (edgar, ; lemoine et al., ) and the alignment was automatically trimmed using trimal with automatic selection of trimming method using the online tool ngphylogeny (capella-gutierrez et al., ) . the trimming contained a total of residues that were used to generate the maximum-likelihood tree using w-iq-tree online version . (the model was automatically selected, being lg+g+i+f the chosen substitution model, branch tests used were ultrafastbootstrap replicates and sh-alrt replicates) (trifinopoulos et al., ) . the sequence database used for this tree, together with the trimmed alignment, and the raw tree are available at zenodo (https://zenodo.org/record/ ). to enable the pharmacological characterisation of a candidate receptor for the a. rubens npy/npf/ prrp-like peptide, a cdna encoding this receptor was cloned into the eukaryotic expression vector pcdna . (+) (invitrogen; . to facilitate expression of the cloned receptor, the forward primer included a partial kozak consensus sequence (acc) and a sequence corresponding to the first bases of the open reading frame of contig (accatgcagatgacaacc) and the reverse primer consisted of a stop codon and a sequence reverse complementary to the ' region of the open reading frame of contig (gcgtcacatagtggtatcatg). pcr was performed using the forward primer and reverse primers, a. rubens radial nerve cord cdna and q polymerase (neb; cat. no. m s). pcr products were ligated into the pcdna . (+) vector that had been cut previously with the restriction enzyme ecorv by performing blunt-end ligation with t dna ligase (neb; cat. no. m s). successful ligation and the direction of the insert was determined by restriction enzyme digestion and sequencing (tubeseq service; eurofins genomics). cell lines and pharmacological characterisation of a candidate receptor for the npy/npf/prrp-like peptide in a. rubens chinese hamster ovary (cho)-k cells stably expressing the calcium sensitive apoaequorin-gfp fusion protein (g a) (baubet et al., ) were used here for receptor assays. these cells have been used previously for neuropeptide receptor deorphanisation (bauknecht and jékely, ) and were generously supplied to us by dr gá spá r jé kely (university of exeter). the cell line was generated using the cho-k cell line from sigma-aldrich ( ), which is certified by the european collection of authenticated cell cultures (ecacc). following transfection with a plasmid encoding g a, cells were selected for stable transfection using geneticin g sulfate (thermo fisher scientific, cat. no. ) . the methods we used for cell culture and receptor assays have been described previously (yañez-guerra et al., ) . upon reaching a confluency of approximately %, cells were transfected with a plasmid containing the ar-snpf/prrp receptor cdna and a plasmid containing the promiscuous gaÀ protein that can couple a wide range of gpcrs to the phospholipase c signalling pathway. the transfection was achieved using mg of each plasmid and ml of the transfection reagents p and lipofectamine (thermo fisher scientific; cat. no. l ), as recommended by the manufacturer. it was not possible to authenticate the cho-k (g a) cells or test the cells for mycoplasma contamination at the time of manuscript submission due to laboratory closure during the covid- pandemic. after transfection with the a. rubens receptor, cells were exposed to the a. rubens npy/npf/ prrp-like peptide pqdrskamqaertgqlrrlnprf-nh (custom synthesised by peptide protein research ltd., fareham, uk), which was diluted in dmem/f nutrient mixture medium at concentrations ranging from À m to À m in clear bottom -well plates (sigma-aldrich; cat. no. cls - ea). luminescence was measured over a s period using a fluostar omega plate reader (bmg labtech; fluostar omega series multi-mode microplate reader) and data were integrated over the s measurement period. for each concentration, measurements were performed in triplicate, and the average of each was used to normalise the responses. the responses were normalised to the maximum luminescence measured in each experiment ( % activation) and to the background luminescence with the vehicle media ( % activation). dose-response curves were fitted with a four-parameter curve and ec values were calculated from dose-response curves based on at least three independent transfections using prism (graphpad, la jolla, usa). editing; ismail moghul, software, formal analysis, writing 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paralogous gnrh and corazonin neuropeptide signalling pathways w-iq-tree: a fast online phylogenetic tool for maximum likelihood analysis neuropeptides and their precursors in the fruitfly, drosophila melanogaster neuropeptide evolution: neurohormones and neuropeptides predicted from the genomes of capitella teleta and helobdella robusta neuropeptide receptor transcriptome reveals unidentified neuroendocrine pathways discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms characterization of a novel lfrfamide neuropeptide in the cephalopod sepia officinalis diversity of the rfamide peptide family in mollusks the neuropeptide y system: pathophysiological and therapeutic implications in obesity and cancer the work reported in this paper was supported by grants from the bbsrc awarded to mre (bb/ m / ) and amj (bb/m / ). layg was supported by a phd studentship awarded by the mexican council of science and technology (conacyt studentship no. ) and queen mary university of london and by a leverhulme trust grant (rpg- - ) awarded to mre. xz was supported by a phd studentship awarded by the china scholarship council and queen mary university of london. we are grateful to gá spá r jé kely (university of exeter) for providing the cho-k (g a) cell line used here for receptor deorphanisation assays. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. luis alfonso yañ ez-guerra, xingxing zhong, conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing -original draft, writing -review and key: cord- -hj s cnu authors: lin, peng; ng, tzi bun title: a novel and exploitable antifungal peptide from kale (brassica alboglabra) seeds date: - - journal: peptides doi: . /j.peptides. . . sha: doc_id: cord_uid: hj s cnu the aim of this study was to purify and characterize antifungal peptides from kale seeds in view of the paucity of information on antifungal peptides from the family brassicaceae, and to compare its characteristics with those of published brassica antifungal peptides. a -da antifungal peptide was isolated from kale seeds. the isolation procedure comprised affinity chromatography on affi-gel blue gel, ion exchange chromatography on sp-sepharose and mono s, and gel filtration on superdex peptide. the peptide was adsorbed on the first three chromatographic media. it inhibited mycelial growth in a number of fungal species including fusarium oxysporum, helminthosporium maydis, mycosphaerella arachidicola and valsa mali, with an ic( ) of . μm, . μm, . μm, and . μm, respectively and exhibited pronounced thermostability and ph stability. it inhibited proliferation of hepatoma (hepg ) and breast cancer (mcf ) cells with an ic( ) of . μm and . μm, and the activity of hiv- reverse transcriptase with an ic( ) of . μm. its n-terminal sequence differed from those of antifungal proteins which have been reported to date. antifungal proteins are a family of proteins employed to combat pathogenic fungi which can cause diseases in plants and animals. those proteins have been purified from a diversity of flowering plants [ , [ ] [ ] [ ] [ ] , ] , animals [ , ] , bacteria [ ] , and fungi [ , ] . plant tissues that produce antifungal proteins and peptides comprise seeds [ , , , , , , , ] , bulbs [ , ] , leaves [ , ] , tubers [ ] , fruits [ ] , shoots [ ] , and roots [ ] . monocots [ , , , , , , , ] , dicots [ , , , , , , , , , ] , and gymnosperms [ , ] have been reported to produce antifungal proteins. plant antifungal proteins are classified, based on structure or activity, into various types [ ] . the different types include chitinases and chitinase-like proteins [ ] , chitin-binding proteins [ , ] , lipid transfer proteins [ , ] , protease inhibitors [ , ] , ribosome inactivating proteins [ , ] , embryo abundant protein-like proteins [ ] , thaumatin-like proteins [ ] , and defensin-like peptides [ ] . to date, only one antifungal peptide has been reported from brassica campestris seeds [ , ] in spite of the presence of multiple brassica species. in new of the structural diversity manifested by the various aforementioned antifungal proteins and the presence of different antifungal proteins even in the same species [ , ] , the intent of the present investigation was to isolate an antifungal peptide from the seeds of kale (brassica alboglabra l.h. bailey), another brassica species, and to compare it with the antifungal peptide from b. campestris seeds [ ] and other plant antifungal proteins. protein concentration was determined by the dye-binding method (bio-red) using bovine serum albumin as a standard. tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-sds-page) it was conducted according to the method of schagger and von jagow [ ] . after electrophoresis using % acrylamide gel, the gel was stained with coomassie brilliant blue. the molecular mass of the isolated antifungal peptide was determined by comparison of its electrophoretic mobility with those of , the extract of kale seeds was applied on an affi-gel blue gel column ( cm t cm). unadsorbed proteins (fraction bg ) were eluted with the same buffer while adsorbed proteins (fraction bg ) were eluted with mm tris-hcl buffer (ph . ) containing m nacl as indicated by the arrows. in (b), fraction bg from the affi-gel blue gel column was dialyzed and applied on an sp-sepharose column ( . cm t cm) in mm nh oac buffer (ph . ). after elution of unadsorbed proteins, the column was eluted stepwise with . m nacl, . m nacl and then with m nacl added to the buffer as indicated by the arrows. in (c), fraction sp from the sp-sepharose column was loaded on a -ml mono s column. following elution of unadsorbed proteins with mm nh oac buffer (ph . ), adsorbed proteins were eluted sequentially, first with a - . m nacl gradient and then with a . - . m and . - m nacl gradient. in (d), fraction s from the mono s column was subjected to gel filtration on a superdex peptide hr / column in mm nh mass spectrometric (ms) analysis of the antifungal peptide was performed on a finnigan lcq-ms, an instrument that essentially consists of an atmospheric pressure electrospray positive-ion source, attached to a triple-quadrupole mass analyzer. the purified peptide ( pmol) was dissolved in water/methanol ( : , v/v) containing % (v/v) acetic acid at a protein concentration of mmol/l, and then applied on the ms instrument [ ] . the n-terminal amino acid sequence of the purified peptide was performed by edman degradation using a hewlett-packard amino acid sequencer [ ] . the assay for antifungal activity was executed using mm  mm petri plates containing ml of potato dextrose agar. the fungal species tested included the following: fusarium oxysporum, helminthosporium maydis, mycosphaerella arachidicola and valsa mali. after the mycelial colony had developed, sterile blank paper disks ( . cm in diameter) were placed around and at a distance of cm away from the rim of mycelial colony. an aliquot ( ml containing mg or mg) of the purified peptide in mm pbs buffer (ph . ) was introduced to a disk. the plates were incubated at c for h until mycelial growth had enveloped peripheral disks containing the control (buffer) and had produced crescents of inhibition around disks containing samples with antifungal activity. to determine the ic value for the antifungal activity of the isolated antifungal peptide, four doses of the peptide were added separately to four aliquots each containing mil potato dextrose agar at c, mixed rapidly and poured into four separate small petri dishes. after the agar had cooled down, a small amount of mycelia, the same amount to each plate was added. buffer only without antifungal peptide served as a control. after incubation at c for h, the area of the mycelial colony was measured and the inhibition of fungal growth determined. inhibition of fungal growth = % reduction in area of mycelial colony = [(area of mycelial colony in absence of antifungal peptide À area in presence of antifungal peptide)/area in absence of antifungal peptide]  %. a graph plotting % reduction in area of mycelial colony caused by antifungal peptide against the concentration of antifungal peptide was then plotted. the concentration of the isolated antifungal peptide that brought about % reduction in the area of mycelial colony is the ic [ ] . to investigate the thermal ( - c) stability, ph ( - and - ) stability and effects of ions, the isolated antifungal peptide was pretreated accordingly and the antifungal assay was then conducted as mentioned above. a solution of the isolated antifungal peptide ( mg/ml) was incubated with an equal volume of trypsin or pepsin ( mg/ml) only the kale and b. campestris antifungal peptides [ ] exhibit antifungal activity. the remaining two proteins [ , ] manifest trypsin inhibitory activity. the napin-like polypeptide also possesses antibacterial and antiproliferative activities [ ] . p e p t i d e s ( ) - at c for h. at the end of the incubation, the reaction mixture was examined for antifungal activity. breast cancer mcf- cell line and hepatoma hepg cell line were suspended in rpmi medium and adjusted to a cell density of  cells/ml. a ml aliquot of this cell suspension was seeded to a well of a -well plate, followed by incubation for h. different concentrations of the antifungal peptide in ml complete rpmi medium were then added to the wells and incubated for h. after h, ml of mg/ml [ -[ , -dimethylthiazol- -yl]- , -diphenyltetrazolium bromide] [mtt] in phosphate buffered saline was spiked into each well and the plates were incubated for h. the plates were then centrifuged at  g for min. the supernatant was carefully removed, and ml of dimethyl sulfoxide was added in each well to dissolve the mtt-formazan at the bottom of the wells. after min, the absorbance at nm was measured by using a microplate reader [ ] . the assay for hiv reverse transcriptase inhibitory activity was carried out according to instructions supplied with the assay kit from boehringer mannhein (germany). the assay takes advantage of the ability of reverse transcriptase to synthesize dna, starting from the template/primer hybrid poly (a) oligo (dt) . the digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule, which is freshly synthesized by the reverse transcriptase (rt). the detection and quantification of synthesized dna as a parameter for rt activity follows a sandwich elisa protocol. biotin-labeled dna binds to the surface of microtiter plate modules that have been precoated with streptavidin. in the next step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigeninlabeled dna. in the final step, the peroxidase substrate is added. the peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. the absorbance of the sample at nm can be determined using a microtiter plate (elisa) reader and is directly correlated to the level of rt activity. a fixed amount ( - ng) of recombinant hiv- reverse transcriptase was used. the inhibitory activity of the antifungal peptide was calculated as percent inhibition as compared to a control without the antifungal peptide [ , ] . assay of ability to inhibit hiv- integrase the assay was conducted as described in ref. [ ] . the ribosome inactivating protein trichosanthin was used as a positive control [ ] . screening for inhibitory effect on severe acute respiratory syndrome (sars) coronavious (cov) protease the assay was conducted as described by leung et al. [ ] . trypsin activity was determined by using n-a-benzoyl-larginine ethyl ester hydrochloride (baee) as the substrate. a similar assay was conducted using casein as substrate instead of baee [ , , ]. the assay was carried out as described earlier. con a was used as positive control and bovine serum albumin as a negative control [ ] . the crude extract was fractionated on affi-gel blue gel into a larger unadsorbed fraction (bg ) eluted by the starting buffer and a smaller adsorbed fraction (bg ) eluted by starting buffer containing m nacl (fig. a) . antifungal activity resided only in fraction bg , which was then resolved on sp-sepharose into an unadsorbed fraction and three adsorbed fractions (sp , sp and sp ) of approximately equal size which were desorbed with . m nacl, . m nacl and m nacl, respectively (fig. b) . antifungal activity was concentrated in fraction sp . fraction sp was separated on mono s into a tiny unadsorbed fraction (s ) and two adsorbed fractions (s and s ) (fig. c) . antifungal activity was detected in the sharp adsorbed fraction s eluted soon after application of the . - . m nacl gradient. final purification of s on superdex peptide resulted in a major and a tiny absorbance peak, the former representing purified antifungal peptide (fig. d ) with a molecular weight of . kda in sds-page ( fig. a ). its molecular weight as determined by mass spectrometry was da (fig. b) . the yields of the chromatographic fractions with antifungal activity from g seeds are as follows: crude extract ( mg), fraction bg ( mg), fraction sp ( mg), fraction s ( mg) and purified antifungal peptide ( mg). it differed from known brassica antifungal proteins, napins and trypsin inhibitors in n-terminal sequence (table ) (fig. ) . the antifungal activity of the peptide was retained after exposure to temperatures in the range - c for min (fig. a) and to the ph ranges - and - for min (fig. b) . it inhibited proliferation of hepg cells (fig. a ) and mcf cells (fig. b) with an ic of . mm and . mm, respectively, and reduced the activity of hiv- reverse transcriptase with an ic of . mm (fig. c) . it was devoid of mitogenic activity, hiv- integrase inhibitory, and sars proteinase inhibitory activities (data not shown). a -kda nonspecific lipid transfer with antifungal activity has been isolated from b. campestris seeds [ ] . the present study constitutes the second report on the isolation of an antifungal seeds did not exhibit trypsin inhibitory activity and also did not resemble b. napus trypsin inhibitor in n-terminal sequence. this is noteworthy in view of the fact that some of the trypsin inhibitors demonstrate antifungal activity [ ] and that trypsin inhibitors and napins are produced by brassica seeds. the two brassica antifungal peptides were also isolated by using similar protocols. ion exchange chromatography on q-sepharose, affinity chromatography on affi-gel blue gel, ion exchange chromatography on mono s, and gel filtration on superdex peptide were used for isolation of b. campestris antifungal peptide, the only difference from the present protocol for kale antifungal peptide being replacement of sp-sepharose by q-sepharose. the antifungal peptide from kale seeds had a smaller molecular mass ( . kda) than that ( kda) of b. campestris antifungal peptide. its yield ( mg/kg) was lower than that of b. campestris antifungal peptide ( mg/kg) [ ] . its hiv- reverse transcriptase inhibitory activity (ic = . mm) was similar to that of b. campestris antifungal peptide (ic = mm), but more potent than many anti-hiv- natural products [ ] . its antiproliferative activity toward hepg cells and mcf- cells (ic = . mm and . mm, respectively) was also analo-gous to that of b. campestris antifungal peptide (ic = . mm and . mm, respectively). both kale and b. campestris antifungal peptide were devoid of mitogenic activity toward mouse splenocytes. it has previously been demonstrated that some [ , , ] but not other [ ] antifungal proteins/peptides exhibit mitogenic activity. neither kale nor b. campestris antifungal peptide demonstrated hiv- integrase inhibitory and sars proteinase inhibitory activities, in line with the observation on french bean defensin-like antifungal peptide [ ] . napin-like polypeptides with trypsin-inhibitory activity but devoid of antifungal activity have been purified from seeds of various brassica species [ ] [ ] [ ] [ ] . a trypsin-inhibitor has also been isolated from brassica napus seeds [ ] . these proteins demonstrate n-terminal amino acid sequences distinctly different from those of the antifungal peptide from kale. the isolation of this antifungal peptide adds to the literature on proteins from brassica seeds. the chromatographic behavior of kale antifungal peptide on ion exchanger and affi-gel blue gel is similar to non-brassica antifungal proteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] . its molecular mass is similar to those of some antifungal peptides from non-brassica plants [ , ] . its remarkable thermostability and ph stability resemble those of leguminous defensins [ ] . its broad spectrum of antifungal activity is interesting in view of the observation that shallot and asparagus antifungal proteins are activity toward only one out of several fungal species tested [ , ] . the antifungal activity of kale antifungal peptide is more potent than that of the antifungal proteins [ , , ] . its potent antiproliferative and hiv- reverse transcriptase inhibitory activities are noteworthy since not all antifungal proteins have been reported to possess these potentially exploitable activities. in summary, the antifungal peptide isolated from kale (b. alboglabra l.h. bailey) seeds has potentially exploitable activities such as stable and broad-spectrum antifungal activity, hiv- reverse transcriptase inhibitory activity and antiproliferative activity toward tumor cells. in contrast, some antifungal proteins like mungbean chitinase [ ] lack the last two activities. there are very few reports on the presence of defense proteins such as antifungal proteins, antiviral proteins, antibacterial proteins and lectins from b. alboglabra except for the demonstration of a napin-like peptide with antiproliferative, antibacterial and translation-inhibitory activities [ ] . the antifungal peptide isolated in this study would add to the existing literature. antimicrobial peptides from amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins a chitin-binding lectin from stinging nettle rhizomes with antifungal properties antimicrobial peptides in insects; structure and function a potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer protein purification, inhibitory properties, amino acid sequence and identification of the reactive site of a new serine proteinase inhibitor from oil-rape (brassica napus) seed antifungal activity of a bowman-birk type trypsin inhibitor from wheat kernel first report of a glutamine-rich antifungal peptide with immunodulatory and antiproliferative activities from family amaryllidaceae isolation, characterization and sequencing of a novel type of antimicrobical peptides, fa-amp and fa-amp , from seeds of buckwheat (fagopyrum esculentum moench) antifungal properties of lectin and new chitinases from potato tuber characteristics and antifungal activity of a chitin binding protein from ginkgo biloba cysteine protease inhibitor from pearl millet: a new class of antifungal protein isolation of a small chitinase-like antifungal protein from panax notoginseng (sanchi ginseng) roots a robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive allium tuberosum biochemical and molecular characterization of three barley seed proteins with antifungal properties concurrent purification of two defense proteins from french bean seeds: a defensinlike antifungal peptide and a hemagglutinin first isolation of an antifungal lipid transfer peptide from seeds of a brassica species lipid transfer proteins from brassica campestris and mung bean surpass mung bean chitinase in exploitability anti-hiv natural products with special emphasis on hiv reverse transcriptase inhibitors inhibitory effects of antifungal proteins on human immunodeficiency virus type reverse transcriptase, protease and integrase the trypsin-inhibitory, immunostimulatory and antiproliferative activities of a napin-like polypeptide from chinese cabbage seeds isolation of a napin-like polypeptide with potent translation-inhibitory activity from chinese cabbage (brassica parachinensis cv green-stalked) seeds a napin-like polypeptide from dwarf chinese white cabbage seeds with translation-inhibitory, trypsin-inhibitory, and antibacterial activities a napin-like polypeptide with translationinhibitory, trypsin-inhibitory, antiproliferative and antibacterial activities from kale seeds isolation and characterization of luffacylin, a ribosome inactivating peptide with antifungal activity from sponge gourd (luffa cylindrical) seeds isolation and partial characterization of two antifungal proteins from barley antifungal proteins tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of protein in the range from to kda synthetic analogues of antimicrobial peptides from the venom of the central asian spider lachesana tarabaevi purification, characterization and differential hormonal regulation of a b- , -glucanase and two chitinases from chickpea (cicer arietinum l.) ginkbilobin, a novel antifungal protein from ginkgo biloba seeds with sequence similarity to embryo-abundant protein isolation of a novel deoxyribonuclease with antifungal activity from asparagus officinalis seeds ascalin, a new antifungal peptide with human immunodeficiency virus type reverse transcriptase inhibitory activity from shallot bulbs isolation of an antifungal thaumatinlike protein from kiwi fruits eryngin. a novel antifungal peptide from fruiting bodies of the edible mushroom pleurotus eryngii alveolarin. a novel antifungal polypeptide from the wild mushroom, polyorus alveolaris isolation of cucurmoschin, a novel antifungal peptide abundant in arginine, glutamate and glycine residues from black pumpkin seeds pilot-scale purification of zeamatin, an antifungal protein from maize gymnin, a potent defensin-like antifungal peptide from the yunnan bean gymnocladus chinensis baill an antifungal protein from escherichia coli a bowman-birk-type trypsinchymotrypsin inhibitor from broad beans we thank miss kathy lau and miss grace chan for excellent secretarial assistance.r e f e r e n c e s key: cord- -iuvigqdx authors: knierman, michael d.; lannan, megan b.; spindler, laura j.; mcmillian, carl l.; konrad, robert j.; siegel, robert w. title: the human leukocyte antigen class ii immunopeptidome of sars-cov- spike glycoprotein date: - - journal: nan doi: . /j.celrep. . sha: doc_id: cord_uid: iuvigqdx the precise elucidation of the antigen sequences for t-cell immunosurveillance greatly enhances our ability to both understand and modulate humoral responses to viral infection or active immunization. mass spectrometry is used to identify unique sequences from sars-cov- spike glycoprotein extracellular domain in a complex with human leukocyte antigen class ii molecules on antigen presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. the identified sequences span the entire spike protein and several sequences are isolated from a majority of the donors sampled indicating promiscuous binding. importantly, many peptides derived from the receptor binding domain used for cell entry are identified. this work represents a precise and comprehensive immunopeptidomic investigation with sars-cov- spike glycoprotein and allows detailed analysis of features which may aid vaccine development to end the current covid- pandemic. severe acute respiratory syndrome coronavirus (sars-cov- ) is a positive-sense single-strand rna virus that is a novel member of the genus betacoronavirus family coronaviridae responsible for the coronavirus disease that emerged in china late and became a global pandemic by march . as of date of manuscript preparation, over , , cases with more than , , fatalities have been reported worldwide (https://coronavirus.jhu.edu/). there are four additional members of the betacoronavius genus; two (hcov-hku and hcov-oc ) that cause mild respiratory symptoms associated with the common cold and two (sars-cov and middle east respiratory syndrome-cov) that can cause fatal respiratory tract infections. the sars-cov- genomic sequence shares . % identity with sars-cov and . % identity with sarsr-covratg isolated from bats supporting a zoonotic origin (zhou et al., a) . at present, no therapeutic interventions to treat or prevent covid- have been approved for use (https://milkeninstitute.org/covid- -tracker). active immunization is an area of intense research with over programs under development and the successful implementation would greatly aid in ending the current pandemic (https://www.who.int/who-documents-detail/draft-landscape-of-covid- -candidate-vaccines). immunization strategies include the use of live-attenuated virus, inactivated virus, non-replicating viral vector, protein subunit, and nucleic acid-based approaches. at the time of manuscript preparation, at least candidate vaccines have progressed into clinical evaluation. an effective immunization would prevent viral entry into cells. the surface spike glycoprotein is the defining feature of all coronaviruses and is critical for internalization by engaging the host receptor and mediation of virus-host membrane fusion (cavanagh, ) . the sars-cov- spike protein, as with sars-cov, interacts with human angiotensin-converting enzyme (ace ) (zhou et al., b) , (walls et al., ) , (li et al., ) . the transmembrane glycoprotein is comprised of two functional subunits and forms homotrimers on the viral cell surface. a defined receptor binding domain (rbd) within the s subunit is responsible for binding to ace , (li et al., ) . the s subunit in all coronaviruses contains heptad repeat segments that form a coiled-coil structure and membrane fusion occurs after proteolytic processing and conformational rearrangement (liu et al., ) , (millet and whittaker, ) . a amino acid polybasic furin cleavage site insertion between the s and s subunits is a unique feature within the sars-cov- spike protein (coutard et al., ) , (walls et al., ) . efficacious sars-cov- vaccine development is dependent on a robust humoral immune response targeting the spike glycoprotein, and j o u r n a l p r e -p r o o f perhaps more specifically the rbd assuming similar results as was observed for sars-cov (buchholz et al., ) . a central feature of the adaptive immune response is the presentation of immunogenic peptides for immunosurveillance by cd + helper t-cells. computational prediction of t-cell epitope candidates has been applied for vaccine discovery and removal of unwanted immune responses against protein therapeutics (griswold and bailey-kellogg, ) . current knowledge of peptide binding motifs is based primarily on data generated using biochemical binding assays (justesen et al., ) , (sidney et al., ) which are compiled in the immune epitope database (iedb) (vita et al., ) . this information is used to train prediction algorithms, such as tepitool resource in iedb (paul et al., ) and netmhciipan (reynisson et al., ) . recently this approach was applied to identify known epitopes from multiple coronaviruses and predict likely b-and t-cell epitopes from several sars-cov- proteins and reactive cd + t-cells obtained from covid- patients were observed when stimulated using a "megapool" comprised of overlapping synthetic -mer peptides spanning the entire spike protein sequence (grifoni et al., b) . however, the precise sequences from the sars-cov- spike glycoprotein for cd + t-cell activation is lacking, and a clear understanding will benefit vaccine development. mass spectrometry-based approaches, often termed immunopeptidomics, have been developed to examine the repertoires of peptides presented by human leukocyte antigen (hla) molecules of the class i or class ii major histocompatibility complex (mhc) used for immunosurveillance by cd + or cd + tcells, respectively (hunt et al., a) (hunt et al., b) . hla class ii molecules are restricted to antigen presenting cells (apcs) and play an essential role in the development of a humoral adaptive immune response via activation of cd + t-helper cells. the hla-ii peptide repertoire is a product of extracellular proteins that are proteolytically processed in the lysosomal compartment after internalization. hla-ii immunopeptidomics has been applied to understand the cd + t-cell epitopes for potential vaccine design from pathogens such as mycobacterium tuberculosis (bettencourt et al., ) , vaccinia virus (strug et al., ) , (lorente et al., ) , measles virus (ovsyannikova et al., ) and human herpes virus b (becerra-artiles et al., ). these approaches have been typically limited in scope, restricted to one or two cell lines, and thus sampling only a very limited subset of hla-dr alleles. mhc-associated peptide proteomics (mapps) is a specific extension of hla-ii immunopeptidomics that incorporates the intentional pulsing of dendritic cells (dcs) with an antigen or protein of interest (rohn et al., ) . more recently, hla-ii mapps has been implemented to investigate and understand the mechanisms of treatment-emergent immunogenicity for biotherapeutic proteins (cassotta et al., ) , j o u r n a l p r e -p r o o f (hamze et al., ) , (jankowski et al., ) , (sekiguchi et al., ) as this approach allows for the facile interrogation of the immunogenic potential from multiple hla class ii alleles. in the present study we sought to identify the naturally processed and presented immunopeptidome of the sars-cov- spike glycoprotein from human antigen presenting cells. dcs from a panel of healthy human subjects representing a large percentage of the hla-drb allele usage within the united states were treated with recombinant spike glycoprotein extracellular domain (ecd). a subset of donors was also selected to represent common alleles from the asia-pacific geography. hla-ii associated peptides were identified by liquid chromatography and nanoelectrospray ionization tandem mass spectrometry after immunoprecipitation. we observed several clusters, or nested sets of peptides, derived from every domain of the sars-cov- spike glycoprotein. we determined the prevalence of these clusters among multiple donors. finally, we sought to compare our observed hla-ii epitopes with those from recent a in silico prediction (grifoni et al., a) and determine regions that are conserved with sars-cov spike protein sequence. the mapps method intentionally pulses human dcs from a panel of donors with a protein of interest. the male and female healthy donors, ranging in age from to years old, used in this study were selected to sample approximately % and % of the hla-ii drb allele frequency from the united states and asian-pacific geographic regions, respectively (table ) . full hla typing of the donors is also available (supplemental table s ). the donors' pbmcs were collected and stored frozen before the outbreak of the sars-cov- pandemic and are expected to be unexposed to potential infection. the sequence used for this work was derived from the sars-cov- wuhan-hu- strain spike glycoprotein ecd spanning residues - taken from genbank accession yp_ . an r a mutation was used to prevent cleavage during recombinant protein production with affinity tags added for purification. the recombinant protein was produced from mammalian cells and is expected to have nlinked glycosylation modifications. the method used to profile the hla-ii peptides is outlined in figure a . monocyte-derived dcs were generated in culture with a cytokine cocktail. the immature dcs were treated with sars-cov- spike glycoprotein ecd and after hours, lipopolysaccharide (lps) was added to mature the dcs. the treated cells were lysed and the hla class ii complex was isolated by immunoprecipitation with a pan-hla class ii antibody. the bound hla-ii peptides were eluted after acidification, filtered to remove high molecular weight co-precipitants, and analyzed by capillary hplc on an orbitrap mass spectrometer. peptide ions were fragmented using multiple fragmentation techniques. peptides were identified using multiple proteomic search engines with a forward and reverse database search. false discovery rate estimates (q-values) were estimated using a null distribution from the reverse database search. the peptides identified from the spike glycoprotein had q-values below . and all fragmentation spectra matching the spike protein were manually reviewed (see methods for details). a total of hla-ii peptides from the sars-cov- spike glycoprotein were identified from the donor panel (supplemental table s ). several peptides with identical sequences were identified from multiple donors. removal of these duplicate peptides resulted in unique sequences. in order to minimize both false positive and negative peptide identifications, the database used for peptide identification was intentionally constructed to contain the spike protein along with approximately background bovine and human proteins previously observed from multiple samples analyzed with this assay system. we also analyzed the data using multiple search engines against a database containing the entire human proteome (downloaded apr ) that also included the sars-cov- spike glycoprotein and did not find any human protein identifications for the unique sars-cov- spike peptides. the primary mass spectrometry data is publicly available for individual analysis (doi: . /c m p). the unique peptide sequences had a distribution of lengths consistent with hla-ii peptides with a mean length of residues ( figure b ) (kampstra et al., ) . there were unique peptides that had modified residues. the modifications observed were consistent with hla-ii peptide processing. the spike protein is heavily glycosylated with putative n-glycosylation sites, presumably to help evade immune detection, and the majority of the regions from the spike ecd not observed were centered around these sites. current limitations of our method prevent detection of glycosylated peptides if, in fact, they were processed and loaded onto hla class ii molecules. interestingly, hla-ii peptides from putative n-linked glycosylation sites were observed which indicates these regions were not modified. we did identify a deamidation modification with the asparagine residue at residue with our search engines (supplemental table s ). this modification is consistent with the conversion of asparagine to j o u r n a l p r e -p r o o f aspartic acid after removal of n-linked glycosylation and is indirect evidence of glycosylation of this residue in the intact protein (khoshnoodi et al., ) . every donor examined produced hla-ii peptides derived from the sars-cov- spike glycoprotein ( figure c ). the number of spike peptides observed per donor ranged from to with a median value of peptides. one donor ( ) did not efficiently produce mature dcs and resulted in a low overall number of peptides relative to all other donors analyzed. repeated analysis with this donor also yielded poor results (data not shown). hla-ii peptides are distributed across entire sars-cov- spike protein extracellular domain and have consensus hla-ii clusters hla-ii peptides derived from the sars-cov- spike glycoprotein were aligned to the ecd sequence and a peptide density map was generated to help visualize both the breadth and depth of sequence coverage. a schematic listing of the various subunits of the spike glycoprotein ecd and the results obtained with each donor are shown on individual rows ( figure ). the shades of red correspond to the number of overlapping peptides encompassing a given amino acid position. from this presentation of the data, it is evident that hla-ii peptides spanning all segments of the spike glycoprotein ecd were obtained from all donors sampled. the heatmap also reveals that hla-ii peptides from several regions of the spike glycoprotein were observed from multiple donors likely reflecting the more promiscuous hla class ii binding epitopes. an expanded view of the rbd encompassing residues - is shown in the bottom panel of figure and clearly demonstrates promiscuous epitopes are also contained in this region of the molecule critical for interaction with ace cell receptor. groups of nested peptides sharing a common core but with ragged n-and c-termini are generated from the multiple proteases and different temporal patterns of processing that occur in the lysosomal compartment (lippolis et al., ) . in order to organize the observed hla-ii peptides from the spike protein into discrete segments for subsequent analysis, we used the iedb epitope cluster analysis tool . with some manual adjustments as noted in star methods to group these into distinct clusters (supplemental table s ). clusters were characterized from both full span and minimal overlap perspectives. the full cluster sequence represents the first start position of the peptides in the cluster to the last position of the peptides in the cluster. the minimum cluster sequence is the smallest common sequence among the peptides in the cluster. clusters with minimum cluster sequences less than residues likely contain overlapping binding cores, which due to their proximity, were unable to be easily separated. as expected, we observed a distribution of the clusters among the donors ( figure ). most of the clusters were observed from or fewer donors and were designated as restricted; whereas, clusters were observed from - donors and were designated as consensus ( figure a and table ). this arbitrary definition of a consensus cluster was set to reflect those sequences observed in at least % of the donors sampled in the study. consensus clusters represent those sars-cov- spike glycoprotein sequences with the most promiscuous, but not necessarily highest affinity, binding to the broadest range of hla class ii alleles. no specific cluster was observed in all donors sampled in this study. the median number of clusters per donor was and all donors displayed at least one of the consensus clusters ( figure b ). we next examined the distribution in the number of peptides within both restricted and consensus clusters ( figure c ). the vast majority of clusters contained less than peptides with most, but not all, of the consensus clusters having or greater members. interestingly, consensus cluster , observed in donors, was comprised of a single peptide sequence in the s subunit (table ) . finally, we examined the location of the clusters within discrete segments of the sars-cov- spike glycoprotein ( figure d ). the clusters were distributed throughout the protein. all regions contained at least one hla-ii peptide cluster with consensus clusters occurring in several different regions. of note, the rbd region responsible for binding ace and a target for vaccination strategies contained a total of clusters in which were consensus (table ) . observed sars-cov- spike protein hla-ii peptides have limited overlap with predicted cd + t-cell epitopes cd + t-cell epitopes from the spike glycoprotein derived from an algorithm designed to predict the dominant hla class ii peptides was recently published (grifoni et al., a) . we sought to compare those predictions with the results from our study. we used the minimum observed cluster sequence for our comparison and considered an overlap of at least residues within the predicted residue peptide sequence as a match with one exception as denoted below. we chose this minimum overlap length based on the number of residues contained within hla class ii peptide binding cleft. we chose to use the minimum cluster sequence in an attempt to reduce matches on the extreme peripheries of the observed peptides that likely do not reflect the likely binding core contained within the cluster. a perfect congruence between prediction and observed results from this donor set would result in matches as one of the predicted peptides resides in the transmembrane portion which was absent from the protein used in this study. in contrast, we observed hla-ii peptide clusters from our donor set that j o u r n a l p r e -p r o o f matched a total of predicted epitopes ( figure a and supplemental table s ). cluster was deemed to match a predicted epitope located from residues - (ylyrlfrksnlkpfe) in the rbd domain even though only a -residue overlap was observed using the criteria defined above. this particular cluster was composed of unique sequences, the most out of all the clusters, and likely contains two closely overlapping allele binding sites. since of the observed peptides from this cluster matched the first residues of the predicted peptide, we included this region as overlapping with the prediction (supplemental table s for cluster details). we also note two instances in which multiple clusters were deemed to sufficiently overlap with a predicted peptide which skews the venn diagram in figure a to show instead of the total observed clusters. comparison of the observed consensus clusters with the predicted peptides reveals that only of the observed promiscuous hla-ii binding regions were predicted (table ). this finding is intriguing given the prevalence of display of these particular sequences and may reflect limitations in the alleles used to generate the predictions and/or the prediction algorithm. the rbd of the sars-cov- spike glycoprotein contained observed hla-ii clusters and predicted peptides. we observed of the predicted epitopes (table and supplemental table s ). the only predicted peptide that was not observed contained a putative n-linked glycosylation site. as denoted above, our mapps method is unlikely to identify a peptide with this modification. in fact, of the predicted peptides contain a putative nlinked glycosylation site (supplemental table s ) possibly reflecting the fact that current predictive algorithms do not consider post-translational modifications. in addition, the glycosylated asparagine residue in many of the predicted peptides is centrally located rather than on the periphery and large complex glycan structures have been shown to interfere with hla or t-cell receptor binding (speir et al., ) , (kario et al., ) , (malaker et al., ) . also striking is the overall number of observed clusters ( ), many obtained from multiple donors, which were not predicted. it is worth noting that the algorithm used to predict sars-cov- spike protein epitopes is largely trained on hla binding affinity data which do not reflect the authentic processing captured by mapps assay. further expansion of mapps-derived data into these training sets will likely be beneficial. the list of unique peptides for each hla class ii cluster was compared with the spike glycoprotein sequence from sars-cov protein (uniprot accession p ). the non-identical residues between the two sequences are denoted in red under the spike glycoprotein schematic in figure b . the s and s ' regions had larger areas of identity between the sequences. the unique hla-ii peptides identified in this study were analyzed for sequence identity with the sars-cov spike glycoprotein. no identical matches were identified in the s subunit. however, clusters match the sars-cov sequence in the s subunit. interestingly, one of the matches, cluster , was a consensus cluster (denoted with blue figure b ). these clusters represent sequence regions from both viruses that are potentially presented by hla class ii molecules for t-cell surveillance. the spike proteins from the other coronaviruses known to infect humans (nl , e, hku , oc , and mers) were also evaluated and no matches were found. to look for potential cross-reactivity to human proteins, each unique identified peptide from the sars-cov- spike glycoprotein was searched against the uniprot human database for an exact match to any human protein. none of the observed peptides had a sequence match (data not shown). finding no matches, we expanded our search to include up to mismatched amino acids without insertions or deletions. a single peptide could be associated with human proteins if residues of non-identity are allowed. these results indicate that the risk from direct sequence cross-reactivity is minimal and any portion of the sars-cov- spike glycoprotein associated with hla class ii molecule is unlikely to be subject to previous tolerization in a vaccinated subject or infected patient. however, we do acknowledge that our analysis does not consider cross-reactivity when strictly limited to putative t cell contact residues given the difficulty to reliably predict such registers. cd + t-cell participation is vital for a robust humoral response to viral infection or active immunization. a clear delineation of the epitopes presented by apcs for t-cell immunosurveillance greatly enhances our understanding of this process. generally, t-cell lines or pbmcs from recovered patients using peptides derived simply by spanning the entire protein(s) of interest or from hla-ii prediction algorithms have been utilized (meunier et al., ) , (grifoni et al., b) , (schulze zur wiesch et al., ) . the ability to automate and miniaturize the mapps assay enables facile identification of 's of naturally processed and displayed hla-ii peptides from human dcs. using this approach, we were able j o u r n a l p r e -p r o o f to determine the precise regions and sequences of peptides from sars-cov- spike glycoprotein ecd derived from a panel of healthy subjects presented for immune surveillance by t-cells. the subjects used in this study enabled sampling of approximately % and % of the hla-drb allele frequency from the united states and asian-pacific geographic regions, respectively (table ) . this work represents, to our knowledge, the most precise and comprehensive immunopeptidomic investigation with sars-cov- spike glycoprotein performed to date and allows detailed analysis of features which may aid vaccine development. we observed a total of unique peptide sequences contained within clusters distributed across each segment of the sars-cov- spike glycoprotein ecd presented by human dcs (figure and supplemental table s ). two of the clusters were in regions that deviated from the reference sequence. one region was the s /s cleavage site in which the novel furin site was eliminated with mutation to enable production of full-length recombinant protein. we speculate that this region of the spike protein containing the native residue could also be presented from those molecules that are not cleaved during virion particle assembly. the other area of deviation was the c-terminal affinity tags used for purification. of particular interest are those peptides from the spike glycoprotein that are presented by multiple donors as these would be sequences likely to elicit a t-cell response from the greatest number of patients or vaccinated subjects. we observed consensus clusters, defined as being present in or more of the donors analyzed in this study, including within the rbd which is essential for binding to ace on host cells (table ) . a majority of the consensus clusters contained or more nested peptides. in the absence of a dedicated assay to quantify the presented hla-ii peptides, we use this metric as a surrogate for peptide abundance but recognize that even a single specific peptide sequence can be presented in sufficient number to elicit a t-cell response. recent reports leveraging either bioinformatics to predict (grifoni et al., a) or a single-pot peptide pools composed of > overlapping peptides spanning the entire open reading frame (grifoni et al., b) , (braun et al., ) have been published attempting to elucidate sars-cov- t-cell epitopes. unfortunately, the latter approach does not allow any insights into the precise sequences capable of eliciting a response. comparisons of the clusters we observed being presented by apcs reflecting the natural processing hla-ii loading processes with the predicted epitopes is illuminating. one of the predicted epitopes resides in the transmembrane domain which was absent from the protein used for our analysis and omitted from further discussion. of the remaining predictions, roughly % ( of ) were observed from our panel of donors selected to represent a sizeable percentage of hla-dr allele j o u r n a l p r e -p r o o f usage from multiple geographies (figure and supplemental table s ). correspondingly, the vast majority of the observed hla-ii clusters were not predicted. of particular interest are the consensus clusters that were observed in or more of the donors and would be expected to represent those sequences with the most promiscuous hla class ii binding. only of these consensus clusters were predicted with only of consensus clusters contained in the rbd predicted. of note and expanded in more detail below, consensus cluster in the s subunit has % sequence identity to sars-cov, was predicted, and has been experimentally shown to be a t-cell epitope (yang et al., ). the " -allele method" hla class ii reference set used for generating the predicted epitopes is restricted to select drb / / / alleles (http://tools.iedb.org/mhcii/) (paul et al., ) . the use of the pan-hla class ii antibody used in our study, which would enrich both hla-dq and hla-dp bound peptides, could explain why some, but certainly not all, of the observed clusters were not predicted. however, given the ample evidence which shows the impact that both hla-dq and hla-dp bound peptides have on t-cell activation for a variety of viral antigens (koelle et al., ) , (mellins et al., ) , (koelle et al., ) , (mellins et al., ) , (lorente et al., ) , (lorente et al., ) , we felt it was important to identify as many of those restricted peptides as possible. nevertheless, this disconnect between promiscuously observed hla class ii clusters and predicted t-cell epitopes accentuates what has been highlighted before that the prediction of t-cell epitopes is an imperfect process that may not reflect what hla class ii molecules preferentially bind (paul et al., ) , (wantuch et al., ) ) and therefore, not the most effective approach for identifying cd + t-cell epitopes. (wantuch et al., ) , (strug et al., ) , (ovsyannikova et al., ) ). mapps has been an instrumental method to preclinically assess the immunogenic potential of protein therapeutics (for review, (quarmby et al., ) ). multiple reports analyzing different therapeutic antibodies approved for clinical use have shown that many, but not all, hla-ii clusters identified from these molecules can elicit t-cell responses from both drug naïve donors, as well as, patients who developed treatment emergent anti-drug antibody responses (walsh et al., ) , (cassotta et al., ) , (hamze et al., ) ). also, unlike the immunogenicity assessment of a majority of protein biotherapeutics which are engineered to j o u r n a l p r e -p r o o f a great extent to be recognized as a self-protein and therefore generally present a very limited number of non-germline residues for scrutiny, each sars-cov- spike protein cluster is fundamentally distinct from any other sequence in the human genome and extremely unlikely to be subject to any tolerization. the list of unique sars-cov- spike glycoprotein peptides identified by mapps searched against the human database (uniprot version _ ) did not identify any significant matches. nevertheless, the characterization of the activation potential of the sars-cov- spike protein clusters with t-cells from healthy donors and, ideally, convalescent patients should be evaluated. the source of dcs used were derived from healthy donors that, due to time of sample collection, had not been exposed to potential sars-cov- infection. therefore, the displayed hla-ii clusters reported here could conceivably deviate from the repertoire obtained from infected individuals. this potential discrepancy would require that the internalization of the virus into apcs would fundamentally alter the proteolysis and/or hla-ii molecule loading mechanism. while examples of hla-ii display interference are known for other viruses that can infect and replicate in immune cells (becerra-artiles et al., ), the authors are not aware of that attribute with sars-cov- at this time and do not consider this to be a fundamental concern. this could be addressed by repeating this study using material obtained from convalescent patients or infection of dcs from healthy donors with live virus. also, the dcs used in this study are derived from monocytes and could potentially have a different processing mechanism from plasmacytoid or follicular dendritic cell lineages. a detailed comparison in the mapps peptides obtained from different dendritic cell types is lacking given the difficulties in obtaining adequate numbers required for investigation. nevertheless, multiple studies have shown monocyte-derived dcs to be as efficient as antigen-specific b-cells in presenting peptides for t-cell surveillance (cella et al., ; sallusto and lanzavecchia, ) . insights into the immunogenic potential of sars-cov- spike glycoprotein can be made from the results obtained in this study. firstly, both the depth and breadth of the hla-ii peptides derived from this critical structural component for viral infectivity indicate that mutational drift as the pandemic continues to spread around the world is not expected to dramatically alter the ability of an infected individual to mount a new b-cell response to replace those antibodies which would be deleteriously impacted by such escape mutations. in the event that a mutation could result in an inability of a particular cluster to bind most hla class ii alleles, the sheer number of clusters distributed across the spike protein, especially the consensus clusters, make the mutation unlikely to enable the virus to escape cd + t-cell activation across a wide portion of the population. secondly, sars-cov spike glycoprotein cross-reactive j o u r n a l p r e -p r o o f hla-ii restricted epitopes seem to be limited to the s domain as all of the sars-cov- clusters identified in this study with complete sequence identity to sars-cov were derived from this region of the molecule (figure b , supplemental table s ). this result is not surprising given the % sequence identity between the two viruses in s but only % identity in the s domain. no significant homology was noted for any of the other human coronavirus spike protein sequences. interestingly, consensus cluster (observed from donors) was one of the clusters from the s subunit with complete identity to sars-cov and has previously been identified as a t-cell epitope from healthy donors using a tetramer guided epitope mapping approach (yang et al., ) . this epitope has significant, but not complete, overlap with the hla-ii derived cluster and indicates, as denoted above, that the approach of using overlapping peptides may reflect imperfect identifications from those that arise due to natural apc processing. limitations of this study include that the identified peptides are restricted to those with suitable biophysical characteristics for ionization and compatibility with reverse phase liquid chromatography. the total number and wide breadth of coverage spanning the entire extracellular domain of the spike protein indicate that any false negative results obtained with the method outlined in this study are likely a small minority. another limit is focusing the analysis to the spike glycoprotein to the exclusion of the other structural components of sars-cov- , the membrane and nucleocapsid proteins. undoubtedly many regions from both of those proteins will also be presented for t-cell surveillance; however, the focus of most immunization strategies seems to target the spike glycoprotein. notwithstanding, the "adjuvant-like" potential of some of these presumed clusters to augment the humoral response to the spike glycoprotein cannot be accounted for with the current results and should be targets of future efforts. additional effort to experimentally identify the actual hla-ii peptides presented from the spike glycoprotein, at a minimum, from all other coronaviruses would prove of interest. the ability to direct humoral immune response to discrete segment(s) of the sars-cov- spike glycoprotein that confer viral neutralization may potentially enable higher protective titers to be achieved with vaccination and limit antibody-dependent enhancement of infection as reported with other coronaviruses ((tseng et al., ) , ). a preliminary report in which the amino acid rbd of sars-cov- spike glycoprotein was used for immunization in rodents suggests that robust neutralizing response can be obtained without ade (quinlan et al., ) . the approach outlined and the results reported in this study can also be applied to developing novel subunit or nucleic acidbased vaccines and/or monitoring response to such vaccines. it also enables the ability to supply the j o u r n a l p r e -p r o o f immune system with synthetic peptide(s) that mirror natural apcs presentation observed from a broad spectrum of the hla class ii alleles from different geographic regions to maximize t-cell responses. the authors grateful acknowledge richard e. higgs, andrea ferrante, and laurent malherbe for critical review and helpful suggestions during preparation of the manuscript. r.w.s dedicates this work to the memory of karen j. haugh. this work was supported by eli lilly and company. the authors declare no competing interests. the donor id is listed on x-axis and the total number of spike protein peptides from that particular donor denoted on the y-axis. see table and supplemental table s for donor information and supplemental table s for all identified sars-cov- spike protein ecd peptides. the various subunits of the spike protein are denoted at top of the schematic. aligned hla-ii presented peptides are displayed as a heatmap with each of the nine donors on an individual row. the shades of red correspond to the number of overlapping peptides encompassing a given amino acid position. the lightest shade represents a single peptide and the darkest red signifies when at least five peptides overlap an amino acid position. the unlabeled yellow region at the n-term corresponds to the signal peptide and the light green region at the c-term corresponds to the affinity tag used for purification. the s /s cleavage site contains an r a mutation (denoted with an asterisk). the rbd portion of the heatmap was expanded and displayed in the bottom panel along with numerical markers to indicate location within the sars-cov- spike protein ecd. data from each donor in the panel was collected from a single biological and technical replicate. see supplemental table s for all identified sars-cov- spike protein ecd peptides. s , subunit ; s , subunit ; ntd, n terminal domain; rbd, receptor binding domain; ctd, c terminal domain. (a) prevalence of peptide clusters across donors. the number of donors from which each of the peptide clusters were observed are shown. the x-axis denotes the number of donors in which a cluster was observed, and the y-axis denotes the number of clusters observed from that particular number of donors. for example, clusters were observed from any given single donor, another clusters were observed from of the donors in the panel, and so forth. peptide clusters that were identified in at least donors were deemed "consensus" and colored red, whereas clusters seen in or less donors are characterized as "restricted" and are colored blue. (b) number and type of cluster by donor. the total number of clusters observed for each donor are shown. the y-axis denotes the total number of clusters from the particular donor listed on the x-axis. clusters designated as restricted or consensus are denoted as blue and red, respectively. the overall median of clusters is indicated with a dashed line. (c) cluster depth. the distribution in the different number of peptides contained within a cluster are shown. the number of peptides within any given cluster are denoted on the x-axis and the number of clusters containing the peptides within the designated bins are denoted on the yaxis. clusters designated as restricted or consensus are denoted as blue and red, respectively. (d) the distribution of the clusters across the spike protein domains. the particular domain is designated on the x-axis and the number of clusters contained with the particular domain denoted on the y-axis. clusters designated as restricted or consensus are denoted as blue and red, respectively. see supplemental table s for further cluster details. (a) prediction comparison. the observed sars-cov- spike protein clusters were compared to the predicted hla-ii peptides. a venn diagram shows the overlap between the observed minimum cluster sequence and the predicted peptides. to view the details of predicted versus observed clusters, see supplemental table s . (b) cluster conservation. the various segments of the sars-cov- spike protein are denoted on the top row and sequence mismatches to the sars-cov spike protein are delineated with a red bar in the second row. the third row is a heatmap of the clusters seen from the sars-cov- spike protein that contain peptides with an exact sequence match to sars-cov spike protein (darker orange represents overlapping clusters). the blue cluster designates a consensus cluster. s , subunit ; s , subunit ; ntd, n terminal domain; rbd, receptor binding domain; ctd, c terminal domain further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, robert siegel (siegel_robert@lilly.com). this study did not generate new unique reagents. frozen pbmcs were obtained from informed consent healthy donors (discovery life sciences) according to local ethical practice. protocol was adapted as described (sallusto and lanzavecchia, ) with the following modifications. pbmcs were selected from the available inventory to have the broadest hla-drb diversity as possible. the cells were lysed on day with ripa lysis and extraction buffer (thermo fisher scientific, cat # , mm tris•hcl ph . , mm nacl, % np- , % sodium deoxycholate, . % sds) containing : of unit/ul dnase (roche, cat# ) and tablet of edta free protease inhibitors (roche, cat# ) per ml of lysis buffer. the lysates were frozen at - °c. an agilent assaymap robot was used to isolate the hla-ii molecules in the lysate. one hundred microliters of mg/ml biotinylated anti-pan hla class ii antibody (in house produced tu clone) was immobilized on streptavidin cartridges (agilent, sa-w, ul) by passing over the cartridge at ul/min. the cartridge was washed with ul of pbs three times. lysates were thawed, passed over a . um filter, and ml of each sample was loaded onto a well polypropylene plate. the lysate was aspirated into the syringes and the antibody loaded cartridge is attached to the syringe tip. the lysate is passed over the affinity cartridge at ul/min at room temperature for minutes. the cartridge is washed x ul with mm ammonium acetate at ul/min and once with ul water at ul/min. the cartridge is eluted with ul of % acetic acid with . % tfa at ul/min into a well polypropylene pcr plate. the eluted peptides were passed over a k mwco spin filter treated with mg/ml bsa (sigma, ) and ug/ml angiotensin i peptide and washed with % acetic acid. the filtered material was loaded in a well polypropylene pcr plate for mass spec analysis. the samples were analyzed with a thermo lumos mass spectrometer using a thermo easy nlc-hplc system. the separation was carried out with a µm x cm ymc-ods c column (new objectives) coupled to a custom nanospray interface with an electrospray potential of . kv. the solvents were a - . % formic acid in water (thermo fisher scientific, optima™ lc/ms grade) and b - % acetonitrile with . % formic acid (thermo fisher scientific, optima™ lc/ms grade). the gradient was minutes using a flow rate of nl/min, starting with a min - %b ramp followed by a min - %b ramp and a min hold at %b). the lumos was run with a full scan at , resolution in the orbitrap followed by a second data dependent ms/ms cycle comprised of ion trap rapid scans where + ions were fragmented by hcd(ce of , , ) and + and + ions were fragmented by hcd (ce of , , ) and ethcd (calibrated charge-dependent etd parameters and supplemental hcd (ce of )). the data were analyzed with the lilly proteomics pipeline (higgs et al. ( ) . the data conditioning steps consisted of extraction from the vendor format, fitting parent ions for data dependent scans to theoretical isotope patterns and correcting the monoisotopic mass and charge of the parent ion, determining the fit of the parent ion isotope to the theoretical isotope pattern and filtering out ms/ms scans if the parent ions did not match the isotope pattern with a score of . or greater. from the filtered scans, an mgf file was created along with a table of spectral features for each spectrum. the spectral identifications were performed with x! tandem version and omssa version . . search engines. a database was used consisting of the sars-cov- spike extracellular domain his-flag tagged protein and common human and bovine proteins identified from hla-ii bound peptides seen from raji cells, dcs, and bovine proteins in the cell media. the search engine parameters included a no enzyme search with a maximum missed cleavage site setting of , ppm tolerance for parent ions, and . m/z tolerance for the fragment ions. potential amino acid modifications included: cysteine mods of free sh; disulfide; mercaptoethanolation; mono,di, and tri oxidation; and cysteinylation; deamidation of glutamine and asparagine; methionine oxidation; tryptophan oxidation, deoxidation, oxidation to kynurenin. hcd spectra were searched for b-and y-ions and ethcd were searched for c-, z-, b-, and y-ions. false positive identifications were controlled by running the searches against a reversed version of the protein database and estimating false discovery rates. an iterative random forest classifier was trained using search results and spectral features to increase identification sensitivity in a manner similar to the percolator algorithm (kall et al., ) . the search results from x! tandem and omssa were pooled and peptides with q-values < . were assigned to the smallest group of proteins that account for all identified peptides. pepnovo plus (frank and pevzner, ) was run on all the spectra with a parent ion tolerance of ppm and a fragment tolerance of . th. modifications included were methionine oxidation, cysteinylation, and disulfide formation. the output was filtered for peptide tags that matched the sars-cov- spike extra cellular domain his flag protein. tag hits were checked against the results from the database search. all tag hits were matched to the database search results indicating that there were no unknown modifications present in the results. the pipeline output was analyzed using knime . (mazanetz et al., ) to merge all the donor search results, manual review of the ms/ms spectra of the identifications to confirm presence of at least contiguous fragment ions to matched peptide sequence, align the peptides to the sars-cov- spike extracellular domain his flag protein, and create an excel file with the alignment. the identified peptides from the sars-cov- spike glycoprotein were clustered with the iedb epitope cluster analysis tool v . using the default settings. the clustering was manually adjusted to group a continuous amino acid run of at least residues after sorting by donor. this adjustment was reflected in the cluster number as a decimal point after the assigned cluster number from the iedb algorithm. predicted sars-cov- spike protein peptides (grifoni et al., a) were matched with the mapps cluster if they shared at least a amino acid overlap to the minimum cluster sequence. the identified hla-ii peptides were checked for exact matches using the unix grep command. the sequences searched were the human database (uniprot version _ ), spike proteins from sars-cov (genbank accession # p . ), human coronavirus nl , e, hku , oc (genbank accession # qed . , ast . , agw . , aar . ), and mers (genbank accession # yp_ . ). an imperfect match search was run using the unix command agrep to look for or mismatched amino acids. the searches were done for each peptide with insertion and deletions scores set to to discount any insertion or deletions during the search. the imperfect match search was run against the human database (uniprot version _ ). table s . hla-ii peptide alignment to sars-cov- extracellular domain, related to figure and figure . naturally processed hla-dr -restricted hhv- b peptides are recognized broadly with polyfunctional and cytotoxic cd t-cell responses identification of antigens presented by mhc for vaccines against tuberculosis sars-cov- -reactive t cells in healthy donors and patients with covid- contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity a single t cell epitope drives the neutralizing anti-drug antibody response to natalizumab in multiple sclerosis patients the coronavirus surface glycoprotein inflammatory stimuli induce accumulation of mhc class ii complexes on dendritic cells the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade pepnovo: de novo peptide sequencing via probabilistic network modeling a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals design and engineering of deimmunized biotherapeutics characterization of cd t cell epitopes of infliximab and rituximab identified from healthy donors label-free lc-ms method for the identification of biomarkers characterization of peptides bound to the class i mhc molecule hla-a . by mass spectrometry peptides presented to the immune system by the murine class ii major histocompatibility complex molecule i-ad peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to fviii products functional recombinant mhc class ii molecules and high-throughput peptide-binding assays semi-supervised learning for peptide identification from shotgun proteomics datasets ligandomes obtained from different hla-class ii-molecules are homologous for n-and cterminal residues outside the peptide-binding cleft n-linked glycosylation does not impair proteasomal degradation but affects class i major histocompatibility complex presentation identification of n-linked glycosylation sites in human nephrin using mass spectrometry antigenic specificities of human cd + t-cell clones recovered from recurrent genital herpes simplex virus type lesions preferential presentation of herpes simplex virus t-cell antigen by hla dqa * /dqb * in comparison to hla dqa * /dqb * structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin-converting enzyme is a functional receptor for the sars coronavirus analysis of mhc class ii antigen processing by quantitation of peptides that constitute nested sets interaction between heptad repeat and regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors the hla-dp peptide repertoire from human respiratory syncytial virus is focused on major structural proteins with the exception of the viral polymerase proteomics analysis reveals that structural proteins of the virion core and involved in gene expression are the main source for hla class ii ligands in vaccinia virus-infected cells identification and characterization of complex glycosylated peptides presented by the mhc class ii processing pathway in melanoma drug discovery applications for knime: an open source data mining platform importance of hla-dq and -dp restriction elements in tcell responses to soluble antigens: mutational analysis impact of human sequences in variable domains of therapeutic antibodies on the location of cd t-cell epitopes host cell proteases: critical determinants of coronavirus tropism and pathogenesis naturally processed measles virus peptide eluted from class ii hla-drb * recognized by t lymphocytes from human blood development and validation of a broad scheme for prediction of hla class ii restricted t cell epitopes tepitool: a pipeline for computational prediction of t cell epitope candidates mapps for the identification of immunogenic hotspots of biotherapeutics; an overview of the technology and its application to the biopharmaceutical arena the sars-cov- receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement. biorxiv netmhcpan- . and netmhciipan- . : improved predictions of mhc antigen presentation by concurrent motif deconvolution and integration of ms mhc eluted ligand data a novel strategy for the discovery of mhc class ii-restricted tumor antigens: identification of a melanotransferrin helper t-cell epitope efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin and downregulated by tumor necrosis factor alpha broad repertoire of the cd <sup>+</sup> th cell response in spontaneously controlled hepatitis c virus infection includes dominant and highly promiscuous epitopes mhc-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity cell entry mechanisms of sars-cov- measurement of mhc/peptide interactions by gel filtration or monoclonal antibody capture crystal structure of an mhc class i presented glycopeptide that generates carbohydrate-specific ctl vaccinia peptides eluted from hla-dr isolated from virus-infected cells are recognized by cd + t cells from a vaccinated donor immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus the immune epitope database (iedb): update structure, function, and antigenicity of the sars-cov- spike glycoprotein post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms molecular mechanism for antibody-dependent enhancement of coronavirus entry isolation and characterization of new human carrier peptides from two important vaccine immunogens searching immunodominant epitopes prior to epidemic: hla class ii-restricted sars-cov spike protein epitopes in unexposed individuals potential therapeutic targets and promising drugs for combating sars-cov- dendritic cells display peptides spanning the entire sars-cov- spike protein ii peptides from regions are presented by a majority of the donors analyzed. • one region with promiscuous hla-ii presentation is conserved with sars-cov • the correlation of presented to predicted peptides is low pulse dendritic cells derived from healthy human donors with sars-cov- spike glycoprotein to determine the precise sequences presented for t-cell surveillance. regions with promiscuous presentation are identified with poor correlation to predicted epitopes. one region with promiscuous presentation is conserved with sars-cov spike glycoprotein sequence key: cord- -vm nh lc authors: perez espitia, paula judith; de fátima ferreira soares, nilda; dos reis coimbra, jane sélia; de andrade, nélio josé; souza cruz, renato; alves medeiros, eber antonio title: bioactive peptides: synthesis, properties, and applications in the packaging and preservation of food date: - - journal: compr rev food sci food saf doi: . /j. - . . .x sha: doc_id: cord_uid: vm nh lc abstract: bioactive peptides are protein fragments which have a positive impact on the functions and conditions of living beings. peptides have shown several useful properties for human health, including antimicrobial, antifungal, antiviral, and antitumor activities. these compounds are produced by almost all species of life. however, they are produced in limited quantities in nature. as a result, researchers have tried to synthesize bioactive peptides to study their properties and applications in various areas. among their applications in food preservation, peptides have been incorporated into packaging materials. this review begins with a brief description of the methods used for the synthesis, purification, and characterization of peptides. also, the main bioproperties and mechanisms of action of peptides are discussed. finally, some applications of peptides are presented, especially their use in active packaging, their effects on the polymeric matrix, and peptide migration. food safety is a growing concern of great importance worldwide. recently, the estimated costs of diseases caused by foodborne pathogens was about $ billion in the united states (scharff ) , and it is estimated that in the united states alone about . million illness cases, hospitalizations and deaths will be caused by foodborne pathogens in . the consumption of processed foods with chemical preservatives has led to increased consumer concern and the demand for more natural and minimally processed foods. as a result, researchers have shown a growing interest in natural antimicrobial agents such as certain peptides. bioactive peptides are defined as specific protein fragments that have a positive impact on the functioning or conditions of living beings, thereby improving their health (korhonen and pihlanto ) . the beneficial effects are attributed to different properties found in peptides such as antimicrobial (reddy and others ; rajanbabu and chen ) , antioxidant (sarmadi and ismail ) , antithrombotic (wang and ng ) , anti-hypertensive (erdmann and others ) , and immunomodulatory activities (st georgiev ; gauthier and others ) , among others. ms submitted / / , accepted / / . authors espitia, soares, coimbra, de andrade, and medeiros are with food technology dept., federal univ. of viçosa, av. p. h. rolfs, s/n, campus univ., - . viçosa, minas gerais, brazil. author cruz is with food technology dept., state univ. of feira de santana, av. transnordestina, s/n, campus univ., - . feira de santana, bahía, brazil. direct inquiries to author soares (e-mail: nfsoares @gmail.com) . peptides with antimicrobial properties are used as the first chemical barrier against microbial attack, being synthesized in response to bacterial infections. they are produced by almost all species of life, from microorganisms, plants and animals, to humans (st georgiev ; hancock and diamond ) . in animals, antimicrobial peptides are produced mainly in those tissues exposed to adverse conditions such as skin, eyes, and lungs, which are more likely to be in contact with microorganisms (zasloff ; papo and shai ) . more than antimicrobial peptides have been reported, showing significant variations with respect to their sequence, length, and structure (papo and shai ) . antimicrobial peptides have found many applications, including those in biomedical devices, food processing equipment, and food preservation. in food preservation, peptides can be incorporated into materials to create antimicrobial packaging (appendini and hotchkiss ) . in this way, antimicrobial packaging plays an important role in maintaining the safety and quality of food, since the aim is to prolong food shelf life and to reduce bacterial growth on the product surface (soares and others a) . this type of active packaging interacts with the product and/or the headspace inside to reduce, inhibit, or retard the growth of microorganisms that may be present (soares and others b) . this review highlights the main methods of peptide synthesis and noteworthy peptide bioproperties. also, specific peptide applications in food preservation are reviewed, focusing on their incorporation in polymeric matrices. finally, the effects of peptide incorporation on packaging characteristics as well as their migration into food are discussed. borgia and fields ( ) . copyright ( ) , elsevier. peptides are biomolecules that contain between to several dozen of amino acid residues joined by peptide bonds. the discovery of the different peptide activities has generated enormous interest in this class of compounds and in the methods of isolation, analysis, purification, identification, and quantification. these methods have been systematically studied and improved. however, most sources of natural peptides are poor in these compounds, thus preventing their isolation in sufficient quantities for research. as a result, there was a growing need to synthesize peptides for application in physiological, chemical, physical, pharmacological, biochemical, and clinical studies. total of methods of peptide synthesis have been developed and improved: chemical synthesis, which uses chemical reagents to mediate peptide bond formation (andreu and rivas ) , enzymatic synthesis, in which the peptide bond formation is catalyzed by enzymes (bongers and heimer ; boeriu and others ) , and the dna recombinant technology synthesis, based on the use of cloning and ribosomal techniques from biological systems for peptide formation (sewald and jakubke ) . research on this synthesis method was first initiated more than y ago. however, the construction of peptides has recently become more accessible due to advances in process efficiency, including the development and use of fast coupling reagents, as well as the minimization of side reactions (borgia and fields ) . the main aspects of chemical synthesis are protection and activation. protection strategies are intended to provide chemical selectivity necessary for the construction of a particular peptide sequence. activation refers to the chemical coupling necessary to ensure quantitative formation of each peptide bond in the sequence (andreu and rivas ) . in chemical synthesis, chemical reagents are used to activate the carboxylic acid (rcooh) of the amino acid, which will donate the acyl group (r-co-) to form the peptide bond. the peptide bond presents a nucleophilic attack of the α-amino group by another amino acid (h n-r). in this synthesis, the reactive functional groups that are not directly involved in peptide bond formation receive prior protection (machado and others ) . there are types of chemical peptide synthesis, synthesis in solution (classical synthesis) and solid-phase synthesis. chemical synthesis in solution is performed with all reagents and reaction products dissolved in the medium (kent ) . in comparison, solid-phase synthesis (sps) is a simple procedure to produce peptides in large quantities on a solid support which remains insoluble in the reaction medium (shigeri and others ) . the solid support is a polymeric resin that has a functional group on its surface (linker) that allows it to form stable bonds in the peptide sequence to the reagent used for the de-protection of the n-amino group. peptide synthesis in the solid phase generally consists on the acylation of an amino acid to be linked to an insoluble support (resin) via a linker ( figure ). after that, the protecting group of the n-terminal is removed (the unprotecting step) to allow the next amino acid of the sequence to be attached to the complex "peptide-linker-resin." the unprotecting-coupling cycle is repeated until the desired sequence is complete. finally, the cleavage reagent is used to separate the complex "peptide-linker-resin." this reagent should also remove the protecting groups of side chains that are stable to unprotecting conditions of the n-terminal group (borgia and fields ) . peptide chemical synthesis can use protocols, boc (tertbutyloxycarbonyl) and fmoc ( -fluorenylmethyloxycarbonyl), named according to the type of protector of the reactive group of the amino acids (n-terminal) involved in the synthesis. the first protocol employs the tert-butyloxycarbonyl (boc) group for n-amino protection. this protocol is based on gradual differences in their sensitivity to acids. thus, the boc group is typically removed with trifluoroacetic acid (tfa), while the protecting groups of the lateral chains (ester, ether, and urethane derivatives based on benzyl alcohol) are specifically designed to be stable to repeated cycles of boc removal and are removed only with a specific reagent, a relatively stronger acid, usually hydrofluoric acid (borgia and fields ) . the second protocol uses a -fluorenylmethyloxycarbonyl (fmoc) as the n-amino protecting group. this protocol provides a greater degree of chemoselectivity than the boc protocol, since the fmoc group is removed under basic conditions (piperidine in n, n-methylpyrrolidone or dimethylformamide), without alteration of the acid-sensitive lateral chains (andreu and rivas ) . protection groups of lateral chains are compatible with the fmoc protection group; these are mainly ether, ester, and urethane derivatives based on t-butanol. protection groups of lateral chains are removed by the end of the synthesis using tfa (borgia and fields ) . in this method, the peptide bond formation is mediated by an enzyme (protease) in free or immobilized form. the enzymatic method is especially useful in the synthesis of very short peptides ( - oligomers) and in the condensation of large peptide fragments (so and others ) . proteolytic enzymes such as chymotrypsin, papain, pepsin, subtilisin, termolisin, trypsin, among others, have been used in the presence of organic solvents as catalysts for the synthesis of peptide bonds (ogino and others ) . the enzymatic synthesis of peptides has several advantages over chemical methods, including good stereoselectivity and regioselectivity. however, it has certain shortcomings, such as peptide synthesis being thermodynamically unfavorable in water, as well as the secondary hydrolysis of synthesized peptide chains, which hinders their use in peptide synthesis with long sequences (so and others ) . thus, the main practical obstacle to employment of a protease for peptide bond formation is finding suitable conditions to allow bond formation without mediating secondary hydrolysis of the peptide or peptide fragments used as reagents (bongers and heimer ) . the formation of a peptide bond by enzyme catalysis can occur through several mechanisms, including the reverse hydrolysis reaction of amides and transpeptidation (machado and others ; boeriu and others ) . the mechanism of the reverse hydrolysis reaction is based on the microscopic reversibility principle. this indicates that the peptide bond formation and hydrolysis reaction come from the same intermediate ( figure ). thus, the reaction conditions are manipulated to shift the equilibrium towards peptide bond formation. the transpeptidation mechanism occurs as a result of the break of a peptide bond, with the formation of an active acyl-enzyme intermediate ( figure ). this intermediate is attacked in the presence of a nucleophile (peptide or amino acid blocked in the α-carboxyl group) and consequently causes the formation of a new peptide bond. for both mechanisms, the equilibrium should shift to the synthesis reaction direction, requiring the use of protective groups of α-amino and carboxyl substrates, the addition of organic solvents to the media reaction, excess substrates, and the removal of products from the reaction medium (machado and others ) . this synthesis uses modern methods of cloning and gene expression in microorganisms, allowing the production of a recombinant peptide or several peptides simultaneously. bacteria are the expression system generally used, with e. coli being the most widely used host. since antimicrobial peptides present a natural destructive activity against the host and relative sensitivity to proteolytic degradation, peptides are often expressed as fusion proteins to neutralize their innate toxic activity and increase their expression levels (wang and others ) . compared with isolation from natural sources and the other synthesis methods, the recombinant approach offers the most costeffective alternative for large-scale peptide production (li ) . peptides are increasingly being produced for various purposes, and these may contain closely related impurities resulting from incomplete reactions or from several side reactions. peptides synthesized for therapeutic and clinical research, as well as for biological and structural studies to explore the structure-activity relationships must have % purity or greater (ridge and hettiarachchi ) . however, there are other applications where low values of purity, between % and %, are tolerable (table ) . peptide purification depends on a series of separation techniques. peptides made on a preparative scale (in gram amounts) can be obtained from a separation process, to isolate one or more individual components from a peptide mixture for future research, or on an analytical scale (about mg of peptide) to identify and determine the relative amounts of some or all components in the mixture. studies on an analytical scale are the first steps for improving separation conditions, which are developed prior to the execution of any preparative separation process (sewald and jakubke ) . after the synthesis process, peptides are submitted to a separation procedure consisting of centrifugation and washing to remove residues of the reagents used, as well as products of side reactions. subsequently, peptides are cleaved and subjected to filtration, as well as lyophilization (dagan and others ) . the most widely used methods used for the purification of peptides are reverse-phase high-performance liquid chromatography (rp-hplc), ion-exchange chromatography, size exclusion chromatography, affinity chromatography, and capillary electrophoresis ( table ) . the purity of a peptide must be verified by a method different from that used for purification, since the results of homogeneity derived from such a system can lead to misinformation and be misleading (ridge and hettiarachchi ) . thus, the characterization should be analyzed by different methods of mass spectrometry. mass spectrometry has different ionization methods, such as electrospray ionization mass spectrometry (esi-ms), fast atom bombardment mass spectrometry (fab-ms) or matrix-assisted laser desorption/ionization mass spectrome-try (maldi-ms) that can be used for peptide characterization ( table ) . the different mass spectrometry techniques are based on the accurate determination of the molecular mass-charge ratio of the peptide, as well as on a chemical structure determination, with high sensitivity and resolution (sewald and jakubke ) . the growing resistance of pathogens against many commonly used antibiotics has led to research of new compounds with the same functions. an interesting approach is the study of molecules of natural origin to replace antibiotics (bechinger and lohner ) . several studies in recent decades have shown that peptides have certain bioactive properties (agyei and danquah ). short peptides ( - amino acids) with cationic and hydrophobic properties are known to be potent defenses of the host organism, providing activity against a wide variety of pathogenic microorganisms such as gram-negative and gram-positive bacteria, fungi, viruses, and parasites (hancock and sahl ) . studies have shown remarkable results of peptide antitumor activity, observed mainly in cancer therapy (korhonen and pihlanto ) . although several peptides have biological activity, antimicrobial activity is one of the most studied. one of the most-used analytical techniques to determine peptide antimicrobial activity is the broth microdilution test. in this test, the microorganisms are cultured in titration microplates and the peptide to be tested is added to each well at different concentrations. the microorganism growth causes turbidity in the wells. however, when a certain concentration of the peptide tested inhibits bacterial growth no turbidity is observed. turbidity is usually read by spectrophotometry, with the greatest frequency at nm, but it can also be seen through visual inspection of the wells (otvos and cudic ) . the standard methods developed by the clinical and laboratory standards inst. (clsi) have been used to test the activity of antimicrobial peptides. among them are the standards for antimicrobial disk susceptibility tests (clsi ) , the method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically m -a (clsi ) , and the method for broth dilution antifungal susceptibility testing of yeast m -a (clsi ) , all of which have been widely used (jang and others ; rubinchik and others ; hwang and others ) . the most studied peptides are those with antimicrobial activity, characterized by their interaction with the cytoplasmic membrane of the microorganism regardless of the final target (powers and hancock ) . factors influencing the antibacterial activity are the electrostatic interactions between the peptide and positively charged and anionic lipids on the surface of the target microorganism. also, the hydrophobicity of the peptide (factor required for insertion into the membrane) and peptide flexibility allow peptide interaction with the microbial membrane (jenssen and others ) . although these characteristics are variable according to each peptide, all of them are essential to the function of peptides as antimicrobials. the exact mechanism of action of antibacterial peptides is not yet fully understood. however, there is a consensus among researchers regarding the first step in the initial interaction between peptide and the target cell (reddy and others ) . exclusion liquid chromatography based on separation process according to the size of the peptide relative to pore sizes in the stationary phase. used primarily in the early stages of purification of the peptide, when performed in multiple steps used to separate low-molecular-weight impurities from a mixture of peptides. however, the separation of the peptide of interest with other closely related peptides is virtually impossible affinity chromatography based on the biological specificity of the peptide. consists of a ligand (small specific biomolecule such as an antibody) that is immobilized in the column. the separation occurs because of highly specific biochemical interactions between the peptide and the ligand used when a high degree of specificity is required, for example, isolation of a target protein present in low concentration in a biological fluid or a cell extract capillary electrophoresis based on the migration of the peptide according to its charge in solution, depending on the application of an electric field. complementary technique to reversed-phase chromatography used for peptides and proteins table -ionization methods used in mass spectrometry. fundamental principle the ions are produced from a peptide contained in a solvent (for example, an organic compound such as methanol or acetonitrile) that is scattered in a fine aerosol fab-ms the peptide analyzed is mixed with a matrix, which is a non volatile reagent of protection (glycerol, diethanolamine, and triethanolamine, among others), and is bombarded with a beam of high-energy atoms ( to ev) in a vacuum. atoms are of an inert gas such as argon or xenon maldi-ms the peptide analyzed is bombarded by a laser beam (nitrogen), while a matrix (sinapinic acid) is used to protect the peptide. the matrix allows avoiding direct contact of the peptide with the beam, facilitating its vaporization, and ionization the initial attraction between the peptide and the target cell occurs via electrostatic binding between the cationic peptide and the components of the negatively-charged outer cell membrane, such as lipopolysaccharides in gram-negative bacteria or lipoteichoic acid on the surface of gram-positive bacteria (jenssen and others ) . this electrostatic interaction removes the native divalent cations (mg + , ca + ) from the cell surface, thus destabilizing the outer membrane and facilitating the entry of the peptide and subsequent peptide contact with the cytoplasmic membrane, a process known as autopromoted uptake (powers and hancock ) . after the peptide is bound to the target cell, an arrangement of the peptide occurs on the surface of the cytoplasmic membrane. this fact is of considerable debate, since several arrangement models have been proposed, such as the barrel-stave or the carpet model among others. depending on the model, the peptide can permeabilize the cytoplasmic membrane and/or translocate through it. thus, antimicrobial peptides can be classified into major groups, the first consisting of those peptides which act on the cytoplasmic membrane, and the second consisting of those which have no action on the cytoplasmic membrane of the target microorganism. this means that the peptide just moves into the cell without causing major disturbances in the membrane (powers and hancock ; jenssen and others ) . peptides acting on the bacterial membrane. several models have been proposed to explain how, after initial attachment, antibacterial peptides are distributed on the surface of the bacterial cytoplasmic membrane to form pores. pore formation results in membrane permeabilization, thereby affecting cellular respiration. it also deprives the microorganisms of their source of energy by interrupting the electrochemical gradient and causing an increase in the flow of water and ions across the membrane, thus leading to cell swelling followed by cellular lysis (bechinger and lohner ) . to explain the formation of pores, the aggregate toroidal pore, barrel-stave, and the carpet models have been proposed. the last models, the barrel-stave and carpet model, have been the most widely studied. barrel-stave model. this model describes the formation of a transmembrane channel (pore) through the binding of amphipathic α-helices. the hydrophobic surface of the peptide interacts with the lipid core of the membrane, while the hydrophilic surface of the peptide is oriented inside, producing an aqueous pore (figure ) . the progressive recruitment of additional peptides to the membrane surface increases the size of the pores, causing the loss of cell content and thus cell death (reddy and others ) . this model has been proposed to explain the activity of antimicrobial peptides, such as magainins (matsuzaki and others ) . carpet model. in this model, the peptide in high concentration is in contact with phospholipids located on the outer surface of the bacterial membrane, a fact that allows the peptide to permeate the membrane ( figure ). the peptides bind to the surface of the target membrane and cover it like a carpet. according to this model, the peptides exhibit a preferential binding for the phospholipid groups. the binding step is followed by the alignment of the peptide on the membrane surface so the hydrophilic surface is in contact with phospholipid or water molecules, causing a reorientation of hydrophilic residues and creating a hydrophobic core. finally, the peptide disintegrates the membrane by deformation of the membrane curvature (reddy and others ) . this model has been proposed to explain the action mechanism of dermaseptins (dagan and others ) . toroidal pore model. this model is considered a variant of the barrel-stave model. it is suggested that a perpendicular inclusion of the peptides to the membrane with their hydrophilic regions is associated with phospholipids, whereas their hydrophobic regions are associated with the lipid core. in this process, the membrane is bent inward so the pores are formed (jenssen and others ) . the main difference between this model and the barrel-stave model is the intercalation of the peptide with phospholipids to form the pore ( figure ). this model has been used to explain the mechanism of action of the peptide melittin (yang and others ) . aggregate model. this model, proposed by wu and others ( ) , has some similarity to the toroidal pore model. this model consists mainly of the arrangement of the peptide in the membrane forming an extension and developing micelle-like aggregate of peptides and lipids, but without adopting a particular orientation (jenssen and others ) . peptides with no activity on the bacterial membrane. these antimicrobial peptides have the ability to translocate into bacterial cells without causing membrane permeabilization. the peptide is accumulated within the cell where it reaches a variety of essential cellular processes that result in bacterial cell death (jenssen and others ) . the target process includes inhibitions of nucleic acid synthesis, protein synthesis, enzyme activity, and cell wall synthesis. peptides such as buforin ii and pleurocidin have shown this mechanism of action (park and others ; patrzykat and others ) . there is a growing need for new antifungal agents due to the increased resistance of molds to therapies with regularly used compounds (de lucca and walsh ) . peptides have emerged as alternative antifungal agents. initially, the antifungal mechanism of action was described as a result of fungal cell lysis or as a result of interferences in fungal cell wall synthesis. however, the discovery of new antifungal peptides in the last decade has led to the identification of new mechanisms of action, including membrane permeabilization, binding to ergosterol/cholesterol in the fungal membrane, the attack of mitochondria or other intracellular organelles, and the deformation of cell membrane structure (jenssen and others ) . according to de lucca and walsh ( ) , fungal peptides can be classified with respect to their mode of action into groups: peptides that act through cellular lysis; peptides that cross the fungal membrane and interact with the intracellular target; and peptides that act by forming pores. peptides acting by cellular lysis. these peptides are characterized by their amphipathic nature, being molecules with faces, one positively charged and the other neutral and hydrophobic. some of these peptides bind only to the membrane surface, damaging the membrane structure, and they may or may not pass through it. peptide smap- (a synthetic peptide derived from the sequence of cathelicidins) has shown antimicrobial activity against the fungus trichosporon beigelii by interaction, penetration, and subsequent damage to the cell membrane (lee and others ) . this result suggests that the main target of smap- peptide is the fungal plasmatic membrane. a similar mechanism was observed for the synthetic peptide ib-amps (an analogue sequence to peptides isolated from seeds of impatiens balsamina), showing antifungal activity by bonding the peptide to the fungal cell membrane and subsequent penetration (thevissen and others ) . peptides that pass into the membrane and interact with intracellular targets. these peptides interfere with cell wall synthesis or the synthesis of essential cellular components such as chitin or glucan. as such, the synthetic peptide omiganan (an indolicin analogue peptide, isolated from bovine neutrophils) has shown antifungal activity against candida albicans, and the main mechanism of action of this peptide is related to its activity in the cytoplasmic membrane, resulting in macromolecules synthesis inhibition of macromolecules and finally cell death (rubinchik and others ) . pore-forming peptides. these peptides are aggregated in a selective way to form pores of varying sizes, which then allow the passage of ions and other solutes. the synthetic peptide di-k hc (a halocidin analogue peptide, isolated from the invertebrate marine animal halocynthia aurantium known as sea peach), has shown antifungal activity against several strains of aspergillus and candida (jang and others ) . the activity of di-k hc results in the formation of pores on the surface of fungal membranes. moreover, these researchers pointed out the specific binding of di-k hc with b- , -glucan, a component of the cell wall of fungi. this mechanism has also been observed with the antifungal peptide psacotheasin, isolated from the yellow-spotted long-horned beetle (psacothea hilaris), which has shown activity against c. albicans (hwang and others ) . the researchers indicated that there was damage to the cell wall, membrane depolarization with the formation of pores ( . - . nm), as well as an increase in membrane permeability, all being responsible for the antifungal activity of this peptide. several studies have shown the ability of cationic peptides to inhibit viral infections. the peptide cecropin a has shown antiviral activity against junin virus (jv-which causes argentine hemorrhagic fever). the peptide melittin inhibited jv and herpes simplex virus (hsv- ) multiplication, as well as magainin i and ii, and has shown inhibitory activity against hsv- and hsv- (albiol-matanic and castilla ). antimicrobial peptides isolated from fish, such as tilapia hepcidin - , have shown activity against the nervous necrosis virus (nn virus), an infectious agent that causes mass mortality of several species of marine fish in the larval stage (chia and others ) . in addition, synthetic peptides consisting of arginine and tryptophan repetitions have shown activity against vaccinia virus (the cause of cowpox) (mohan and others ). the antiviral activity of peptides is often related to virus adsorption and its entry into the host cell or, in other cases, is the result of a direct effect on the viral envelope. thus, the antiviral activity of peptides may result from multiple mechanisms of action, the most important being blocking virus entry through interaction with the host cell and blocking viral entry through interaction with the virus. blocking viral entry through interaction with the host cell. peptides can interact directly with specific viral receptors on the host cell, thus preventing the virus from binding to the cell membrane or binding intracellularly (jenssen and others ) . proteoglycans are proteins found in all types of tissue, in intracellular granule secretions as well as in the extracellular matrix and cell surface. proteoglycans are covalently linked to one or more chains of glycosaminoglycans (gag), long polysaccharide unbranched structures, which have a sugar that contains nitrogen and are usually sulfated. gag chains are present on the surface of mammalian cells and their degree of sulfation makes these compounds more anionic. this network of strong negative charges allows gag to attract and bind to small cations, such as enzymes and proteins, and also pathogens such as viruses (spillmann ) . heparan sulfate, one type of gag chain, is one of the most important molecules related to viral binding (spillmann ) . thus, by blocking heparan sulfate molecules can be inhibited viral infection. jenssen and others ( ) have suggested that antimicrobial peptides which interact with heparan sulfate have the ability to block a number of viral infections. due to the large number of amino acid residues positively charged peptides can interact electrostatically with negatively charged heparan sulfate molecules on the cell surface. studies on lactoferrin (lf) have shown that this peptide prevents infection of the host cell rather than inhibiting virus replication after infection of the target cell. the interaction of lf with heparan c institute of food technologists ® vol. , r comprehensive reviews in food science and food safety sulfate molecules has been proposed as the mechanism responsible for lf antiviral activity (van der strate and others ). similarly, jenssen and others ( ) showed the antiviral activity of synthetic peptides (consisting of arginine and lysine residues) against herpes simplex virus and (hsv- and hsv- ). the peptides presented higher affinity in binding to heparan sulfate with an increasing number of cationic residues, thereby blocking the entry of hsv (- or - ). in addition, luganini and others ( ) reported the inhibition of cytomegalovirus by binding synthetic peptide dendrimers with molecules of heparan sulfate on the surface of fibroblasts and endothelial cells. thus, cytomegalovirus infection was blocked by the interaction of synthetic peptide binding sites with heparan sulfate. blocking viral entry through interaction with the virus. the interactions of peptides with the glycoproteins (gp) in the viral envelope have been proposed as another mechanism that influences the process of viral entry and virus inactivation. in this way, peptides generated from chemical modification of milk proteins, such as α-lactalbumin, β-lactoglobulin, and lysozyme with -hydroxyphthalic anhydride ( -hp) inhibited infection of vero cells with hsv- (oevermann and others ) . according to those researchers, the antiviral activity of these peptides is based on their direct interaction with viral glycoproteins (gb, gc, gd), which are responsible for adsorption and penetration of the virus into the host cell. similarly, lf has shown the ability to bind to the gp glycoprotein (a protein present in the outermost layer of the hiv virus) with antiviral effects, since the gp glycoprotein plays an important role in the adsorption and entry of hiv into target cells (van der strate and others ; pan and others ) . on the other hand, other peptides, such as magainins, have shown antiviral effects through direct interaction with virus cells. egal and others ( ) have indicated that the effect of magainins is the result of the peptide acting on the viral envelope. a similar mechanism was suggested for the activity of mucroporin-m , a defense cationic peptide present in scorpion venom, which has shown activity against the measles virus, the coronavirus that causes severe accurate respiratory syndrome (sars), and flu virus h n (better known as the bird flu virus) (li and others ) . the researchers have suggested that the antiviral activity of the peptide is the result of direct interaction with the virus envelope, thereby reducing viral activity in the host cell. cancer, also known as malignant neoplasm, is a general term that refers to more than different diseases affecting various tissues and different types of cells. all forms of cancer are characterized by abnormal cell growth, that is, they lack the mechanisms that control normal cell division. this lack of regulatory mechanisms is the result of a multistep process involving genetic mutations induced by inheritance or environmental changes (hütter and sinha ) . despite major advances in cancer therapy, there is considerable interest in the development of antitumor agents with a novel mode of action, since the cells have shown carcinogenic development of resistance to current chemotherapy (hoskin and ramamoorthy ) . carcinogenic cells often become resistant to chemotherapy. this mainly occurs as a result of increased expression of intracellular enzymes for the detoxification of antitumor agents, the correction of dna damage, generation of intracellular organelles with the ability to eliminate and/or transport the drugs out of the tumor, and irreversible defects in the cellular machinery that mediates apoptosis (hütter and sinha ) . thus, recent studies have shown peptides as an alternative to conventional cancer treatments. however, not all peptides have selective activity against carcinogenic cells. according to hoskin and ramamoorthy ( ) peptides that have antitumor activity can be classified into major groups: peptides with selective activity, and peptides with non selective activity, that is, those that have activity against bacteria, carcinogenic cells, and healthy cells. peptides with selective activity toward carcinogenic cells. these peptides have activity against bacteria and carcinogenic cells, but not against normal cells. several peptides, such as the cecropins, buforins, and magainins have shown antitumor activity without affecting normal eukaryotic cells (cruciani and others ; cho and others ) . studies with magainin ii have shown to inhibit the proliferation of carcinogenic cells (in bladder cancer) without any effect on normal cells (lehmann and others ) . similar results were observed by chen and others ( ) in the study of the synthetic peptide th - (isolated from tilapia and analogous to the peptide hepcidin), with antitumor activity shown primarily by direct interaction and lysis of target carcinogenic cells (human fibrosarcoma cells). these researchers indicated that the lytic activity of the peptide and proliferative cells were restricted mainly to carcinogenic cells, since normal cells showed no significant effects. likewise, the synthetic peptide th - (isolated from tilapia and an analogue to the peptide hepcidin) has shown antitumor activity against carcinogenic cells, due to interaction with and penetration of the membrane. this peptide has less toxicity toward normal cells supposedly because it can discriminate between healthy cells and carcinogenic ones (chang and others ) . researchers have also indicated that the interaction with the cell membrane and its subsequent damage is caused by the formation of pores on its surface. it has been suggested that the internalization of the peptide and the subsequent damage to the mitochondrial membrane activates apoptotic pathways (chang and others ) . according to hoskin and ramamoorthy ( ) there are fundamental differences between the membranes of malignant cells and normal cells which allow the selectivity of certain peptides to attack carcinogenic cells without affecting healthy cells. electrostatic interactions between cationic peptides and anionic components of the cell membrane have also been considered an important factor. carcinogenic cells typically have a negative charge due to a higher expression than normal of anionic molecules such as phosphatidylserine (ps) and mucin (glycoprotein) (oren and shai ) . however, normal cells are not affected, since these cells have a neutral surface charge, conferred by the zwitterionic nature of most membrane components such as phosphatidylethanolamine (also known as cephalin), phosphatidylcholine, and sphingomyelin (sok and others ) . membrane fluidity and the surface area of the cell are also considered factors that contribute to the selectivity of peptides for carcinogenic cells. the fluidity of carcinogenic cells is greater than that of normal cells, which may increase the activity of lytic peptides through the easy destabilization of the membrane. in addition, the carcinogenic cells have a higher surface area than healthy cells due to the presence of greater numbers of microvilli, which are small projections of the cell membrane, irregular in size and shape. the microvilli may allow the bonding between peptide and carcinogenic cells (hoskin and ramamoorthy ) . peptides with nonselective activity. this group is comprised of peptides with activity against bacteria, carcinogenic cells, and against normal eukaryotic cells (hoskin and ramamoorthy ) . according to papo and shai ( ) non selective activity of these antimicrobial peptides results from their ability to interact with and cause damage to negatively charged membranes and those of a zwitterionic nature. dathe and others ( ) have indicated that the hydrophobic moment of antimicrobial peptides exerts a substantial influence on the neutral lipidic membranes, although it has a small role in the permeabilization of highly charged lipid membranes. peptides of this group include melittin, isolated from bee venom; taquiplesin ii, isolated from the horseshoe crab; defensins, isolated from insects; and plantaricin, a bacteriocin isolated from lactobacillus plantarum (schweizer ). plantaricin has shown activity against carcinogenic cells and against normal lymphocytes and neuronal cells (sand and others ) . the mechanism of action of antitumor peptides consists of permeabilization of the cell membrane mediated by electrostatic interaction. the electrostatic interaction is generated by the negatively charged phospholipids in the cell membrane and the positively charged peptide (schweizer ). unlike carcinogenic cells, eukaryotic cells have most of their negatively charged phospholipids, particularly ps, in the inner membrane, while neutral lipids are positioned on the outside (zhao and others ) . however, the result obtained by sand and others ( ) suggests that in addition to the mechanism of action related to the electrostatic interaction, there is another mechanism which explains the sensitivity of normal eukaryotic cells to plactaricin. probably another negatively charged macromolecule present on the membrane surface of healthy cells is also involved in plantaricin activity. similar results were observed by nan and others ( ) in the study of synthetic peptides consisting of lysine or arginine enriched with tryptophan. the peptide with arginine residues showed higher toxicity against human erythrocytes and mammalian cells. the hydrophobicity of the peptides has been suggested as an important factor in the increase of hemolytic activity and cytotoxicity in mammalian cells, as hydrophobic regions are required for direct interaction between peptide with membrane lipid components. the peptide with arginine residues was slightly more hydrophobic than the peptide with lysine residues. thus, these researchers suggested that small differences in hydrophobicity of these peptides may be responsible for the cytotoxic activity of this peptide in mammalian cells. the growing problem of microorganism resistance to conventional antibiotics, as well as the need for new agents with antibiotic properties has stimulated interest in developing antimicrobial peptides aiming for their application in the medical field (zasloff ) . most of the studies are devoted to the development of topical agents with antibacterial and antifungal activities. also, due to their antiviral activity, antimicrobial peptides have also been proposed as chemical preservatives. in the food industry, antimicrobial peptides, especially those produced by bacteria, have been widely researched in recent years due to their potential use as natural preservatives (papagianni ; coma ; settanni and corsetti ) . the direct application of antimicrobial peptides in food preservation can be achieved by methods: the direct addition of peptide to the food matrix, or the inoculation of the food matrix with the bacteriocin producer strain under the conditions favorable for the in situ production of the antimicrobial peptide. bacteriocins can be obtained ex situ by the cultivation of the producer strain at an industrial scale in a food-grade substrate, followed by a series of separation and purification techniques. these ex situ bacteriocins are commercially available in concentrated form, such as alta tm or microgard tm , and can be added directly to the food matrix. the production of bacteriocins in the food matrix offers several legal and cost advantages. the use of bacteriocin producer strain requires careful selection depending on the particular food intended for inoculation to ensure the producer strains will produce bacteriocins in the necessary amounts to inhibit the target microorganism. in addition to the peptides being studied as antimicrobial agents for direct addition to foods, they also have shown potential for being incorporated into food preparation surfaces (such as cutting surfaces) and processing equipment, as well as in food packaging (appendini and hotchkiss ) . active packaging includes the incorporation of antimicrobial agents in the packaging material to control and extend the shelflife of food (soares and others a). these types of packaging are considered an innovative technology in food preservation, since they allow better antimicrobial efficiency on food surfaces, thus improving stability. the development of active packaging by incorporating antimicrobial peptides in food packaging material can be done either to prolong the life of the product or to reduce the microbial load of the packing before use (steven and hotchkiss ) . the development of active packaging with antimicrobial peptides can be accomplished by main methods of incorporation: direct peptide incorporation in the polymer; peptide coating on the polymeric surface; and peptide immobilization in the polymer. numerous studies have reported the incorporation of antimicrobial peptides directly in the polymeric material, especially bacteriocins. the peptides are relatively resistant to heat (appendini and hotchkiss ) . however, their antimicrobial activity may be greater when heat is not used in the incorporation process. moreover, bioactive peptides incorporated in polymer films must be able to diffuse to the package surface over time to be effective. thus, polymers such as cellulose acetate, alginate, chitosan, and soy protein, among others, have been widely used to develop films with direct incorporation of these antimicrobials (marcos and others ; pires and others ; sivarooban and others ; santiago-silva and others ). researchers have studied the antimicrobial activity of bacteriocins incorporated into polymeric materials in synergy with other antimicrobial agents. synergistic activity against staphylococcus aureus, listeria monocytogenes, and bacillus cereus has been observed for nisin with potassium sorbate and garlic oil when incorporated into chitosan films (pranoto and others ) . in addition, soy protein films incorporated with nisin, grape seed extract, and ethylenediaminetetraacetic acid (edta) have shown inhibitory synergistic activity against pathogenic microorganisms such as l. monocytogenes, e. coli o : h , and salmonella typhimurium (sivarooban and others ) . the activity of bacteriocins incorporated into polymeric materials in synergy with other conservation technologies has also been reported. films incorporated with enterocins a and b (bacteriocins produced by enterococcus faecium) have shown synergistic activity when used together with high-pressure processing. thus, the use of antimicrobial packaging developed in conjunction with the high-pressure process allowed the control of l. monocytogenes at below detectable levels after d of storage at • c (marcos and others ) . this is an alternative method when the polymer requires extreme processing conditions during packaging material manufacture, such as high pressure and temperature, which can result in inactivation of the antimicrobial agent (appendini and hotchkiss ) . in some cases, the antimicrobial coating is done by contacting the film with or immersing it in the peptide solution. in this way, linear low-density polyethylene (lldpe) has been coated with lactocin and lactocin al (both bacteriocins produced by lactobacillus curvatus crl ), by direct contact of the film with a bacteriocin solution, showing antimicrobial activity in vitro against lactobacillus plantarum crl and listeria innocua (massani and others ) . similarly, scannell and others ( ) used alternatively lacticin and nisin adsorbed on the surface of plastic bags (polyethylene/polyamide) through direct contact of the polymeric material with bacteriocin solution. the film coated with nisin showed inhibitory activity against l. innocua and s. aureus, maintaining its activity for mo either at room temperature or under refrigeration. however, the film coated with lacticin did not show antimicrobial activity. the researchers suggested that lacticin was not retained by the polymer (scannell and others ) . proper handling of solvents and polymeric structures has been suggested to increase the adsorption of the peptide into the polymer matrix (appendini and hotchkiss ) . for example, the polymeric surface can be coated by applying a filmogenic solution that can be deposited on the film surface by the casting method. accordingly, chollet and others ( ) developed a laminated film of polyethylene (pe) and polyamide (pa), with the structure pe/pa/pe, coated with a filmogenic solution of hydroxypropyl methyl cellulose (hpmc) and adsorbed with nisin. the developed film presented antimicrobial activity in vitro against kocuria rhizophila (chollet and others ) . peptides can be immobilized or attached to solid supports by physical methods, such as layer-by-layer assembly, or by chemical methods, such as covalent bonding (onaizi and leong ) . layer-by-layer assembly. in this process, the peptide is sandwiched between polyionic polymers and the number of peptides and polymers is flexible (figure ) . the effectiveness of the peptide depends on its relative mobility. the advantage of this method is that it allows the slow release of the peptide embedded in the surface of the polymer. however, a key drawback of this method is that the peptide immobilized in the layers closest to the solid support will not be in direct contact with the target surface, thus reducing peptide activity. this peptide must be able to diffuse through the different layers of the assembly to the interface (onaizi and leong ) , to ensure efficient release and consequent bioactivity. the diffusion process of the peptide in the different layers is more complex than its diffusion in solution, since additional factors such as tortuosity of the diffusion path, the number of layers, and the polymer-peptide interactions can affect the diffusion process (appendini and hotchkiss ; sukhishvili ) . covalent bonding. in this process, the antimicrobial peptide will react chemically with a given surface to form a stable bond, which results in the formation of an antimicrobial coating on the polymeric surface (haynie and others ) . covalent bonding offers several advantages, including a more stable attachment between the peptide and the polymer surface (goddard and hotchkiss ) . covalent bonding reduces attached peptide ability to destabilize and improves its bioactivity, by protecting it from denaturation. due to the inert nature of most polymers, they must be subjected to a functionalization process on the surface before bonding with the peptide. the polymers can be functionalized with different spacers, which are reactive functional groups that allow peptide attachment on the spacer surface (humblot and others ) . the quantity of reactive functional groups generated in the functionalization process results in a restricted number of covalent bondings, which limits the amount of peptide that can be attached to the packaging. according to goddard and hotchkiss ( ) , direct bioactive compound applications require small amounts to be effective. however, for the applications of peptides in polymeric matrixes it is necessary to maximize the amount of peptides per unit area. to accomplish this, the functionalization technique must be optimized with the objective of linking the desired type and quantity of reactive functional groups. stiff or flexible spacers have been used as reactive groups for the functionalization of polymers (figure ). stiff spacers, such as polymethyl-methacrylate (pmma) and polyvinyl chloride (pvc), restrict the lateral mobility of the peptide bond, keeping the peptide firmly in a specific orientation. on the other hand, flexible spacers, such as polyethylene glycol (sand and others ) allow lateral mobility of the peptide bound, which can result in different orientations of the peptide molecules at the interface (onaizi and leong ) . among the different types of spacers, polyethylene glycol (peg) is widely used in the immobilization of peptides. the use of peg has several advantages, such as rapid and free peptide orientation, promoting peptide-bacteria interactions (costa and others ) . although the potential penetration and translocation of peptides through the microorganism cytoplasmic membrane is low due to the covalent bond that attaches the peptide to the polymer, it has been reported that peptides have sustained their bioactivity after attachment to polymeric matrixes. the synthetic peptide k l (a peptide sequence derived from magainin) was covalently bound to polystyrene (ps) resin by functionalization with peg and showed antimicrobial activity against foodborne pathogenic microorganisms, including e. coli o : h , l. monocytogenes, and pseudomonas fluorescens (appendini and hotchkiss ) . similarly, the synthetic peptide e lkk was covalently immobilized in ldpe film after chromium oxidation and functionalization with peg. this active packaging had antimicrobial activity against e. coli showing a reduction of log cycles when compared with the control (steven and hotchkiss ) . the sustained bioactivity of attached peptides is caused by the presence of spacers which allow sufficient freedom of motion for the active portion of the peptide to contact microorganisms on the food surface (appendini and hotchkiss ) . haynie and others ( ) have previously demonstrated that peptide-bacteria interactions are sufficient for peptide bioactivity. moreover, costa and others ( ) have indicated that the efficacy of attached peptides could possibly result from a higher peptide-relative surface availability, contrary to the other methods of peptide applications in which peptide aggregation can occur, producing uneven distribution. on the other hand, the diffusion of attached peptides into the food surface is restricted due to the covalent bonding. however, diffusion to the food product can occur in extreme conditions, such as high temperatures, which can promote hydrolysis reactions. the characterization of active packaging involves processes: structural analysis and measurement of their properties (table ) . according to goddard and hotchkiss ( ) , the type of analytical tool used in the structural characterization of polymers depends on the kind of modification, the specificity required, and available resources. some of the techniques used in structural analysis of active packaging with antimicrobial peptides are the contact angle, x-ray photoelectron spectroscopy (xps), fourier transform infrared spectroscopy (ftir), and scanning electron microscopy (sem). steven and hotchkiss ( ) used the techniques of contact angle and xps to assess changes in the surface of ldpe films after treatment with chromic oxide and functionalization with peg as spacer, subsequently covalently binding a synthetic peptide. the contact angle for the film before being subjected to any process of functionalization showed values of • . however, after chromic oxidation and peg bonding, films presented values of • and • , respectively; which indicated that the film surface became hydrophilic. these researchers concluded that the decrease in the contact angle is the result of an increased ionization at the film surface after oxidation, due to the presence of functional groups, such as carboxylic acid (cooh); and a value even lower after functionalization was observed due to solubility of peg. the xps technique showed changes in chemical composition on the film surface, resulting in the detection of nitrogen ( . %) and an increased percentage of oxygen (initially from . % to . % after oxidation and . % after functionalization). the oxygen increase was due to the presence of carboxylic acid after chromic oxidation. also, this increase was the result of functionalization with peg due to the main presence of o in the peg chain backbone. in addition, the functionalization with peg also introduced nitrogen originating from its amino-terminal functions (nh -peg-nh ). the ftir spectroscopic technique has been used by pranoto and others ( ) to study the interactions between chitosan films and nisin. they observed an increase in the band of the amide i corresponding to the wave number cm − related to the increased concentration of nisin incorporated in the film. according to the researchers, this is probably due to the interaction between the amine functional groups of chitosan and functional groups of nisin, which resulted in covalent bonds, and consequently in a larger peak. microscopic techniques have also been widely used to evaluate morphological changes in the surface of films that have been incorporated with antimicrobial peptides. pires and others ( ) used sem and observed that cellulose-based films incorporated with nisin, or a mixture of nisin and nantamicin, showed crystals deposited on the surface. these results indicated a heterogeneous distribution of the peptide in cellulosic films, while the control film presented a homogeneous structure. similarly, santiago-silva and others ( ) used sem to observe changes in surface morphology of cellulose acetate film incorporated with pediocin. when the concentration of peptide was increased, the films incorporated with pediocin had a rough surface c institute of food technologists ® vol. , r comprehensive reviews in food science and food safety table -characterization techniques of packaging incorporated with antimicrobial peptides. technique factor studied structural analysis contact angle quantifies surface hydrophobicity by measuring how far a droplet of water spreads on a surface x-ray photoelectron spectroscopy (xps) determines the atomic composition of the top several nanometers of a solid. this technique can be used to quantify the percent atomic composition and stoichiometric ratios fourier transform infrared spectroscopy (ftir) detects and identifies the chemical functional groups present in the polymer scanning electron microscopy (sem) allows the characterization of the polymer surface morphology and the observation of the dispersion quality of the peptide in the polymeric matrix property measurements mechanical properties measurement of the mechanical performance of the polymer. generally according to the standard method astm d (astm a) barrier properties measurement of water vapor permeability. generally according to the standard method astm e /e m (astm b); astm f (astm ) due to large amounts of pediocin granules dispersed in the matrix. this resulted from the lack of peptide solubility. on the other hand, the control film showed a homogeneous and transparent surface. in addition to the analysis of structural changes and interactions between the peptide and the polymeric matrix, the study of packaging properties is important as well. these properties show the performance of the developed material and how it will relate to the primary functions of food packaging, such as physical integrity. thus, mechanical and barrier properties have become increasingly relevant and are more frequently studied. the mechanical properties of films incorporated with antimicrobial peptides serve as the basis for assessing the effects on the mechanical performance resulting from the modification made to the polymer (table ) . guiga and others ( ) investigated the effect of nisaplin ® ( . % purity of nisin) coating on the mechanical properties of laminated films (pe/pa/pe), studying the mechanical properties of elongation at break and young's modulus. their results showed a significant difference in mechanical properties between the films incorporated with the peptide and the control treatment; peptideincorporated films showed an increase of young's modulus and a decrease in elongation at break. the polymer-based coating (hpmc) applied in laminated film, as well as the interaction between proteins and salts present in nisaplin, may have modified the mechanical behavior of the manufactured packaging, thereby increasing the rigidity of the film with the consequent decrease in its elongation. a similar result was observed by santiago-silva and others ( ) in their study of cellulosic films incorporated with pediocin. the researchers indicated that the addition of % of the peptide increased the maximum load required for film rupture when compared to the control. the researchers pointed out that a possible interaction between the pediocin and the polymeric matrix allowed the development of a more resistant film. however, a significant drop in the maximum load value was observed at a % concentration. according to the researchers, there was an excessive amount of the peptide incorporated, which weakened the cellulose chains of the film and resulted in a reduction of film resistance. a decrease in the mechanical strength of films incorporated with antimicrobial peptides has also been observed. sivarooban and others ( ) reported a decrease in puncture resistance and tensile strength values of soy protein films incorporated with nisin. similarly, pires and others ( ) indicated that nisin incorporated into cellulose-based films affected the film structure, reducing maximum load and elongation values. these resulted from the heterogeneous distribution of the peptide in the polymeric matrix, which consequently lead to the formation of stress points and reduced film resistance. although several studies have indicated changes in film properties, in some cases peptide incorporation into polymeric matrices had no significant effects. massani and others ( ) reported no significant difference in tensile strength, elongation, and water vapor permeability of ldpe films coated with lac-tocin and lactocin al . similar results were observed by chollet and others ( ) who indicated that the incorporation of nisin into pe/pa/pe laminated films, by coating with hpmc, showed neither changes in tensile strength at break nor in water vapor permeability. similarly, guiga and others ( ) reported that the direct incorporation of nisin into multilayer films of ethyl cellulose (ec) and hpmc (ec/hpmc/ec) did not alter the properties of tensile strength, young's modulus, or elongation at break. barrier properties of packaging include resistance to water vapor or gases (o and co ). the water vapor barrier property of the packaging can be determined by calculating the water vapor permeability (wvp) or the water vapor transmission rate (wvtr). both parameters cover the determination of the passage of water vapor through a polymeric material. however, the wvp considers vapor pressure difference between specific surfaces (internal and external) of the analyzed packaging (astm b). both parameters, wvp as well as wvtr, have been used to indicate that the addition of nisin in different polymeric matrixes, such as ldpe film coated with a cellulose-based solution (grower and others ; massani and others ) , sodium caseinate films (kristo and others ) and pe-film coated with hpmc (chollet and others ), causes no significant changes in the water vapor barrier property. on the other hand, studies regarding gas permeability in active packaging have been limited due to their applications as films or coatings for food products. during the evaluation and characterization of antimicrobial packaging it is important to research the transference of antimicrobial substances from the packaging material into the food, since this information allows the determination of how the antimicrobial agent is released from the active packaging. the mass transfer can occur by the diffusion mass transfer mechanism or by the convective mass transfer mechanism. the convective mass transfer mechanism occurs in a moving fluid, known as natural convection, if the movement is caused by differences in the density, or as forced convection, if the movement is caused by external agents or when a fluid is flowing on the solid surface by forced movement. on the other hand, diffusion mass transfer consists of a random motion of individual molecules as a result of a concentration gradient (crank ; geankoplis ) . the migration of substances from packaging materials takes place through the diffusion mass transfer mechanism, since the active packaging and the food contain a concentration gradient for the antimicrobial agent incorporated in the packaging. unlike the research on the release of active substances from drugs or the release of solvents from polymers, the study of antimicrobial release from active packaging is still limited (buonocore and others ; bastarrachea and others ) . the knowledge of diffusion parameters allows an efficient design of active packaging. several factors must be considered when studying the migration from antimicrobial packaging, including the release rate of the antimicrobial molecules from the packaging. if this rate is high, the active packaging would release the antimicrobial rapidly, resulting in a large concentration at a determined time. however, the large concentration would not be maintained over time, depending on the solubility of the antimicrobial in the selected food. if the solubility is very high, the antimicrobial will migrate rapidly to the food matrix, and therefore result in a decreased concentration of the antimicrobial on the food's surface along time. on the other hand, if the release rate is low, the antimicrobial agent will be slowly released in a desired concentration and if it presents a low solubility in the selected food, the antimicrobial can accumulate on the food surface and slowly migrate into the food matrix. in this situation the release rate should not be slower than the microbial growth (bastarrachea and others ) . in either case, the release of the antimicrobial agent from the packing material is indicated by the diffusion coefficient (d). thus, the diffusion characteristics of the antimicrobial agent can be used to determine the amount needed to maintain the proper concentration on the food surface (buonocore and others ) . the literature reports a few migration studies of antimicrobial peptides incorporated in active packaging. most evaluate the migration of nisin, probably due to the fact that this is the only antimicrobial peptide substance indicated as generally recognized as safe (gras) for direct contact with food in the united states (fda ). nisin is also widely accepted as a food preservative in the european community where it is classified as a safe preservative for food contact, coded as e (fsa ), as well as in brazil where the use of nisin is permitted by brazilian law as a natural preservative for biological products (anvisa ) . diffusion of several antimicrobial agents, such as potassium sorbate or lysozyme incorporated in active packaging, has been explained by fick's second law (han and floros ; gemili and others ) : where, c is the diffusing substance concentration; d is the apparent diffusion coefficient; t is the diffusion time; and x the distance. depending on the conditions of the migration test, different analytical solutions have been applied to solve fick's second law and to calculate the d-value in the migration of the antimicrobial peptide nisin incorporated in different types of packaging materials (table ) . different analytical solutions of fick's second law have been used in previous studies to calculate the d-value of nisin at a specific temperature ( table ) . some of these studies were conducted at different temperatures to characterize the d-value as a function of this parameter. protein films (corn zein or wheat gluten) and poly(butylene adipate-coterephthalate) (pbta) films incorporated with nisin showed an increase in d-value with increasing temperature, indicating that the peptide concentration was higher in the simulant at equilibrium state with increasing temperature (teerakarn and others ; bastarrachea and others ) . similarly, at low temperatures, lower d-values of nisin diffusivity indicate that the film retains larger amounts of the peptide in the polymer matrix while in contact with the simulant. the arrhenius activation energy model (eq. ) has been shown to confirm the dependence of the diffusivity with respect to temperature. where, d is a constant; e a is the activation energy for the diffusion process (j/mol); r is the universal gas constant ( . j/mol k); and t abs is the absolute temperature (k (− ) n ierfc nh √ d t m , is the initial amount of nisin in the film; m t , released amount of nisin at time t; h, film thickness; d, diffusion coefficient; ierfc, associated function of the mathematical error function teerakarn and others ( ) paper coated with acrylic polymer and ethylene-vinyl acetate co-polymer (eva) is the amount of nisin released at time t; m ∞ , is the migration in a state of equilibrium; l p coating layer thickness, d, diffusion coefficient kim and others ( ) hydroxypropyl methyl cellulose (hpmc) films is the amount of nisin in the simulant at time t; m f, , amount of nisin in the film when t = ; α, mass ratio between the amount of nisin in the simulant and in the film at equilibrium; q n , is the ''n" root of tanq n = −αq n ; l, is a half of the film's thickness bastarrachea and others ( ) teerakarn and others ( ) indicated e a values of . and . kj/mol for corn zein and wheat gluten films, respectively, and bastarrachea and others ( ) obtained an e a value of . kj/mole for pbta film incorporated with nisin. the value of e a represents the degree of molecular interactions between the antimicrobial substance incorporated and the polymeric matrix. thus, higher e a values represent stronger antimicrobial-polymer interactions, which is reflected in a lower d-value due to the greater energy level required for antimicrobial release (bastarrachea and others ) . the relationship between temperature and diffusivity of the antimicrobial agent is the result of structural changes in the polymer matrix, since above the glass transition temperature (t g ) the molecular mobility in the system increases along with temperature, which leads to an increase in the ability of the packaging material to transport substances through its polymeric matrix (teerakarn and others ) . in addition to the interactions between polymer and antimicrobial agent, the d-value is also influenced by interactions between the antimicrobial and the food matrix. thus, the food nh -r-w-c-f-r-v-c-y-r-g-i-c-y-r-k-c-r-conh miyata and others ( ) * x = means that the specific identity of an amino acid cannot be determined unambiguously. * * dhb = (z)- , -didehydrobutyrine. * * * abu = -aminobutyric acid. composition, as well as the solubility of the antimicrobial in these components also affects the d coefficient. in the study of paper coated with eva incorporated with nisin, the d-values varied according to the composition of the food in contact with the active packaging (kim and others ) . the highest d-value was observed when the film was in contact with a % citric acid solution, and the lowest value was observed when in contact with a % nacl solution. characteristic parameters of each solution, such as ph and ionic strength, have been shown to influence nisin solubility. nisin has a high solubility (up to mg·ml − at ph ) at low ph, but at high concentrations of nacl (above m) nisin solubility dependence on ph almost disappears and the solubility decreases to values below mg·ml − at any ph (rollema and others ) . chollet and others ( ) also investigated the influence of food composition on the migration of nisin incorporated in pe/pa/pe films coated with hpmc by changing the fat percentage. they found that increasing the fat content in the food resulted in an increased d-value and, therefore, in a greater diffusion of incorporated nisin. in their experiment, nisin diffusion mechanism was governed by the fat content. the increase in fat content resulted in microstructural changes, such as enlargement of pore size in the food matrix, which favored nisin diffusion into it. consumer demand for minimally processed foods and additivefree products has led to the development of antimicrobial packaging. peptides have shown various bioproperties, among them antimicrobial activity, leading to the application of these compounds in the food preservation area by either direct addition or incorporation into packaging materials (table ) . active packaging materials incorporated with antimicrobial peptides have shown effectiveness in inhibiting pathogenic microorganisms, an improvement in food safety. moreover, antimicrobial peptides incorporated into the polymeric matrix may af-fect the engineering characteristics of the packaging material, and lead to differentiated diffusion performance. this review highlights the characteristics of pure peptides, as well as their incorporation into polymeric matrices. several studies have indicated significant changes in mechanical properties and surface morphology of the films incorporated with antimicrobial peptides. however, research related to the study of barrier properties to gases and water vapor is still limited. more studies on the release of other peptides, different from nisin, from food packaging materials are needed to better understand the mechanism of dissemination of antimicrobial agents. finally, in the years ahead, the advent of nanotechnology will lead to research on the synergistic effects of antimicrobial peptides and nanoparticles, such as metals, metal oxides, and nanoclays, with the objective being to improve the mechanical and barrier properties of antimicrobial packaging. bioactive peptides: synthesis, properties, and applications shortened cecropin a-melittin hybrids significant size reduction retains potent antibiotic activity uso da nisina com a função de conservador para queijos pasteurizados. portaria deten/ms n • , de de janeiro de surface modification of poly(styrene) by the attachment of an antimicrobial peptide review of antimicrobial food packaging in: standard test method for water vapor transmission rate through plastic film and sheeting using a modulated infrared sensor astm d in: standard test method for tensile properties of thin plastic sheeting astm e /e m in: standard test methods for water vapor transmission of materials biochemical and genetic characterization of enterocin a from enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins engineering properties of polymeric-based antimicrobial films for food packaging: a review release kinetics of nisin from biodegradable poly(butylene adipate-co-terephthalate) films into water detergent-like actions of linear amphipathic cationic antimicrobial peptides optimized enzymatic synthesis of c-terminal peptide amides using subtilisin a from bacillus licheniformis recent applications of enzymatic peptide synthesis chemical synthesis of proteins a general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications tilapia (oreochromis mossambicus) antimicrobial peptide, hepcidin - , shows antitumor activity in cancer cells a fish antimicrobial peptide, tilapia hepcidin th - , shows potent antitumor activity against human fibrosarcoma cells antimicrobial peptides (amp) with antiviral activity against fish nodavirus buforins: histone h a-derived antimicrobial peptides from toad stomach monitoring nisin desorption from a multi-layer polyethylene-based film coated with nisin-loaded hpmc film and diffusion in agarose gel by an immunoassay (elisa) method and a numerical modeling reference method for broth dilution antifungal susceptibility testing of yeast performance standards for antimicrobial disk susceptibility tests methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically bioactive packaging technologies for extended shelf life of meat-based products covalent immobilization of antimicrobial peptides (amps) onto biomaterial surfaces the mathematics of diffusion antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation in vitro antiplasmodium effects of dermaseptin s derivatives hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides antifungal peptides: novel therapeutic compounds against emerging pathogens antiviral effects of synthetic membrane-active peptides on herpes simplex virus, type the possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease direct food substances affirmed as gras -electronic code of the bactericidal activity of pediocin pa- is specifically inhibited by a -mer fragment that spans the bacteriocin from the center toward the c terminus food standards agency. current eu approved additives and their e numbers defensins: antimicrobial peptides of innate immunity immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins principles of mass transfer development of cellulose acetate-based antimicrobial food packaging materials for controlled release of lysozyme polymer surface modification for the attachment of bioactive compounds development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of listeria monocytogenes antimicrobial plastic film: physico-chemical characterization and nisin desorption modeling innovative multilayer antimicrobial films made with nisaplin ® or nisin and cellulosic ethers: physico-chemical characterization, bioactivity and nisin desorption kinetics simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films the role of cationic antimicrobial peptides in innate host defences antimicrobial and host-defense peptides as new anti-infective therapeutic strategies antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin a coating for use as an antimicrobial and antioxidative packaging material incorporating nisin and [alpha]-tocopherol studies on anticancer activities of antimicrobial peptides the antibacterial activity of magainin i immobilized onto mixed thiols self-assembled monolayers proteomics for studying cancer cells and the development of chemoresistance antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from psacothea hilaris antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, halocynthia aurantium peptide antimicrobial agents chemical synthesis of peptides and proteins properties of nisin-incorporated polymer coatings as antimicrobial packaging materials bioactive peptides: production and functionality thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on listeria monocytogenes antifungal mechanism of smap- ( - ) isolated from sheep myeloid mrna against trichosporon beigelii antitumor activity of the antimicrobial peptide magainin ii against bladder cancer cell lines virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses recombinant production of antimicrobial peptides in escherichia coli: a review peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate sínteses química e enzimática de peptídeos: princípios básicos e aplicações high-pressure processing and antimicrobial biodegradable packaging to control listeria monocytogenes during storage of cooked ham development and 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potential challenges selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study broth microdilution antibacterial assay of peptides purification and amino acid sequence of lactocin , a bacteriocin produced by lactobacillus casei crl antiviral properties of milk proteins and peptides ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes mechanism of action of the antimicrobial peptide buforin ii: buforin ii kills microorganisms by penetrating the cell membrane and inhibiting cellular functions a novel antimicrobial peptide from bufo bufo gargarizans sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in escherichia coli development and evaluation of active packaging for sliced mozzarella preservation the relationship between peptide structure and antibacterial activity enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin applications of antimicrobial peptides from fish and perspectives for the future antimicrobial peptides: premises and promises peptide purity and counter ion determination of bradykinin by high-performance liquid chromatography and capillary electrophoresis improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering antimicrobial and antifungal activities of a novel cationic antimicrobial peptide, omiganan, in experimental skin colonisation models plantaricin a, a peptide pheromone produced by lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells antimicrobial efficiency of film incorporated with pediocin (alta ® ) on preservation of sliced ham antioxidative peptides from food proteins: a review development of bioactive food packaging materials using immobilised bacteriocins lacticin and nisaplin ® bioactive peptides: synthesis, properties, and applications health-related costs: from foodborne illness in the united states. produce safety project. available from: www.producesafetyproject.org. accessed cationic amphiphilic peptides with cancer-selective toxicity controlled diffusion of an antimicrobial peptide from a biopolymer film application of bacteriocins in vegetable food biopreservation synthesis and application of caged peptides and proteins physical and antimicrobial properties of grape seed extract, nisin, and edta incorporated soy protein edible films lipase-catalyzed synthesis of peptides containing d-amino acid: facts and artifacts recent patents on active packaging for food application active and intelligent packaging for milk and milk products membrane fluidity characteristics of human lung cancer heparan sulfate: anchor for viral intruders? immunomodulatory activity of small peptides covalent immobilization of an antimicrobial peptide on poly(ethylene) film responsive polymer films and capsules via layer-by-layer assembly nisin diffusion in protein films: effects of film type and temperature influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by lactococcus lactis antiviral activities of lactoferrin natural products with hypoglycemic, hypotensive, hypocholesterolemic, antiatherosclerotic and antithrombotic activities expression and purification of antimicrobial peptide buforin iib in escherichia coli lantibiotics: peptides of diverse structure and function mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of escherichia coli barrel-stave model or toroidal model? a case study on melittin pores antimicrobial peptides of multicellular organisms interaction of the antimicrobial peptide pheromone plantaricin a with model membranes: implications for a novel mechanism of action the authors would like to thank to nicholas j. walker for providing language help and writing assistance. financial support for this research was provided by coordenação de aperfeiçoamento de pessoal de nível superior (capes) and the conselho nacional de desenvolvimento científico e tecnológico (cnpq). key: cord- -v xue ha authors: xu, yongtao; yu, shui; zou, jian-wei; hu, guixiang; rahman, noorsaadah a. b. d.; othman, rozana binti; tao, xia; huang, meilan title: identification of peptide inhibitors of enveloped viruses using support vector machine date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: v xue ha the peptides derived from envelope proteins have been shown to inhibit the protein-protein interactions in the virus membrane fusion process and thus have a great potential to be developed into effective antiviral therapies. there are three types of envelope proteins each exhibiting distinct structure folds. although the exact fusion mechanism remains elusive, it was suggested that the three classes of viral fusion proteins share a similar mechanism of membrane fusion. the common mechanism of action makes it possible to correlate the properties of self-derived peptide inhibitors with their activities. here we developed a support vector machine model using sequence-based statistical scores of self-derived peptide inhibitors as input features to correlate with their activities. the model displayed % prediction accuracy with the matthew’s correlation coefficient of . , obviously superior to those using physicochemical properties and amino acid decomposition as input. the predictive support vector machine model for self- derived peptides of envelope proteins would be useful in development of antiviral peptide inhibitors targeting the virus fusion process. fusion process is the initial step of viral infection, therefore targeting the fusion process represents a promising strategy in design of antiviral therapy [ ] . the entry step involves fusion of the viral and the cellular receptor membranes, which is mediated by the viral envelope (e) proteins. there are three classes of envelope proteins [ ] : class i e proteins include influenza virus (ifv) hemagglutinin and retrovirus human immunodeficiency virus (hiv- ) gp ; class ii e proteins include a number of important human flavivirus pathogens such as dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), west nile virus (wnv), hepatitis c virus (hcv) and togaviridae virus such as alphavirus semliki forest virus (sfv); class iii e proteins include vesicular stomatitis virus (vsv), herpes simplex virus- (hsv- ) and human cytomegalovirus (hcmv). although the exact fusion mechanism remains elusive and the three classes of viral fusion proteins exhibit distinct structural folds, they may share a similar mechanism of membrane fusion [ ] . a peptide derived from a protein-protein interface would inhibit the formation of that interface by mimicking the interactions with its partner proteins, and therefore may serve as a promising lead in drug discovery [ ] . enfuvirtide (t ), a peptide that mimicks the hr region of class i hiv- gp , is the first fda-approved hiv- fusion drug that inhibits the entry process of virus infection [ ] [ ] [ ] . then peptides mimicking extended regions of the hiv- gp were also demonstrated as effective entry inhibitors [ , ] . furthermore, peptides derived from a distinct region of gb virus c e protein were found to interfere with the very early events of the hiv- replication cycle [ ] . other successful examples of class i peptide inhibitors include peptide inhibitors derived from sars-cov spike glycoprotein [ ] [ ] [ ] and from pichinde virus (picv) envelope protein [ ] . recently, a peptide derived from the fusion initiation region of the glycoprotein hemagglutinin (ha) in ifv, flufirvitide- (ff- ) has progressed into clinical trial [ ] . the success of developing the class i peptide inhibitors into clinical use has triggered the interests in the design of inhibitors of the class ii and class iii e proteins. e.g. several hydrophobic peptides derived from the class ii denv and wnv e proteins exhibited potent inhibitory activities [ ] [ ] [ ] [ ] [ ] . in addition, a potent peptide inhibitor derived from the domain iii of jev glycoprotein and a peptide inhibitor derived from the stem region of rift valley fever virus (rvfv) glycoprotein were reported [ , ] . examples of the class ii peptide inhibitors of enveloped virus also include those derived from hcv e protein [ , ] and from claudin- , a critical host factor in hcv entry [ ] . moreover, peptides derived from the class iii hsv- gb also exhibited antiviral activities [ ] [ ] [ ] [ ] [ ] [ ] , as well as those derived from hcmv gb [ ] . computational informatics plays an important role in predicting the activities of the peptides generated from combinatorial libraries. in silico methods such as data mining, generic algorithm and vector-like analysis were reported to predict the antimicrobial activities of peptides [ ] [ ] [ ] . in addition, quantitative structure-activity relationships (qsar) [ ] [ ] [ ] [ ] [ ] and artificial neural networks (ann) were applied to predict the activities of peptides [ , ] . recently, a support vector machine (svm) algorithm was employed to predict the antivirus activities using the physicochemical properties of general antiviral peptides [ ] . however, the mechanism of action of antiviral peptides is different from antimicrobial peptides; in fact, various protein targets are involved in the virus infection. e.g. hiv- virus infection involves virus fusion, integration, reverse transcription and maturation, etc. thus it is difficult to retrieve the common features from general antiviral peptides to represent their antiviral activities. virus fusion is mediated by e proteins. although e proteins are highly divergent in sequence and structure, they share a common pathway of membrane fusion dynamics. i.e. e proteins experience significant conformational change to form a-trimer-of-hairpin, which drives the fusion of viral membrane and host membrane [ ] . the antiviral peptides derived from enveloped proteins function by in situ binding to their respective accessory proteins, disrupting forming of the trimer-of-hairpin and membrane fusion, and therefore inhibiting the virus infection. in view of the important role of e proteins in virus fusion process and common mechanism of action of self-derived peptides, we developed a svm model to predict the antiviral activities of self-derived peptides using sequence-based statistical scores as input features. the sequencebased properties were calculated by a conditional probability discriminatory function which indicates the propensity of each amino acid for being active at a specific position. our model exhibited remarkably higher accuracy in predicting the activities of self-derived peptides, compared to the previous models developed for general antiviral peptides using classical physicochemical properties as descriptors [ ] . the method would be useful in identification of entry inhibitors as a new generation of antiviral therapies. peptide virus entry inhibitors of enveloped viruses were collected, among them, are active peptides and are non-active peptides. these peptides comprised the p+ n training set of svm models. the remaining active peptides and non-active peptides inhibitors were used as the test set. amino acid composition. amino acid composition is the fraction of each amino acid in a peptide. the fraction of the amino acids was calculated using the following equation: fraction of amino acid x ¼ total number of x = peptide length five physicochemical properties were used in svm models. isoelectric point (pi), molecular weight (mw) and grand average of hydropathicity (gravy) [ ] were calculated using the protparam tool implemented in expasy web server. solvent accessibility and secondary structure features were calculated using sspro and accpro packages implemented in the scratch protein predictor server [ ] . sequence-based statistical scoring function. the knowledge-based statistical function is developed from the concept of residue-specific all-atom probability discriminatory function (rapdf) [ ] . rapdf is a structure-based statistical scoring function. it is based on the assumption that averaging over different atom types in experimental conformations is an adequate representation of the random arrangements of these atom types in any compact conformation. here we developed a sequence-based statistical scoring function, where we presume that averaging over different amino acid sequences with experimental validated inhibitive activities is an adequate representation of the random amino acid sequences with any inhibitory activity. the basis of this assumption is that the peptides share a common mechanism of action, i.e. the peptides derived from e proteins bind competitively to their partner proteins, disrupt the forming of a-trimer-of-hairpin, and therefore inhibit the virus membrane fusion. the sequence-based scoring function is described in the following form: sðfq i a gÞ ¼ Àln here, q i a factiveg. pðq i a jcÞ is the probability of observing amino acid i in an active peptide sequence; pðq i a Þ is the probability of observing amino acid i in any peptide sequence, active or nonactive. they are approximately estimated using the following forms: similarly, we employed a dataset of experimentally verified non-active peptides in developing the statistical function, where q i a finactiveg. for a given amino acid sequence, columns of input are generated, corresponding to the occurrence of twenty natural amino acids at each position. each column is assigned a value of n à (−log-likelihood), where n is the number of amino acid and −log-likelihood is derived from the statistical function score. each of the features thus combines the propensity of the amino acid for being active or non-active with the corresponding amino acid composition. below is an example of calculating the statistical scores for a given peptide sequence: the amino acid order for svm input features is set as: acdefghiklmnpqrstvwy. if the amino acid sequence of an active peptide inhibitor is: svm models combined with radial basis function (rbf) kernel parameters were developed using the c-svc module in libsvm (version . ) [ , ] and executed under the matlab interface. the performance of svm depends on two parameters, gamma -g and cost-c [ ] . the default value is for -c and /k for -g, where k is the number of input entries. various pairs of (c, g) values were converted to exponential values (i.e. x ; y ) and optimized using cross-validation and the pair with the best cross-validation accuracy was selected. -fold cross validation was performed to evaluate the performance of svm models. in the evaluation process, dataset was partitioned randomly into five equally sized subsets. the training and testing were carried out five times, each time four distinct subsets being used as training sets and the remaining subset as test set. the results were averaged over all five rounds of validation. the following equations were used to evaluate the prediction quality of the svm models [ , ] : in the above equations, tp is the number of true positives, tn is the number of true negatives, fp is the number of false positives and fn is the number of false negatives. matthew's correlation coefficient (mcc) reflects the performance of the model. it ranges between - to and a larger mcc value indicates a better prediction. svm learning algorithm is a powerful machine learning method that has been widely used in pattern recognition and classification. svm trains a dataset of experimentally validated positive and negative samples and generates a classifier to classify unknown samples into two distinct categories (positive or negative). we performed an exhaustive literature search on self-derived peptide inhibitors of enveloped proteins and collected experimentally validated peptides derived from the three classes of e proteins. for those peptides with overlapping segments, only one peptide sequence was kept. peptides were found, among them, are active peptides and are non-active peptides ( table ) . active peptide inhibitors and non-active peptides ( p+ n) of e proteins were used as the training dataset in svm learning; the remaining active and non-active peptides ( p+ n) were used as the test set. svm input features. three svm models were developed using different features as input descriptors, namely physicochemical properties (denoted as eapphysico), amino acid composition (eapcompo) and statistical scoring function amino acid composition (eapscoring). knowledge-based statistical functions are rooted in the bayesian (conditional) probability formalism and derived directly from properties observed in the known folded proteins [ ] [ ] [ ] . in knowledge-based scoring function, it was presumed that averaging over different atom types in experimental conformations is an adequate representation of the random arrangements of these atom types in any compact conformation [ ] . because the three classes of e proteins have different structural folds, it is difficult to retrieve a structure-based feature that is relevant to their antiviral activities. generally speaking, any property associated with folded proteins can be converted into an energy function [ ] . since amino acid sequence determines the structural folds and properties of proteins/peptides, we presumed that a sequence-based statistical scoring function averaging over different amino acid sequences exhibiting inhibitive activities is an adequate representation of the random combinations of all twenty amino acid exhibiting any activity. in this approach, a peptide sequence derived from e protein is represented by twenty features each corresponding to the propensity of observing each of the twenty natural amino acids to be either active or non-active. a vector space of twenty sequence-based statistical scores was used as the eapscoring input entries in the svm learning. we also built a svm model using physicochemical properties as input features. because of the feature of membrane fusion process, it was suggested that functional regions in glycoproteins need to be solvent accessible, hydrophobic and flexible [ ] . actually the majority of known peptide entry inhibitors share a common physicochemical property of being hydrophobic and amphipathic with a propensity for binding to lipid membranes [ ] . therefore, here the properties of e peptide inhibitors were described by five physicochemical parameters: pi, mw, gravy index (positive and negative gravy values indicate hydrophobic and hydrophilic peptides, respectively), solvent accessibility (exposed or buried) and secondary structure features (propensity for adopting α-helix, β-sheet or turn structure). these physicochemical features were calculated for each of the peptides and used as the eapphysico input entries in the svm learning. a third svm model eapcompo was also built where the fractions of amino acids in a peptide were used as input features in the machine learning process. svm training. the svm models were trained using the experimentally validated p+ n data sets. during -fold cross validation, the training set was randomly partitioned into four subsets with equal size of ( p+ n) and a remaining subset ( p+ n). three svm models were built using sequence-based statistical scores, physicochemical properties and amino acid composition, respectively. the performances of the three models are shown in table . it can be seen that the eapscoring model performed best among the three models during -fold cross validation. a "grid-search" combined with cross-validation was adopted to search for the optimal parameters -c and -g in svm models [ ] . the result of the grid search is shown in the support information (s file). it is shown that the performances of three eap models during -fold cross validation have been improved significantly using the optimized parameters ( table ) . the performance of the svm models was evaluated using an independent dataset of experimentally validated peptides that were not contained in the learning dataset (table ). in the eapphysico model where physicochemical properties of peptides were used as input features, an accuracy of % with a mcc value of . was observed (table ). in the eapcompo model where amino acid composition features were used, the predictive accuracy and the mcc value are slightly higher. when the sequence-based statistical function scores were used as input in the eapscoring model, a remarkable accuracy of % was achieved with a mcc value of . . thus the sequence-based statistical scores developed in the present research are predominantly superior to the conventional physicochemical properties or amino acid decomposition features in identifying active peptides derived from enveloped proteins. avppred is a web server for prediction of the activities of general antiviral peptides (avps) based on a number of experimentally validated positive and negative data sets [ ] . the peptide inhibitors employed in avppred target a variety of biological targets involved in virus infection. in contrast, the self-derived peptides of enveloped proteins being studied in the present research competitively bind to e proteins so as to mediate the virus fusion process. because the self-derived peptides share similar mechanism of action, it is feasible to retrieve common features from them to build predictive svm models. in order to evaluate the performance in predicting peptide inhibitors of the enveloped virus, we compared the avppred models with our eappred models using an independent p+ n dataset as test set. the results are shown in table . four different features were employed in the avppred models, namely conserved motif search using meme/mast, amino acid composition, sequence alignment using blast and physicochemical parameters including secondary structure, charge, size, hydrophobicity and amphiphilic character [ ] . when the avpmotif model was used to predict the activities of the self-derived peptide inhibitors, it performed rather poorly with accuracy of % and mcc of . . this is not surprising because avpmotif was developed based on general antiviral peptide motifs. however, the self-derived peptide inhibitors may not share a conserved motif with the general antiviral peptides since the latter interact with various biological targets with different mechanisms of action. in the avpalign model, the peptide sequences were classified into active and non-active databases and the query peptide sequences were matched against the active and non-active databases using the blast program. compared with avpcompo and avpphysico, avpalign performed better with a predictive accuracy of % and mcc value of . . fusion mechanism is highly conserved among related viruses and entry of viruses into host cells has been inhibited by peptides derived from various regions of envelope glycoproteins [ ] . self-derived peptides would inhibit interactions of their original domain by mimicking its mode of binding to partner proteins [ ] . because similar sequences are often associated with similar structure and function, the sequence-based property avpalign would account for the activities of the self-derived peptide inhibitors which regulate the virus fusion by mimicking the binding to e proteins. in the avpphysico model, best performing physicochemical properties were selected out of the properties to build the svm model [ ] . antiviral peptide inhibitors are generally amphiphilic [ ] and the activities of peptide entry inhibitors are dependent on their interfacial hydrophobicity [ ] . therefore we only employed five physicochemical properties reflecting hydrophobicity, solvent accessibility and secondary structure features as svm input features. it was demonstrated that the accuracy and mcc of eapphysico is comparable to that of avpphysico model, indicating the five properties used in current modeling building are critical for their activities. the mcc value of the avpcompo models is . , indicating that the antiviral activities of the peptides are related to amino acid composition. when the amino acid composition was used as input, the predictive accuracy of the eapcompo model was higher than that of the avpcompo model, indicating the peptide inhibitors of e proteins employed in the training set is sufficient to represent the contribution of amino acid composition to their inhibitive activities. in the eapcompo model, the preference of the amino acid composition was ranked as: p, r, q, d, f, w, e, l, t, i, n, h, y, c, a, s, m, v, k, g (fig ) . the role of arginine-arginine pairing and its contribution to protein-protein interactions has been investigated by computational approaches [ ] . the higher abundance of r at protein-protein interfaces compared to k may be attributed to the formation of cation-π-interactions and the greater capacity of the guanidinium group in r to form hydrogen bonds (compared to k) [ ] [ ] [ ] . furthermore, it was suggested that the interface regions are enriched in aliphatic (l, v, i, m) and aromatic (h, f, y, w) residues and depleted in charged residues (d, e, k) with the exception of arginine [ , [ ] [ ] [ ] [ ] [ ] . this is in agreement with our amino acid composition analysis, where higher population of aliphatic leu residue as well as aromatic residues trp and phe was observed, whereas positively charged lys was hardly observed. the predominant occurrence of proline and glutamine residues is characteristic for the unique protein-protein interactions for e proteins. e.g. a conserved proline-rich motif was suggested to be engaged in monomer-monomer interactions in dengue e proteins [ ] . a conserved glutamine-rich layer is involved in the extensive hbond network in hiv- gp e proteins [ ] . thus the preference of the amino acid composition identified from the eapcompo model is generally in accordance with the predominant residues involved in protein-protein interactions, manifesting the amino acid composition of the self-derived peptide inhibitors are closely related to their potential activities in mediating the protein-protein interactions in the virus fusion process. because the antiviral activities of peptides are dependent on amino acid composition, we presume amino acid composition discriminated by the propensity of their activities would be an intrinsic feature in the self-derived peptide inhibitors which share a common mechanism of action. when statistical function scores were employed in the svm model (eapscoring), a remarkable predictive accuracy of % with an ideal mcc value of . was achieved, significantly better than any avp models. the logarithm form of the discriminatory function (eq ) can be deemed as the pseudo energy of the system. in our previous study, we suggested that the stability of proteins is related to their in situ binding potential to the partner regions [ ] . the prominent performance of eapscoring model indicates the sequence-based stability feature of self-derived peptides may reflect their potential of binding to e proteins so as to regulate the virus entry process. we developed three svm models using physicochemical properties, amino acid composition and statistical discriminative function as input features. the prediction accuracy and the mcc value of the eapphysico model where five physicochemical properties were employed are comparable with the previous avpphysico model where physicochemical properties were used. the avpcompo and eapcompo models demonstrated that the activities of antiviral peptides are dependent on amino acid composition. a sequence-based scoring function was developed for the self-derived peptide inhibitors of e proteins. the outperformance of the eapscoring models supports our hypothesis that an intrinsic feature, represented by the propensity of each amino acid for being active in self-derived peptides, is responsible for the activities of the peptides to regulate virus fusion by mimicking the binding to their accessory proteins. the sequence-based statistical scoring function would be useful in development of novel antiviral therapies to target the initial step of viral infection. supporting information s file. parameters optimization by grid-research combined with -fold cross validation. x-axis is log g , y is log c and z-axis represents accuracy(%) ( figure a targeting cell entry of enveloped viruses as an antiviral strategy. molecules class iii viral membrane fusion proteins virus membrane-fusion proteins: more than one way to make a hairpin can self-inhibitory peptides be derived from the interfaces of globular protein-protein interactions? characterization of a putative cellular receptor for hiv- transmembrane glycoprotein using synthetic peptides propensity for a leucine zipperlike domain of human immunodeficiency virus type gp to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex a synthetic peptide from hiv- gp is a potent inhibitor of virusmediated cell-cell fusion hiv gp c-terminal heptad repeat contains multifunctional domains. relation to mechanisms of action of anti-hiv peptides inhibition of human immunodeficiency virus type entry in cells expressing gp -derived peptides peptides derived from a distinct region of gb virus c glycoprotein e mediate strain-specific hiv- entry inhibition inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein synthetic peptides outside the spike protein heptad repeat regions as potent 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regions in antimicrobial proteins optimization of antibacterial peptides by genetic algorithms and cheminformatics antibp : improved version of antibacterial peptide prediction application of 'inductive' qsar descriptors forquantification of antibacterial activity of cationic polypeptides qsar analysis of antimicrobial and haemolytic effects of cyclic cationic antimicrobial peptides derived from protegrin- design of novispirin antimicrobial peptides by quantitative structure-activity relationship qsar modeling and computer-aided design of antimicrobial peptides identification of novel antibacterial peptides by chemoinformatics and machine learning de novo design of potent antimicrobial peptides evaluating different descriptors for model design of antimicrobial peptides with enhanced activity toward p. aeruginosa avppred: collection and prediction of highly effective antiviral peptides structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme a simple method for displaying the hydropathic character of a protein scratch: a protein structure and structural feature prediction server an all-atom distance-dependent conditional probability discriminatory function for protein structure prediction support-vector networks working set selection using second order information for training svm a practical guide to support vector classification. initial version comparison of the predicted and observed secondary structure of t phage lysozyme distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction a distance-dependent atomic knowledge-based potential for improved protein structure selection statistical potential for assessment and prediction of protein structures comparison of database potentials and molecular mechanics force fields scoring functions for de novo protein structure prediction revisited peptide inhibitors against herpes simplex virus 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depends on interhelical interactions computational identification of self-inhibitory peptides from envelope proteins the authors are grateful for the computing resources from qub high performance computing centre. the authors declare no conflict of interest. key: cord- - ovqhypt authors: iqbal, umar h.; zeng, emma; pasinetti, giulio m. title: the use of antimicrobial and antiviral drugs in alzheimer’s disease date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: ovqhypt the aggregation and accumulation of amyloid-β plaques and tau proteins in the brain have been central characteristics in the pathophysiology of alzheimer’s disease (ad), making them the focus of most of the research exploring potential therapeutics for this neurodegenerative disease. with success in interventions aimed at depleting amyloid-β peptides being limited at best, a greater understanding of the physiological role of amyloid-β peptides is needed. the development of amyloid-β plaques has been determined to occur – years prior to ad symptom manifestation, hence earlier interventions might be necessary to address presymptomatic ad. furthermore, recent studies have suggested that amyloid-β peptides may play a role in innate immunity as an antimicrobial peptide. these findings, coupled with the evidence of pathogens such as viruses and bacteria in ad brains, suggests that the buildup of amyloid-β plaques could be a response to the presence of viruses and bacteria. this has led to the foundation of the antimicrobial hypothesis for ad. the present review will highlight the current understanding of amyloid-β, and the role of bacteria and viruses in ad, and will also explore the therapeutic potential of antimicrobial and antiviral drugs in alzheimer’s disease. alzheimer's disease (ad) is a progressive neurological disorder that accounts for the greatest number of dementia cases. as of , . million people were living with ad, with its prevalence predicted to increase to . million by [ ] . the vast majority of cases are concentrated in ages over , impacting % of people in this age group. in addition, the economic toll of ad on the united states economy is significant, estimated to be roughly usd billion in [ ] . as the number of cases is only expected to rise over the coming decades, research in this field is critical in order to understand the pathology of this disease, as well as potential therapeutics. the understanding and characterization of ad can be traced back over years to alois alzheimer, from whom the disease takes its name. after completing an autopsy of a patient with progressive dementia, alzheimer noticed a severe amount of cortical degeneration and an accumulation of protein deposits, specifically extraneuronal plaques and intraneural tangles [ ] . by , the buildup of extraneuronal amyloid-β (aβ) plaques became the hallmark trait of the pathogenesis of alzheimer's disease [ ] , initiating the development of the amyloid cascade hypothesis [ ] . in parallel to aβ plaques formation, the accumulation of other naturally unfolded proteins is central to ad and other cerebral proteopathies [ ] . the intracellular aggregation of tau proteins in the form of neurofibrillary tangles (nfts) is also an essential trait in the pathogenesis of alzheimer's disease [ , ] . a recent study alzheimer's disease [ , ] . a recent study found that neuroinflammation could play a role in the aggregation of tau, as dna extracted from various bacterial species promoted tau misfolding [ ] . whereas aβ plaques are more critical to ad pathogenesis, the tau protein appears to be more responsible for subsequent cognitive impairment and dementia symptoms associated with ad [ ] . indeed, tau hyperphosphorylation and nft levels are closely correlated with cognition, and exhibit potential as therapeutic targets for ad treatment [ ] . furthermore, tau protein production has been shown to have a positive correlation with the production of aβ plaques [ ] , with the formation and lack of clearance of aβ plaques also being proposed to induce tau protein formation into nfts [ ] . coupling this with the bi-directional relationship between aβ plaques and neuroinflammation [ ] would cement aβ's key role in driving ad pathology. aβ formation begins with the breakdown of the amyloid precursor proteins (app) embedded in the membranes of cells, such as neurons, as a type transmembrane glycoprotein [ ] . aβ peptides are produced through a two-step cleavage process, in which app is metabolized into smaller fragments. in the first step, app is cleaved by β-secretase into a membrane-bound ctfβ fragment (containing amino acids) and an extracellular fragment sappβ. ctfβ is then further cleaved by γsecretase to create the final aβ peptide [ ] [ ] [ ] , as illustrated in figure . the length of aβ peptides is not fixed, and can consist of anywhere between and amino acids, depending on where the cleavage was done by β-secretase and γ-secretase [ , ] . the most abundant length is aβ - , representing approximately - % of aβ peptides, whereas the least soluble of the aβ peptides, aβ - , represents roughly - % [ , ] . as a greater number of aβ peptides form, they begin to aggregate into oligomers, which then form fibrils, and eventually the insoluble plaques characteristic of ad [ ] . of the different isoforms of aβ peptides, aβ - and aβ - are the most common in plaques. regarding the comparative role of aβ - and aβ - peptides in the pathogenesis of ad, aβ - peptides have been found in higher concentrations in ad. furthermore, aβ - peptides have been found to be more prone to forming insoluble amyloid fibrils than aβ - [ ] . this is further supported by a study using transgenic mice that expressed either aβ - (bri-aβ ) or aβ - (bri-aβ a). the authors found that the mice that selectively expressed aβ - did not develop ad pathology at any age. however, the same did not hold true for bri-aβ a mice, which had developed aβ deposits [ ] . in addition, another study found aβ - peptides to promote aβ plaques formation, and aβ - to decrease aβ deposition [ ] . these findings would indicate the key role aβ - peptides play in the pathogenesis of ad. even with the tremendous effort that has been put into developing potential therapies for ad over the past few decades, there has been little success in reaching an effective therapy, with no new drug being approved in over a decade. while cholinesterase inhibitors and memantine are fda-approved drugs for ad, and do address some of its symptoms, they lack the ability to attenuate disease progression. over the past years, a majority of the therapies have been based on the amyloid cascade hypothesis, and hence have focused on depleting aβ peptides. theses therapies often aim to inhibit γ-secretase or β-secretase activity in order to limit aβ peptide production. therapies that use such methods have, however, seen an increase in the rate of infection during clinical studies, with one study seeing % of participants develop meningoencephalitis [ , ] . tarenflurbil, for instance, had been administered clinically, after it was shown to modulate γ-secretase and increase production of the less toxic aβ - peptide, rather than aβ - peptide [ , ] . however, in addition to not showing any significant benefit in individuals with mild ad, participants in the treatment group experienced an increase in upper respiratory infections and dizziness compared to the placebo group [ ] . additionally, in the time of covid- infection, respiratory-related side effects, such as the ones related to tarenflurbil, are of growing concern. the γ-secretase inhibitor semagacestat has not only been associated with increased levels of infection, but also with a failure to provide any cognitive improvement in patients with probable ad [ ] . furthermore, when patients with mild to moderate ad were administered elnd , a compound that inhibits aβ fibrils and plaque formation, it was observed that higher doses of this treatment led to serious infection. this led to lower dosage recommendations for future trials [ , ] . lastly, the b-site abpp cleaving enzyme (bace )-inhibitor e has also been associated with oralabial herpes relapse [ , ] . a rise in infection occurring in tandem with the reduction of aβ peptide production could indicate these peptides' potential role in immune function. through these past clinical trials, it is evident that therapies largely based on the amyloid cascade hypothesis, which in turn aim to eradicate aβ peptides in the brain, have historically been ineffective. these failures could imply that current approaches either intervene at a stage that is too late, or possess a therapeutic target that is not as relevant to disease progression [ ] . this would make sense in the context of ad especially, as aβ deposition occurs - years before the occurrence of clinical symptoms [ ] . therefore, these treatments that target aβ peptides specifically may already be too late. to create a successful therapy, it may be necessary to consider intervention in the presymptomatic stage of the ad instead. to do so, it would be crucial to identify biomarkers for early identification of ad. in march of , a meeting was convened in which international, interdisciplinary experts identified a list of biomarkers that could be used for identifying ad early on [ ] . csf levels of aβ - and aβ , and the ratio of aβ - /aβ - , were determined to be among the candidates [ , ] . in addition, plasma levels of the same biomarkers were determined to decrease in ad patients when compared to healthy subjects [ ] . other biomarkers to consider would include plasma levels of tau protein and neurofilament light [ ] . plasma levels of the latter have been shown to be able to detect neurodegeneration in presymptomatic ad [ ] . other highly sensitive methods that have shown promise in the early identification of ad include protein misfolding cyclic amplification (pmca) and real-time quaking-induced conversion (rt-quic), to determine aβ oligomers and tau protein levels in csf [ , ] . taking a more preventative approach to ad treatment, and understanding and addressing factors that contribute to the progression of ad, may be favorable for identifying therapeutic targets earlier in the disease. based on evidence of the association between bacterial/viral infection and ad progression, the antimicrobial hypothesis suggests that aβ peptides may be produced as a protective mechanism by the innate immune system, and act as an antimicrobial peptide (amp) against foreign agents. if aβ peptides do in fact play a beneficial role in immunity, then the aim of treatment should not be to eradicate the compound entirely. rather, it should be to target the root cause of its over production, and reduce its deleterious effects and general neuroinflammation in ad. neuroinflammation is inflammation within the brain or spinal cord due to infection, toxins or injury [ ] . in the brain specifically, resident glial cells, such as microglia and astrocytes, along with endothelial cells and mast cells, all aid in defending the brain against foreign pathogens [ ] . microglia, the main immune effector cells of the central nervous system (cns), are constantly surveying their environment for potential threats to the brain [ , ] . when an invading agent is detected, microglia change into an activated state, characterized by an enlarged soma and the production of inflammatory cytokines and chemokines [ ] . astrocytes also play a critical role in mediating neuroinflammation as they are responsible for many neuroprotective functions, such as maintaining blood brain barrier (bbb) integrity and buffering neurotransmitters [ ] . upon injury, astrocytes likewise undergo morphological changes, and exhibit increased reactivity and secretion of cytokines and chemokines [ ] . while acute inflammatory responses are common to healthy individuals, chronic inflammation is damaging to the natural balance of pro-and anti-inflammatory signaling in the brain, and can lead to the development and progression of neurodegenerative diseases like ad [ ] . over the years, there has been increasing evidence of neuroinflammation's role in ad. in addition to aβ plaques and nfts, markers of sustained inflammation and microglial activation have repeatedly been found in ad brain samples [ ] . the cytokines interleukin and interleukin are especially elevated [ ] . one source of the neuroinflammation in ad patients could be the response to invading microorganisms and viruses. in fact, researchers have found evidence pointing to the presence of pathogens, such as viruses, bacteria and fungi, in ad brains [ ] [ ] [ ] [ ] [ ] [ ] . this notion draws parallels with the measles virus, which can lead to the development of the neurological disease known as subacute sclerosing panencephalitis [ ] . these findings include the identification of viral [ ] and bacterial dna in post-mortem brain samples, and the detection of pathogens [ ] and/or their respective antibodies [ ] in the serum or cerebrospinal fluid of patients. furthermore, detection of lipopolysaccharide is commonly used by researchers to measure the presence of gram-negative bacteria, like p. gingivalis specifically, as it is found in their cell walls and can stimulate an inflammatory response in the immune cells [ ] . herpes simplex virus- (hsv- ) [ , [ ] [ ] [ ] [ ] was the first pathogen found to be present in ad brain samples [ ] , and it thereafter became the most widely-researched pathogen regarding the linkage between viral infection and ad. since then, other viruses have been identified in leading to the progression of ad, including human cytomegalovirus [ ] and epstein barr virus [ ] . a recent study found that, in addition to hsv- , herpesvirus types hhv- [ , ] and hhv- were highly present in ad patients [ ] . in this study, by readhead et al., hhv- and hhv- were also observed to be involved in regulatory processes critical to characteristic features of the disease [ ] . bacterial infection has likewise been associated with the progression of ad. the presence of bacteria in the brain has been determined in previous studies, suggesting the presence of a brain microbiome [ ] [ ] [ ] . even though bacterial presence has been seen in the brains of healthy individuals, tissue samples from ad brains have greater levels of bacterial species [ ] , indicating a greater level of infiltration. chlamydia pneumoniae is the most widely-studied bacteria regarding association to ad [ , ] . a clinical investigation, made up of a healthy control group and an ad group, detected c. pneumoniae in % of the ad patients, whereas the control group were all negative [ ] . escherichia coli, likewise identified in ad brains [ ] , has been found to be capable of synthesizing extracellular amyloid [ ] . stains such as borrelia burgdorferi [ ] , spirochetes [ ] , and porphyromonas gingivalis, a pathogen commonly linked to chronic periodontitis, have also been identified in ad brain samples [ ] . interestingly, fungal infection from species primarily associated with periodontal disease has recently been suggested to be involved in ad progression. researchers in a study detected multiple fungal species in ad brain samples, including saccharomyces cerevisiae, malassezia globosa, malassezia restricta, penicillium and phoma [ ] . pisa et al. have since followed up on this initial discovery by analyzing the presence of these species between brain regions [ ] . with any of these studies that have been conducted, however, it is important to recognize the technical limitations that arise when studying microorganisms and neurodegenerative disease. many of these studies are limited to the use of post-mortem brain samples, and thus present the risk of contamination due to death or the passage of microbes from other areas of the body, such as the gut to the brain, due to the lack of a functioning bbb to prevent this leakage. depending on the organism, there are several ways that pathogens can infiltrate the cns and potentially further the progression of ad. the first is through a compromised bbb. whereas a healthy and functional bbb normally provides a selective barrier to the passage of cells and molecules into the brain, a compromised bbb can allow direct entry into the cerebral spinal fluid via the bloodstream [ ] . this places aging populations and those with weakened immune responses especially at risk, as some viruses, such as herpesvirus, can remain latent after initial infection and then reactivate in aging individuals long after, to introduce delayed adverse complications [ ] . even with a healthy bbb, however, bacteria and viruses are still able to be introduced into the brain through various mechanisms. hiv, for example, is carried from the immune system to the brain by infected leukocytes that are able to cross the bbb. p. gingivalis and other oral spirochetes have also been suggested to be capable of invading the cns via the oral cavity, through the trigeminal nerves and ganglia [ ] . additionally, pathogens such as bacteria and viruses can bypass the bbb altogether by entering through the olfactory system, as the nasal cavity connects the peripheral environment to brain regions such as the olfactory bulb [ ] , the entorhinal cortex and the hippocampus, which traditionally receive smell sensory signals. c. pneumoniae, a respiratory pathogen, has specifically been suggested to enter the brain through the olfactory system, with its presence detected in the entorhinal cortex and hippocampal formation of ad patients [ ] . a recent study model demonstrated that exposure to c. pneumoniae via the olfactory system was sufficient to induce aβ plaque and nft formation in the olfactory cortex and hippocampus of immunocompromised individuals [ ] . this is further evidenced by little et al., who found that intranasal inoculation of c. pneumoniae was sufficient to induce ad-like traits in mice [ ] . once in the brain, there are several ways in which these pathogens contribute to the aβ production that is characteristic to ad. one mechanism is through the alteration of gene expression. the study previously mentioned by readhead et al. found that hhv- and hhv- interact with known regulatory genes responsible for amyloid processing, such as the amyloid beta a precursor protein-binding family (apbb ), clusterin (clu), and gamma-secretase subunit presenilin- (psen ) [ ] . similarly, infection of c. pneumoniae within human neuronal cell cultures possibly alters calcium-related gene expression such that they express patterns similar to those reported in ad brain samples [ ] . another way viruses can influence aβ production is through protein misfolding. specifically, viruses such as hhv, cytomegalovirus and epstein barr virus have been shown to contain prion-like domains that may trigger the misfolding of proteins like aβ [ ] . through these varying mechanisms, many microorganisms and viruses have been found to initiate aβ plaque formation. in-vivo studies have noted a correlation between viral and bacterial infections and the accumulation of aβ peptides. mice infected with hsv- [ ] , pseudorabies virus [ ] , c. pneumoniae [ ] and p. gingivalis [ , ] were found to have a significantly increased level of aβ - in the brain. in addition, aβ expression was found to be upregulated in rats exposed to bacterial pathogens. hsv- has also been found to infect the hippocampus region at a greater rate; the same area found to have greater amounts of aβ plaques in ad [ ] . in vitro studies observed cells co-cultured with either hsv- , hsv- , p. gingivalis or b. burgdoferi to have increased intracellular concentrations of aβ [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, hsv- has been associated with the inhibition of the non-amyloidogenic pathway of app metabolism, and the increased expression of β-secretase. this is evidenced by a study indicating the direct and frequent interaction between hsv- and aβpp [ ] . aβ plaques have also been identified in the brains of hiv- -infected individuals. autopsies performed on hiv positive individuals found roughly half of them to contain aβ plaques [ ] . cell culture studies observed an increase in aβ production and secretion following exposure to mrna and proteins from the hiv nef gene [ ] . it must be noted, however, that the association between infection and aβ plaque formation is not consistent across all populations. for example, in the study previously mentioned by sundar et al., younger and healthier individuals exposed to c. pneumoniae did not exhibit the same ab peptide and nft formation as older and immunocompromised subjects [ ] . moreover, it has been observed that genetic discrepancies, especially in the apoe gene, influence one's susceptibility to hsv- infection and subsequent ad development. specifically, the apoeε allele places individuals at a greater risk of developing hsv- -associated ad, with a combination of apoeε and hsv- comprising % of all ad cases [ , ] . this finding has been recapitulated in animal studies, where mice with the apoeε allele display a greater viral load than mice with other allele types after hsv- infection [ ] . it is clear that a relation exists between bacterial and viral infections and aβ production rate, as described in the previous section. aβ peptides have long been thought to lack any physiological function; however, this notion has been challenged in recent years. clinical studies have observed the depletion of aβ peptides, through anti-aβ therapies, to increase the rate of infections in some participants. furthermore, aβ plaques have been found to contain microbial and viral dna, such as hsv- . one study identified hsv- virus dna in roughly % of aβ plaques [ ] . in addition, ad brains have been associated with -to -fold increases in bacterial read compared to control brains [ ] . in the presence of bacterial lipopolysaccharides, microglial cells have also been shown to upregulate aβ production [ ] . from these and similar findings, it has been suggested that the pathogenesis of ad could be triggered by viral and/or microbial infections. these observations led to the recent development of the antimicrobial protection hypothesis for ad, which explores the notion of aβ peptides having a role in innate immunity as an amp that aids in the entrapment and degradation of invading bacteria and viruses. the innate immune system utilizes amps to target invading microorganisms, such as bacteria, viruses, fungi, and in some instances cancerous cells. mammalian amps exist in three main families: defensins, histatins and cathelicidins [ ] . similar to how aβ peptides are generated through the two-step cleavage of app, amps are also formed from the breakdown of larger precursor proteins. examples of amyloid amps that have a role in immunity are present in the human body. amyloidogenic major basic protein- (mbp- ) is implemented in eosinophils against pathogens [ ] . like aβ peptides, mbp- also forms aggregates, specifically at the surface of the bacteria to limit its spread. further support of aβ peptides being amp stems from their similarity to amp ll- , the only cathelicidin identified thus far in humans. both compounds exhibit tendencies to form cytotoxic soluble oligomers and insoluble fibrils, characteristic features of tinctorial amyloid [ ] . additionally, deficiency in the latter can result in kostmann syndrome, an immunodeficiency disorder that, if left untreated, can result in death due to infection within the first year of life [ ] . high levels of ll- are likewise dangerous, as they has been associated with the development of plaques in atherosclerosis and other non-infectious diseases [ ] . protein analyses comparing known amps and aβ peptides demonstrate structural similarities between these peptides, as well sequential similarities pointing to a shared homology between aβ peptides and a specific family of bacteriocins [ ] . this is particularly notable as bacteriocins are traditionally synthesized by bacteria as part of an antimicrobial response to contact with closely-related strains [ ] . if verified as an amp, aβ would not be the only amp suggested to be involved in ad. often expressed in epithelial cells, β-defensin- is significantly elevated in astrocytes of the hippocampus, the choroid plexus, and the granulovacuolar degeneration structures of ad brain samples [ ] . in vitro studies suggest the ability of aβ peptides to be an amp, and inhibit growth of a number of bacteria and viruses. in respect to the latter, aβ has been shown to have antiviral activity against both hsv- and the influenza virus a by inhibiting the infectivity of hsv- [ ] , influenza virus a [ ] , h n [ ] and h n [ ] . researchers found that in mouse and human neural cell cultures, aβ peptide deposition was accelerated in response to hsv- and hhv infection, with the oligomers binding to the viruses as part of a protective entrapment mechanism [ ] . aβ peptides have also been shown to have antimicrobial properties against both gram-positive and gram-negative bacteria, including enterococcus faecalis, escherichia coli, listeria monocytogenes, salmonella typhimurium, staphylococcus aureus, staphylococcus epidermidis, streptococcus agalactiae and streptococcus pneumoniae [ ] . in a study comparing aβ peptides and ll- , soscia et al. found that, against eight different bacteria and viruses, aβ peptides demonstrated an antimicrobial activity equivalent to, and sometimes even greater than, the known amp ll- . the same study also found that, when comparing the brain homogenates of aβ-enriched areas from both ad and non-ad brains, the ad brain samples had elevated antimicrobial and antiviral activity. these discrepancies were eliminated once the ad brain tissues were immunodepleted using anti-aβ antibodies. it is important to consider the sequence length of the aβ peptide when examining its antimicrobial capabilities, as aβ - was shown to be capable of binding to the surface of bacteria and aggregating into clusters, whereas other peptide lengths were not [ ] . animal studies have further evidenced the important potential role that aβ peptides have in protecting against infections. in a study, researchers tested aβ's functionality as an amp in mice and nematode models [ ] . kumar et al. found that transgenic xfad mice, which constitutively express human aβ peptides, survived significantly longer than wild-type mice after the injection of salmonella typhimurium into their brains. the xfad mice were observed to have accelerated aβ deposition that closely co-localized with the bacteria, reducing their cerebral viral load compared to wild-type mice. these findings were recapitulated again in worm models, as nematodes expressing aβ were found to have increased survival following fungal infection of c. albicans, compared to nematodes that did not produce aβ peptides. additionally, it has been observed that impaired mice that lacked the ability to generate aβ peptides have been shown to have increased postpartum mortality, which was only reversed by maintaining a sterile environment [ ] . furthermore, app knockout mice were also observed to have increased rates of mortality. altogether, these studies support the notion that aβ peptides are an amp, and an integral part of the brain's innate immune response against invading pathogens. the mechanism by which aβ peptides have been suggested to exert their antimicrobial and antiviral effect has been based on entrapment and lytic activity, as illustrated in figure . being a self-complimentary peptide containing two distinct hydrophobic and hydrophilic surfaces, aβ peptides can self-assemble into oligomers. as oligomerization continues, a fibril network is created which targets, captures and agglutinates microbes, limiting their proliferation and impact on their environment. aβ peptide affinity towards microbes has been suggested to be due to its positive charge and the microbe's negatively charged membrane [ , , ] . furthermore, the ability of aβ peptides to agglutinate microbes stems from its heparin-binding activity, which is able to target carbohydrates present on the surface of microbes. once entrapped, it is suggested that aβ peptides induce cell membrane disruption by forming cation channels. these channels cause ion dyshomeostasis and subsequent cell death. this mechanism is similar to the activity observed in amps such as ll- , which assert protection through microbial agglutination and entrapment. the oligomerization activity observed with aβ peptides is a common trait of amps, which mediates their ability to entrap and lyse pathogens while maintaining their resistance to protease activity. the entrapment of microbes and viruses can also enhance their uptake by neutrophils and macrophages. in respect to hsv- , aβ peptides have been proposed to interfere with its ability to fuse with the plasma membrane of cells, hindering its infective ability [ ] . int. j. mol. sci. , , x for peer review of in aligning with the antimicrobial hypothesis of ad, the use of antimicrobial and antiviral therapeutics could prove to be effective in targeting the root cause of ad. it should be noted, however, that the chronic over-production of aβ peptides, which form numerous insoluble plaques, would also need to be addressed. as such, the primary aim of these drugs would be to target bacteria and viruses, but a secondary aim would be to reduce the already-present aβ burden that the brain is under. acyclovir is an antiviral drug that is used for hsv- infections, which has been found to be well tolerated and safe [ ] . by inhibiting the virus's replication, this antiviral agent is able to reduce the viral load exhibited by hsv- . administration of acyclovir in hsv- -infected cells has been found to cause a significant reduction of hsv- proteins, along with a reduction of roughly % in aβ accumulation, compared to untreated cells. the study associated this with reduced levels of βsecretase and a component of γ-secretase which metabolizes app into aβ [ ] . acyclovir administration has also shown to prevent hsv- -related neuronal death [ ] . in relation to the cognitive impairment observed in ad, a study by hui et al. investigated the effects of acyclovir on aβ oligomer-induced spatial cognitive impairments. the study found that the co-administration of in aligning with the antimicrobial hypothesis of ad, the use of antimicrobial and antiviral therapeutics could prove to be effective in targeting the root cause of ad. it should be noted, however, that the chronic over-production of aβ peptides, which form numerous insoluble plaques, would also need to be addressed. as such, the primary aim of these drugs would be to target bacteria and viruses, but a secondary aim would be to reduce the already-present aβ burden that the brain is under. acyclovir is an antiviral drug that is used for hsv- infections, which has been found to be well tolerated and safe [ ] . by inhibiting the virus's replication, this antiviral agent is able to reduce the viral load exhibited by hsv- . administration of acyclovir in hsv- -infected cells has been found to cause a significant reduction of hsv- proteins, along with a reduction of roughly % in aβ accumulation, compared to untreated cells. the study associated this with reduced levels of β-secretase and a component of γ-secretase which metabolizes app into aβ [ ] . acyclovir administration has also shown to prevent hsv- -related neuronal death [ ] . in relation to the cognitive impairment observed in ad, a study by hui et al. investigated the effects of acyclovir on aβ oligomer-induced spatial cognitive impairments. the study found that the co-administration of acyclovir with dexamethasone attenuated impairments in spatial cognition. furthermore, this combination reduced the levels of neuroinflammation markers such as tnf-α and il- , along with microglia activation. interestingly, the study found these effects to only occur when acyclovir and dexamethasone were administered together [ ] . penciclovir is another antiviral drug that targets hsv- dna replication by blocking chain elongation. cell cultures infected with hsv- displayed a reduction of virus and aβ accumulation when penciclovir was administered. this was paralleled with a reduction in β-secretase and a component of γ-secretase [ ] . foscarnet has been tested for its ability to reduce hsv- levels in vitro. a study found it was able to reduce aβ accumulation, although only at higher doses. it was also unable to significantly reduce virus levels. furthermore, foscarnet was not as effective as acyclovir or penciclovir, and hence currently is not seen as the optimal antiviral drug available for ad [ ] . valacyclovir, an antiviral medication used in hsv- and hsv- infections, has been determined to positively impact cognition by improving visual object learning, verbal memory and working memory in patients with schizophrenia [ ] . due to its effects on working memory, its effectiveness against hsv- and hsv- , and its generally safe consumption, valacyclovir has been suggested as a potential therapeutic for ad. a clinical study is currently underway in which patients that both have mild ad and tested positive for hsv- or hsv- will receive valacyclovir. the aim of the study is to determine the impact of this treatment on cognition and the accumulation of amyloid and tau [ ] . numerous studies have determined the antiviral agent bay - to be effective in combating hsv- [ ] [ ] [ ] [ ] [ ] . by targeting the helicase-primase complex, bay - can inhibit viral dna replication, and has been found to be more potent than acyclovir. the severity and frequency of recurring hsv was also found to be reduced by use of this drug [ ] . furthermore, it was able to decrease levels of aβ and reduce p-tau production in vero cells infected with hsv- [ ] . our lab has investigated the use of bioflavonoids, including ginkgetin, isoginkgetin and ginkgolic acid, derived from the leaves of ginkgo biloba. the antiviral capabilities of these compounds has been well established in previous studies [ ] [ ] [ ] [ ] [ ] . hayashi et al. determined ginkgetin to successfully inhibit the viral replication of hsv- , hsv- and the human cytomegalovirus, while also suppressing viral protein synthesis [ ] . additionally, a study by miki et al. found ginkgetin to have anti-influenza virus activity [ ] . ginkgetin has been studied for use in ad by zeng et al., who administered the drug to app/ps transgenic mice. they observed a significant reduction in aβ plaques and an improvement in inflammation [ ] . borenstein et al. have demonstrated the ability of ginkgolic acid to limit virus infectivity by inhibiting its fusion. the study found ginkgolic acid to be successful in inhibiting hsv- , human cytomegalovirus and zika virus. furthermore, it was effective in inhibiting viral protein synthesis and genome replication, in hsv- and human cytomegalovirus, respectively [ ] . ginkgolic acid has also demonstrated antimicrobial properties, specifically against e. coli and staphlylococcus aureus [ ] . isoginkgetin has been shown to provide neuroprotection against the cytotoxic effects of excessive aβ accumulation [ , ] , while also having anti-microbial and anti-fungal activity [ ] . our lab's preliminary work in testing these three compounds in ad determined their effectiveness in reducing aβ load in vitro, further supporting their therapeutic potential in ad. doxycycline is a tetracycline antibiotic that has been studied for its therapeutic efficacy in ad models. contrary to other tetracyclines, doxycycline has been determined to be safe and is able to penetrate the bbb [ ] , allowing it to exert its effect directly in the cns. in vivo models, in which doxycycline was administered to mice, observed its accumulation in amyloid deposits, including aβ plaques [ ] . with respect to the production and formation of aβ oligomers, it was observed that although doxycycline administration in transgenic mice did not cause a shift in aβ monomers, there was a significant reduction in aβ -mer levels when compared to control [ ] . the same study also observed a significant memory recovery in animals that received treatment; however, there was no reduction in aβ plaque size [ ] . the paper suggested this was possibly due to the short two-month period of the study, as a previous three-month study found plaque size to be significantly reduced [ ] . in respect to neuroinflammation, a reduction in microglia activation has also been associated with doxycycline administration [ ] . a drosophila model, which administered doxycycline to aβ - -expressing flies, observed that the treated group's locomotor deficits developed slower than the control group. the same study also observed doxycycline administration to be associated with reduced aβ fibrilization, suggesting the production of smaller amyloid structures [ ] . another study associated doxycycline with the destabilization of aβ fibrils [ ] . clinical trials, however, were not as successful. one study, which administered doxycycline and rifampicin, observed improvements in cognitive function, as assessed by the standardized alzheimer's disease assessment scale-cognitive subscale (sadascog) score [ ] . however, a second study did not find any improvements in the cognition or function of patients with mild to moderate ad with doxycycline/rifampicin administration [ ] . further investigations would be needed to understand why the benefits seen in murine models do not translate into clinical trials. propranolol hydrochloride, an antihypertensive drug shown to have antimicrobial properties [ ] , has also been found to impact aβ production. cortico-hippocampal neuronal cultures treated with this drug manifested reduced levels of aβ production. furthermore, the one-month treatment of tg mice resulted in roughly a % reduction of aβ - and aβ - levels in the brain. when administered over a period of months, aβ peptide levels were still reduced in the brain; however, no improvement in spatial memory function was observed [ ] . rifampicin is an antibiotic derived from nocardia mediterranei, which has been investigated for use in neurodegenerative diseases such as parkinson's and ad [ , ] . rifampicin has been found to provide neuroprotection through its anti-oxidant and anti-inflammatory properties [ , ] . furthermore, in vitro studies found that its administration improved neuronal survival and reduced microglial activation [ ] . studies by tomiyama et al. found rifampicin to protect neurons from cytotoxicity by scavenging free radicals [ , ] . in relation to the antimicrobial hypothesis, rifampicin has been previously studied for use in bacterial cerebral infections [ ] . as rifampicin is able to cross the bbb [ ] , it can exert its antimicrobial effect directly in the brain. in the presence of rifampicin, a reduction of aβ fibril formation [ ] has been observed in addition to augmented aβ clearance [ ] . a study by umeda et al., in which rifampicin was administered to apposk mice, found the treatment to reduce aβ accumulation, provide synaptic protection, and reduce microglial activation [ ] . clinical studies exploring the impact of rifampicin on cognitive function have also been investigated, as mentioned in previous sections. even with its many benefits, the oral intake of rifampicin has also been associated with liver injury in humans. to circumvent this limitation, administering rifampicin intranasally or subcutaneously has been suggested [ ] . these routes of rifampicin administration have been shown to be more effective in improving memory than oral administration [ ] . the use of gingipain inhibitors in ad is another approach that has been taken to alleviate the negative impact of the disease. gingipains are virulence factors that are produced by p. gingivalis [ ] . they are made up of a group of cysteine proteinases, specifically arginin-gingipain a, arginine-gingipain b, and lysign-gingipain [ , ] . given the key role gingipains play in host colonization [ ] and the inactivation of host defenses [ ] [ ] [ ] , they are essential for the survival and pathogenicity of p. gingivalis. regarding aβ - peptide production, p. gingivalis infection was found to increase aβ - levels. furthermore, incubating p. gingivalis with aβ - peptides led to a significant increase in p. gingivalis death. these two findings further support the antimicrobial hypothesis for aβ peptides [ ] . gingipain inhibitors, such as cor , cor and cor , have been found to be effective in inducing p. gingivalis death and reducing the bacterial load in the brain, more so than other antibiotics, such as moxifloxacin [ , ] . in addition, cor was found to provide some level of neuroprotection as well [ ] . the administration of gingipain inhibitors has also been associated with a decrease in host aβ - response to p. gingivalis infection [ ] . even with the benefits associated with the antimicrobial and antiviral drugs listed above, insights into their mechanism of action and their impact on aβ peptide levels are needed. a greater understanding as to whether their administration indirectly reduces the presence of aβ peptides by reducing the viral/bacterial load on the brain, or if they act directly in reducing aβ peptides level, is needed. if it is the latter, and the antimicrobial hypothesis for aβ peptides holds true, their efficacy might not be as positive as hoped. in addition, it is important that the chosen antimicrobial or antiviral drug does not have any adverse effects that could take away from its benefits. for example, cefepime is an antibiotic that has shown to be able to cross the blood-brain barrier and cause neurotoxic symptoms [ ] . the findings of the numerous studies highlighted in this review present a clear indication of the role bacteria and viruses can have in ad development. even with this conclusion, it is clear that a specific bacteria or virus alone is not responsible for ad development, as no specific bacteria or virus has been identified to be universally present in all ad brains. rather, a number of viruses and bacteria could exacerbate the progression of neurodegenerative diseases, either independently or along with other pathogens. by exploring the presence of multiple viruses/bacteria in ad brains, future investigations can give insights into which microorganisms are most present, and whether all ad brains have both a detected and increased level of selected bacteria/virus. the use of antiviral and antimicrobial drugs early on, while the patient is still in the presymptomatic phase of ad, could have potential effectiveness in targeting the root cause of ad pathogenesis and alleviating the viral/microbial load on the brain. further investigations into their use in ad would give greater insight regarding their efficacy and limitations. funding: this study was supported by the altschul foundation and in part by grant number p at - from the nccih and ods. we acknowledge that the contents of this review do not represent the views of the nccih, the ods, the nih, or the united states government. the authors declare no conflict of interest. alzheimer's disease aβ amyloid-β amp antimicrobial peptide app amyloid precursor proteins bace b-site abpp cleaving enzyme bbb blood brain barrier cns central nervous system hsv- herpes simplex virus- mbp- major basic protein- nfts neurofibrillary tangles alzheimer's disease facts and figures alzheimer's disease amyloid deposition as the central event in the aetiology of alzheimer's disease alzheimer's disease: the amyloid cascade hypothesis new α-and γ-synuclein immunopathological lesions in human brain neuronal loss correlates with but 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protective mechanism against alzheimer's disease. j. alzheimer's dis rifampicin is a candidate preventive medicine against amyloid-β and tau oligomers intranasal rifampicin for alzheimer's disease prevention gingipains from porphyromonas gingivalis-complex domain structures confer diverse functions molecular genetics and nomenclature of proteases of porphyromonas gingivalis dichotomy of gingipains action as virulence factors: from cleaving substrates with the precision of a surgeon's knife to a meat chopper-like brutal degradation of proteins role of bacterial proteinases in matrix destruction and modulation of host responses cleavage of the human c a receptor by proteinases derived from porphyromonas gingivalis: cleavage of leukocyte c a receptor treatment of porphyromonas gulae infection and downstream pathology in the aged dog by lysine-gingipain inhibitor cor cefepime-induced neurotoxicity: a systematic review key: cord- -e pb mj authors: zunszain, patricia a.; knox, stephen r.; sweeney, trevor r.; yang, jingjie; roqué-rosell, núria; belsham, graham j.; leatherbarrow, robin j.; curry, stephen title: insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus c protease complexed with a peptide substrate date: - - journal: journal of molecular biology doi: . /j.jmb. . . sha: doc_id: cord_uid: e pb mj abstract picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by c protease(s) ( cpro) at multiple specific sites with related but non-identical sequences. to investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus cpro with peptide substrates. the x-ray crystal structure of the foot-and-mouth disease virus cpro, mutated to replace the catalytic cys by ala and bound to a peptide (apakq|llnfd) corresponding to the p –p ′ region of the vp - a cleavage junction in the viral polyprotein, was determined up to . Å resolution. comparison with free enzyme reveals significant conformational changes in cpro on substrate binding that lead to the formation of an extended interface of contact primarily involving the p –p ′ positions of the peptide. strikingly, the deep s ′ specificity pocket needed to accommodate p ′-leu only forms when the peptide binds. substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the p –p ′ region of synthetic substrates. the structure of the enzyme–peptide complex explains the marked substrate preferences for particular p , p and p residue types, as well as the relative promiscuity at p and on the p′ side of the scissile bond. furthermore, crystallographic analysis of the complex with a modified vp - a peptide (apake|llnfd) containing a gln-to-glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus cpro to cleave sequences containing either p -gln or p -glu. structure-based mutagenesis was used to probe interactions within the s ′ specificity pocket and to provide direct evidence of the important contribution made by asp of the cys-his-asp catalytic triad to proteolytic activity. our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by cpro. foot-and-mouth disease is a serious widespread viral disease of cloven-hoofed animals, including important agricultural species such as cattle, sheep, pigs and goats. , the virus spreads rapidly, and although endemic and epidemic situations can be controlled using vaccines that are based on inactivated virus particles, political and technical difficulties with the maintenance and use of vaccine stocks have stimulated the search for alternative means of tackling the disease, such as anti-viral drugs. the development of such treatments will demand a detailed knowledge of the molecular basis of viral replication. in this article, we focus on the structural basis of the cleavage activity of foot-and-mouth disease virus (fmdv) c protease(s) ( c pro ). as a highly conserved viral enzyme, fmdv c pro is a potential drug target. fmdv is a single-stranded positive-sense rna virus belonging to the picornavirus family that also includes human pathogens such as poliovirus (pv), human rhinovirus (hrv) and hepatitis a virus (hav). picornaviruses share a common replication strategy in which the viral rna genome is translated in the cytoplasm of infected cells as a long polyprotein precursor that is cleaved by virally encoded proteases to release functional proteins needed for the synthesis of new virions. in all cases, c pro performs the majority of these cleavages ( out of for fmdv) by targeting specific sequences within the polyprotein. , , structural studies on picornaviral c pro revealed an overall fold that closely resembles the architecture of trypsin-like serine proteases. [ ] [ ] [ ] [ ] [ ] the peptidebinding cleft, which contains the active site at its centre, is located at the interface between two βbarrels. unusually, picornaviral c pro possess a cys-his-asp/glu catalytic triad at the centre of this cleft, instead of the ser-his-asp arrangement of active-site residues that is commonly found in serine proteases. [ ] [ ] [ ] most investigations of the cleavage specificity of picornaviral c pro have relied on extensive sequence analyses of cleavage junctions within the polyprotein , or on in vitro cleavage assays using protein or peptide substrates. , [ ] [ ] [ ] [ ] [ ] there is a surprising degree of variability in the sequences of polyprotein junctions that are cleaved by picornaviral c pro , but these studies have nevertheless been valuable in identifying residues in the substrate that are important determinants of cleavage. most picornavirus c pro cleave the peptide bond between a highly conserved p -gln/p ′-gly pair , (where p and p ′ denote the first amino acids on the nterminal and c-terminal sides of the scissile bond, respectively; in this notation, the corresponding subsites on the enzyme are labelled s and s ′). fmdv c pro is an interesting exception to this general pattern, since it exhibits a preference either for substrates in which p -gln is followed by a relatively large apolar amino acid in the p ′ position or for p ′glu/p ′gly junctions. hav c pro can also tolerate larger p ′ amino acids in substrates with p -gln. other positions in the peptide also contribute to substrate recognition; in general, there is a preference for a hydrophobic residue at p , , , and the nature of the p and p ′ amino acids, which vary between different picornavirus families, can also be important. extensive structural analysis of trypsin-like serine proteases has revealed a common mode of substrate binding in which the polypeptide sequence recog-nised by the enzyme lies in an extended conformation (similar to a β-strand) within the peptide-binding cleft. these enzymes typically recognise the alternating positions of the side chains along the sequence that are characteristic of an extended backbone conformation, and this serves to place the scissile bond in the correct orientation at the active site. the primary specificity determinants in trypsin-like serine proteases are commonly located within the p -p sequence, [ ] [ ] [ ] but can extend well beyond this core region for enzymes that are highly specific. crystallographic analysis of enzyme-substrate complexes reveals that viral c-like proteases, such as tobacco etch virus nia protease (tev pro ) or severe acute respiratory syndrome coronavirus main protease, fall into the latter group, since contacts with the enzyme can involve up to five or six amino acids on either side of the cleavage site. however, although both of these proteases are ' c-like' in structure and have the common trypsin-like fold, they exhibit substantial differences from one another and from genuine picornaviral c pro , especially in the loops that define the peptide-binding surface. previous structural analyses of picornavirus c pro -peptide complexes have used relatively short covalently attached peptides or peptidemimicking inhibitors. in most cases, inhibitors were designed to bind to the s -s subsites, [ ] [ ] [ ] although there is one report of the structure of an inhibitor bound to the s ′-s ′ subsites. these studies are a powerful complement to cleavage assays, since they have helped to elucidate the structural basis of amino acid recognition by these enzymes, particularly on the n-terminal side of the scissile bond. however, they cannot give a complete picture of protease-substrate interactions since, in each case, the peptide is covalently attached to the protease and represents, at most, one-half of the peptide substrate recognised by the enzyme. here we report the first crystallographic analysis of a picornaviral c pro complexed with an intact peptide substrate that spans p -p ′. our structural analysis of fmdv c pro is combined with peptide cleavage assays that have examined the effects on the cleavage rate of amino acid variations within p -p ′ and of mutations in the peptide-binding cleft of the enzyme. this concerted investigation provides a detailed and insightful description of the interactions between enzyme and substrate. most strikingly, we find that significant conformational adaptation by the enzyme is important for substrate recognition. this helps to explain several important aspects of specificity differences between c pro from different picornaviruses. in conjunction with our structural analysis, we performed an extensive investigation of the impact of sequence variation in the peptide on the rate of cleavage by fmdv c pro . for these assays, we synthesised a series of variants of fret , a modified peptide based on the p -p ′ region of the vp - a cleavage junction -one of the sites cleaved by fmdv c pro in the viral polyprotein. cleavage of fret produces a readily detectable increase in fluorescence (materials and methods). the fret variants, which contained single amino acid substitutions at positions p -p ′ and p ′ or a double substitution at p and p ′, were used in cleavage assays to determine the relative value of the specificity constant (k cat /k m ) in each case (fig. a) . a detailed account of the results of these experiments will be presented alongside a description of the structural features of peptide-protease interactions at each position in the substrate sequence (see the text below). overall, it is notable that, within the context of the vp - a sequence, variation at all positions from p to p ′ (inclusive) can reduce the rate of peptide cleavage by fmdv c pro to between % and % of the rate observed with the control peptide (fig. a) . in contrast, variation at the p and p ′ positions at the extremities of the peptide had little impact on peptide cleavage. with one exception, these results are consistent with a previous alanine-scanning analysis of the peptide. they confirm the importance of positions p , p , p and p ′ for substrate recognition, but reveal, for example, that residues at the p position can also contribute to cleavage specificity. the only inconsistency noted is that substitution of p ′-phe (by arg, pro or ala) had little effect on peptide cleavage in the fluorescence assay, whereas it had been found previously in an hplc-based assay that substitution by ala abrogated cleavage. the origin of this discrepancy is unknown, but the structural results are consistent with more recent findings indicating that the p ′ residue is unlikely to contribute significantly to substrate specificity (see the text below). recombinant-type a fmdv c pro , inactivated by mutation of the active site cys to ala (c a) and containing the c k and c l substitutions necessary for enhanced solubility, was purified from escherichia coli and co-crystallised with a -fold molar excess of the decameric peptide vp - a (materials and methods). this peptide spans the p -p ′ positions and contains the sequence apakq|llnfd from the vp - a junction in the polyprotein that was cut most rapidly in peptide cleavage assays. the protease-peptide complex yielded crystals in space group p that diffracted x-rays to . Å. the structure was determined by molecular replacement phasing using the structure of the free enzyme [protein data bank (pdb) id j ] as search model. there are two molecules in the asymmetric unit, and the initial-difference electron density map revealed clear density for the peptide bound to each molecule. the density was of high quality, consistent with full occupancy of the binding site, and readily allowed determination of the side-chain conformations for of the amino acids (p -p ′) in the peptide. the electron density at the c-terminal end of the peptide was too weak to permit incorporation of the p ′-asp residue into the refined model ( supplementary fig. a ). atomic structures of the two c pro -peptide complexes in the crystal were built using o and refined with cns to yield a model with an r free of . % and excellent stereochemistry (table ). an identical approach was applied to the preparation and structure determination of the complex of c pro with the modified vp - am peptide, which contains a gln-to-glu substitution at the p position. although this peptide differs by only one amino acid from vp - a, the c pro -vp - am complex yielded crystals of a different space group (p ) that diffracted to a slightly lower resolution ( . Å). the data set is only % complete, but averaging across the four molecules in the asymmetric unit yielded a good-quality electron density map ( supplementary fig. b ) that provided a clear indication of the bound conformation of residues p -p ′ of the vp - am peptide. the refined model has an r free of . % ( table ) . the overall structures of the c pro -peptide complexes obtained with vp - a and vp - am are very similar to one another ( supplementary fig. ). there are differences in the backbone conformations of residues - at the c-terminal end of the polypeptide linking the two β-barrels of the enzyme, indicative of flexibility in this portion of the backbone. however, this is unlikely to impact peptide recognition, since this part of the linker runs along the underside of the c-terminal β-barrel, opposite to the surface containing the peptide-binding cleft. when viewed with the interdomain cleft aligned vertically (as in fig. b and c) , the peptide is observed to bind largely within a deep surface groove that is oriented diagonally and intersects the cleft at the active site. consequently, residues p -p on the amino-terminal side of the scissile bond mainly contact the c-terminal β-barrel, while residues p ′-p ′ interact primarily with the nterminal β-barrel. the length of the recessed groove is sufficient to accommodate residues p -p ′ of the peptide, although residues outside this core are also in contact with the surface of the protease. comparison of the bound and free forms of fmdv c pro reveals several conformational changes that can be ascribed to peptide binding because they are observed for the six independent structures found in the asymmetric units of the crystals of both c pro -peptide complexes, all of which have different packing environments in the crystal. these differences extend across both β-barrels ( fig. e ; supplementary movie ). on the p -p side, the β-ribbon (residues - ) moves in to contact the peptide, and there are adjustments in the backbone of the polypeptide that form the flanks of the s subsite (β-strand e and the c-terminal end of the c -d loop) (fig. e) ; on the p ′-p ′ side of the peptide, the most significant changes in backbone conformation occur in residues - and in the nearby a -b pair of β-strands (residues - ) that are in closest contact with the peptide. these two segments of polypeptide are altered due to a propagated series of side-chain movements initiated by the rotation of the side chain of leu that is necessary to accommodate the p ′-leu residues of the peptide (see the text below). the extensive malleability of the peptide-binding surface allows the protease to form intimate contacts with the substrate along most of its length. in common with other trypsin-like proteases, there is a network of hydrogen bonds between the peptide and the protease backbones that mimics the interactions between protein β-strands. in the case of the complex between fmdv c pro and the vp - a peptide, there are nine such main-chain hydrogen bonds in total, extending from p -pro to p ′-asp (fig. e) . these main-chain contacts are bolstered by hydrogen bonds and apolar contacts between the peptide side chains and the protease. we will consider each of these in detail. p -ala at the n-terminus of the peptide is largely solvent exposed (fig. a) . the side chain points towards the hydrophobic side chain of met , but is too far away ( . Å) to make contact. this lack of contact accounts for the finding that substitution by arg, leu or pro at this position has only a modest effect on substrate cleavage, reducing the rate by fold at most (fig. a) . p -pro, by contrast, is accommodated in a shallow apolar depression that marks the beginning of the peptide-binding groove and is formed by the side chains of leu (cys in the wild-type protein ), val and tyr (fig. a) . the p -pro side chain is not completely buried, since one flank containing the c δ atom faces outward and is solvent exposed. this part of the side chain is quite close to the carbonyl oxygen of val and to the side-chain hydroxyl of tyr ( . Å in each case). intriguingly, mutation of the p residue to val, leu or ile completely abrogated cleavage of the fluorescent substrate ( fig. a) , consistent with a previous observation that an ala substitution at this position also prevented cleavage. these results suggest that, at least within the context of the vp - a peptide, there is a strict requirement for p -pro. although the structure suggests that ala and val side chains, which are similar in size to pro, should be accommodated in the s pocket, they may nevertheless affect the binding conformation sufficiently to prevent proper presentation of the scissile bond to the active site. this is a rather surprising result given the evident malleability of the protease. it is also unexpected, since ala, val and ile are observed at p in other fmdv polyprotein cleavage junctions, albeit within the context of different sequences (see table in birtley et al. ). the side chain of p -ala points towards the solvent, but packs against met at the apical tip of the β-ribbon that alters its conformation on peptide binding to contact the p side of the substrate ( fig. a ; supplementary movie ). this explains why, in common with other similar proteases, there is no strong preference for particular residues at this position in natural cleavage junctions. in sequences cleaved by fmdv c pro , val, gln, glu, his, phe, ser, arg and ala-all of which could make some hydrophobic contact with the side chain of met -are found at p . nevertheless, variation of this the activity is normalised with respect to the cleavage rate observed with wild-type substrate (materials and methods). the amino acids in the wild-type sequence are given at the top of each section; the identity of the substituted amino acid is given along the bottom. note that, in the p -p ′ panel, the glu-leu and gln-gly data bars are for single p and p ′ mutants, respectively, but are duplicated here for ease of comparison with the glu-gly double mutant. (b) schematic view of the co-crystal structure of c pro (with nterminal and c-terminal β-barrels shown in lilac and green, respectively) complexed with the vp - a peptide. the peptide is depicted in sticks colour coded by atom type (carbon, orange; oxygen, red; nitrogen, blue), with its van der waals surface shown as a semitransparent surface. (c) surface representation of fmdv c pro complexed with the vp - a peptide (sticks), coloured as in (b). (d) surface representation of tobacco etch virus nia c-like protease complexed with a p -p ′peptide. the n-terminal and c-terminal β-barrels are shown in cyan and tan, respectively. (e) superposition of the free (gray) and peptide-bound (purple) forms of fdmv c pro . the viewpoint is similar to that of (b). backbonebackbone hydrogen bonds are shown as purple broken lines; other hydrogen bonds are shown in orange. selected side chains are shown as sticks; secondary structural features that show greatest movement on peptide binding are labelled. note that the unbound structure of fmdv c pro has a ser at position . residue to gln, glu or val within the context of the vp - a peptide reduces cleavage rates -fold to fold, demonstrating that variation at the p position can affect cleavage (fig. a) . it may be that these larger side chains affect the positioning of the βribbon, which then has a knock-on effect on peptide interactions at other subsites. the p -lys side chain inserts into the cleft between the β-ribbon and the body of the nterminal β-barrel of the protease (fig. a) . on one side, the lys side chain primarily contacts the side chain of his (the central residue of the catalytic triad), whereas on the other side, the aliphatic portion of the lys contacts the apolar side chains of leu and met . at the distal end of the pocket, which is open to solvent, there are salt bridges from the amine group at the tip of the lys side chain to two acidic residues from the β-ribbon, asp ( . Å) and asp ( . Å) (fig. a) . formation of the s pocket appears to be dependent on peptide binding, since leu , asp and asp all adjust their positions in the presence of substrate (fig. e) . the salt bridges to the pair of asp residues in the β-ribbon explain the strong preference for p -lys in natural substrates (although it is not yet clear why this preference is strongest in substrates with p -gln ). consistent with the acidic nature of the distal end of the s pocket, incorporation at p of norleucine (which is structurally similar to lys but lacks the amine group) abrogates cleavage, whereas substitution by arg or ornithine (both of which have a positive charge at the tip of the side chain) only modestly reduces the rate of peptide cleavage (fig. a) . curiously, substitution by thr also abrogates cleavage, even though this residue is found in other natural fmdv c pro substrates; it may be that thr is tolerated at p only in the context of the variation of the amino acids at other positions in the peptide substrate. the side chain of p -gln is accommodated by small ( . Å) movements of the two backbone segments that form the flanks of the s pocket (residues - and - ) (fig. e) . the p side chain makes three hydrogen bonds to the pocket: two between the carbonyl group and the side chains of his and thr ( . - . Å) and one from the amide group to the backbone carbonyl of thr ( . - . Å) (fig. a and c) . the pocket therefore displays good chemical complementarity to the p -gln side chain. however, peptide cleavage assays previously showed that incorporation of glu at this position only reduces the cleavage rate by a factor of , consistent with the common occurrence of p -glu in fmdv c pro substrates. to investigate this finding in more detail, we determined the crystal structure of c pro bound to a modified vp - a peptide, in which p -gln is substituted by p -glu (vp - am). the structure reveals that the p -glu side chain binds in a manner that is almost identical with that observed for p -gln in the vp - a peptide ( fig. c and d) . one oxygen atom from the p -glu side-chain carboxylate (oe ) makes two hydrogen bonds (ranging in length from . to . Å over the four independent structures) to his and thr , but the other carboxylate oxygen (oe ) is more distant from the main-chain carbonyl oxygen of thr ( . - . Å) and-assuming that the sidechain carboxylate is deprotonated-cannot form a hydrogen bond. the fact that the oe oxygen is also exposed to solvent (fig. d ) may also attenuate any negative impact on binding by allowing access to positive counterions in the cytoplasm of infected cells. it therefore appears that the primary interactions with the p side chain of the substrate are the two shorter hydrogen bonds made with the side chains of his and thr . the longer separation between the carbonyl oxygen of thr and the p -gln amide or the p -glu carboxylate oxygen (oe ) is likely to reduce the impact of these groups on peptide binding, thus allowing fmdv c pro to cleave substrates containing either type of residue in the p position. the importance of the hydrogen bonds to the side chains of his and thr is underscored by the finding that incorporation of uncharged (norleucine), longer (adipic acid) or shorter (asp) carboxylated side chains at p prevents cleavage of the substrate by fmdv c pro (fig. e) . the binding of the p ′-leu side chain is unusual because it is accommodated in a pocket that forms only when the protease interacts with the peptide substrate (fig. b) . in the free enzyme, leu packs against cys to form the floor of a shallow s ′ pocket, but peptide binding was found to induce the rotation of the side chain of leu towards the core of the n-terminal β-barrel, thereby creating a much deeper compartment to accommodate the peptide side chain (fig. e) . the largely apolar sides of the s ′ pocket are formed on one side by pro , his , leu and the aliphatic flank of glu , and by ala and cys on the other side. the displacement of leu has significant knock-on effects, leading to displacement of phe , tyr , leu and ile ; there is also alteration of the main-chain conformation of residues - ( fig. e ; supplementary movie ). as noted previously, large hydrophobic side chains at the p ′ position are preferred in natural fmdv c pro substrates containing p -gln. this correlation of p and p ′ amino acids seems to be necessary for efficient cleavage, since substitution of p ′-leu by gly yielded a -fold reduction in cleavage rate, whereas introduction of val or cys at p ′ reduced the rate of cleavage by only -fold (fig. a) . examination of the structure suggests that a p ′-gly side chain would be too small even to make stabilising contacts with the shallow form of the s ′ pocket. since the bound conformations of vp - a (p -gln) and vp - am (p -glu) peptides are very similar, it was no surprise to find that incorporation of p ′-gly within the context of the vp - am sequence also gave a -fold reduction in the rate of cleavage (fig. a) . however, these results cannot explain the observation that natural fmdv substrates with p -glu are most frequently observed to have gly at p ′. we suggest that sequences beyond the residues on either side of the scissile bond are important in ensuring that it is presented optimally to the active site. p ′-leu binds at one end of the recessed peptidebinding groove (figs. b and b) . the side chain packs against ala , gly , ile and the c β group of asp , and against the side chain of p ′-phe from the peptide, but is still only partially shielded from solvent (fig. b) . the interaction between the p ′ and the p ′ side chains may not be replicated in other peptides, since neither position is particularly well conserved in polyprotein junctions cleaved by c. the apolar nature of the s ′ binding pocket explains the general preference for p ′ residues with some hydrophobic functionality (ser, lys, thr and leu in p -gln substrates; pro, gly, leu and ile in p -glu substrates). the p ′-asn side chain is almost completely solvent exposed, but is positioned to form a hydrogen bond with glu on the enzyme surface ( fig. b) . however, there is little sequence conservation at this position in fmdv c pro substrates, and the most common alternative p ′ residues (tyr, ala and ile) would not maintain this interaction. the exposed position of p ′-asn explains why a -(( aminoethyl)amino)naphthalene- -sulfonic acid group can be added to an asp side chain at this position in the fret peptide substrate without compromising its ability to be cleaved by fmdv c pro . p ′-phe is also largely solvent exposed and primarily contacts a small apolar patch on the upper surface of the c-terminal β-barrel formed by the side chain of ile and the methyl group of thr (fig. b) . the hydrophobic nature of this patch explains the general preference for residues with apolar side chains at p ′. consistent with this notion, substitution of p ′-phe by arg, pro or alaall of which have significant apolar features-only modestly reduced the rate of peptide cleavage (fig. e) . the position of p ′-asp is unknown, since there is no electron density for this residue. it appears to make no stabilising contacts with the protein, in agreement with the observation that mutation to ala at p ′ had no effect of the rate of peptide cleavage. fig. . mutagenic analysis of s ′ specificity. (a) schematic diagram of the inactive polyprotein precursor, bc, (containing three cleavage junctions) used as substrate in these cleavage assays (see materials and methods). the p -p ′ sequences of the three cleavage junctions in this polyprotein are: b / b , lpqqe|gpyag; b / b , pvvke|gpyeg; b / c, livte|sgapp. the sizes of the primary cleavage products are indicated. (b) sds-page analysis of polyprotein precursor digestion by fmdv c pro s ′ mutants at and min. labels on the right-hand side indicate the substrate (sub.), the active c pro ( c act ) and the primary cleavage products (prod.). (c) comparative activity of fmdv c pro s ′ mutants measured in the fluorescent peptide cleavage assay using the synthetic fret substrate, mutagenic analysis of the s ′ specificity pocket as described above, the co-crystal structure of c pro with the vp - a peptide revealed that binding of the peptide necessitated movement of leu to create a pocket deep enough to accommodate the p ′-leu side chain (fig. e) . this s ′ pocket is the site of an interesting difference in specificity between fmdv c pro , on one hand, and c pro from hrv and pv, on the other hand. whereas fmdv c pro preferentially cleaves gln-xaa junctions (where xaa is predominantly leu, ile or thr), hrv and pv c pro have a marked preference for cleavage at gln-gly junctions. comparison of the protease structures readily accounts for this difference, since the cys -leu residues that form the floor of the shallow s ′ pocket in the free form of fmdv c pro are replaced by phe -ala in c pro from hrv (pdb id cqq ) and pv (pdb id l n ) ( fig. c and e; supplementary fig. ) . examination of these structures suggests that steric hindrance would prevent the movement of phe that would be necessary to accommodate a large p ′ side chain. we explored this idea by mutating the s ′ pocket on fmdv c pro to make it resemble hrv c pro and by testing the effect on the cleavage activity of a bc polyprotein substrate that contains glu-ser and glu-gly cleavage junctions (i.e., they have a small p ′ side chains) ( fig. a ; materials and methods). in this assay, the primary cleavage observed is located at the glu-ser junction; further cleavage at the glu-gly junctions within b - b - b may be impeded by the low solubility of this fragment ( fig. a and b) . under the assay conditions used, wild-type fmdv c pro cleaves over % of the substrate within min. the single mutation c f drastically reduced the ability of the protease to cleave this protein substrate; no cleavage was evident after a h incubation (fig. b) , and very partial cleavage was observed after h ( supplementary fig. ). we estimate that the reduction in activity is approximately -fold. in contrast, mutation to the much smaller ala side chain (c a) had essentially no effect on the proteolytic activity of c pro in this assay (fig. b) . the considerable reduction in protease activity due to the c f substitution is probably due to the steric strain that arises from the close packing of the large side chains of phe and leu in the s ′ binding pocket of the mutant, which may have a knock-on effect on the position of his , a key component of the catalytic triad (compare fig. c and e; supplementary fig. ). this interpretation is strongly supported by the finding that the c f/ l a double mutation, which reduces the size of the side chain opposite phe and generates an s ′ pocket that resembles that found in hrv and pv c pro , restores near-wild-type cleavage activity (fig. b) . the c a/l a double mutant is slightly impaired compared to the wild-type protease, perhaps because the p ′ residue is bound less tightly in the enlarged s ′ pocket. we also investigated the impact of these s ′ mutations on the cleavage of the fluorescent fret substrate, which is based on the sequence of the vp - a peptide and contains a gln-leu cleavage junction; this substrate therefore places a large hydrophobic side chain (p ′-leu) in the s ′ pocket. as found previously, the c f mutant showed the greatest impairment in cleavage activity, although the -fold reduction in cleavage rate compared to that in wild type was much less than the ∼ -fold reduction observed in the polyprotein cleavage assay (fig. b ). the precise reason for the difference in the magnitudes of the effects is not clear, but may relate to the different sequences that are being cleaved in the two experiments. strikingly, and in contrast to the result obtained with a substrate containing a small p ′ amino acid (fig. b) , the double mutation c f/l a did not rescue wild-type activity with the fret substrate (although, as expected, the enzyme was slightly more active than the c f single mutant) (fig. c) . overall, these results are consistent with structural data: the c f/l a double mutation would be predicted to make the s ′ pocket on fmdv c pro resemble the inflexible shallow depression found in the hrv enzyme ( supplementary fig. ), which has a preference for substrates with a small p ′ residue. curiously, the single c a mutation reduced the rate of cleavage of the fluorescent vp - a peptide by about -fold (fig. c) . this loss of activity possibly occurred because the p ′-leu of the substrate was bound less snugly by the enzyme. as before, the c a/l a double mutation caused only a very slight further reduction in cleavage activity. the crystal structure of the c pro -peptide complex reveals that the conformation of the catalytic triad is unchanged by peptide binding (fig. e) . in fig. . mutagenic analysis of d from the catalytic triad. sds-page analysis of polyprotein precursor digestion by wild-type fmdv c pro and the d a and d e mutants at and min. the substrate is the same as depicted in fig. a. particular, it shows that asp , the acidic member of the catalytic triad, maintains a hydrogen-bond interaction with his , supporting the idea that this unusual class of trypsin-like cysteine proteases requires a full cys-his-asp triad for catalytic activity. , we tested this hypothesis by mutating asp (fig. ) . the mutation d e, which maintains a carboxylate side chain, reduced cleavage activity by about orders of magnitude under our experimental conditions: whereas wild-type c pro cleaved ∼ % of the polyprotein in min at °c, the d e mutant cleaved b % of the substrate after min (fig. ) . the mutation d a was even more deleterious: there was no discernable cleavage of the substrate after h, consistent with a reduction in activity of at least orders of magnitude. these findings were confirmed in more quantitative assays using the fluorescent fret peptide substrate ( supplementary fig. ). cleavage by the d e mutant was about times slower than cleavage by wild-type fmdv c pro . cleavage by the d a mutant was at least times slower and was beneath the detection level of the assay. our results indicate that mutation of asp is even more deleterious to c pro activity than was reported previously. this apparent discrepancy is likely due to the greater sensitivity of our assay, which examined cleavage over a range of time points (fig. ) . our findings confirm the importance of asp for cleavage activity by fmdv c pro and explain the strict conservation of this residue in all serotypes of fmdv. the contacts that the side chain of leu in the βribbon makes with apolar features on p -pro and p -lys in the co-crystal structure explain the importance of this residue to catalytic activity (figs. e and a) . in fmdv c pro , this residue is strictly conserved as cys, which is also apolar. previously, we mutated c to prevent aggregation of the protein due to formation of intermolecular disulphide bonds, but found that substitution by a large hydrophobic side chain was necessary to preserve the catalytic activity of the protease. , although slightly larger, leu is a reasonable structural mimic of cys, and the structure of the c pro -peptide complex confirms our previous conclusion that the apolarity of c in the wild-type protein is necessary for its interaction with the p and p residues to ensure a correct presentation of the polypeptide substrate to the active site. consistent with this idea, introduction of the c l mutation into a type o k fmdv had no significant effect on the infectivity of the virus in tissue culture (fig. ) . this observation and the fact that the equivalent residue in hrv and pv c pro is a leucine (leu ) nevertheless beg the question, "why is cys strictly conserved in fmdv c pro ?" we speculate that this may confer a selective advantage during infection of natural hosts by permitting inactivation of the protease on cell lysis. we have determined the crystal structure of fmdv c pro in complex with a specific peptide substrate, providing the first detailed view of enzyme-substrate interactions for this important group of picornavirus proteases. the mode of peptide binding is similar to that observed in hrv and hav c pro complexed with covalently attached peptide-like inhibitors [ ] [ ] [ ] and with peptide complexes of other trypsin-like proteases in that the peptide adopts a linear extended conformation that places the p side chain into the s specificity pocket within the c-terminal β-barrel, immediately adjacent to the catalytic triad. the mode of binding in fmdv c pro involves the five main-chain-main-chain hydrogen bonds between the enzyme and the p -p ′ positions on the peptide that are conserved in trypsin-like proteases and appear to be necessary for proper positioning of the scissile bond in the active site. however, the structure reveals that the enzymesubstrate interaction extends well outside this core region; all side chains from p -p ′ are involved to a greater or lesser extent in contacts with the surface of c pro . overall, the structure of the complex is highly consistent with the results of experiments performed to probe the effect of variation in side chains at different positions within the peptide substrate on cleavage rates, which suggest that the region p -p ′ contributes most to peptide specificity. this is probably because these six residues occupy the deepest part of the surface groove that forms the peptide-binding cleft (fig. c) . there are, nevertheless, some unexpected results from the cleavage assays that are not readily explained by the structure. for example, it is notable that, within the context of the vp - a cleavage junction sequence, conservative substitution of p -pro with residues such as val or ala that occur at this position in other junctions leads to complete abrogation of peptide cleavage (fig. a) . the effect of these substitutions is all the more surprising given the evident flexibility displayed by the protease (fig. e ; supplementary movie ), but it is perhaps a reflection of the important role played by sites remote from the scissile bond in peptide cleavage efficiency. , the extensive interface of contact between the peptide and the protease in fmdv c pro exhibits similarities to that observed in the c-like tev pro , which was solved in complex with a peptide that spanned positions p -p ′ ( fig. c and d) . superposition of the two structures shows that the backbone geometry is similar over the common p -p ′ region. structural similarity is closest for the core segment centred on the scissile bond (p -p ′; root-mean-squared deviation over c a = . Å), but there are significant structural deviations outside this region, dictated by the differing topologies of the peptide-binding cleft. in particular, tev pro provides a much more enclosed binding site for the p -p residues due to the close apposition of the c-terminal β-ribbon with the e -f loop in the cterminal β-barrel (fig. d) . the fmdv c pro -peptide interaction is also reminiscent of the extended interface observed in the structures of specific protein inhibitors of trypsin and trypsin-like proteases such as chymotrypsin and collagenase. [ ] [ ] [ ] [ ] [ ] these inhibitors are generally composed of a small stable domain that interacts with the target enzyme by inserting an extended internal loop, constrained at both ends, into the peptide-binding cleft of the target enzyme. the extent of the interaction varies from p -p ′ in the complex of trypsin with pancreatic trypsin inhibitor to p -p ′ for ecotin bound to collagenase. these inhibitors strongly mimic highly specific substrates, but are usually cleaved extremely slowly because the rigidity of the enzyme-peptide interface prevents the conformational changes necessary to attain the tetrahedral intermediate at the scissile bond and because the tightly packed interface prevents access of water molecules that would be necessary for the deacylation step of catalysis. in contrast, in c pro , extensive sequence recognition must also be allied with sufficient flexibility to permit catalysis. although the residual flexibility in the c propeptide complex necessary for catalysis is not detectable by crystallography, our results do reveal that structural adjustments are made across the peptide-binding surface on substrate binding. particularly notable are the movements of the β-ribbon to form the s -s subsites, the strands flanking the s pocket and the large rotation of leu within the s ′ pocket to accommodate the large p ′ side chain (fig. e) . these observations are broadly consistent with previous nmr analyses of hrv c pro , which revealed flexibility throughout the enzyme that was attenuated by interactions between the protein and a peptide-like inhibitor spanning p -p . these marked conformational changes associated with peptide binding to fmdv c pro are in contrast to the relatively rigid lock-and-key binding of some protein inhibitors to trypsin-like proteases , and underscore the importance of the structural analysis of the peptide-bound form in obtaining a more complete picture of the substrate basis of cleavage specificity. this information is of particular relevance to inhibitor design, although further structural analyses of fmdv c pro bound to different peptide sequences will be needed to map out the full extent of structural changes that accompany substrate binding. the conformational changes propagated by the rotation of leu to accommodate p ′-leu in the fmdv c pro substrate were unanticipated. our structural and mutagenic analyses suggest that such side-chain movements are very unlikely to occur in hrv or pv c pro , since the f /a pair of residues that lie on either side of the pocket entrance (equivalent to c /l in fmdv c pro ) does not have the flexibility to accommodate large p ′ side chains (fig. c-e) . this explains the strict requirement for small p ′ side chains (most often gly) in substrates for c pro from hrv and pv. , in contrast, hav c pro , like fmdv c pro , is tolerant of larger p ′ residues (e.g., r, v and m). since it has an m /a pair of residues flanking the s ′ pocket, we suggest that the met side chain may have the ability to move to accommodate larger p ′ side chains. the crystal structures of fmdv c pro , in complex with the vp - a and vp - am peptides, show that p -gln or p -glu binds similarly within the s specificity pocket, with both making hydrogen bonds from a side-chain oxygen atom to the side chains of thr and his ; tyr also plays an important role in determining p specificity, since it stabilises the orientation of his (fig. c and d) . this structural arrangement accounts well for the observation that a gln-to-glu substitution at p within the vp - a peptide only modestly reduces peptide cleavage (fig. a and e) . however, the bound conformation of p -gln in fmdv c pro is also very similar to the conformation of the modified glutamine observed in the s pocket when the peptide-like inhibitor ag is bound to hrv c pro . although the glutamine side chain was altered to incorporate a lactam ring, glutaminespecific hydrogen bonds were observed in s . it is therefore difficult to understand why fmdv c pro readily cleaves substrates with p -gln or p -glu, while hrv c pro exhibits a strong preference for p -gln substrates. , , the discrepancy is all the more remarkable, since the residues primarily responsible for p -gln or glu recognition in fmdv c pro (thr , tyr and his ) are conserved in the p -gln-specific hrv c pro and in the p -gluspecific v protease from staphylococcus aureus. given the close structural similarity between fmdv and hrv c pro in the s pocket, why does hrv c pro discriminate so strongly against p -glu? in the coronavirus main protease from tobacco etch virus, the selectivity for p -gln is enforced by an interaction between the gln side-chain amide and the carboxylate of asp (fig. f) ; clearly, the presence of this negatively charged side chain strongly disfavours binding of substrates with p -glu. however, hrv c pro lacks an equivalent repulsive group (fig. e) , and it remains unclear how discrimination against p -glu substrates is achieved. it may be worth considering the impact of remote sites on p selectivity in these enzymes. as noted previously, fmdv c pro substrates with p -glu most commonly have a small residue at p ′ and are less likely to conserve lys at p . perhaps the correlated amino acid variations at these and other positions have a positive impact on the cleavage of p -glu substrates. further exploration of this notion will benefit from a structural analysis of a complex of fmdv c pro with a natural p -glu substrate. the original construct that expresses a c-terminally truncated, catalytically inactive form of the type a fmdv c pro containing c k, c s and c a substitutions (see table in birtley and curry ) was modified for cocrystallisation trials using quikchange (stratagene) to engineer an s l mutation. this generates a form of c pro that has wild-type binding activity but remains soluble at purified protein concentrations in excess of mg/ml. this modified construct was used to generate additional c pro variants by the same technique to add mutations to the active site and the s ′ subsite. fmdv c proteins were produced in e. coli and purified essentially as reported previously, , although the gel-filtration step was omitted for samples that were used in cleavage assays. the purified proteins were concentrated to - mg/ml in mm hepes (ph . ), mm nacl and mm βmercaptoethanol, and stored in small aliquots at − °c before use in protease cleavage assays. a modified pet-fmdv cxs construct, , inactivated by the introduction of the c a mutation in c, was used to express the -kda fragment ( bc) of the fmdv a polyprotein that corresponds to b - b - b - bc (fig. a) . the protein, which has a non-cleavable cterminal his-tag, was produced in bl (de ) e. coli and purified to about % homogeneity on talon beads (bd biosciences) using the same protocol as for the active c pro mutants. the peptides for co-crystallisation (vp / a, apakqllnfd; vp / am, apakellnfd)-synthe-sised by standard fmoc solid-phase synthesis as described previously, with acetylation of the n-terminus and amidation of the c-terminus to block charges-were purified by reverse-phase hplc. sequences were confirmed by mass spectroscopy. the fret substrate (and sequence variants) used in peptide cleavage assays was prepared as reported. , complex formation and crystallisation c pro -peptide complexes were prepared at a : . molar ratio of enzyme to peptide, since this has worked well for related proteases. typically, μl of c pro at mg/ml protein in mm hepes (ph ), mm nacl, mm ethylenediaminetetraacetic acid, mm β-mercaptoethanol and . % (wt/vol) sodium azide was mixed with μl of mm peptide dissolved in the same buffer, yielding a final protein concentration of . mg/ml. the complex was incubated with rotation at room temperature for h and used immediately in sitting-drop vapour diffusion crystallisation trials. optimised crystals of c pro bound to apakqllnfd were obtained by mixing μl of complex with μl taken from a -ml reservoir composed of - . % polyethylene glycol , . crystals were soaked for a few seconds in mother liquor incorporating % (vol/vol) glycerol and flash cooled on beamlines id - and id - at the european synchrotron radiation facility (esrf; grenoble, france) in a stream of n at k. diffraction data were processed with mosflm and scaled using scala from the ccp suite of programs. the data for the c pro -vp - a complex crystals were phased with phaser (version . . ) using the unliganded structure of c pro (pdb id j ) as search model after removal of the most mobile surface loops (residues - and - ). this procedure located two molecules in the asymmetric unit, with an overall log likelihood gain of . the protease structure from this model was then used to solve the structure of the c pro -vp - am complex, again using phaser. in that case, four molecules were identified in the asymmetric unit; the overall log likelihood gain was . manual model adjustment and refinement of both models were performed with o and cns, respectively. the cleavage activity of talon-purified c pro mutants was assessed using a protolytically inactive form of fmdv strain a bc precursor as substrate. , working stocks of c pro and the bc substrate (fig. a) were prepared at mg/ml in mm hepes (ph . ), mm nacl and mm dtt. in each assay, equal volumes of substrate and c pro were mixed to give a final concentration of each of mg/ml and incubated at °c for varying times up to h. digestion reactions were stopped by direct addition of μl of × sds sample buffer to μl of the reaction mixture, immediately followed by heating at °c for min. assays were performed in -well plates essentially as described previously, with minor modifications. fifty to μl of varying concentrations of the fret fluorogenic substrate were added to an equal volume of enzyme. the final enzyme concentrations were typically - μm (wild type), μm (c a, c f, c a/l a and c f/ l a mutants) and μm (d a and d e mutants). reactions were performed in triplicate at °c in mm nah po /na hpo (ph . ), mm ethylenediaminetetraacetic acid, mm tris( -carboxyethyl)-phosphine hcl and % glycerol. relative hydrolysis rates were determined by monitoring fluorescence at -min intervals for - min in a cytofluor microplate reader or a molecular devices spectramax m e . the excitation wavelength used was nm, and emission was measured at nm. initial hydrolysis rates were recorded (typically from to min) for each substrate concentration, and specificity constants were calculated by fitting to the michaelis-menten equation. the generation of the c l sequence change within the whole virus (type o k) was achieved by overlap pcr using the pt s infectious copy cdna plasmid as template, using the flanking primers aecofor (agg-cagcaattgaattctttga) and cpstrev (cgttg-cctcctgcagagtga) plus the degenerate mutagenic oligonucleotides cmutc lfor (acttacaagga-cattgtggttctnatggacggagacaccatg) and cmutc lrev (ccgtccatnagaaccacaatgtc-cttgtaagt). up to four different codons (ctn) for leucine (l) could have been generated to replace the cys (c) residue codon (tgc) in this procedure, but only two were used. the mutant pcr product, including the ecori (nt )-psti (nt ) region (numbered according to accession no. x ), was inserted into the pcr-xl-topo vector (invitrogen), and individual plasmids were sequenced. the ecori-psti fragments containing the cta (c l ) and ctg (c l ) codons were excised and reconstructed into pt s via an intermediate plasmid containing the bamhi fragment (nt - ). the fulllength wild-type and mutant plasmids were linearised with hpai, and rna transcripts were prepared using t rna polymerase (t megascript kit; ambion). the transcripts were introduced into bhk cells by electroporation, essentially as described previously. complete cytopathic effect was apparent following overnight incubation. rescued virus harvests were passaged once in bhk cells, and then rna was extracted using a viral rna extraction kit (qiagen). rt-pcrs, with or without reverse transcriptase, were performed using the primers ( aecofor and cpstrev, as described above) for cycles. the expected products were only observed in the presence of the reverse transcriptase (this ensured that products were derived from the viral rna and not from residual cdna template), then the amplicons were sequenced using the same primers and maintained the respective wild-type or mutant sequences as anticipated (data not shown). a single-step growth curve of the rescued viruses was performed within bhk cells, which were infected with wild-type or mutant viruses in parallel, and the cells were harvested at t = , , and h postinfection directly into rlt buffer (qiagen), the first stage of the rna extraction procedure. fmdv rna was quantified by a standard diagnostic real-time rt-pcr assay, as described previously, , using amounts of rna and cdna that ensured that both cdna synthesis and real-time pcr were within the linear range of the assays. the numbers of viral genomes in equal aliquots of rna were determined by reference to a dilution series of rna transcripts. all structural images were prepared with pymol . the morph animation shown in supplementary movie was generated using emovie . coordinates and structure factors have been deposited in the pdb with accession numbers wv and wv . control and eradication of footand-mouth disease foot-and-mouth disease foot-and-mouth disease virus c protease: recent structural and functional insights into an antiviral target comparative genomics of foot-and-mouth disease virus structure and function of picornavirus proteases translation and replication of fmdv rna picornaviral c cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases crystal structure of foot-and-mouth disease virus c protease: new insights into catalytic mechanism and cleavage specificity structure of human rhinovirus c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein refined x-ray crystallographic structure of the poliovirus c gene product structural basis of inhibition specificities of c and c-like proteases by zinccoordinating and peptidomimetic compounds structural and mutagenic analysis of foot-and-mouth disease virus c protease reveals the role of the beta-ribbon in proteolysis crystallization of footand-mouth disease virus c protease: surface mutagenesis and a novel crystal-optimization strategy dual modes of modification of hepatitis a virus c protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site cleavage site analysis in picornaviral polyproteins: discovering cellular targets by neural networks substrate requirements of human rhinovirus c protease for peptide cleavage in vitro cleavage of small peptides in vitro by human rhinovirus c protease expressed in escherichia coli hepatitis a virus c proteinase substrate specificity cleavage of synthetic peptides by purified poliovirus c proteinase a consensus sequence for substrate hydrolysis by rhinovirus c proteinase the structures of picornaviral proteinases on the size of the active site in proteases: i. papain structural basis of substrate specificity in the serine proteases nmr solution structures of the apo and peptide-inhibited human rhinovirus c protease (serotype ): structural and dynamic comparison structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus c protease with potent antiviral activity against multiple rhinovirus serotypes an episulfide cation (thiiranium ring) trapped in the active site of hav c proteinase inactivated by peptide-based ketone inhibitors evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold structural basis for the substrate specificity of tobacco etch virus protease structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design crystal structure of an inhibitor complex of the c proteinase from hepatitis a virus (hav) and implications for the polyprotein processing in hav a continuous assay for foot-and-mouth disease virus c protease activity improved methods for building protein models in electron density maps and the location of errors in these maps version . of the crystallography and nmr system structural analysis of foot-and-mouth disease virus c protease: a viable target for antiviral drugs? cleavage of translation initiation factor ai (eif ai) but not eif aii by foot-and-mouth disease virus c protease: identification of the eif ai cleavage site identification of the active-site residues of the c proteinase of foot-andmouth disease virus serine protease mechanism and specificity crystal and molecular structure of the bovine alpha-chymotrypsineglin c complex at . Å resolution crystal structure of an ecotin-collagenase complex suggests a model for recognition and cleavage of the collagen triple helix structure of the trypsin-binding domain of bowman-birk type protease inhibitor and its interaction with trypsin structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor: ii. crystallographic refinement at . Å resolution crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at . Å resolution protein inhibitors of proteinases the structure of a universally employed enzyme: v protease from staphylococcus aureus the ccp suite: programs for protein crystallography phaser crystallographic software crystallography and nmr system: a new software suite for macromolecular structure determination evidence for the role of his- of protein c in the acid-induced disassembly of footand-mouth disease virus capsids role of rna structure and rna binding activity of foot-andmouth disease virus c protein in vpg uridylylation and virus replication dynamics of picornavirus rna replication within infected cells evaluation of automated rt-pcr to accelerate the laboratory diagnosis of foot-and-mouth disease virus the pymol molecular graphics system emovie: a storyboard-based tool for making molecular movies we would like to thank the staff on beamlines id - and id - at the esrf for assistance with xray data collection. s.c. and r.j.l. are grateful for grant support provided by the biotechnology and biological sciences research council. we also thank preben normann, inge nielsen and tina frederiksen (national veterinary institute, lindholm), and imperial college students siew lian low and george wong for excellent technical assistance. s. r.k. was supported by an engineering and physical sciences research council phd studentship. n.r.-r. was funded by a marie curie host fellowship for early stage research training. t.r.s. was funded by a phd studentship awarded by imperial college. supplementary data associated with this article can be found, in the online version, at doi: . / j.jmb. . . key: cord- -lknyehsa authors: da mata, Élida cleyse gomes; mourão, caroline barbosa farias; rangel, marisa; schwartz, elisabeth ferroni title: antiviral activity of animal venom peptides and related compounds date: - - journal: j venom anim toxins incl trop dis doi: . /s - - - sha: doc_id: cord_uid: lknyehsa viruses exhibit rapid mutational capacity to trick and infect host cells, sometimes assisted through virus-coded peptides that counteract host cellular immune defense. although a large number of compounds have been identified as inhibiting various viral infections and disease progression, it is urgent to achieve the discovery of more effective agents. furthermore, proportionally to the great variety of diseases caused by viruses, very few viral vaccines are available, and not all are efficient. thus, new antiviral substances obtained from natural products have been prospected, including those derived from venomous animals. venoms are complex mixtures of hundreds of molecules, mostly peptides, that present a large array of biological activities and evolved to putatively target the biochemical machinery of different pathogens or host cellular structures. in addition, non-venomous compounds, such as some body fluids of invertebrate organisms, exhibit antiviral activity. this review provides a panorama of peptides described from animal venoms that present antiviral activity, thereby reinforcing them as important tools for the development of new therapeutic drugs. considering the most common pathologies in humans and other animals, cardiovascular and infectious diseases and cancer are among the leading causes of deaths. the cultural and educational background of affected people largely influences the prevention and treatment of human diseases; nevertheless, the availability of new drugs contributes greatly to mitigating diseases. more than viruses are known to cause human diseases [ , ] . some of them present high public health importance, such as cytomegalovirus (cmv), epstein-barr virus (ebv), hepatitis b and c viruses (hbv and hcv, respectively), herpes simplex virus (hsv), human immunodeficiency virus (hiv), rabies virus and ebola virus. the most recent worldwide estimates presented by the world health organization (who) reported . million deaths caused by hiv in , million people living with hepatitis b or c, % of liver cancer deaths caused by hepatitis viruses, thousand cases of cervical cancer caused by hpv infection, and over thousand cervical cancer deaths each year [ ] . the very few antiviral drugs commercially available can induce severe and considerable adverse effects, especially to those patients receiving lifelong treatment for diseases such as hiv. furthermore, viruses possess rapid mutational capacity to trick and infect host cells. all these facts together have propelled the prospection for new antiviral drugs, particularly from natural products, as they constitute more than % of the new drug prototypes approved in the last decades [ ] . among sources of natural products, animal venoms have revealed a great potential for drug discovery [ ] [ ] [ ] , and despite the harmful action mechanism of animal venoms, most of them have components holding potential medicinal properties to cure diseases. it is widely reported in the literature that animal venoms are rich sources of antimicrobial substances, and contain a vast array of active biological compounds with distinct chemical structures [ ] . thus, antimicrobial peptides (amps)a diversified group of peptides that exert essential function in the innate immune host response, when invaded by pathogenic organisms, such as bacteria, fungi and virusare considered the first line of defense of many organisms, including plants, insects, bacteria and vertebrates [ , ] . some peptides exhibit direct virucidal activity; others disturb attachment of virus particles to the cell membrane surface or interfere with the virus replication. because of the limited efficiency of commonly used drugs and emerging resistance of viruses, antiviral peptides may have the potential for development as putative therapeutic agents [ ] . in addition to their reduced market availability, the collateral effects and toxicity of the synthetic antiviral drugs have triggered an expanded search for natural compounds displaying antiviral activities [ , ] . any compound to be utilized as an antiviral should comply with the virus pathways during the cellular infectious cycle. initially, any rna or dna virus, enveloped or not, expresses glycoproteins that are responsible for the interaction with surface molecules, receptors, usually glycosylated proteins, integrated in the host cell membrane. at this step, any potential antiviral candidate must compete for the cell receptor by inhibiting the virus attachment to the cell membrane, thereby aborting the viral infection. other candidates may act intracellularly by interacting with the virion capsid to prevent its decapsidation; therefore, the viral nucleic acid would not be freed and transcribed. concerning retroviruses, the antiviral candidates can act by inhibiting (i) the viral reverse transcriptase activity; (ii) the pre-integration complex, thus avoiding the transport of circular viral dna to the nucleus; (iii) and also by inhibiting the action of the viral integrase, which would not allow the viral dna to integrate into the cellular chromosome. the proviral dna, after transcription, is transduced into a polyprotein that requires the viral protease in order to generate small proteins to assemble the viral capsid. in this manner, an antiviral compound could inhibit the viral protease by blocking the retroviral morphogenesis ( fig. ) [ ] . some retroviral proteins play a major role in the pathogenesis, by down regulation of cd and mhc molecules of the host cell, driving them to the proteasome for degradation. if supposed antiviral candidates target these viral proteins, hiv- nef, tat and vpr, their actions can be restrained. all the mentioned mechanisms are directly performed by retroviral molecules [ ] , but other mechanisms could also be triggered, such as those involved in the innate immune system, e.g. (i) the induction of toll-like receptor expression, that interacts fig. action mechanism of animal venom peptides or derivatives at different retrovirus replication cycle phases. ( ) the chtx and scyllatoxin-based mimetics, such as cd m , inhibit the attachment of the viral glycoprotein (gp ) to the host cell receptor cd . ( a) the peptides cecropin a, magainin , papuamide a, dermaseptin ds , caerins . and . and maculation . disintegrate the viral envelope. ( b and c) the peptides cd m , bmkn , kn - , polyphemusin, tachyplesin, immunokine and p bv obstruct the interaction of the viral gp to the cxcr and ccr co-receptors. ( ) the peptides miramides a-h inhibit the fusion of the viral envelope to the host cell membrane. ( ) the peptides melittin, didemnis a, b and c interfere with the reverse transcription process, aborting the synthesis of double-stranded viral dna. ( ) the peptides hecate and tvs-lao act in the post-translation process, in the cleavage of the gag/pol protein precursor thus interfering in the assembly of the viral capsid and in the organization of the polymerase complex with viral nucleic acid, or (ii) production of cytokines that stimulate the action of t cytotoxic cells, and nk cells, and even host cell expression of the major histocompatibility complex molecules, in order to present viral peptides to the other cells of the immune system [ ] . furthermore, antiviral compounds may activate innate restriction factors coded by the host cell [ ] . the viral dna integration in the host cell chromosome represents the major problem to be overcome in a retroviral infection. until now, there is no available drug capable of completely clearing the virus from the host [ ] . furthermore, silent retroviral infection is hidden at anatomical sites that are difficult to reach by drugs, such as the gut-associated lymphoid tissues, lymph nodes and central nervous system. infected cells, including macrophages, are quiescent in these tissues and it is not known when they will activate and release new viral progenies. another challenge for an antiviral candidate is posed by the mutation rate of viral genes, mainly among rna virus, due to the polymerase synthesis error. this is much more intriguing among retroviruses, as the initial virion genome, maintained in quiescent cells in "sanctuary niche", are distinct, mutated from each round of cell infection. thus, in each cycle of viral infection, the hijacked cell produces a growing number of recombinant new virions [ ] . the arachnid venoms, utilized as a tool for defense and attack, by killing or immobilizing their prey for feeding or their possible competitors and predators, are composed of a rich molecular diversity and complex mixture, with an intricate protein and peptide expression by mechanisms of gene regulation still under investigation [ , ] . scorpion venoms have been exhaustively studied, mainly due to the clinical effects after envenomation in humans, which sometimes lead to death [ ] . paradoxically, biotechnological applications are devised by the increased understanding of the action mechanisms of venom components, and therefore, many research works deal with the generation of new drugs based on the structure and function of molecules found in these venoms [ ] [ ] [ ] . with the rapid increase in the number of characterized scorpion venom compounds, many new drug candidates have been identified as potential medicines to deal with emerging medical global threats [ , ] . in scorpions the biologically active peptides are classified as disulfidebridged peptides (dbps) and non-disulfide-bridged peptides (ndbps) [ , ] , with the former being the main components of scorpion venoms, responsible for the neurotoxic symptoms and signs observed during scorpionism. usually these dbps target the ion channels of excitable and non-excitable cell membranes. these properties make these molecules interesting prototypes of drugs for the treatment of diverse diseases, particularly those affecting the neural system [ ] . in relation to the activity of scorpion venom compounds against retroviruses, such as hiv/siv, it has been reported that some dbps can bind to hiv gp glycoprotein due to molecular mimicry of lentiviruses host cell cd + receptor. as a result, they abolish the gp -cd interaction, which is essential to initiate the conformational changes in the viral envelope that trigger viral entry into host cells [ ] . these cd mimetic scorpion toxins contain about amino acid residues, with three or four disulfide bridges, characterized by the cysteine-stabilized α/β motif (cs-α/β), in which a β-turn between the two β-strands in these peptides resembles the cdr loop of cd . both charybdotoxin (chtx) and scyllatoxin, isolated from leiurus quinquestriatus hebraeus venom, present the cs-α/β motif and are capable of blocking k + channels [ ] [ ] [ ] [ ] . these toxins have been used effectively as molecular scaffolds for gp -cd interaction assays [ , , ] . since the amino acid residues phe and arg of cd were shown to be critical for cd binding to gp , equivalent amino acid residues were added to the new compounds. examples of mimetic peptides using chtx as a scaffold include cd m and txm , with and amino acid residues, respectively [ , ] . among the main modifications, the cd cdr loop sequence qgsf was inserted in the equivalent position of the β-turn of chtx. thus, phe of cd m, or phe of txm , would function as phe in cd . the remaining sequence is similar between the two analogs, except in two positions: arg in txm (arg in chtx) is replaced by lys in cd m, and txm has a gly as the n-terminal residue in place of val -ser residues in cd m. thus, the charged n-terminus of the gly residue in txm is in a position similar to that of the charged side-chain of arg in cd [ ] . cd m was able to inhibit gp binding to cd with an ic value of μm [ ] . likewise, txm also competed with cd for gp binding, besides causing a cd -like enhancement in gp binding to the antibody b [ ] . subsequently, other cd mimetics exhibiting gp affinity were successfully generated by phage epitope randomization of the β-turn loop in a chtx-based scaffold [ ] . as to scyllatoxin scaffold-based mimetics, a -amino acid residue miniprotein named cd m was constructed, which inhibited cd binding to gp with an ic value of μm [ ] . structural and functional analysis performed with cd m suggested additional mutations that, once incorporated in the new compound (cd m ), caused an increased affinity for gp , with ic values of . - . μm, depending on the viral strains. additionally, cd m inhibited infection of cd + cells by different hiv- strains [ ] . its β-turn sequence ( agsf ) is similar to that of txm . after that, based on cd m structural analysis, a potent mimetic with bona fide cd -like properties was synthesized [ ] . denominated cd m , it inhibited cd -gp binding in different viral strains with . - . nm ic , with these values being comparable to those obtained with cd . cdm also inhibited hiv- cell-cell fusion and infection of cells expressing cd and either the ccr or cxcr co-receptors at similar concentrations to cd [ ] . its three dimensional structure was further analyzed in complex with gp [ ] . then, another analog was designed, denominated f , which differs from cd m due to the presence of phe in replacement by biphenylalanine in position (bip ). the authors showed that f had higher mimicry of cd than cd m . in addition, f presented increased neutralization against isolates of phylogenetically related primate lentiviruses [ ] . the scorpion venom amps belong to ndbps; many of them and their analogs exert strong antiviral activity, as shown in table . some of these compounds act by direct rupture of the viral envelope, thereby decreasing viral infectivity [ ] . amps could also prevent or block the virion from entering into the cell by occupying cell receptors utilized by the viral glycoproteins [ ] . other amps do not compete with viral glycoproteins to get attached to cell receptors. instead, they can cross the cell lipoprotein membrane and internalize themselves in the cytoplasm and organelles, yielding alterations in the profile of host cells that can enhance the defense against the virus or may also block the expression of viral genes in the host cell, halting viral dissemination to other cells [ ] . mucroporin is a cationic -amino-acid residue amp isolated from lychas mucronatus venom. one of its derivatives, named mucroporin-m , has an enhanced net positive charge, and besides having antibacterial activity, presented antiviral activity against measles, sars-cov and influenza h n viruses (table ) , possibly through a direct interaction with the virus envelope [ ] . additionally, it has been shown to reduce the production of hbv antigens and viral dna in cell culture microenvironment and also to hinder hbv infection in mouse models [ ] . the molecular mechanism implicated reveals the specific activation of mitogen-activated protein kinases (mapks) leading to down-regulation of hnf α expression and consequently less binding to the hbv pre-core/core promoter region [ ] . mucroporin-m also presented anti-hiv- activity [ ] . an amphipathic α-helical peptide, hp , was screened from the cdna library of heterometrus petersii venomous gland. this -amino-acid residue ndbp inhibited the hcv infection (table ) , acting as a viricide against hcv particles and preventing the initiation of hcv infection by permeabilizing the viral envelope and decreasing virus infectivity [ ] . also from h. petersii venom gland cdna library, other α-helical ndbps were synthesized. two of them, hp and hp , exhibited potent virucidal activity against hsv- (table ) [ ] . they showed inhibitory effects on multiple steps of the virus replication cycle, caused the destruction of the viral morphology and also entered the infected cells where they reduced viral infectivity. from the cdna library of mesobuthus martensii venom gland, a compound denominated bmkn with amino acid residueswas cloned and synthesized. based on its sequence, kn - was designed by making ctry -h hcv . μg/ml [ ] ctry -h hcv . μg/ml [ ] the substitutions g k, a r and s r, enhancing its net positive charge and α-helix structure [ ] . both compounds exerted anti-hiv- activity through inhibition of chemokine receptors ccr -and cxcr -mediated activities and replication of the viruses, of which kn - was the most potent (table ) [ ] . another ndbp, screened from chaerilus tryznai scorpion venom gland, ctry , was able to inhibit initial hcv infection in huh . . cells by inactivating infectious viral particles (table ) [ ] . however, due to the low bioavailability of this -amino-acid residue peptide, ctry could not suppress an established infection. thus, in order to enhance the helicity, amphiphilicity and endosomal escape of peptides, the authors designed histidine-rich peptides based on a ctry template. denominated ctry -h and ctry -h , they were more effective against hcv than ctry (table ) , significantly reducing intracellular viral production. unlike ctry , these analogs reduced the viral rna by and %, respectively; however, ctry diminished viral infectivity in a manner similar to that of wild-type peptide [ ] . recently, the antiviral activities of scorpio maurus palmatus and androctonus australis crude venoms were shown against hcv. they presented ic values of . ± . and . ± . μg/ml, respectively. s. maurus palmatus venom was considered a good natural source for characterizing new anti-hcv agents targeting the entry step, since it impaired hcv infectivity in cell culture, but not intracellularly, through a virucidal effect. this effect was not inhibited by a metalloprotease inhibitor or heating at °c [ ] . snake venoms are composed of a mixture of proteins, peptides ( - %), free amino acids, nucleotides, lipids, carbohydrates and metallic elements coupled to proteins ( %) [ ] . some studies have reported the antiviral activity of snake venoms and their components against measles virus, sendai virus, dengue virus (denv), yellow fever virus (yfv) and hiv [ ] [ ] [ ] [ ] [ ] . thus, snake venoms are sources of promising candidates for new antiviral drugs ( table ). in relation to antiretroviral activity, the benefits of treating a patient with multidrug-resistant hiv with a snake venom preparation in addition to the antiretroviral therapy were demonstrated in clinical practice [ ] . the response was a decreased viral load and elevated t cd + cell count. the authors suggest that this activity may be related to the presence of some snake venom molecules that are homologous to hiv- glycoprotein or proteases [ , ] . this homology occurs between the - highly conserved amino acid residues of snake venom neurotoxins long loop and the sequence - of short segment hiv- gp . as a result, both may compete for the same receptor or binding site and present anti-hiv activity [ ] . the sequence homology between hiv gp and snake neurotoxins, such as cobratoxin and bungarotoxin, had generated some antiretroviral patents [ ] [ ] [ ] . linking the gp fragment to the hiv peptide fusion inhibitors (fragments of gp ectodomains) was shown to improve their anti-hiv efficacy [ ] . besides structural homology, other action mechanisms of snake venoms against hiv are also discussed in the literature, such as catalytic/inhibitory activity through enzymes, binding interference (receptor/enzyme), and induction/interaction at the membrane level [ ] . the l-amino acid oxidases (laaos or laos, ec . . . ), which constitute one of the most studied main components of snake venoms, are oxidoreductase flavoenzymes with molecular masses around to kda and are usually non-covalently linked homodimeric glycoproteins [ , ] . these compounds are widely distributed in other organisms and play an important role in biological activities such as apoptosis induction, cytotoxicity, inhibition or induction of platelet aggregation, hemorrhaging, hemolysis and edema, as well as anti-hiv, antimicrobial and antiparasitic activities [ ] . tsv-lao, characterized from trimeresurus stejnegeri snake venom, seems to be the first snake venom lao reported to present antiviral activity ( table ) [ ] . tsv-lao is a glycoprotein with a molecular weight of about kda that also forms homodimers, similarly to laos from other snake venoms. its precursor sequence, obtained by cdna analysis, codes for a polypeptide of amino acid residues, including an -amino-acid potential signal peptide that is identical to those of laos from other snake species. tsv-lao inhibited hiv- infection and replication in a dose-dependent manner, and seems to act at nanomolar concentrations by inhibiting syncytium formation (ec of . nm) and hiv- p antigen expression (ec of . nm) [ ] . additionally, another lao, isolated from bothrops jararaca venom and denominated bjarlaao-i (table ) , reduced the viral load in cells infected with dengue virus type strain exposed to the toxin in comparison to controls [ ] . its cdna-deduced sequence has amino acid residues and is similar to other snake venom laos. these flavoenzymes also produce hydrogen peroxide (h o ) as a free radical, which appears to enhance their antiviral activity [ ] . other compounds found in snake venoms that exhibit antiviral activity are the phospholipases a (pla ). among their biological effects, they seem to interact with the host cells and prevent the intracellular release of virus capsid protein, suggesting that they block viral entry into the cells before virion uncoating [ , , ] . the pla isolated from crotalus durissus terrificus venom (pla -cdt, hiv human immunodeficiency virus, hsv herpes simplex virus, iav influenza virus, vsv vesicular stomatitis virus, denv dengue virus. adapted from jenssen et al. [ ] and mulder et al. [ ] inhibited both denv and yfv in vero e cells [ ] . this pla is part of crotoxin, a heterodimeric protein composed of two different subunits non-covalently linked: the basic pla (~ . kda) and the acidic protein crotapotin (~ . kda) [ ] . the mechanism proposed for pla -cdt antiviral activity involves the cleavage of the glycerophospholipid virus envelope and protein destabilization on the virion surface, which partially exposes the genomic rna and culminates with viral inactivation, making it unable to access the cell receptor [ ] . pla -cdt also showed in vitro activity against hiv (table ) [ , ] , as well as the snake venom pla s nmmcm iii from naja mossambica mossambica, taipoxin from oxyuranus scutellatus, and nigexine from naja nigricollis [ ] . additionally, the pla variants, lys and asp , denominated blk-pla and bld-pla , from bothrops leucurus venom (table ) , reduced dengue viral rna in cells treated with these compounds, and presented cytotoxic activity against denv-infected cells in vitro [ ] . blk-pla and bld-pla have and amino acid residues, respectively, including seven disulfide bonds. another example of the antiviral effect of biomolecules extracted from snake venoms are the metalloprotease inhibitors, which could prevent the production of new hiv particles by inhibiting the viral proteases [ ] . in addition, immunokine® (oxo chemie, thailand), an oxidized derivative of the α-toxin extracted from naja siamensis venom (table ) , has been shown to inhibit infection of lymphocytes by hiv through the chemokine receptors ccr and cxcr [ , ] . many reports detail potent antiviral activity of amphibian skin secretions. such skin secretions constitute the amphibians' first line of defense, consisting of their innate immunity. the secretions produced by the anuran skin granular glands have been screened for many biological activities, including antimicrobial, antineoplastic, antiviral, contraceptive and anthelminthic activities [ , ] . the dermaseptin family of antimicrobial peptides comprise - amino acids, exhibiting a linear polycationic molecule disposed as an amphiphilic α-helical structure when associated with a lipid cell bilayer. bergaoui et al. [ ] described the dermaseptin s , a chemically synthesized -amino-acid drug derived from an amphibian skin antimicrobial peptide, exhibiting anti-herpetic activity (hsv type ), with reduced cytotoxic effects after biochemical modifications of the original peptide. it also reduced in vitro hiv- infection of an established cell line, p -ccr , expressing cd , ccr , and cxcr hiv- cell receptors and, primary t lymphocytes, being capable of acting on both r and x tropic hiv- virions. upon insertion in the viral envelope, the dermaseptin s disrupts the virion [ ] . caerin . , caerin . and maculatin . , peptides also derived from the skin secretions of the amphibians litoria caerulea, litoria chloris and litoria genimaculata, respectively, completely abolished hiv infection of t cells, after a few minutes of virion exposure to these modified peptides, which disintegrates the viral envelope, preventing viral fusion to the cell membrane. furthermore, these molecules obstructed viral transfection from dendritic cells to t cells. caerin peptides are composed of amino acid residues in their structure, including four central amino acid residues not present in maculatin peptides. in lipid bilayer membranes, these peptides are adjusted to two α-helices, interlinked by a flexible hinge region limited by pro and pro , which determine the disruption of viral envelope and cell membrane [ ] . mastoparan is a tetradecapeptide present in wasp (vespula lewisii) venom [ ] that forms amphipathic helical structures that insert into lipid bilayers of bacteria, erythrocytes, mast cells and others, forming pores [ , ] . mastoparan- , a mastoparan analogue, displayed a wide spectrum of antiviral activity against enveloped viruses of five different families (rhabdoviridae, poxviridae, flaviridae, paramyxoviridae and herpesviridae) in in vitro assays ( table ). structural studies have indicated pore formation by the insertion of the mastoporan amphiphilic α helix into the viral lipidic envelope, causing its disruption [ ] . hiv virions usually infect the host cells in the genital mucosae, by infecting macrophages, being denominated m-tropic virus; after migrating to the lymph nodes, they infect t lymphocytes, changing into t-tropic virus [ ] . based on the hiv tropism, a phospholipase a from bee venom, bvpla , blocked the replication of both m and t-tropic hiv virions [ ] , while a small peptide derived from bvpla , the p bv, exclusively inhibited the replication of t-tropic virus, behaving as a ligand for the hiv- co-receptor cxcr [ , ] (table ) . amps isolated from invertebrate organisms presented augmented antiviral activity in human diseases. such peptides enclose melittin, cecropin and alloferon molecules [ ] (table ) . melittin, isolated from honey bee (apis mellifera) venom, is an amphipathic peptide composed of amino acid residues, arranged in two α helical segments. inserted in nanoparticles, melittin exhibited virucidal activity against hiv- in the vk cell line, an epithelial vaginal cell line, and also inhibited hiv infection in tzm-bl reporter cells (hela cell line expressing hiv receptors) [ ] [ ] [ ] . among other antiretroviral mechanisms, melittin complemented the azidovudin reverse transcription inhibition [ , ] . hecate, an analogue of melittin, selectively reduced the protein biosynthesis of virus-specified glycoproteins b, c, d, and h of the hsv type [ ] . the mechanism is similar to the one detected among hiv- infected lymphoblastic cells, previously treated with melittin, by the intervention in the processing of the gag/pol protein precursor. therefore, specific intracellular events are targeted by melittin and its derivatives [ , ] . cecropins, isolated mostly from the hemolymph of infected pupae of the silk moth hyalophora cecropia, but also from other insects, tunicates and ascaris nematodes, are a family of amps, containing - amino acid residues arranged in two amphiphilic α-helices linked by a gly-pro hinge. synthetic hybrid peptides, namely cecropin a ( - )-magainin ( - ), exhibited potent antiviral activity by a mechanism mainly based on the compound hydrophobicity and α-helical content, inhibiting the virushost cell fusion [ ] (table ) . alloferon and are peptides constituted of - amino acid residues, isolated from the hemolymph of the blowfly calliphora vicina. alloferons exert immunomodulatory activities to control infection by the human influenza virus in mice model of lethal pulmonary infection [ ] , whereas their derivatives also inhibited in vitro hsv replication in vero cells [ , ] (table ). these peptides also displayed a relevant role in the innate immunity, being considered prospective peptides for the pharmaceutical industry [ , ] . sea organisms are also promising sources of antiviral cationic peptides. they present a broad spectrum of antiviral activity, while one single peptide may present activity against different viruses and other pathogens. the promiscuous antifreeze pa-map peptide, which consists of an α-helix composed of amino acid residues, was isolated from the polar fish pleuronectes americanus ( table ). the pa-map exerted antimicrobial activity against bacteria, fungi, neoplastic cells, and also interacted with the viral envelope of the hsv types and , inhibiting the infection of susceptible cells [ , [ ] [ ] [ ] . some sponge species contain linear or cyclic bioactive peptides composed of atypical amino acid residues, generating unique structures that are rarely found in terrestrial organisms [ , ] . these compounds, particularly the cyclic depsipeptides mirabamides a-h, isolated from siliquaria spongia mirabilis and stelletta clavosa, obstruct the hiv- virion entry into tzm-bl cells, thus neutralizing the viral glycoprotein fusion for expressing cd and ccr hiv cell receptors [ , ] (table ). peptide concentrations between and nm were sufficient to inhibit infection by % (ic ). another cyclodepsipeptide, homophymine a, obtained from homophymia sp., conferred % cell protection at nm concentration against hiv- infection in vitro [ ] (table ) . discovered in the early s, didemnins a, b and c from the caribbean tunicate trididemnum solidum were the first antiviral marine depsipeptides described. didemnins were effective against vaccinia virus, hsv type and , coxsackie virus a- and equine rhinovirus, presenting strong activity at low doses [ ] . furthermore, these peptides were active in in vivo assays in a rat model infected with herpes simplex virus, reducing the skin lesions after topical administration [ ] . didemnins inhibit protein, dna and rna synthesis in cells [ , ] . the protein synthesis inhibition mechanism may be related to the binding of didemnins to the elongation factor alpha (ef- alpha) [ ] . didemnin b underwent phases i and ii of clinical trials in the s, but presented low selectivity and therapeutic index, as well as toxic side effects [ ] . dehydrodidemnin b (aplidin®, pharma mar sa, spain) is currently under phase iii of clinical trials as an anticancer drug against multiple myeloma and t-cell lymphoma [ ] . several antiviral peptides and depsipeptides have been described in marine sponges from the genus theonella sp.: koshikamides f and h isolated from t. swinhoei and t. cupola [ ] ; papuamides a and b, and theopapuamide a from theonella sp. and t. swinhoei, respectively [ ] [ ] [ ] . all of them inhibited hiv entry into t cells. theopapuamide b was isolated from an indonesian sponge, siliquariaspongia mirabilis, and was also able to inhibit hiv- entry into host cells [ ] . papuamide a presented antiviral activity not only against hiv- , but also against vesicular stomatitis virus and amphotropic murine leukemia virus. due to its tyrosine residue and the presence of a hydrophobic tail, the peptide may insert into the viral membrane, causing its rupture [ ] . other peptides from marine sponges that inhibit hiv- entry into host cells are: callipeltin a, isolated from sponges of the genus callipelta, which displayed antiviral activity with a high selectivity index ( ) between the virus and host cells (si ratio % cytotoxic dose [cd ]/ed ) [ ] ; celebesides a-c from siliquariaspongia mirabilis [ ] ; neamphamide a, from neamphius huxleyi, a compound with structural similarities to callipeptins and papuamides that exhibited low toxicity to host cells and a selectivity index above [ ] ; and microspinosamide, isolated from sidonops microspinosa [ ] . marine arthropod species have also yielded antiviral peptides, tachyplesin and polyphemusin (t ), and shown anti-hiv- activity by attachment to the chemokine receptor, cxcr , which is also the viral t cell co-receptor. hemocytes of horseshoe crabs (tachypleus tridentatus and limulus polyphemus) are an abundant source of tachyplesin and polyphemusin. the tachyplesin consists of - amino acid residues, primarily arranged in three tandem repeats of a tetrapeptide, hydrophobic amino acid-cys-aromatic amino acid-arg and an amidated c-terminus, while the polyphemusin analog, t , is composed of amino acid residues, exposing an antiparallel β-sheet conformation stabilized by a disulfide bridge between cys and cys [ , ] . as a consequence of the scarcity of new families of antiviral drugs, pharmaceutical companies have strengthened their efforts to increase developments of known current drugs, resulting in little or even no improvement to the existing therapies. these new patent protections guarantee the rights to the same stakeholders who are charging high consumer prices due to the lack of competition [ ] . at the same time, the growing demand for new drugs and natural therapeutic products is a matter of extreme necessity to face the emergency of multiresistant viral pathogens. more than compounds obtained from vertebrate and invertebrate organisms presented in vitro or in vivo antiviral activity. although none of those has yet been launched on the market as an antiviral drug, they present chemical structures completely different from the current drugs used in therapy, despite acting on similar targets. those compounds may lead to new classes of therapeutic drugs after additional chemical and pharmacological studies. emerging and reemerging viruses of medical relevance challenge health authorities all around the planet. some viral vaccines have taken too long to be designed and approved for human and animal utilization, and even in some cases could not be developed. preventive and curative measures should always be in the hands of health authorities to ensure control of epidemics, such as the recent ebola virus in africa or arboviruses, particularly in brazilrepresented by the dengue, chikungunya and zika virusesor worldwide pandemics, such as influenza and hiv. therefore, prospection, screening and all other phases of biological activity, validation, clinical development of animal peptides represent an essential scientific investment for protecting and perpetuating humankind. human viruses: discovery and emergence outbreaks of ebola virus disease in africa: the beginnings of a tragic saga world health organization (who) antiviral drug discovery: broad-spectrum drugs from nature bufotenine is able to block rabies virus infection in bhk- cells synergic effects between ocellatin-f and bufotenine on the inhibition of bhk- cellular infection by the rabies virus mechanisms of virus resistance and antiviral activity of snake venoms scorpion peptides: potential use for new drug development peptide antimicrobial agents antimicrobial peptides peptides -a new strategy for combating viral infections incidence rate of modifying or discontinuing first combined antiretroviral therapy regimen due to toxicity during the first year of treatment stratified by age aids -an old disease with new challenges illustrations of the hiv life cycle hiv life cycle, innate immunity and autophagy in the central nervous system innate immunity against hiv- infection retroviral restriction factors and infectious risk in xenotransplantation targeting hiv transcription: the quest for a functional cure overcoming pharmacologic sanctuaries scorpion venom components as potential candidates for drug development molecular diversity of spider venom epidemiology of scorpionism: a global appraisal chlorotoxin: structure, activity, and potential uses in cancer therapy toxin modulators and blockers of herg k + channels from the stretcher to the pharmacy's shelf: drug leads from medically important brazilian venomous arachnid species scorpion venom components that affect ion-channels function scorpion venom peptides with no disulfide bridges: a review phage randomization in a charybdotoxin scaffold leads to cd -mimetic recognition motifs that bind hiv- envelope through non-aromatic sequences charybdotoxin, a protein inhibitor of single ca + -activated k + channels from mammalian skeletal muscle structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent k + channel purification and characterization of a unique, potent inhibitor of apamin binding from leiurus quinquestriatus hebraeus venom leiurotoxin i (scyllatoxin), a peptide ligand for ca + -activated k + channels. chemical synthesis, radiolabeling, and receptor characterization conformational changes of gp in epitopes near the ccr binding site are induced by cd and a cd miniprotein mimetic rational engineering of a miniprotein that reproduces the core of the cd site interacting with hiv- envelope glycoprotein engineering a cd mimetic inhibiting the binding of the human immunodeficiency virus- (hiv- ) envelope glycoprotein gp to human lymphocyte cd by the transfer of a cd functional site to a small natural scaffold rational design of a cd mimic that inhibits hiv- entry and exposes cryptic neutralization epitopes scorpion-toxin mimics of cd in complex with human immunodeficiency virus gp crystal structures, molecular mimicry, and neutralization breadth anti-hiv- activity of a new scorpion venom peptide derivative kn - virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses mucroporin-m inhibits hepatitis b virus replication by activating the mitogen-activated protein kinase (mapk) pathway and down-regulating hnf α in vitro and in vivo a new natural a-helical peptide from the venom of the scorpion heterometrus petersii kills hcv inhibitory activity and mechanism of two scorpion venom peptides against herpes simplex virus type design of histidine-rich peptides with enhanced bioavailability and inhibitory activity against hepatitis c virus virocidal activity of egyptian scorpion venoms against hepatitis c virus snake venomics. strategy and applications inhibitory potential of crotalus durissus terrificus venom on measles virus growth selective lysis of virus-infected cells by cobra snake cytotoxins: a sendai virus, human erythrocytes, and cytotoxin model crotoxin and phospholipases a from crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses secreted phospholipases a , a new class of hiv inhibitors that block virus entry into host cells hypothesis of snake and insect venoms against human immunodeficiency virus: a review snake venom preparation for drug-resistant human immunodeficiency virus re: snake venom preparation for drug-resistant human immunodeficiency virus modified venom and venom components as antiretroviral agents pan-antiviral peptides for protein kinase inhibition modified venom and venom components as antiretroviral agents peptide fusion inhibitors targeting the hiv- gp : a patent review snake venom l-amino acid oxidases: trends in pharmacology and biochemistry crystal structure of laao from calloselasma rhodostoma with an l-phenylalanine substrate: insights into structure and mechanism past decade study of snake venom l-amino acid oxidase molecular characterization of trimeresurus stejnegeri venom l-amino acid oxidase with potential anti-hiv activity antiviral and antiparasite properties of an l-amino acid oxidase from the da mata et al snake bothrops jararaca: cloning and identification of a complete cdna sequence secreted phospholipases a (spla ): friends or foes? are they actors in antibacterial and anti-hiv resistance? phospholipase a isolated from the venom of crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope use of a phospholipase a for the preparation of pharmaceutical and/or cosmetic compositions for the local and/or systematic treatment and/or prevention of diseases and/or processes caused by intraand extracellular pathogens expressing membrane phospholipids molecular characterization of lys and asp phospholipases a from snake venom and their antiviral activities against dengue virus therapeutic alternatives from venoms and toxins bioactive molecules from amphibian skin: their biological activities with reference to therapeutic potentials for possible drug development host-defense peptides of the skin with therapeutic potential: from hagfish to human in vitro antiviral activity of dermaseptin s and derivatives from amphibian skin against herpes simplex virus type antimicrobial peptides from amphibian skin potently inhibit human immunodeficiency virus infection and transfer of virus from dendritic cells to t cells a new mast cell degranulating peptide "mastoparan" in the venom of vespula lewisii new insight into the mechanism of action of wasp mastoparan peptides: lytic activity and clustering observed with giant vesicles the effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and cd spectroscopy a mastoparan-derived peptide has broad-spectrum antiviral activity against enveloped viruses m-tropic hiv envelope protein gp exhibits a different neuropathological profile than t-tropic gp in rat striatum a peptide derived from bee venom-secreted phospholipase a inhibits replication of t-cell tropic hiv- strains via interaction with the cxcr chemokine receptor current scenario of peptide-based drugs: the key roles of cationic antitumor and antiviral peptides nanoparticulate-based contraceptive/anti-hiv. composition and methods cytolytic nanoparticles attenuate hiv- infectivity recent advances in developing insect natural products as potential modern day medicines. evid based method and composition for the treatment of mammalian hiv infection three valuable peptides from bee and wasp venoms for therapeutic and biotechnological use: melittin, apamin and mastoparan an amphipathic alpha-helical synthetic peptide analogue of melittin inhibits herpes simplex virus- (hsv- )-induced cell fusion and virus spread influence of amphipathic peptides on the hiv- production in persistently infected t lymphoma cells structure-antiviral activity relationships of cecropin a-magainin hybrid peptide and its analogues studies of insect peptides alloferon, any-gs and their analogues. synthesis and antiherpes activity novel analogs of alloferon: synthesis, conformational studies, pro-apoptotic and antiviral activity antiviral and antitumor peptides from insects anti-tumor activity of immunomodulatory peptide alloferon- in mouse tumor transplantation model marine peptides: bioactivities and applications structural and functional characterization of a multifunctional alanine-rich peptide analogue from pleuronectes americanus in vivo antimicrobial evaluation of an alanine-rich peptide derived from pleuronectes americanus bioactive peptides from marine sources: pharmacological properties and isolation procedures mirabamides e-h, hiv-inhibitory depsipeptides from the sponge stelletta clavosa mirabamides a-d, depsipeptides from the sponge siliquariaspongia mirabilis that inhibit hiv- fusion homophymine a, an anti-hiv cyclodepsipeptide from the sponge homophymia sp didemnins: antiviral and antitumor depsipeptides from a caribbean tunicate didemnins a and b. effectiveness against cutaneous herpes simplex virus in mice biochemical and cellular effects of didemnins a and b mechanism of action of didemnin b, a depsipeptide from the sea gtp-dependent binding of the antiproliferative agent didemnin to elongation factor alpha the discovery and development of marine compounds with pharmaceutical potential an overview of the marine natural products in clinical trials and on the market mutremdamide a and koshikamides c-h, peptide inhibitors of hiv- entry from different theonella species characterizing the anti-hiv activity of papuamide a sponges theonella mirabilis and theonella swinhoei collected in papua new guinea insights into an unusual nonribosomal peptide synthetase biosynthesis: identification and characterization of the ge biosynthetic gene cluster celebesides a-c and theopapuamides b-d, depsipeptides from an indonesian sponge that inhibit hiv- entry callipeltin a, an anti-hiv cyclic depsipeptide from the new caledonian lithistida sponge callipelta sp neamphamide a, a new hiv-inhibitory depsipeptide from the papua new guinea marine sponge neamphius huxleyi microspinosamide, a new hiv-inhibitory cyclic depsipeptide from the marine sponge sidonops microspinosa a lowmolecular-weight inhibitor against the chemokine receptor cxcr : a strong anti-hiv peptide t natural products with anti-hiv activity from marine organisms panorama patentário dos medicamentos antirretrovirais no brasil antiviral activity of antimicrobial cationic peptides against junin virus and herpes simplex virus evaluation of the inactivation of infectious herpes simplex virus by host-defense peptides the antimicrobial peptide dermaseptin s inhibits hiv- infectivity in vitro in vitro antiviral activity of dermaseptins against herpes simplex virus type the authors thank patrícia souza wanderley for fig. authors' contributions ecgm was a major contributor in writing the manuscript. all authors contributed in writing the manuscript, read and approved the final document. the authors declare that they have no competing interests. key: cord- - yozebv authors: vitiello, mariateresa; finamore, emiliana; falanga, annarita; raieta, katia; cantisani, marco; galdiero, francesco; pedone, carlo; galdiero, marilena; galdiero, stefania title: viral fusion peptides induce several signal transduction pathway activations that are essential for interleukin- and beta-interferon production date: - - journal: intervirology doi: . / sha: doc_id: cord_uid: yozebv objectives: the deciphering of intracellular signaling pathways that are activated by the interaction between viral fusion peptides and cellular membranes are important for the understanding of both viral replication strategies and host defense mechanisms. methods: fusion peptides of several enveloped viruses belonging to different virus families were prepared by standard -fluorenylmethoxycarbonyl polyamine solid-phase synthesis and used to stimulate u cells in vitro to analyze the phosphorylation patterns of the signaling pathways (pkc, src, akt, and mapk pathways). immunoprecipitation and western blotting were carried out by using phosphospecific antibodies. all samples were also assayed for the presence of il- and ifn-β by elisa and activation of nuclear factors (ap- and nf-κb). results: we have demonstrated that hydrophobic domains of fusion proteins are able to induce several transduction pathways that lead to cytokine (ifn-β and il- ) production, an event that appears to be dependent on early activation of ap- and nf-κb. conclusions: the results obtained on the signaling activity of fusion peptides from different viruses enabled us to shed some light on the complex mechanism of viral entry and more precisely we focused on the exact signaling event induced by hydrophobic domains characteristic of fusion peptides interacting with the cell membrane. cytokine synthesis and several accessory cell functions of macrophages, il- is a potent suppressor of the effector functions of macrophages, t cells and nk cells [ ] . many viruses as well as several viral envelopes or particles can induce the activation of innate host defense pathways that results in the production of type i ifns, among these ifn-␣ and ifn-␤ . in particular, induction of ifn-␤ gene expression is a tightly regulated process, and previous studies have identified the signal transduction pathway tank-binding kinase- (tbk- )/ifn regulatory factor- (irf- ) as essential to the activation of ifn-␤ gene expression [ ] . in addition to irf- activation, efficient induction of ifn-␤ usually requires the activation of the nuclear transcription factor-b (nf-b) [ ] and of the transcription factor- (atf- /cjun) [ ] , suggesting that it is an essential component in the innate immune response to virus infection. the activator protein- (ap- ) transcription factor belongs to a large family of structurally related transcription factors that includes c-fos, c-jun and c-myc. ap- consists of various combinations of fos and jun family members (i.e. c-fos, fosb, fra- , fra- , c-jun, junb and jund) that dimerize via a leucine zipper domain and bind to dna via a specific target dna sequence [ ] . in particular, these transcription factors become activated by tyrosine and serine/threonine phosphorylation, and involve primarily non-receptor protein tyrosine kinase (nt-ptk), protein kinase c (pkc), and janus-activated kinase (jak), initiating further downstream signaling cascades, such as the mitogen-activated protein kinase (mapk) and phosphatidylinositol -kinase (pi k) that regulate several responses including mitosis, apoptosis, motility, proliferation, differentiation and many others. it is not surprising, therefore, that many viruses target the pi k and mapk pathways as a means to manipulate cellular function [ , ] . four different members of the mapk family, which are organized in separate cascades, have been identified to date. three of them, extracellular-signal-regulated kinase (erk), jun n-terminal kinase (jnk), and p have been reported to be activated upon viral infection [ , ] . erks are activated by many viruses including hcmv, kaposi sarcoma-associated herpesvirus (kshv), hepatitis b and c viruses, papilloma virus, adenovirus, influenza virus, respiratory syncytial virus (rsv), and human immunodeficiency virus (hiv). p / jnk mapks activation has been documented for infections with rhinovirus, herpesviruses, hiv, adenovirus, influenza virus, and hepatitis b virus. many enveloped viruses trigger upon binding to their specific cellular receptors a fusion reaction between the viral envelope and the cell membrane, a process that involves structural modifications and exposure of small stretches of hydrophobic amino acids. the fusion reactions are directed by fusion proteins that undergo consistent structural modifications that lead to the exposure of small stretches of hydrophobic aminoacids, the fusion peptides, which interact with the opposing lipid bilayer and are involved in the initial stages of virus penetration. three classes of fusion proteins have been identified so far. class i comprises many unrelated virus families such as paramyxoviruses, orthomyxoviruses, retroviruses and filoviruses class ii applies to the flaviviridae family, which comprises the tick-borne encephalitis virus (tbe), the dengue virus, the yellow fever virus, the west nile virus, and the hepatitis c virus and the togaviridae family, of which the best known are the semliki forest virus and the rubella virus; class iii fusion proteins comprises rhabdoviruses and herpesviruses [ , ] . in this study, the effect of the binding on the cell surface of peptides with known fusogenic activity on the activation of signaling pathways was examined. a set of known fusion peptides from different virus families was synthesized and the activation of several signal transduction pathways investigated. cell lines u monocytes (atcc crl- . ) were cultured at ° in % co in rpmi (gibco) with % heat-inactivated fetal calf serum, glutamine ( m m ), penicillin ( u/ml), streptomycin ( u/ml) and differentiated as previously described [ ] . before treatment of the cells, the serum concentration was reduced to % for h at ° and then further reduced to serum-free media for at least - h. this should prevent most of the interference from serum factors on the phosphorylation state of the proteins in the signaling cascade. fusion peptides corresponding to a set of different viruses were prepared by standard -fluorenylmethoxycarbonyl polyamine solid-phase syntheses, using a pssm multispecific peptide synthesizer (shimadzu corporation biotechnology instruments department, kyoto, japan). the tga resin (substitution . mmol/g) was used as the solid-phase support, and syntheses were performed on a scale of mol. all amino acids, equivalents relative to resin loading, were coupled according to the tbtu/hobt/diea method: equivalent of fmoc-amino acid, equivalent of tbtu, equivalent of hobt ( m hobt in dmf) and equivalents of diea ( m diea in dmf). the fmoc-protecting group was removed with % piperidine in dmf (v/v). peptides were fully deprotected and cleaved from the resin by hydrofluoric acid treatment ( % tfa solution containing . % thioanisole, . % ethandithiol and . % anisole as scavengers); the crude peptides were precipitated with ice-cold ethyl ether, filtered, re-dissolved in water and lyophilised. the crude peptides were purified to homogeneity by preparative reverse-phase highpressure liquid chromatography (hplc) on a waters delta prep chromatographic system, equipped with an uv lambda max mod. detector. the samples were injected on a jupiter (phenomenex) c column ( . mm ! cm, m) eluted with a h o/ . % tfa (a) and ch cn/ . % tfa (b) solvent mixture. a linear gradient from to % of b over min at a flow rate of ml min - was employed. the collected fractions were lyophilised to dryness and analyzed by analytical reverse-phase hplc on a shimadzu class-lc equipped with a diode array detector spd-m av using a phenomenex c analytical column ( ! mm, m); a linear gradient from to % of b over min at a flow rate of ml/min was used. the identity of purified peptides was confirmed by maldi spectrometry. all purified peptides were obtained with high yields ( - %). a scrambled peptide of hiv- fusion peptide was also synthesized as a control to evaluate the sequence dependency of results. table shows the sequences of all the synthesized peptides. all the peptides were detoxified before being tested on cells. detoxification was performed by using detoxy gel affinity pak columns supplied by pierce (rockford, ill., usa). the peptides have been used to stimulate cells in vitro to analyze the phosphorylation patterns of the signaling pathways. u cells ( ! cells/ml) were stimulated with each fusion peptides at concentrations comprised between and m and at different time points. the time points and concentrations have been determined in preliminary experiments. in the further analysis we tested peptides ( m ) for min. after incubation, the cells were washed twice with ice-cold pbs without ca + and mg + , and resuspended in l of appropriate kinase lysis buffer. lysates were transferred to ice-cold microcentrifuge tubes, vortexed, incubated for min in ice and centrifuged at , g for min at ° to remove insoluble components. cell lysates were precleared with protein a/g plus-agarose (santa cruz biotechnology, inc., calif., usa) ( l) for min. immunoprecipitation was done with the appropriate antibodies and beads at ° overnight with gentle rotation. after incubation, the beads were pelleted ( , g ), washed three time with l of specific lysis buffer, and boiled in l of laemmli sample buffer with % ␤ -mercaptoethanol for min. lysate samples containing g of protein were separated on sds % page gels as described by laemmli [ ] . following electrophoresis, the separating gel was soaked in transfer buffer ( m m tris, m m glycine, % methanol) for min, and then the proteins were transferred to polyvinylidene difluoride membranes (pore size, . m) overnight at v and ° . blots were blocked for h at room temperature in tris-buffered saline (tbs; m m nacl, m m tris-hcl [ph . ]) containing % bovine serum albumin (bsa) plus % blotting-grade blocker nonfat milk (bio-rad laboratories), and subsequently membranes were washed twice with tbs containing . % tween (ttbs) before incubation with anti-phosphorylated kinase antibodies diluted : , in tbs containing % bsa for h at room temperature. after being washed six times with ttbs for min, the polyvinylidene difluoride membranes were incubated at room temperature for h with specific immunoglobulin g (igg)-horseradish peroxidase secondary antibodies diluted : , . they were then washed six times with ttbs and twice with pbs for min. thereafter, proteins were visualized with enhanced chemiluminescence on kodak x-omat ls film. immunoprecipitation and western blotting were carried out by using phospho-specific antibodies as follows: anti-phospho-pkc antibody detects endogenous levels of pkc ␣ , ␤ i, ␤ ii, ␥ , ␦ , , and isoforms only when phosphorylated at a residue homologous to thr of human pkc ␣ (cell signaling technology, inc.); anti-phospho-src family antibody recognizes a synthetic phosphopeptide corresponding to residues surrounding tyr of human src (cell signaling technology, inc.); anti-phospho-akt, which recognizes phosphorylation on ser (cell signaling technology, inc.); anti-phospho-p /p which is a mouse monoclonal antibody raised against thr of p (erk ) and tyr of p (erk ) (cell signaling technology, inc.); anti-phospho-p antibody (santa cruz biotechnology, inc.) which is a rabbit polyclonal antibody raised against a peptide mapping at the amino terminus of p of mouse origin identical to the corresponding human sequence and is directed against thr and tyr -phosphorylated p (santa cruz biotechnology, inc.); and anti-phospho-jnk antibody, which is a mouse monoclonal igg antibody raised against a peptide corresponding to a short amino acid sequence phosphorylated on thr and tyr of jnk of human origin (santa cruz biotechnology, inc.). all assays were carried out using u cells ( ! cells/ml) stimulated with viral fusion peptides ( m ) and incubated for h at ° in % co . after incubation, samples were centrifuged at , rpm at ° for min and the supernatants were collected and stored at - ° . all samples were assayed for the presence of il- and ifn-␤ by elisa, according to the manufacturer's instructions. we used human interferon-␤ (hu-ifn-␤ ) elisa kit from pbl biomedical laboratories and human il- elisa kit from bender medsystem. standard and sample dilutions were performed at least four times for each individual cell-stimulation assay. to detect and quantify ap- and nf-b activation in u cells, we used elisa-based trans-am transcription factor kits (active motif, carlsbad, calif., usa) according to the manufacturer's recommendations. g of protein was added to the wells of a -well plate coated with immobilized oligonucleotide containing a tpa-responsive element (tre; -tgagtca- ) or the nf-b consensus site ( -gggactttcc- ) according to the transcription factors analyzed [ , ] . after a -hour incubation period at room temperature, the wells were washed times with the washing buffer included on the kit and l of the provided anti c-fos and anti c-jun or anti-p and anti-p antibodies was added at a : , dilution. the plate was incubated at room temperature for h and the wells were washed times. hrp-conjugated anti-rabbit igg was added at a : , dilution and incubated for h at room temperature. cells were washed times and developing solution was added, followed by the stop solution. the amount of ap- or nf-b activation was measured at nm in an hts bioassay reader (perkin elmer, norwalk, colo., usa). in preliminary experiments, the trans-am kits showed a good correlation with an emsa in detecting the dna binding capacity of ap- and nf-b. the optimal time of stimulation ( h) and peptides concentration ( m ) used in the ap- and nf-b eli-sa were determined in pilot assays. lactate dehydrogenase (ldh) assay was carried out according to the manufacturer's instructions by using a cytotoxicity detection kit (roche diagnostic gmbh, roche molecular biochemicals). ldh is a stable cytoplasmic enzyme present in all cells and is rapidly released into cell culture supernatant when the plasma membrane is damaged. ldh activity was determined by a coupled enzymatic reaction whereby the tetrazolium salt (int) was reduced to formazan. an increase in the number of dead or damaged cells resulted in an increase in ldh activity in the culture supernatant. the amount of ldh shows that treated and untreated cells are healthy. all solutions and peptide preparations used in our experiments were tested for the presence of endotoxin using a limulus amoebocyte lysate (lal) gel-clot assay (associates of cape cod, inc.; distributed by pbi international, milan, italy) as described by yin et al. [ ] . the lower detection limit of this assay was . iu/ml. gels were scanned for densitometry analysis by sigma gel software and the results shown are an average of triplicate experiments. the results were expressed as mean values ses of three independent observations. specific ligand-receptor interactions result in activation of signaling pathways; thus, we selected fusion peptides belonging to each of the known viral classes of fusion glycoproteins in order to verify whether during the interaction with the cell surface, they were sufficient to induce phosphorylation of pkc, src, akt, and mapk pathways, activation of nuclear factor (ap- and nf-b), and cytokine release (il- and ifn-␤ ). to address this question we synthesized fusion peptides of several viruses which had already been identified and their sequences are reported in table . in all experiments, we tested a scrambled sequence of the hiv- fusion peptide (scrambled: nh -flagivgalgaafgl-conh) and the lipopolysaccharide (lps) from escherichia coli (sigma, st. louis, mo., usa) in order to verify the reliability of our experiments demonstrating that the activation of signal transduction pathways and cytokine release were only inducted by active sequences. in addition, a peptide derived from the porin p of haemophilus influenzae type b (peptide l : nh -ns-tvdnqkqqhgalr-conh ) and a peptide derived from a non-fusion viral glycoprotein, namely sendai virus glycoprotein hn (peptide non-fp: nh -nsevdldhpf-salyp-conh ) were included in our study. by using the lal test, endotoxin levels of cell culture and peptide solutions resulted lower than . iu/ml (data not shown). in order to investigate peptide-induced protein phosphorylation in whole-cell lysates from u cells, we assayed immunoprecipitation of phosphorylated proteins followed by western blot analysis. to determine the dose response and kinetics, u cells ( ! cells/well) were treated with peptides at a concentration comprised between and m and for different time periods ( , , , min). the duration of the treatment with peptides at the concentrations used was not toxic for the cells; in fact, the treatment did not induce any significant release of ldh in the cell supernatants (data not shown). concentrations of m were still not toxic for cells, as verified by ldh release, and did not further increase the enzyme phosphorylation. pkc activation being an early marker of cell response to interacting external molecules, we assayed pkc phosphorylation after cell treatment with viral fusion peptides. all fusion peptides studied induced rapid pkc phosphorylation as early as min after treatment. we followed the phosphorylation status of pkc for up to min (data not shown) after treatment and found that the response reached its maximum after min. we next examined pkc phosphorylation at different peptide con-centrations (data not shown). the dose-response experiments showed that the optimal peptide concentration to induce kinase phosphorylation resulted m (data not shown). a standard concentration of m and stimulation time of min were chosen for subsequent experiments ( fig. a, b) . we observed that pkc phosphorylation was induced by almost all peptides (except fp-vsv) and that fp-sev caused the most significant increase. moreover, a significant phosphorylation of akt (pkb) was evident after treatment with fp-sev, fp-mev, fp-ndv, fp-rsv and fp-sfv. to assess the eventual activation of nt-ptk cascade as a consequence of the interaction of fusion peptides with the lipid domain of cell surface, we assayed src phosphorylation. as shown in figure a, src was significantly activated by fp-hiv- , fp-mev, fp-rsv, fp-iv, and fp-ebov, but mainly by fp-sfv, fp-tbev and fp-mev. to further determine the involvement of mapk cascades, we performed a kinase assay using cell lysates upon fusion peptide treatment. the results indicate that erk / is the most activated kinase. all peptides (except fp-vsv) strongly increased erk / phosphorylation. in particular fp-ndv, fp-iv and fp-ebov caused an activation ϳ -fold higher than the control ( fig. b) . moreover, as shown in figure b, jnk was slightly activated by all tested peptides, and only fp-rsv resulted in an activation -fold higher than the control. similar assays to determine p activity indicated no change in the levels of its phosphorylation as a results of peptide stimulations ( fig. b) . to selectively analyze the regulation of virus-induced ap- and nf-b activation, an elisa-based trans-am technology from nuclear lysates of u cells stimulated by viral fusion peptides was performed. these transcription factors are supposed to be involved in the expression of proinflammatory cytokine genes. therefore, in order to demonstrate ap- and nf-b activation, we investigated fusion peptides induction of ap- c-fos/c-jun subunits and nf-b p /p subunits in whole-cell extracts using abs specific for epitopes that are accessible only when the nuclear factors are phosphorylated and bound to their target dna. following treatment of u cells with synthesized viral fusion peptides at m , ap- and nf-b binding significantly increased by min, was maintained at the same level by min, and returned to background levels by min (data not shown). in particular, we found that all the peptides were able to activate significantly both ap- ( fig. ) and nf-b ( fig. ). supernatants from by viral fusion peptides stimulated u cells were collected at h and il- and ifn-␤ measured by elisa. in figure , we report data obtained using u cells ( ! cells/ml) stimulated with a concentration of m for each peptide; the results obtained show that all the peptides were able to induce a significant release of ifn-␤ and il- . the concentrations of peptides used as well as the duration of the treatment were not toxic for the cells; in fact, the treatment did not induce any significant release of ldh in the cell supernatants. cell lysates ( g/ml) were tested for binding of the activated c-fos or c-jun subunits to an ap- consensus sequence using the trans-am ap- elisa kit. the experiment was performed in the presence of soluble wild-type or mutated consensus oligonucleotides. the results are expressed as specific binding (absorbance measured in the presence of the mutated oligonucleotide minus that measured in the presence of the wild-type oligonucleotide). the results are shown as means ses of triplicate determinations. fig. . nf-b activation by viral fusion peptides. effect of peptides on binding of nf-b subunits to an nf-b-binding consensus sequence. u cells were stimulated with viral peptides at m . cell lysates were tested for binding of the activated p or p subunits to an nf-b consensus sequence using the trans-am nf-b elisa kit. the experiment was performed in the presence of soluble wild-type or mutated consensus oligonucleotides. the results are expressed as specific binding (absorbance measured in the presence of the mutated oligonucleotide minus that measured in the presence of the wild-type oligonucleotide). the results are shown as means ses of triplicate determinations. among the first consequences of viral binding to surface receptors there are changes in the structure of many virus particles. for many enveloped viruses, including retroviruses and herpesviruses, receptor binding can trigger a fusion reaction between the viral membrane and the plasma membrane; for other viruses, including influenza virus, vesicular stomatitis virus and semliki forest virus, the penetration starts with the fusion of viral membranes with cellular endosomes upon exposure to low ph. as a consequence, fusion glycoproteins undergo consistent structural modifications that lead to the exposure of small stretches of aminoacids, the fusion peptides, which interact with the opposing lipid bilayer and initiate the fusion reaction [ , ] . to our knowledge, the present study is the first one addressing the role of fusion peptides in the complex events of signaling that are activated by viral infections. the analysis of the role played by fusion peptides from different virus families may also help in understanding the complex mechanism of viral entry. the experimental results demonstrated that hydrophobic domains of fusion proteins are able to induce several transduction pathways and cytokine production (such as il- and ifn-␤ ) during the earliest events of the viral life cycle. activation of these pathways seem to be directly related to the sequence and conformation of the viral fusion peptides, in fact the hiv-scrambled peptide did not induce activation of any of the analyzed pathways. several viral proteins, such as hepatitis b virus hbx [ ] , epstein-barr virus latent membrane protein- [ ] , hcmv ie [ ] , hsv- icp pk [ ] , and sars coronavirus nucleocapsid protein [ ] , have been shown to activate ap- . also for nf-b, the expression of a single viral protein is sufficient to its activation as seen with tax from htlv- [ ] , e / k from adenovirus [ ] , and hbx from hepatitis b virus [ ] . however, the underlying molecular mechanisms appear to be different among them and, hence, previous works on signaling cascades during viral entry have mainly focused on either whole viruses or viral proteins. pleschka [ ] showed that several human pathogenic rna viruses including influenza, ebola, hepatitis c and sars corona virus induced the raf/mek/erk signal transduction cascade. erk pathway activation is required at different levels during hiv infection [ ] as well as in rsv-induced early gene expression [ ] . monick et al. [ ] reported that rsv infection caused two separate peaks of erk activity both immediately following viral binding ( - min) and later during active viral replication ( - h) matched by activation of multiple pkc isoforms. among such kinases, the pkc superfamily is responsible for diverse regulatory roles in many cellular processes and has also been implicated in virus entry. in fact, the entry of several enveloped viruses, including rhabdoviruses, alphaviruses, herpesviruses and influenza virus, requires pkc activation upon binding to host cell surface receptors [ , ] . taken together, all literature data highlight the role of pkc and erk pathways in the efficient infection and replication of certain viral species and are consistent with the results reported in this study of fusion peptides. the results show that immediately following treatment of u cells with peptides of class i (fp-hiv, fp-sev, fp-mev, fp-ndv, fp-rsv, fp-iv, fp-ebov) and class ii (fp-sfv, fp-tbev) pkc and erk / are activated; moreover, class i peptides also induce the activation of jnk. our results show that none of the peptides was able to induce activation of p . moreover, fusion peptides of both class i and ii are able to induce activation of ap- ; in particular all the peptides were able to activate more significantly c-fos and less significantly c-jun. though the predominant mechanism of activation of the transcription factor ap- is usually through phosphorylation of c-jun by jnk, recently erk was also shown to phosphorylate c-fos and thereby activate ap- [ , ] . all the peptides significantly activate the src signaling pathway. src family kinases are proto-oncogenic enzymes that were initially characterized in the context of cell growth and differentiation. the activation of src should result in the activation of downstream cellular targets and their physiological effects. here a higher erk / activity was demonstrated in u cells according to other investigators that have shown that activation of mapk in cells follow the src-dependent pathway. akt affects multiple cellular targets that increase metabolism, growth, synthetic processes and proliferation and suppress apoptosis. all of these processes are beneficial to viral lytic replication, thus it is not surprising that viruses have developed means to activate akt during lytic infections. sun et al. [ ] have recently reported that akt activity is required for optimal replication of paramyxovirus and other nonsegmentated negative-strand viruses; moreover, akt has been demonstrated to play a fundamental role also in other viruses such as herpesviruses, adenoviruses, polyomaviruses and poxviruses [ ] . the results show that akt phosphorylation is evident following treatment with fp-mev, fp-ndv and fp-rsv fusion peptides (all members of the paramyxoviridae family). particularly interesting appear the results obtained for a segment of vsv glycoprotein g, which has the charac-teristic of a class iii fusion protein. the peptide is not able to induce significantly the activation of any of the kinases studied here, although it is able to induce the release of cytokines. fp-vsv correspond to one of the two predicted fusion loops of vsv glycoprotein g [ , ] . the resolution of the crystal structure of the protein resembles the structure of glycoprotein b of hsv- and points out to a possible cooperation of two nonconsecutive segments both on the tip of their relative loop in order to induce lipid destabilization during the event of membrane fusion [ , ] . class iii of fusion proteins seems to operate with a different mechanism from the other two, therefore it is interesting to observe that cytokine production is obtained through different signaling pathway. the results strongly suggest that the analyzed fusion peptides activate a signaling cascade that leads to the activation of different components, regulating the transcription factors ap- and nf-b. it has been well established that these transcription factors are a hallmark of most infections including viral infections and play a central role in virus-dependent cytokine expression and pathology. thus, the release of il- and ifn-␤ , following treatment of u cells with peptides, has also been analyzed. the results demonstrate that both cytokines are significantly released after stimulation with all the selected peptides. the data reported show the presence of a strong correlation between the fusion peptides, and the activation of signaling pathways and the release of cytokines, and further support the fundamental role played by fusion peptides in viral infections. hepatitis b virus x protein enhances nfkappab activity through cooperating with vbp erk- / activity is required for efficient rsv infection cleavage of structural proteins during the assembly of the head of bacteriophage t galphai protein-dependant extracellular signal-regulated kinase- / activation is required for hiv- reverse transcription hunninghake g: respiratory syncytial virus infection results in activation of multiple protein kinase c isoforms leading to activation of mitogen-activated protein kinase phosphorylation of the carboxyl-terminal transactivation domain of c-fos by extracellular signal-regulated kinase mediates the transcriptional activation of ap- and cellular transformation induced by platelet-derived growth factor molecular interpretation of erk signal duration by immediate early gene products strategies for use of il- or its antagonists in human disease activation of transcription factor nf-kap-pab by the adenovirus e / k protein requires its er retention rna viruses and the mitogenic raf/mek/erk signal transduction cascade development of a sensitive multi-well colorimetric assay for active nfkb crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g structure of the perfusion form of the vesicular stomatitis virus glycoprotein g role of protein kinase c betaii in influenza virus entry via late endosomes the herpes simplex virus type protein icp pk: a master of versatility role of map kinase-dependent apoptotic pathway in innate immune responses and viral infection akt plays a critical role in replication of nonsegmented negative-stranded rna viruses the hepatitis b virus x protein enhances ap- activation through interaction with jab viral targeting of the interferon-{beta}-inducing traf family member-associated nf-{kappa}b activator (tank)-binding kinase- structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme picogram-sensitive assay for endotoxin: gelation of limulus polyphemus blood cell lysate induced by purified lipopolysaccharides and lipid a from gram-negative bacteria role of epstein-barr virus encoded latent membrane protein in the carcinogenesis of nasopharyngeal carcinoma the hbz factor of human t-cell leukaemia virus type i dimerizes with transcription factors junb and c-jun and modulates their transcriptional activity the pivotal role of phosphatidylinositol -kinase-akt signal transduction in virus survival assembly of a functional beta interferon enhanceosome is dependent on atf- -c-jun heterodimer orientation induction of cytokine mrna expression in u cells by salmonella typhimurium porins is regulated by different phosphorylation pathways viral membrane fusion activation of ap- signal transduction pathway by sars coronavirus nucleocapsid protein crystal structure of glycoprotein b from herpes simplex virus ap- subunits: quarrel and harmony among siblings convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response the immune response induced by hepatitis b virus principal antigens hepatitis c virus (hcv): a review of immunological aspects human cytomegalovirus ie protein activates ap- through a cellular protein kinase(s) key: cord- -xc osdx authors: qureshi, abid; thakur, nishant; tandon, himani; kumar, manoj title: avpdb: a database of experimentally validated antiviral peptides targeting medically important viruses date: - - journal: nucleic acids res doi: . /nar/gkt sha: doc_id: cord_uid: xc osdx antiviral peptides (avps) have exhibited huge potential in inhibiting viruses by targeting various stages of their life cycle. therefore, we have developed avpdb, available online at http://crdd.osdd.net/servers/avpdb, to provide a dedicated resource of experimentally verified avps targeting over medically important viruses including influenza, hcv, hsv, rsv, hbv, denv, sars, etc. however, we have separately provided hiv inhibiting peptides in ‘hipdb’. avpdb contains detailed information of peptides, including modified peptides experimentally tested for antiviral activity. in modified peptides a chemical moiety is attached for increasing their efficacy and stability. detailed information include: peptide sequence, length, source, virus targeted, virus family, cell line used, efficacy (qualitative/quantitative), target step/protein, assay used in determining the efficacy and pubmed reference. the database also furnishes physicochemical properties and predicted structure for each peptide. we have provided user-friendly browsing and search facility along with other analysis tools to help the users. entering of many synthetic peptide-based drugs in various stages of clinical trials reiterate the importance for the avp resources. avpdb is anticipated to cater to the needs of scientific community working for the development of antiviral therapeutics. viruses are the causative agents of various dreadful diseases in humans and animals ( , ) . for majority of viruses like hepatitis c virus (hcv), influenza, dengue virus (denv), severe acute respiratory syndrome (sars), herpes simplex virus (hsv), etc., antiviral therapeutics are limited or lacking ( ) . moreover, owing to increasing drug resistance, conventional antiviral therapy is continuously challenged these days ( , ) . therefore, scientific efforts are underway to search for novel antivirals ( , ) . antiviral peptides (avps) are being regarded as such new promising entities to combat the viral infections. avps are a subset of antimicrobial peptides (amps) which act as the first line of defence in many organisms as innate immune response and are the hosts' defence peptides generated in response to pathogenic disease condition ( ) ( ) ( ) . avps are known to act either directly or by eliciting immune response ( ) . they usually inhibit directly one or more stages in the life cycle of a virus, viz., entry, attachment, replication, transcription, translation, maturation, release, etc.; thereby exhibiting the antiviral effects ( , ) . one of the earliest reports stating the direct involvement of peptides in inhibiting herpes simplex virus (hsv) multiplication dates back to ( ) . since then researchers have been extensively working on peptidebased antiviral development. bultmann et al. ( ) used fgf signal peptide derivatives to inhibit hsv- entry and the best performing avp had a half maximal inhibitory concentration (ic ) of . mm. budge and graham ( ) used r-a derived peptides to inhibit respiratory syncytial virus (rsv) replication and achieved a maximum ic of . mm. also, a peptide derived from spike (s) protein of sars-cov has been proved to be effective against sars virus entry with an efficacy of mm ( ). the peptide 'flupep' inhibits influenza virus attachment to the cells with an ic of . mm ( ) . similarly, xu et al. ( ) were able to inhibit denv protease using avps with a minimum ic of . mm. an avp named 'ctry ' has been synthesized, which possesses anti-hcv activity with an ec of . mg/ml ( ) . therapeutic potential, mode of action and importance of avps has been further reviewed ( , , ) . peptide-based drugs are advantageous over conventional drugs in having lesser molecular weight, higher efficiency, lower toxicity and minor side effects ( ) . avps are usually derived from natural sources but they can be readily modified by adding chemical groups or non-natural amino acids to further enhance their activity and stability ( ) . due to high potential, an estimated peptidebased therapeutics as antimicrobial/immunomodulatory are under clinical trials ( ) . the first avp to pass the clinical trials was 'enfuvirtide' (t ), an hiv fusion inhibitor that is being sold under the name of 'fuzeon' ( , ) . bioinformatics resources are required to accommodate and analyse the enormous data being generated on avps. although a number of resources exist for general antimicrobial peptides like apd ( ) , camp ( ) , dampd ( ) , yadamp ( ) , lamp ( ), etc., yet, specific resources on avps are lacking. therefore, to fill this void we have recently developed avppred ( ) and hipdb ( ) . avppred is the first avp prediction algorithm developed using support vector machine (svm). whereas hipdb is a specific database of experimentally validated hiv inhibiting peptides, which is freely available at http://crdd.osdd.net/servers/hipdb. hipdb harbours information of peptides and modified peptides experimentally tested for hiv inhibiting activity. besides above, no other resource is available for avps. hence, we developed avpdb-a comprehensive resource of peptides experimentally validated for their antiviral activities. relevant data were retrieved from the pubmed database, a free repository of abstracts and references on biomedical and life sciences. exhaustive literature search was accomplished by building search queries having combination of many keywords including virus, viral, peptide, inhibit, block, etc. a typical text mining query is given below: full text search returned articles as on july . in the initial screening, we found that majority of the articles were not furnishing the desired data. this could be due to the fact that the above keywords are quite frequent in the literature. therefore, we limited our query to the title/abstract fields and retrieved articles using the advanced search option of pubmed. these articles were manually examined in detail based on their abstracts/full paper to fish out the desired data. besides, we have also searched these keywords in the patentlens database and included data from eight relevant patents. reviews, general methodological and non-english articles were not considered. besides these, there were number of articles in which information on only predicted peptides or peptide structures or analogues was given were excluded. also, dendrimeric peptides, complex peptide conjugates and peptide/drug combinations were removed. similarly, articles that were lacking peptide sequence or experimental efficacy were also not considered. in addition, peptides targeting hiv were also left out of the database, as these data were already published in our recent database, hipdb ( ) . papers that were limited in giving information only on predicted peptides or design, peptide structural studies, peptide analogues, dendrimeric peptides, complex peptide conjugates, peptides used in combination with drugs, emphasis was laid on to articles having experimentally validated peptides and covering all or most of the avpdb fields. after filtering out the above articles, remaining research articles were finally used to collect peptides experimentally tested for virus inhibiting activity. further modified peptides were also extracted and have been provided separately in avpdb. in our database, complete avp data of almost all human viruses reported in the literature have been included. avpdb is a manually curated, open source database of avps targeted against diverse viruses of therapeutic importance. the database comes with easy-to-operate browsing as well as searching with sorting and filtering functionalities. avpdb also provides physicochemical properties and predicted structure of avps along with more informative tools for data analysis such as blast and map as well as links to major peptide resources. physicochemical properties displayed are charge, polarity, composition, hydrophobicity and secondary structure preference. the values used for calculating these properties were retrieved from the aaindex database. structures of the peptides were predicted using the pepstr algorithm ( ) and pep-fold ( ) server. structures are displayed in jmol applet. to view the structures, java plugin should be installed in the browser and javascript to be enabled. blast and map tools help in finding the similar peptides reported in the database. overall database architecture is shown in figure . avpdb currently archives the following fields extracted from the literature: sequence: all peptide sequences are formatted in standard one letter amino acid notation along with their respective string length. figure . these facts were also separately calculated for modified peptides as shown in figure . further analysing the overall amino acid composition of the database, it was noticed that some amino acids like leu, lys, ala, arg and val were found to be more abundant while some amino acids like his, met, trp and tyr were present less frequently. these results are shown in supplementary figure s . peptide efficacy statistics for natural as well as modified peptides are presented in table . also, the top sources of the natural and modified peptides are given in table . the 'avpdb map' is a user friendly tool to fetch the perfectly matching peptide available in our database. so, it helps the user to find how many peptides against the user-provided protein sequence are available in our database. the output of this tool displays the avpid, its source, sequence and its target. also mentioned is the start position where the match is found in the user-provided sequence. (ii) avpdb blast additionally, the blast allows alignment of a userprovided peptide sequence against all the peptide sequences available in our database. this helps the user to confirm whether a given peptide sequence or similar one has already been reported or not. the output is given in the standard format with the blast score and e-value. the alignment is shown for the peptides found to be identical or similar in the database. the output can be formatted based on the options provided by user. various important physicochemical properties such as amino acid composition, hydrophobicity, preference for b-sheets, frequency of a-helix, amino acid charge and polarity can been calculated using aaindex ( ) . these properties can be calculated for any user-provided peptide sequence by submitting it on the analysis page available under tools column. a user-friendly 'browse by' option allows to explore the data for normal peptides by any of the fields categorized in the database, viz., virus, family, peptide source, cell line, target and assay. for modified avps also, a separate browse option is provided where the data can be sought by virus, modification, peptide source, cell line, target and assay. to specifically retrieve hiv inhibiting peptides, extensive links of hipdb are provided from avpdb pages. avpdb has been incorporated with four different searches: (i) field search: here the user can enter the query in the box and can specify any of the fields against which one wishes to search or else keep the default 'all' option which will search against all the fields in the database. besides the option to choose the fields, search type allows to retrieve either an exact match or the match containing the query. the results obtained from this search display fields where first nine contain the experimental data and the last one, 'analysis', has links to blast results, physicochemical properties and predicted peptide structure. as more and more avps are being published, the interested workers may submit the desired data into avpdb via the online submission form provided in the database. once the information is cross-verified by our team, it will be included in the updates of database. avpdb database is implemented using the open source lamp solution stack on red hat enterprise linux (ibm sas  machine) with mysql ( . . b) and apache ( . . ) in back-end and front-end of web interface is implemented with php ( . . ). the database is freely available at http://crdd.osdd.net/servers/avpdb. a vast amount of data regarding avps both natural as well as modified is reported every year. to cope with these valuable data, we would like to include more viruses or newly discovered unique peptides to our database as appropriate information becomes available in the scientific literature. also, a tool to predict the ic value of virus inhibitory peptides shall be plugged in the database in near future. supplementary data are available at nar online. mechanisms of viral emergence emerging viral diseases rates of evolutionary change in viruses: patterns and determinants herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy antiviral resistance and the future landscape of hepatitis c virus infection therapy hivsirdb: a database of hiv inhibiting sirnas virsirnadb: a curated database of experimentally validated viral sirna/shrna anti herpes simplex virus activity of lactoferrin/ lactoferricin -an example of antiviral activity of antimicrobial protein/peptide inhibition of respiratory syncytial virus by rhoa-derived peptides: implications for the development of improved antiviral agents targeting heparinbinding viruses antimicrobial and hostdefense peptides as new anti-infective therapeutic strategies the gamma interferon (ifn-gamma) mimetic peptide ifn-gamma ( - ) prevents encephalomyocarditis virus infection both in tissue culture and in mice hipdb: a database of experimentally validated hiv inhibiting peptides avppred: collection and prediction of highly effective antiviral peptides inhibition of virus multiplication by immunoactive peptides modified fgf signal peptide inhibits entry of herpes simplex virus type identification of a new region of sars-cov s protein critical for viral entry a novel family of peptides with potent activity against influenza a viruses critical effect of peptide cyclization on the potency of peptide inhibitors against dengue virus ns b-ns protease design of histidine-rich peptides with enhanced bioavailability and inhibitory activity against hepatitis c virus antimicrobial peptides of multicellular organisms phage display of combinatorial peptide libraries: application to antiviral research chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization designing antimicrobial peptides: form follows function a phase ii clinical study of the long-term safety and antiviral activity of enfuvirtide-based antiretroviral therapy enfuvirtide (fuzeon): the first fusion inhibitor apd : the updated antimicrobial peptide database and its application in peptide design camp: a useful resource for research on antimicrobial peptides dampd: a manually curated antimicrobial peptide database yadamp: yet another database of antimicrobial peptides lamp: a database linking antimicrobial peptides pepstr: a de novo method for tertiary structure prediction of small bioactive peptides pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides aaindex: amino acid index database, progress report conflict of interest statement. none declared. these peptides are comprised of non-natural or chemically modified amino acids. key: cord- -f xcnuw authors: wang, guangshun title: bioinformatic analysis of amphibian antimicrobial peptides uncovers multiple length-dependent correlations for peptide design and prediction date: - - journal: antibiotics (basel) doi: . /antibiotics sha: doc_id: cord_uid: f xcnuw amphibians are widely distributed on different continents, except for the polar regions. they are important sources for the isolation, purification and characterization of natural compounds, including peptides with various functions. innate immune antimicrobial peptides (amps) play a critical role in warding off invading pathogens, such as bacteria, fungi, parasites, and viruses. they may also have other biological functions such as endotoxin neutralization, chemotaxis, anti-inflammation, and wound healing. this article documents a bioinformatic analysis of over amphibian antimicrobial peptides registered in the antimicrobial peptide database (apd) in the past years. these anuran peptides were discovered in africa, asia, australia, europe, and america from to . genomic and peptidomic studies accelerated the discovery pace and underscored the necessity in establishing criteria for peptide entry into the apd. a total of . % of the anuran antimicrobial peptides are less than amino acids with an average length of and a net charge of + . . interestingly, the various amphibian peptide families (e.g., temporins, brevinins, esculentins) can be connected through multiple length-dependent relationships. with an increase in length, peptide net charge increases, while the hydrophobic content decreases. in addition, glycine, leucine, lysine, and proline all show linear correlations with peptide length. these correlations improve our understanding of amphibian peptides and may be useful for prediction and design of new linear peptides with potential applications in treating infectious diseases, cancer and diabetes. . discovery timeline of the major families of frog antimicrobial peptides . peptide source species continent count ref xenopus laevis africa [ ] xpf xenopus laevis africa [ ] dermaseptin phyllomedusa bicolor s. america [ ] brevinin rana brevipoda porsa asia [ ] caerin litoria splendida, australia [ ] esculentin rana esculenta europe [ ] ranalexin rana catesbeiana n. america [ ] rugosin rana rugosa asia [ ] amolops loloensis asia [ ] hymenochirin hymenochirus boettgeri africa [ ] frenatin sphaenorhynchus lacteus s. america [ ] defensin theloderma kwangsiensis asia [ ] based on the antimicrobial peptide database (apd) (http://aps.unmc.edu/ap) in june . the year indicates the discovery of the first member in the peptide family on an indicated continent. some peptide families such as temporin and brevinin have been subsequently found on other continents. this reference first reports the peptide in the family based on the apd. antibiotics , , of the interest in antimicrobial peptides in the s was driven by the desire to better appreciate immune systems by taking innate immunity into consideration [ , ] . there is also the desire to develop new types of antibiotics to meet the challenge of antibiotic resistance [ ] [ ] [ ] , ] . the advance of the amp research is facilitated by technological development. solid-phase synthesis [ ] allowed scientists to make a sufficient amount of newly isolated peptides for detailed characterization. in the s, d nmr method was established to determine protein structure [ , ] and timely utilized to determine the three-dimensional structure of host defense peptides in membrane-mimetic conditions [ ] [ ] [ ] . it became clear that amphibian peptides adopt an amphipathic helix with distinct hydrophobic and hydrophilic faces [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the hydrophobic surface is flanked with basic amino acids, usually lysine. such an interface provides a molecular basis for amphibian amps to target anionic bacterial membranes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the bacterial killing mechanism of amps is complex and not well understood. it can also be concentration-dependent. at the minimal inhibitory concentration (mic, usually at micromolar or µg/ml), a pore may form on bacterial membranes. supporting evidence for the pore formation is the detection of ions or molecules leaked from cells after peptide treatment. different models have been proposed to explain the pore formation. the barrel-stave model suggests that peptides assemble into a pore in bacterial membranes [ ] . shorter peptides such as gramicidin a may stack to form an ion channel [ ] . as a permanent pore on bacteria is not readily observed, there was also a proposal that such pores are transient, allowing indicator dyes to enter the cell to associate with internal molecules, such as dna or rna. then, the carpet model was also proposed [ ] . in this model, cationic peptides bind to the anionic membrane surface. a detergent model may have a similar implication [ ] . since all these are interactions which occur in the membrane interface, the interfacial activity model was also proposed [ ] . earlier, we proposed the membrane perturbation potential for cationic amps based on three-dimensional ( d) structure. a peptide with more basic charges and a wider hydrophobic surface tends to be more powerful in perturbing anionic membranes [ ] . also proposed are lipid clustering [ ] , membrane curvature and thinning [ ] . all these interfacial actions may lead to the formation of a toroidal pore with a mixture of peptide and lipids [ , ] . at even higher peptide concentrations, bacteria may be lysed and the culture becomes clear [ ] . however, at a sublethal concentration where no visible damage to bacteria can be observed, the peptide, as a dangerous signal, can still upset bacteria and trigger various response mechanisms [ ] . in addition, at a non-lethal concentration, peptides may also affect host cells. this results from the association of the peptide with cell receptors to trigger signal transduction events, including the release of molecules such as cytokines [ , ] . these events could be related to a variety of biological processes and therefore play a role in human health or disease. the information on amphibian peptides is broad and scattered in different journals in the literature. to facilitate the research in the field, we entered peptide information into the antimicrobial peptide database (apd). as of june , amphibian peptides ( frog amps, toad peptides, and salamander peptides) were registered into the apd [ ] [ ] [ ] . this article focuses on the bioinformatic analysis of frog amps with an attempt to identify the unifying theme behind numerous peptide families discovered and named by scientists on different continents. it will address the following questions: ( ) how many amps were discovered per year since the publication of magainins? ( ) when was the first member of each amphibian peptide family reported? ( ) how will the averaged amino acid signature of amphibian peptides alter from continent to continent? ( ) with so many peptide families, is there any theme that connects them? also discussed are peptide length, charge, hydrophobicity, and post-translational modification, which are key parameters for designing potent peptides and their mimics to combat drug-resistant pathogens. for this study, we found frog antimicrobial peptides in the apd , ranging from to amino acids (an average length of ) [ ] . however, . % of these frog peptides are less than amino acid residues. they have a net charge in the range of − to + with an average of + . . finally, frog amps have a hydrophobic content that ranges from % to % (on average . %). the annual total for frog peptides discovered from to is presented in figure . the number of frog amps discovered per year steadily increased from to and then started to decline. there are two exceptional years. the first occurred in with frog peptides reported. the second appeared in with amps registered into the apd. an analysis of the database revealed that the peak in resulted mainly from the contributions of american and australia scientists [ , , [ ] [ ] [ ] . the peak in was primarily due to the contributions of chinese scientists. as many as experimentally validated amps from a single paper were registered into the apd [ ] . it is evident that proteomic technologies greatly accelerated the discovery of amphibian peptides [ ] [ ] [ ] [ ] . based on the apd, we counted the total frog antimicrobial peptides contributed by scientists from different continents. a total of amphibian peptides ( %) were discovered in asia. while originated from south america ( %), were found in north america ( %). a total of amps, including additional members in the magainin family, were identified in african frogs. finally, australians characterized amphibian peptides ( %), european scientists isolated and purified such peptides ( %) (figure a ). antibiotics , , x for peer review of for this study, we found frog antimicrobial peptides in the apd , ranging from to amino acids (an average length of ) [ ] . however, . % of these frog peptides are less than amino acid residues. they have a net charge in the range of − to + with an average of + . . finally, frog amps have a hydrophobic content that ranges from % to % (on average . %). the annual total for frog peptides discovered from to is presented in figure . the number of frog amps discovered per year steadily increased from to and then started to decline. there are two exceptional years. the first occurred in with frog peptides reported. the second appeared in with amps registered into the apd. an analysis of the database revealed that the peak in resulted mainly from the contributions of american and australia scientists [ , , [ ] [ ] [ ] . the peak in was primarily due to the contributions of chinese scientists. as many as experimentally validated amps from a single paper were registered into the apd [ ] . it is evident that proteomic technologies greatly accelerated the discovery of amphibian peptides [ ] [ ] [ ] [ ] . based on the apd, we counted the total frog antimicrobial peptides contributed by scientists from different continents. a total of amphibian peptides ( %) were discovered in asia. while originated from south america ( %), were found in north america ( %). a total of amps, including additional members in the magainin family, were identified in african frogs. finally, australians characterized amphibian peptides ( %), european scientists isolated and purified such peptides ( %) (figure a ). figure b depicts the discovery timeline for the major amphibian peptide families [ ] when they were first reported from different continents. peptide source species, family counts, and reference for each family can be found in table . magainins are the first amps found in the african frog xenopus laevis [ ] . subsequently, caerins were discovered in australia in the s [ ] . with the isolation of dermaseptins in south america in [ ] , ranalexin was also discovered in north america in [ ] . the first member of brevinin was discovered in in asia [ ] . esculentins appeared to be the first frog amp discovered in europe in [ ] . additional members for some frog amp families were subsequently discovered on different continents. the peptide members in different families vary substantially in the apd, ranging from two to over . these numbers can be regarded as minimal since many predicted/isolated amps without antibiotics , , of antimicrobial activity data or those tested to be inactive (mic > µm) are not included in our analysis. we found - members for the ranatuerin, esculentin, and dermaseptin families. palustrins, nigrocins, phylloseptin, and odorranains also had about peptide members each (table ) . depending on the presence or absence of cysteine (cys), frog amps can be classified into two major classes: peptides with and without cys. a typical cys-containing family is brevinin, which is also the largest frog peptide family in the apd with peptide entries. the lengths of brevinins ranged from to amino acids. the majority of brevinins contain a pair of cysteines at the c-terminal region, forming a rana box. there are also exceptions. some brevinins contain only one cysteine, while others have five cysteines [ , ] . the odd number of cysteines in brevinin may lead to a different functional form by establishing a disulfide bond with either itself or a different molecule with an unpaired cysteine. a typical family of frog amps without cysteine is temporin [ ] . after validation, we found temporins in the apd. these peptides are relatively small with to amino acids. remarkably, temporins contain amino acids. australians characterized amphibian peptides ( %), european scientists isolated and purified such peptides ( %) (figure a ). of note, cathelicidins and defensins, initially discovered in mammals [ , ] , have also been identified in frogs. we found cathelicidins [ ] and three defensins from amphibians [ ] . the discovery of these peptides enriched the reservoir of amphibian peptides. antimicrobial activities include antibacterial, antifungal, antiviral, and anti-parasitic effects on pathogens. in addition, amphibian amps may have other functions such as neutralization of lipopolysaccharides (lps), chemotactic, anti-inflammatory and wound healing [ ] [ ] [ ] . the amphibian peptide counts with a variety of activities are listed in table . in the following, we briefly describe these activities/functions. antibacterial activity. magainin is recognized as the first frog peptide with demonstrated antibacterial activity [ ] . it is used as a model peptide for mechanistic studies. it kills bacteria rapidly. there is a consensus that this peptide acts on bacterial membranes (see introduction). by the time this manuscript was completed, there were amphibian peptides in the apd annotated to be antibacterial. these peptides usually have a broad activity spectrum and kill both gram-positive (g+) and gram-negative (g−) bacterial pathogens. this is one of the driving forces to develop such peptides into potential antibiotics. however, one cannot generalize this to all amphibian peptides as some members show a narrow-spectrum activity at least based on the data in the literature (below). antig-. gram-negative bacteria consist of both outer and inner membranes, making them more challenging to eliminate. while lipopolysaccharides (lps) are a key component of the outer membranes, phosphatidylglycerols (pgs) and phophatidylethanolamine (pes) are frequently found in the inner membranes of bacteria such as e. coli [ ] . there were amphibian amps that are mainly active against gram-negative pathogens. typically, e. coli is used as a model strain for antibacterial assays. out of the antig-amphibian peptides, are active against e. coli. cationic amps can penetrate the outer membranes and exert their damaging effects on inner membranes [ , ] . antig+. gram-positive bacteria do not have an outer membrane, but possess a cell wall [ ] . most amps are believed to target cell membranes. however, there are already amps that target the cell wall [ ] . we found that amphibian amps were primarily active against gram-positive bacterial pathogens. many temporins are such examples [ ] . staphylococcus aureus is often used as a model species for this assay. antig+ peptides were active against s. aureus and were annotated to kill methicillin-resistant s. aureus (mrsa), usually by disrupting membranes. antifungal activity. fungi also have a cell wall (chitin) and inner membranes (ergosterol). the cell wall components are unique and do not exist in human cells, they constitute excellent drug targets [ ] . antifungal activity could be essential to amphibians considering the recent decline. batrachochytrium dendrobatidis was identified as the culprit for this decline [ ] . it is found that non-declining amphibians have more effective amps than those declined species in the same niche [ ] . a total of amphibian peptides were antifungal. candida albicans has been widely utilized as a model organism to test antifungal activity of amps. among these peptides, could eliminate c. albicans. in the apd, amphibian peptides (e.g., magainin , pgla, temporin- p, brevinins, dermaseptin-l , phylloseprin, and ranatuerins) are known to inhibit b. dendrobatidis. amphibian peptides are likely to kill fungi by damaging membranes [ ] [ ] [ ] ] . antiviral activity: viruses are challenging pathogens, as can be seen from the current covid- pandemic. viruses may, and may not, be surrounded by membranes (enveloped and non-enveloped). they can be rna or dna viruses. host defense peptides such as human cathelicidin ll- probably plays a critical role in protecting humans from microbial infection [ ] . amps are likely to be essential in protecting amphibians from ranaviral infection [ ] . there were amphibian peptides with demonstrated antiviral activity [ ] , mostly against human immunodeficiency virus type (hiv- ) due to the interest in search of anti-hiv peptides [ ] [ ] [ ] . the assay is usually conducted in viral infected cell lines by determining the viral plaque changes with and without treatment. the host cells used depend on the viral type. caerin . , caerin . , and maculentin . . are demonstrated to inhibit hiv infection rapidly. they appear to inhibit the initial stage of viral fusion with host cells [ ] . of note is that frog urumin is shown to specifically inhibit influenza a virus h n via binding to hemagglutinin [ ] . temporin b is found to inhibit herpes simplex virus (hsv- ) by disrupting viral membranes. it could influence other stages of viral infection ranging from attachment to replication [ ] . the potential multiple hits could reduce the resistance development of pathogens. antiparasitic activity: protozoa are in the category of neglected tropical diseases (ntds). malaria is one of such diseases. due to the complex life cycle and rapid mutation of parasites, it remains challenging to eradicate such pathogens. amphibian amps also have an effect on parasites in infected cells [ ] . in the apd, amphibian peptides were demonstrated to have activity against parasites. dermaseptin is able to lyse most of the protozoan cells after incubation for h [ ] . dermaseptins also showed anti-malarial potency with % growth inhibitory concentrations (ic ) in the micromolar range [ ] . there are different strains responsible for human malaria, but plasmodium falciparum is most virulent [ ] . in the apd, peptides, including amphibian magainin and dermaseptin-s , are known to inhibit p. falciparum. a natural concern is the eukaryotic nature of parasites, which may limit cell selectivity. however, it is found that membranes of infected human red blood cells more resemble parasites and contain increased amounts of anionic phosphatidylinositol (pi) and phosphatidic acid (pa), which may endow the desired selectivity [ ] . anticancer activity. there is a high interest in the anticancer effect of amps [ , , ] . in the apd, amphibian peptides were demonstrated to have an effect on cancer cells. the classic idea is that cancer cells are transformed and have anionic phosphatidylserine (ps) exposed. such a bacteria-like membrane property may provide a molecular basis for cell selectivity of cationic peptides. however, we found limited cell selectivity for frog temporins with high hydrophobicity [ ] . there are also other anticancer mechanisms. dermaseptin-ps inhibits human glioblastoma u- mg cells (from cells frozen in uppsala in ) via inducing apoptosis at a low micromolar concentration and disrupts cancer cell membranes at -fold the concentration [ ] . moreover, brevinin- ghd was shown to inhibit proliferation of human cancer lines [ ] . as a means to improve cell selectivity of anticancer peptides, some compounds may be utilized to potentiate peptide potency [ ] . anti-diabetic activity. type diabetes (t d) is a condition where insufficient insulin is released to keep the glucose balance in a human body. it is hypothesized that stimulating the production of insulin may provide an avenue of treatment. amphibian peptides can induce the release of insulin from β-cells. these peptides may be useful to treat t d. in the current database, amphibian amps were annotated to have this property [ , ] . further studies are ongoing to improve peptide selectivity and stability of amphibian peptides and their analogs [ ] . this will pave the way to test in vivo efficacy of the promising candidates in animal models. insecticidal activities. pesticides play an important role in suppressing pests harmful to crops. they also caused environmental pollution and are perhaps part of the reasons for the decline of amphibians. in this regard, alternatives are sought to minimize the impact of pesticides on our environment. naturally occurring amps may be sprayed to kill pests. alternatively, frog amps, when expressed in plants, also showed insecticidal effects [ ] . spermicidal activities. amps may also be developed into novel contraceptives. a few dermaseptins were found to have spermicidal activity [ , ] . this is because anionic sulfogalactosylglycerolipid antibiotics , , of (sgg) and sulfogalactosylceramide (sgc) in the head region of sperms make the surface negative and can become a preferred target of amps [ ] . ongoing research is attempting to improve peptide selectivity and stability to avoid the cleavage by host proteases. endotoxin neutralization, immune modulation and wound healing. some frog peptides such as temporins [ ] are known to associate with lps (endotoxin), which can regulate the release of cellular cytokines and lead to an anti-inflammatory effect [ ] . most of these effects can promote wound healing [ ] [ ] [ ] . antioxidant and protease inhibitory activities. in the apd, amphibian peptides were found to have antioxidant effects [ ] . notably, some frog amps can inhibit proteases [ ] . the protease inhibitory properties of amphibian amps is remarkably interesting and deserves further study. is this property necessary for antimicrobial activity? if not, which functional role requires the stability of these peptides in innate immune systems? synergistic effects between amphibian amps. we have assumed that one peptide performs the above tasks. some amphibian amps such as uperin . are capable of forming amyloid fibrils in solution at ph , which are toxic to neuronal cells. since this peptide has the tendency to form different oligomers, these aggregates may play a role in bacterial membrane damage [ ] . moreover, a single peptide chain may be covalently linked via a disulfide bond to achieve other advantages. the dimerization of distinctin to a helix bundle confers stability to the peptide as the monomer is equally active [ ] . furthermore, two different peptides (e.g., pgla and magainin from the same african clawed frog xenopus laevis) can work together to produce a synergistic antimicrobial effect. the most recent results suggest the formation of heterodomains and enhanced peptide affinity toward bacterial phosphatidylethanolamine [ ] . such an assembly and synergy of amphibian peptides further widens our view on innate immune peptides and suggests the benefit of combined therapy. identification of amphibian peptides with the antimicrobial activities or other functions above is an important step. to develop any peptide for therapeutic use, it is essential to know its toxicity to mammalian cells. ideally, the peptide should be not toxic. hemolysis of red blood cells (rbc) is usually the first test since it is convenient to conduct. different blood cells are used in the literature, adding difficulty to compare cytotoxicity of these peptides. we recently compared several types of blood cells and the results are similar to those obtained from human rbcs [ ] . a more thorough toxicity analysis requires the use of different host tissues. in our recent study, we compared the toxicity of the same peptide on kidney, lung, spleen, and liver cells [ ] . more importantly, the food drug administration of the united states (fda) requires toxicity evaluation of the lead compound in at least two different animal models. our recent toxicity evaluation of designer antimicrobial peptides in both mice and rats provides an example for this [ ] . we have annotated hemolysis of amps in the first version of the apd [ ] . it may be reasonable to set µm as a threshold for toxicity, but there is no consensus. perhaps, a definition of the required cell selectivity is more useful considering the differences in mic values of amps. cell selectivity is usually defined as the ratio of % hemolytic concentration (hc ) and mic. at present, amphibian amps were annotated to be hemolytic. because peptide hydrophobicity is known to be important for hemolysis, we calculated the averaged hydrophobic content of these peptides as an approximation. indeed, the total hydrophobic content ( . %) of the hemolytic peptides is higher than that ( . %) summed for all the antibacterial peptides, while both averaged net charge and lysine% are similar (table ) . such a picture has not changed since we observed the higher hydrophobic content for hemolytic peptides in our original database than any antimicrobial group [ ] . this database observation is in line with our discovery that reducing hydrophobicity is a general avenue for improving cell selectivity of membrane-targeting cationic peptides [ , ] . we first defined frequently occurring (abundant) amino acids in the signature of amps (i.e., a profile of amino acids for one or more peptides, see figure ) [ ] . amino acids leucine (l), alanine (a), glycine (g), and lysine (k) are frequently occurring in amphibian amps [ ] . our classification of the amphibian peptides from different continents revealed subtle differences in the proportions of these amino acid residues [ ] . for instance, alanine is more abundant in frog peptides from south america, while leucine is slightly higher in peptides discovered in europe. to validate our observation, we also analyzed brevinins discovered in asia, europe and north america (figure ). the european brevinins also had a higher level of leucine ( %) than the two groups from asia and north america. in contrast, alanine was higher in frog amps from north america ( . %) than those from europe ( . %). it is possible that the amino acid signatures for some frog amps deviate from those of brevinin. on average, temporins ( - residues) are shorter than brevinins ( - residues) [ ] . they have been identified in asian ( peptides), african ( peptides), european ( peptides), and north american frogs ( peptides in figure ) . surprisingly, temporins were especially abundant in leucine with the highest ( %) from european peptides and the lowest ( %) from african amphibian peptides. in fact, all temporins in the apd contain at least one leucine. both alanine and lysine had reduced levels in this special peptide family (below %). the variation in the levels of these four residues (l, a, g, and k) in different peptide families modulates peptide activity. while % of temporins were active only against gram-positive bacteria, only % brevinins were annotated to have the same activity spectrum. there are also common features in the sequences of temporins and brevinins. a hydrophobic cluster or motif, usually containing a proline at position , exists at the n-terminus of temporins and brevinins, while charged residues (usually lysine) are located in the center or both the center and the c-terminus. the hydrophobic amino acids (x) involved in this cluster xxpxx are usually leucine (l), isoleucine (i), valine (v), phenylalanine (f), and occasionally alanine (a). frequently, the second x is a leucine. based on our recent structural analysis [ ] , the proline in this n-terminal motif introduces a bend so that all the four hydrophobic amino acids come together to extend the hydrophobic surface of the three-turn helix. , exists at the n-terminus of temporins and brevinins, while charged residues (usually lysine) are located in the center or both the center and the c-terminus. the hydrophobic amino acids (x) involved in this cluster xxpxx are usually leucine (l), isoleucine (i), valine (v), phenylalanine (f), and occasionally alanine (a). frequently, the second x is a leucine. based on our recent structural analysis [ ] , the proline in this n-terminal motif introduces a bend so that all the four hydrophobic amino acids come together to extend the hydrophobic surface of the three-turn helix. the change in the amino acid signatures for amps with different lengths (brevinin vs. temporins) (figures and ) prompted us to investigate the factors that connect different frog peptide families. we reasoned that frog amps would be ideal for this study since ( ) there is a large number (over ) in the apd and ( ) they are relatively homogenous in sequence, usually forming an amphipathic helix [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to get insight into the length-dependent design of frog amps, we separated these peptides into numerous length groups at a step size of five (e.g., - , - , - , - , - , - , - , - , and - ) . the last group is - because . % of these frog amps in the apd have a peptide length less than amino acids. we excluded the - length group from this analysis because it is a small group with only four peptides and contains the only two frog defensins, which are drastically different from linear helical peptides. the averaged net charge and hydrophobic content for each group of frog amps were then calculated by using the database statistical analysis after each peptide search [ ] . in the apd, the hydrophobic content (pho) is the sum of the percentages for leucine, isoleucine, valine, methionine, alanine, cysteine, phenylalanine, and tryptophan. figure shows the charge-length and pho-length plots. with an increase in peptide the change in the amino acid signatures for amps with different lengths (brevinin vs. temporins) (figures and ) prompted us to investigate the factors that connect different frog peptide families. we reasoned that frog amps would be ideal for this study since ( ) there is a large number (over ) in the apd and ( ) they are relatively homogenous in sequence, usually forming an amphipathic helix [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to get insight into the length-dependent design of frog amps, we separated these peptides into numerous length groups at a step size of five (e.g., - , - , - , - , - , - , - , - , and - ) . the last group is - because . % of these frog amps in the apd have a peptide length less than amino acids. we excluded the - length group from this analysis because it is a small group with only four peptides and contains the only two frog defensins, which are drastically different from linear helical peptides. the averaged net charge and hydrophobic content for each group of frog amps were then calculated by using the database statistical analysis after each peptide search [ ] . in the apd, the hydrophobic content (pho) is the sum of the percentages for leucine, isoleucine, valine, methionine, alanine, cysteine, phenylalanine, and tryptophan. figure shows the charge-length and pho-length plots. with an increase in peptide length, we observed a proportional increase in peptide net charge ( figure a ). on contrary, the hydrophobic content decreased with an increase in peptide length ( figure b ). in other words, longer frog peptides tend to have more charged amino acids at the expense of hydrophobic amino acids. to illustrate, the long frog peptides in the apd are mostly esculentins with - amino acids, an averaged net charge of + . and a hydrophobic content of . %. on the opposite side, the short peptides with - amino acids are mostly temporins and aureins with an average peptide length of . , hydrophobic content of . % and net charge of + . . we then asked which amino acids are proportionally changed with peptide length, leading to the linear correlations in figure . for this purpose, we also calculated the average amino acid contents for each amino acid in the different length groups at the same step size of . we scanned through the plots of the residue content versus peptide lengths for all the amino acids. the top four amino acids that displayed a linear relationship are plotted in figure . they are (a) leucine; (b) glycine; (c) proline; and (d) lysine, three of which are the frequently occurring amino acids of frog amps mentioned above. this is interesting considering leucine, glycine, and lysine are sufficient to design active peptides [ ] . while both glycine and lysine are proportional to peptide length, leucine and proline are inversely proportional to peptide length. the correlation coefficients (r ) for these four plots are in the range of . - . (figure ), indicating a linear relationship. moreover, phenylalanine shares a similar inverse relationship with leucine, although the correlation coefficient is . (not shown). the next amino acid with a correlation coefficient greater than . is cysteine. the rest of the amino acids are less correlated with r ranging from . to . . it is likely that the decrease in proline in longer peptides is to retain the helical structure required for peptide function. we propose that these linear relationships uncovered herein in figures and might be utilized to design new linear peptides at different lengths. to illustrate this, we analyzed dermaseptins (drs), one family of amphibian peptides discovered mostly in south america [ ] . these peptides were separated into three length groups ( - , - , - amino acids) and the features of each group are listed in table . it is remarkable that there is a proportional increase of alanine, lysine, and net charge with peptide length, while leucine and glycine are inversely proportioned to peptide length in dermaseptins. these relationships are essentially the same as we found above based on all frog peptides. the only exception is glycine. this may result from the abundance of a small alanine, which can be regarded as an analog of glycine (table ) . indeed, many dermaseptins start with either the glw or alw sequence motif. w is a highly conserved aromatic residue in dermaseptins, facilitating peptide quantification by uv [ ] . our analysis here in table uncovers the design parameters of dermaseptins at various length groups, yielding novel insight into these amphibian peptides. we then asked which amino acids are proportionally changed with peptide length, leading to the linear correlations in figure . for this purpose, we also calculated the average amino acid contents for each amino acid in the different length groups at the same step size of . we scanned through the plots of the residue content versus peptide lengths for all the amino acids. the top four amino acids that displayed a linear relationship are plotted in figure . they are (a) leucine; (b) glycine; (c) proline; and (d) lysine, three of which are the frequently occurring amino acids of frog amps mentioned above. this is interesting considering leucine, glycine, and lysine are sufficient to design active peptides [ ] . while both glycine and lysine are proportional to peptide length, leucine and proline are inversely proportional to peptide length. the correlation coefficients (r ) for these four plots are in the range of . - . (figure ), indicating a linear relationship. moreover, phenylalanine shares a similar inverse relationship with leucine, although the correlation coefficient is . (not shown). the next amino acid with a correlation coefficient greater than . is cysteine. the rest of the amino acids are less correlated with r ranging from . to . . it is likely that the decrease in proline in longer peptides is to retain the helical structure required for peptide function. we propose that these linear relationships uncovered herein in figures and might be utilized to design new linear peptides at different lengths. to illustrate this, we analyzed dermaseptins (drs), one family of amphibian peptides discovered mostly in south america [ ] . these peptides were separated into three length groups ( - , - , - amino acids) and the features of each group are listed in table . it is remarkable that there is a proportional increase of alanine, lysine, and net charge with peptide length, while leucine and glycine are inversely proportioned to peptide length in dermaseptins. these relationships are essentially the same as we found above based on all frog peptides. the only exception is glycine. this may result from the abundance of a small alanine, which can be regarded as an analog of glycine (table ) . indeed, many dermaseptins start with either the glw or alw sequence motif. w is a highly conserved aromatic residue in dermaseptins, facilitating peptide quantification by uv [ ] . our analysis here in table uncovers the design parameters of dermaseptins at various length groups, yielding novel insight into these amphibian peptides. amphibians produce numerous active peptides for a variety of biological functions such as antioxidant, hormone, growth factor, antibiotics, immune modulation, and wound healing [ , ] [visit also database of anuran defense peptides (dadp); http://split .pmfst.hr/dadp/]. this study focuses on amphibian peptides with demonstrated antimicrobial activity collected in the apd database. however, amphibian amps can have other functions such as anticancer, anti-diabetes, and spermicidal functions ( table ). the expression of such peptides is not understood, but can depend on pathogen types, amphibian species [ , ] , sex [ ] , life stages [ ] , season [ , ] , and geographic regions [ ] . cultivation of frogs under sterilized conditions reduces amp expression and can be restored after exposure to the natural environment [ ] . it is clear that amphibian amps discovered from different frog species are rather different. occasionally, the same sequence was "found in multiple species". a search of the apd using the quoted phrase returned shared amphibian amps. this is relatively small compared to the total of amphibian amps examined during our analysis. male and female frogs may not express the same set of amps. for example, caerin . is expressed in the male australia magnificent tree frog, litoria splendida, but not in female [ ] . the level of such peptides also varies with season [ , ] . cathelicidin-bg peaks in august and september [ ] . modern systems biology, genomics, proteomics, lipidomics, metabolomics and amphibians produce numerous active peptides for a variety of biological functions such as antioxidant, hormone, growth factor, antibiotics, immune modulation, and wound healing [ , ] [visit also database of anuran defense peptides (dadp); http://split .pmfst.hr/dadp/]. this study focuses on amphibian peptides with demonstrated antimicrobial activity collected in the apd database. however, amphibian amps can have other functions such as anticancer, anti-diabetes, and spermicidal functions ( table ). the expression of such peptides is not understood, but can depend on pathogen types, amphibian species [ , ] , sex [ ] , life stages [ ] , season [ , ] , and geographic regions [ ] . cultivation of frogs under sterilized conditions reduces amp expression and can be restored after exposure to the natural environment [ ] . it is clear that amphibian amps discovered from different frog species are rather different. occasionally, the same sequence was "found in multiple species". a search of the apd using the quoted phrase returned shared amphibian amps. this is relatively small compared to the total of amphibian amps examined during our analysis. male and female frogs may not express the same set of amps. for example, caerin . is expressed in the male australia magnificent tree frog, litoria splendida, but not in female [ ] . the level of such peptides also varies with season [ , ] . cathelicidin-bg peaks in august and september [ ] . modern systems biology, genomics, proteomics, lipidomics, metabolomics and microbiota aim at integrating such information for a more complete understanding of peptide functional dynamics in a defined ecological niche. in particular, peptidomic studies led to the discovery of up to peptides in a single frog, which presumably conduct a variety of functions beyond antimicrobial activity. this study shines light on frog amps from the angle of bioinformatics. our study was made possible due to our continued registration of the peptide data into the apd database for over a decade. the reliability of the data has been increasing with time, owing to the establishment of peptide registration criteria and its continued updating. our analysis reveals that four amino acids are abundant in amphibian amps: leucine, alanine, glycine, and lysine. these amino acids are useful in designing new amps [ ] . the variations of such amino acids modulate activity of different peptide families and may offer a living advantage on a continent [ ] . in the following, we discuss peptide length, hydrophobicity, charge, and post-translational modification, which are the key parameters for designing amps. there appears to be a minimal requirement for peptide length in order for it to be antimicrobial. during isolation of novel amps, some truncated peptides were found to be inactive [ , , ] . likewise, further truncation of the minimal antibacterial peptide kr- of human cathelicidin ll- led to inactive peptides such as ri- [ ] . on a different track, we found a non-toxic bacterial membrane-targeting sequence ( figure a ) [ ] could be converted to an antibacterial peptide when d was changed to f [ ] based on sequence homology to the antibacterial and anticancer peptide aurein . ( figure b ), isolated from an australia frog [ ] . however, some amphibian amps, such as esculentins, are much longer and can be up to residues. little is known why and how frog amps are constructed at varying lengths. in terms of the simplicity principle, shorter peptides would be advantageous to reduce synthesis cost. then why bother with long peptides? one possibility is that the short and long peptides confer different antimicrobial capabilities. in particular, longer peptides may be able to form two structural domains that work synergistically against the pathogen membranes. maximin has a helix-break-helix structure in either micelles or organic methanol [ ] . a different model was found in human cathelicidin ll- ( figure c) , where a long helix is separated into two hydrophobic domains for synergistic binding to bacterial lps [ , ] . another possibility is that long sequences can encode additional sequence elements for specific molecular recognition that triggers signal pathways for the functional regulation and coordination required for the survival and health of frogs. future studies may elucidate the exact functions of these peptides in anurans, including a possible role as poisons against predators [ ] . once the peptide length is decided, both hydrophobic and cationic amino acids are considered to generate the popular amphipathic sequences of amphibian amps [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our recent analysis of all the amps in the apd uncovered a linear relationship of the peptide hydrophobic content (pho) with the percentage of arginine, but not lysine [ ] . however, there is a clear linear relationship between lysine and pho when pho is below % in the plot. we found here different requirements for hydrophobic contents depending on the bacterial gram type. the hydrophobic content is higher for amps against gram-positive bacteria than gram-negative bacteria (table ) . leucine is a frequently occurring hydrophobic amino acid that can be used to represent all hydrophobic amino acids. in addition, phenylalanine (phe) appears to be an important aromatic residue to anchor such peptides onto membranes. in several cases (figure ) , we have demonstrated direct magnetic dipole-dipole interactions between aromatic rings of phe and anionic phophatidylglycerol (pg) [ , , ] . this may explain why phe increases with a decrease in peptide length in helical peptides [ ] . a typical example is temporin-shf (sequence: ffflsrif) [ ] , an eight residue peptide with % phe. the four phe residues in this natural frog peptide are reminiscent of human cathelicidin ll- . the four phe aromatic rings (f , f , f , and f ) of ll- ( figure c ), as well as arginine, all interact with anionic lipid pg [ ] . such features are remarkably conserved in temporin-shf, making it a minimal ll- like peptide. in contrast, the averaged net charge is slightly higher for peptides active against only gram-negative bacteria than those against gram-positive bacteria. this basic amino acid is usually lysine in frog peptides. that explains why lysine can be utilized to represent basic amino acids in amphibian amps in general. this database observation is in line with our results obtained from the structure-activity relationship study of a broad-spectrum helical peptide gf- , the major antibacterial peptide of ll- [ ] . a decrease in peptide hydrophobicity by disrupting the helical backbone or by sequence truncation eliminated its activity against gram-positive s. aureus but not gram-negative e. coli. however, conversion of three arginines to alanines in biphe (a stable, selective and potent peptide designed based on gf- ) made the peptide active against s. aureus but not e. coli [ , ] . hence, the ratio between hydrophobic and charged amino acids is a determinant of the peptide activity spectrum (table ) . these results may open the door to engineering peptides with a desired activity spectrum to selectively eliminate the unwanted pathogens with no harm to commensal bacteria. [ , , ] . the importance of the aromatic rings for membrane targeting is indicated by an intermolecular nuclear overhauser enhancement (noe) between the aromatic protons and bacterial anionic phosphatidylglycerols (pgs). once the peptide length is decided, both hydrophobic and cationic amino acids are considered to generate the popular amphipathic sequences of amphibian amps [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our recent analysis of all the amps in the apd uncovered a linear relationship of the peptide hydrophobic content (pho) with the percentage of arginine, but not lysine [ ] . however, there is a clear linear relationship between lysine and pho when pho is below % in the plot. we found here different requirements for hydrophobic contents depending on the bacterial gram type. the hydrophobic content is higher for amps against gram-positive bacteria than gram-negative bacteria (table ) . leucine is a frequently and (c) human cathelicidin ll- determined by multidimensional nmr spectroscopy [ , , ] . the importance of the aromatic rings for membrane targeting is indicated by an intermolecular nuclear overhauser enhancement (noe) between the aromatic protons and bacterial anionic phosphatidylglycerols (pgs). this study shines light on the deployment of charged and hydrophobic amino acids in amphibian peptides with varying peptide lengths. with an increase in the peptide length of anuran peptides, there is a decrease in peptide hydrophobic content and an increase in averaged net charge ( figure ) . moreover, the decrease in peptide hydrophobic content is related to a decrease in leucine. in contrast, the increase in net charge with an increase in peptide length is mainly contributed by lysine ( figure ). the increase in glycine with peptide length may be important for the peptide flexibility required for peptide activity. the decrease in proline in long peptides is likely to avoid the loss of the amphipathic helical structure critical for function. proline can confer special structural and biological function. in buforin, the proline appears to be critical for the peptide to enter the cell [ ] . in a hybrid cecropin-magainin peptide, p , a central proline is critical for antifungal activity rather than a more helical structure [ ] . the linear relationships discovered herein (figures and ) provide one mode to unify a variety of amphibian peptides ( figure b ). these relationships might be useful to design new peptides at different lengths. it is relevant to point out the limitations of these relationships derived from linear anuran peptides, including those stabilized by a rana box. anionic amphibian peptides ( in the apd) do not obey these equations. moreover, these relationships are not applicable to non-linear antimicrobial peptides, such as multiple disulfide-bonded defensins and circular cyclotides [ , , ] . those peptides are more complex and restricted in sequence length and amino acid composition (summarized in ref. [ ] ). as a fourth parameter to understand amphibian amps, post-translational modification can also modulate peptide structure and activity [ ] . although types of chemical modifications have been annotated in the apd [ ] , such modifications are very limited in amphibian peptides [ , [ ] [ ] [ ] [ ] . here we only describe c-terminal amidation and rana box. c-terminal amidation results from chemical modification of the last glycine in the peptide. in the apd database, % of amphibian amps are c-terminally amidated to increase the peptide net charge by + (for accuracy, this modification is considered in the apd when calculating the net charge of peptides). c-terminal amidation can be essential for the antimicrobial activity of amphibian peptides [ , ] . a plausible reason is stabilization of the helical structure to allow for the formation of one additional hydrogen bond. in addition, amphibian peptides in the apd contain a rana box, usually at the c-terminus where - residues are bracketed between a pair of cysteines that presumably form a disulfide bond [ ] [ ] [ ] [ ] [ ] [ ] . this disulfide bond can stabilize the helix and may be a prototype for stapled helices [ ] . disruption of the rana box can reduce peptide activity. note that both c-terminal amidation and a rana box can co-exist in the same frog peptide (seven such peptides in the current apd). it is likely that the rana box is broken in reduced physiological conditions. then, the free cysteines can enhance anti-oxidant ability of the peptide [ ] or perform other functions yet to be elucidated. for decades, scientists have been attempting to understand nature's design principles for amphibian amps [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] so that we can design useful peptide therapeutics. focus has been placed on designing a new generation of antibiotics to overcome the microbial resistance problem. the rapid killing, broad antimicrobial spectrum and low chance of resistance development are regarded as advantages over conventional antibiotics. however, narrow-spectrum peptides are also of outstanding interest nowadays in the era of microbiota. to design an effective peptide, one needs to determine its length, charge, hydrophobic content, and chemical modification. it is to our advantage that the helical backbone can be decorated with different amino acids to confer the desired antimicrobial activity or mechanism of action by varying the type and ratio of charged/hydrophobic amino acids [ ] . in addition, the classic helical structure may be distorted locally or globally to improve protease stability and to reduce peptide toxicity without a substantial loss of antibacterial activity [ , ] . the peptides made entirely using d-amino acids are known to be more resistant to proteases [ ] . magainins have been extensively explored as potential membrane-targeting antibiotics [ ] . buforins may be a useful template to design peptides to attack intracellular pathogens [ ] . the n-terminal region (residues - ) of esculentin- a has also been investigated for various applications [ , , ] . using a computing approach, juretić and colleagues designed a selective peptide [ ] . quantitative structure-activity relationship (qsar) studies are also applied to the design of antimicrobial and antibiofilm peptides [ , ] . we developed database-guided methods [ ] . although our database screening identified potent peptides against mrsa or hiv- [ , ] , it is more inspiring to develop the database filtering technology [ ] . this technology illustrates nicely how to derive the above discussed peptide parameters step-by-step by following the most probable principle. since the target pathogen was mrsa, we arrived at a new peptide template with a high hydrophobic content and low cationicity. remarkably, our database designed dftamp peptide kills mrsa both in vitro and in vivo. however, insertion of additional lysines to the optimized dftamp peptide is detrimental to in vivo efficacy [ ] . our results reveal the importance of balanced charged and hydrophobic residues for systemic efficacy, implying potential systemic uses of certain amphibian peptides with special properties. along the classic path to antibiotic development, there is the desire to make peptides stable so that they can work longer in the presence of host/pathogen proteases. deployment of multiple rana box like stapled motifs along the helix is a state of the art design to confer stability to helical peptides [ ] . distortion of the peptide backbone can also confer stability [ , ] . as an alternative strategy, peptide mimics, such as oligomers of acylated lysines (oaks) and arylamide oligomers, are made to improve peptide stability (reviewed in refs [ ] [ ] [ ] ). they can be normal α-peptide analogs, β-peptides or their combinations [ ] , or non-peptide polymers [ , ] . it is also possible to make small molecules to mimic cationic amps. dftamp mimics are such examples [ ] . the successful synthesis of a variety of mimics underscores that the classic regular amphipathic structure is not a must for antimicrobial activity. irrespective of peptide scaffold or size, however, both charged and hydrophobic moieties are deployed in peptide mimics. systematic studies of these mimics enabled the identification of lead candidates for in vivo tests. in addition, one can consider numerous application strategies to enhance the chance of success. these include ( ) induction of the amp expression in the host, ( ) the use of probiotic microbes to deliver the peptides, ( ) the design of the prodrug, ( ) the construction of peptide conjugates to improve cell targeting, and ( ) combination with conventional antibiotics to improve efficacy and to reduce toxicity [ ] . this study was conducted based on the apd (website: http://aps.unmc.edu/ap; accessed june ). our database project was initiated in as the thesis work of a graduate student in our laboratory. the first version of our database with entries was published in [ ] . the second version reported peptide entries in [ ] . the number of amps increased to in the apd [ ] . the apd database was continuously updated [ ] . as of june , there were amps in this database, mainly from natural sources. in the literature, antimicrobial activity can be obtained by different methods such as broth dilution and the agar diffusion method [ ] . the microdilution method is most widely utilized due to convenience. the results are expressed as mic in either micromolarity (µm) or µg/ml when bacteria do not grow in the -well microplates. the advantage of micromolarity over µg/ml is that it allows us to compare the activity of peptides at different sizes. for those determined by the diffusion method, they will be accepted as long as they are active below the defined concentration. because these amps are tested by different laboratories under different media conditions, it poses a challenge to classify them into strong, medium and weak on a universal activity scale. the apd is attempting to assign "strong" to amps if the mic values are below µm against all testing bacterial strains and "weak" if the mic values are above µm. finally, all the activity annotations were based on the literature data. therefore, the activity spectrum for each peptide entry could change with time when more data become available. the apd also annotated structural information for amphibian peptides during - [ ] . circular dichroism (cd) is frequently utilized to provide secondary structural information. as a helix has a characteristic feature, we included cd information in the apd [ ] as evidence for helix. to date, all the d structures of amphibian peptides in the apd were determined by two-dimensional ( d) nuclear magnetic resonance (nmr) spectroscopy in membrane-mimetic environments such as organic solvents and micelles. the structure can be directly viewed via the protein data bank (pdb at https://www.rcsb.org/) link in the apd. when multiple structures are available, the link usually points at the better-defined structure to reduce redundancy. the biological sources of amps in the apd are systematically separated into six kingdoms: bacteria, archaea, protists, fungi, plants, and animals [ ] . the majority of the peptides originate from the animal kingdom ( %) and amphibian amps account for almost half of animal peptides in this database. in , peptide d structures were unified into four classes: α, β, αβ, and non-αβ based on the presence or absence of an α-helix and a β-sheet in the peptide structure [ ] . in the case of amphibians, it is interesting to note that all the currently known structures are α-helical (determined by solely by nmr or implied by cd). a unified peptide classification scheme that does not depend on peptide source, d structure, and activity was also introduced into the apd in [ ] . many anuran peptides were isolated from skin secretions after electrical stimulation. in the first two versions of our database, we attempted to register all the antimicrobial peptides into the apd, including peptides without activity data. with the application of genomic and peptidomic approaches, peptides were reported in one paper from nine frog species ( each species) [ ] . it became a question whether all the peptides should be registered into the apd. the antimicrobial testing results in that paper gave us the answer. nearly all the isolated peptides show a good activity, whereas chemically synthesized predicted peptides are largely not antimicrobial [ ] . to further verify this, we synthesized selected amphibian peptides, which were previously isolated from frogs without activity data due to a limited quantity, and tested their antibacterial, antifungal, and anticancer activities. these isolated peptides are indeed antimicrobial [ ] . in addition, colleagues have noticed that not all amphibian peptides are antimicrobial [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a peptide might have been isolated earlier, but its antimicrobial status will not be conferred until the antimicrobial activity is determined [ ] . as a consequence, the apd currently registers only peptides with demonstrated antimicrobial activities (mic < µm or µg/ml) [ ] . consequently, some old amphibian peptide entries were replaced during this study due to mic values greater than µm. our rigor in data registration led to a reliable data set. the majority of the peptides are linear, helical [ , ] , and relatively homogeneous, making the results of this analysis useful for peptide design and prediction. the amp analysis was conducted in the apd and linear regression was performed using excel to search for correlated peptide parameters. figures were made using excel/powerpoint and figure using molmol [ ] . amphibian peptides are diverse. as of june , amphibian antimicrobial peptides were registered in the antimicrobial peptide database. these peptides are predominantly linear and usually fold into a classic amphipathic helix in membranes. the deployment of different proportions of hydrophobic and cationic amino acids modulates peptide activity. thus, it is likely that not all amphibian peptides display a broad-spectrum antimicrobial activity (table ) . peptides with a high hydrophobicity and low cationicity tend to be potent primarily against gram-positive pathogens such as mrsa. narrow-spectrum peptides can be important for future personalized antibiotics that selectively eliminate pathogens of concern. our bioinformatic analysis of the frog peptides uncovers multiple length-dependent correlations. short frog peptides tend to be more hydrophobic and less charged. with an increase in peptide length, charged lysine and glycine increase, whereas hydrophobic residues such as leucine and phenylalanine decrease in frog peptides. proline, a helix breaker, is less common in longer peptides discovered in frogs. these relationships may be utilized to design new types of peptides with different sizes. they may also 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supported by the nih grant r ai , r gm , and by the state of nebraska proof of concept award. the author declares no conflict of interest. key: cord- - jup v authors: gouveia, duarte; miotello, guylaine; gallais, fabrice; gaillard, jean-charles; debroas, stéphanie; bellanger, laurent; lavigne, jean-philippe; sotto, albert; grenga, lucia; pible, olivier; armengaud, jean title: proteotyping sars-cov- virus from nasopharyngeal swabs: a proof-of-concept focused on a min mass spectrometry window date: - - journal: j proteome res doi: . /acs.jproteome. c sha: doc_id: cord_uid: jup v [image: see text] rapid but yet sensitive, specific, and high-throughput detection of the severe acute respiratory syndrome coronavirus (sars-cov- ) in clinical samples is key to diagnose infected people and to better control the spread of the virus. alternative methodologies to pcr and immunodiagnostics that would not require specific reagents are worthy to investigate not only for fighting the covid- pandemic but also to detect other emergent pathogenic threats. here, we propose the use of tandem mass spectrometry to detect sars-cov- marker peptides in nasopharyngeal swabs. we documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. in this proof-of-concept study, simili nasopharyngeal swabs spiked with different quantities of purified sars-cov- viral material were used to develop a nanolc–ms/ms acquisition method, which was then successfully applied on covid- clinical samples. we argue that peptides adetqalpqr and gfyaqgsr from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a min window in the tested conditions. these results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry. the new severe acute respiratory syndrome-related coronavirus (sars-cov- ) is the causative agent of covid- , the coronavirus disease that was first reported in december in the city of wuhan, china. because of its easy interhuman transmission, sars-cov- has since quickly spread worldwide, causing more than million covid- diagnosed infections and more than thousand deaths officially reported as off mid-july (https://covid .who.int/). the rapid, sensitive, and specific detection of the sars-cov- virus in large cohorts of clinical samples is of utmost importance to identify infected people and control the propagation of the virus by specific containment measures. at the same time, being able to catch the numerous sars-cov- variants represents an opportunity to identify attenuated forms of the virus. however, the occurrence of specific mutations, especially deletions, may challenge current molecular detection methodologies. the research community has been placing great efforts in the development of quick and accurate detection tests. − the gold standard in diagnostics relies on the amplification and measurement of the viral rna by reverse transcription polymerase chain reaction (rt-pcr). rt-pcr is highly specific and achieves a good compromise between speed ( − min) and sensitivity. however, due to the great demand for pcr-based testing, shortage of rna extraction kits and pcr reagents may have limited the testing capacity in some countries at the early stage of the pandemic. besides, rt-pcr testing of clinical samples may be in some case less efficient due to nucleic acid variations in the targeted regions, primers or their close vicinity, that could affect the amplification rate. , for these reasons, alternative detection strategies that address these concerns should be developed to complement conventional tools. immunoassays, whole-genome sequencing and mass spectrometry (ms) technologies are commonly suggested alternatives to pcr-based assays. among these, new generation ms offers a highly sensitive technology that allows the rapid identification of thousands of proteins present in a single sample. the typing of organisms by tandem mass spectrometry (ms/ms), commonly referred to as "proteotyping", is based on the identification of specific peptide sequences that allow the unambiguous identification of organisms. − the uniqueness of the mass to charge ratios and fragmentation patterns measured in ms/ms allows identification of peptides that differentiate organisms at the subspecies level. although classical ms-based identification of pathogens in the clinical setting is based on whole-cell maldi-tof technology, the field has thrived with the increases in speed, sensitivity, and accuracy of new ms instrumentation in the past decade. the coupling of new generation instruments with the separation power of liquid chromatography makes lc−ms/ms a valuable technology to implement in the routine of clinical laboratories. despite their high potential, the application of lc−ms/ms approaches for virus proteotyping is still scarce. among the few examples available in the literature, lc−ms/ms was shown to be able to detect purified influenza virus and human metapneumovirus in clinical samples. because of the considerable damages of the covid- pandemic, the mass spectrometry community quickly proposed to mobilize its efforts at helping to understand the molecular mechanisms of infection − and at improving detection methods. several research groups started investigating msbased quantification of peptides for the detection of sars-cov- in clinical samples, but most of these results are not yet published. , , these preliminary results indicate that targeted ms, in which the mass spectrometer is programmed to precisely detect and quantify a limited number of peptides of interest, can be successfully applied to virus detection. targeted ms is considered as the gold standard for peptide quantification due to its higher sensitivity when compared to shotgun proteomics approaches. nevertheless, this approach has a much lower throughput and is commonly used to test hypotheses on a subset of proteins of interest, in contrast to discovery shotgun proteomics. by being more flexible, the latter provides a more comprehensive picture of the viral peptidome including the detection of variant sequences because of the possibility of detecting peptides without any previous knowledge of their sequences. here we established the proof-of-concept of the use of ms/ ms for the rapid proteotyping of sars-cov- from clinical samples. we recently published a data set from a shotgun lc− ms/ms experiment performed with sars-cov- infected cells and proposed a list of specific viral peptides that could be used for the development of targeted approaches. interestingly, we observed that some sars-cov- specific peptides eluted from the lc column at narrow windows of retention time. here, we used lc−ms/ms with an orbitrap instrument (q exactive hf) for analyzing the peptidome from nasal swabs spiked with different quantities of viral material. by using a short lc gradient focusing on the region of interest identified in our previous study, we tested the detection of the virus in samples containing different quantities of viral peptides, as well as covid- clinical samples, paving the way for the development of time-efficient viral diagnostic tests based on an alternative platform. two nasopharyngeal swabs were collected using a sterile polyester swab with semiflexible polystyrene handle (puritan) from two healthy volunteers (swabs r and r ). each swab was soaked into a tube containing μl of sterile water, incubated for min at room temperature, and then rinsed with μl of sterile water. the biological material from the μl of solution was precipitated with the addition of μl of trichloroacetic acid at % (w/v) and centrifugation at g for min. the supernatant was discarded. the hardly visible pellet was dissolved into μl of lds x containing % beta-mercaptoethanol, heated for min at °c, and centrifuged briefly. for each swab sample, a volume of μl of lds x sample was deposited on a sds-page gel and run for min. after migration, the gel was rinsed with water, stained with simply blue safestain (invitrogen), and destained overnight in water. the two polyacrylamide gel bands corresponding to the whole proteome of each matrix were excised, processed as described, and then subjected to trypsin gold proteolysis (promega) using . % proteasemax surfactant (promega). the nasal matrix peptide fractions were μl for each swab. peptides from the nasal swab matrices were analyzed with a q-exactive hf mass spectrometer (thermo) coupled with an ultimate lc system (dionex-lc) and operated in datadependent mode as previously described. a volume of μl of peptides was injected, desalted onto an acclaim pepmap c precolumn ( μm, Å, μm id × mm), and then resolved onto a nanoscale acclaim pepmap c column ( μm, Å, μm id × cm) with a min gradient at a flow rate of . μl/min. the gradient was developed from to % of ch cn, . % formic acid in min, and then from to % in min, washed, and re-equilibrated. peptides were analyzed with scan cycles initiated by a full scan of peptide ions in the orbitrap analyzer, followed by high-energy collisional dissociation and ms/ms scans on the most abundant precursor ions (top method). full scan mass spectra were acquired from m/z to at a resolution of with internal calibration activated on the m/z . signal. ion selection for ms/ms fragmentation and measurement was performed applying a dynamic exclusion window of s and an intensity threshold of × . only ions with positive charges + and + were considered. vero e (atcc, clr- ) cells were cultured at °c in % co in dulbecco's modified eagle's medium (dmem, gibco, themofisher) supplemented with % fetal calf serum (fcs) and . % penicillin−streptomycin. the sars-cov- strains -ncov/italy-inmi (genbank mt ) was provided by the lazzaro spallanzani national institute of infectious diseases (rome, italy) via the evag network (european virus archive goes global). sars-cov- stocks used in the experiments had undergone two passages on vero e cells and were stored at − °c. virus titer was . × plaque forming units (pfu)/ml, as determined by standard plaque assay (three dilutions in duplicates). all experiments entailing live sars-cov- were performed in our biosafety journal of proteome research pubs.acs.org/jpr article level facility and strictly followed its approved standard operating procedures. vero e cells ( × ) seeded into cm flasks were grown to cell confluence in ml of dmem supplemented with % fcs and . % penicillin−streptomycin for one night at °c under % co . they were infected at multiplicity of infection (moi) of . . cells were harvested at days post infection (dpi), and viral suspension was recovered after centrifugation at rpm for min to remove cell debris. thirty-three milliliters of the viral suspension was laid on ml of % (w/ v) sucrose cushion prepared in nacl . m, edta mm, and mm tris hcl buffer (ph . ) (tne buffer) in ultra-clear ml tubes (beckman coulter). samples were centrifuged at rpm for h at °c. pellets were solubilized in μl of cold tne buffer, and a volume of . ml was laid on a five step − % (w/v) sucrose gradient prepared in ultra-clear ml tubes (beckman coulter). the tubes were centrifuged at rpm for h at °c in a beckman sw rotor. after recovery of the virus band, the viral suspension was inactivated by incubation with betapropiolactone at a final concentration of . % for h at °c. plaque assay titration was used to quantify the purified virus and validate the viral inactivation. the inactivated purified virus sample (equivalent to . × pfu/ml) was quantified in terms of protein concentration ( . mg/ml) by uv spectrophotometry. a volume of μl was mixed with μl of lds x to obtain a protein fraction of . mg/ml. after denaturation at °c for min, a volume of μl ( . μg of proteins) was deposited on a nupage − % gel (invitrogen) and subjected to min electrophoretic migration. the whole proteome was excised as a single polyacrylamide gel band and subjected to trypsin proteolysis as previously described. an aliquot of μl of peptides was extracted. ms/ms analysis was performed to confirm the high content of viral proteins in this sample (gallais and armengaud, unpublished results). sars-cov- viral peptides ( μl) were diluted in μl of h o, . % tfa. after mixing, μl of this tube was removed and diluted with μl of h o, . % tfa. this was repeated several times to obtain a one-third dilution cascade of viral peptides. two series of simili swabs were prepared in parallel. the two peptide fractions obtained from nasopharyngeal swabs ( μl) were diluted with μl of h o, . % tfa. a volume of μl of this diluted matrix was added to each simili swab samples, giving a final volume of μl per sample. thus, each simili swab contained the equivalent of . % of the proteins harvested by a nasal swab. two biological replicates were prepared using each nasal swab matrix. a volume of μl per sample was injected in the q-exactive hf tandem mass spectrometer. they were analyzed in the same conditions as above except that the gradient was developed from to . % of ch cn, . % formic acid for min at a flow rate of . μl/min. the min ms/ms acquisition started min after injection. nasopharyngeal swabs were collected from covid- diagnosed adult patients as routine medical controls to monitor virus clearance after their hospital isolation. they were tested by rt-pcr assay for detecting sars-cov- in a nasopharyngeal sample (swabs t -t ). this study was approved by the institutional review boards of the university hospital of nimes, france ( − − ). patients have been previously be informed that part of these samples could be used for research purpose and agreed. each swab was soaked into a tube containing ml of phosphate buffered saline (ph . ) sterile solution and transferred in the biosafety level facility. the biological material was precipitated with the addition of . ml of trichloroacetic acid at % (w/v). after centrifugation, the supernatant was discarded and the pellet was dissolved into μl of lds x containing % betamercaptoethanol, heated for min at °c, and deposited on a nupage − % gel (invitrogen). the proteins were subjected to min electrophoresis and treated as described here above to obtain tryptic peptides. ms/ms acquisition was done as for the simili swabs. the min ms/ms acquisition started min after injection with an inclusion list comprising m/z values corresponding to viral peptides. ms/ms spectra from the nasopharyngeal swabs were searched against the generalist ncbinr database ( sequences totalling amino acids) with the mascot daemon . . search engine (matrix science). the search parameters were as follows: full-trypsin specificity, maximum of two missed cleavages, mass tolerances of ppm on the parent ion and . da on the ms/ms, carbamidomethylated cysteine (+ . ) as a fixed modification, and oxidized methionine (+ . ) and deamidation of asparagine and glutamine (+ . ) as variable modifications. psms with an fdr < % were selected for peptide inference. peptides were assigned to taxa using the unipept . web interface with default parameters (equate i/l, filter duplicate peptides). ms/ms spectra from the simili sars-cov- contaminated swabs and from the covid- nasopharyngeal swabs were assigned with the mascot daemon . . search engine (matrix science) as follows: the spectra were first queried against the crap_contaminants_ − − .fasta file and then against the swissprot_human_isl_ _ − − database ( sequences totalling amino acids) in follow-up mode and with the decoy option activated. this last database is the merge of the sars-cov- viral proteins and the swissprot human proteome. the mascot search was performed with the same parameters as above. all peptide matches presenting a mascot peptide score with a fdr lower than % were assigned to protein sequences. ms peak areas were evaluated with skyline. briefly, we created spectral libraries based on the dat files from each mascot search (cut-of . ) and uploaded the ms full scan information contained in the raw files. the protein database previously used for the mascot search was used as background proteome. only the viral proteins were added to the target panel. peptide settings were matched to those used in the mascot search. peak peaking was manually checked for all peptides. the mass spectrometry and proteomics data acquired on simili swabs have been deposited to the proteomexchange consortium via the pride partner repository with the data set identifiers pxd and . /pxd . to assess the performance of shotgun ms-based proteomics in detecting sars-cov- peptides in a background matrix consisting of nasopharyngeal swab protein material, we experimentally created tryptic peptidomes from (i) a purified virus solution obtained from vero e cells infected with a sars-cov- reference strain, and (ii) nasopharyngeal swabs obtained from two healthy volunteers ( figure ). we first characterized the nasal peptidomes and searched for the presence of detectable microorganisms by metaproteomic data analysis. then the virus peptidome was serially diluted into nasopharyngeal swab peptidomes to obtain two sets of seven tubes containing from ng (equivalent to infectious particles) to . ng (equivalent to infectious particle) of viral protein material. the samples were subsequently analyzed by lc−ms/ms. a window of min of acquisition within a min lc gradient was adjusted to target the region of elution of five previously identified virus-specific peptides. the rationale for focusing the mass spectrometry measurements on these peptides was their remarkable sequence conservation among the numerous sars-cov- strains sequenced to date or their specificity to the novel coronavirus. these peptides were the following: eitvatsr, gfyaegsr, htpinlvr, iaghhlgr, and adetqalpqr. while known variants exist for the latter, the other four peptides are conserved along the several sars-cov- sequenced genomes. the ms/ms spectra acquired over min on the two nasopharyngeal swabs were analyzed to infer the main microbial components present in these samples as such presence should be taken into account for creating an ad hoc database for ms/ms interpretation. swabs r and r yielded and ms/ms spectra, respectively, from which and were attributed to and peptide sequences from organisms present in the ncbinr database (fdr < %). these peptide sequences were analyzed with the unipept tool to assess the biodiversity present in each sample through their taxon-specificity characteristics based on the lowest common ancestor approach. only a small proportion of the peptide sequences mapped by unipept belonged to microorganisms (table s ) . a rather low number ( and ) of peptides from the r and r swabs, respectively, were attributed to bacteria, archaea, or fungi. these corresponded to . % and . % of the mapped peptide sequences, respectively. to exclude false positive identifications, we applied a threshold of at least three-taxon specific peptides for organism validation at the species level, corresponding to . % of the total number of species-specific peptides ( and in each sample), as suggested by ref . thus, one low-abundant corynebacterium was identified in sample r , namely corynebacterium accolens, with specific peptides. in swab r , corynebacterium propinguum, corynebacterium pseudodiphtheriticum, and dolosigranulum pigrum could be identified at the species taxonomical rank with , , and specific peptide sequences, respectively. simili swabs containing specific quantities of sars-cov- virus and the equivalent of . % of the nasal matrix protein material collected during sampling were analyzed by ms/ms with a short gradient. we first confirmed on the most diluted fraction that the bacterial signal was negligible for both fractions, thus not to consider at the ms/ms attribution search stage. for this, the two data sets were searched against the generalist database ncbinr to check for the presence of nonhuman peptides in the swab peptidomes. the unipept analysis of the detected peptide sequences showed that only and peptides, from replicate and , respectively, were attributed to bacteria and no bacterial species could be confidently identified (table s ). the results from the short gradient ms analysis on the simili swabs against the specific human/virus database yielded ms/ms spectra recorded in the samples. from these, were attributed to peptide sequences with a journal of proteome research pubs.acs.org/jpr article fdr below % (table s ) . these data allowed for the identification of protein groups (table s ) . a small fraction of peptide-to-spectrum matches (psms), corresponding to . % of the total psms, allowed identification of different viral peptide sequences including the five peptides of interest. the peptides report for structural proteins from the virus: peptides from the nucleocapsid protein (n), peptides from the spike protein (s), and peptides from the membrane glycoprotein (m). at least one viral peptide was identified in all samples independently of the concentration of the viral material, from ng ( pfu) to ng ( pfu). however, no peptide from the virus was identified in the sample containing viral peptides corresponding to . ng ( pfu). the heatmap in figure displays the ms peak areas, the number of psms attributed to each peptide in each sample, and the number of viral peptides identified in each sample. the five peptides with the highest ms peak areas across samples were the following: eitvatsr, gfyaegsr, lnqlesk, adetqalpqr, and kadetqalpqr. among them, eitvatsr, gfyaegsr, and adetqalpqr are between the five peptides of interest. peptide htpinlvr was the seventh most abundant. inversely, peptide iaghhlgr was among the peptides with the lowest ms peak areas, along with peptides msecvlgqsk, lddkdpnfk, and eidrlnevak. as expected, the number of identified peptides decreased as the viral load decreased in the sample. while all peptides were identified in the initial dilution containing ng of viral proteins ( pfu), in highly diluted samples containing ng of viral proteins ( pfu) and ng ( pfu), the virus was proteotyped with only and peptides, respectively. in these samples, only peptides from protein n were detected (adetqalpqr, kadetqalpqr, and gfyaegsr). generally, the peptides from protein n were the most consistently detected across samples. despite being among the peptides with higher peak areas in the chromatograms, peptides of interest htpinlvr and eitvatsr were only detected in the simili swabs containing an estimated ng of viral proteins ( pfu). on the other hand, the two other peptides of interest from protein n, gfyaegsr and adetqalpqr, allowed virus proteotyping in the sample containing ng of viral proteins ( pfu). of note, the peptide identified in the condition with ng of viral proteins ( pfu) is a miss-cleaved version of the adetqalpqr peptide: kadetqalpqr. peptide asanlaatk, that had not been previously selected among the "best" candidates for sars-cov- proteotyping, was detected in the dilution with ng of viral proteins ( pfu) and was the most sensitive peptide from protein s. figure represents the retention times of viral peptides from to min of ms acquisition, and their intensities in the nasopharyngeal swabs were sampled from nine covid- diagnosed patients with different clinical manifestations (moderate symptoms and asymptomatic) and at different postdiagnostic stages (table ). these patients were monitored for virus clearance before hospital discharge, thus the viral load in some of these samples was particularly low. the cohort was not established for assessing the performances of the methodology in terms of clinical diagnostic. because of the complexity of the samples, an inclusion list of m/z signals corresponding to the five peptides of interest as well as other sars-cov- peptides detectable in this gradient region was added to the acquisition method to increase the likelihood of their detection. table s reports this inclusion list, which contained m/z values for different precursors from different viral peptide sequences. the short gradient ms analysis on these clinical samples yielded between and ms/ms spectra recorded per sample. sixty-five spectra were attributed to viral peptide sequences with a fdr below % (table s ) . these data allowed for the detection of six peptides reporting for two viral proteins (table s ) : lddkdpnfk, kadetqaipqr, kkadetqaipqr, adetqaipqr, gfyaegsr from protein n, and eitvatsr from protein m. the heatmap in figure displays the ms peak areas, the number of psms attributed to each peptide in each sample, the number of viral peptides identified in each sample, and the result from the pcr testing performed on the same sample. the virus was confidently proteotyped in clinical swab t moderated ct negative swab t asymptomatic negative negative swab t moderated negative negative swab t asymptomatic negative negative swab t asymptomatic ct negative swab t asymptomatic negative negative swab t moderated ct positive swab t asymptomatic ct positive swab t moderated ct negative a moderated severity with radiological visible signs or asymptomatic. b pcr control done within h after control sampling. ct stands for "cycle threshold". to foster the development of alternative detection methods for sars-cov- , we performed a proof-of-concept study to assess the potential of ms/ms for proteotyping sars-cov- : (i) in simulated nasal swabs containing different quantities of viral peptides and (ii) in nasopharyngeal swabs from covid- diagnosed patients. the two nasal peptidomes collected from healthy donors for the first experiment were first analyzed with a gradient of min to check for the presence of detectable microorganisms from the natural microbiota. a search against a generalist database such as ncbinr detected only trace levels of very low abundant bacteria commonly found in the nasal tract, thus confirming the absence of a measurable microbiome in the swab samples. on the basis of this metaproteomic analysis, we used a human-only database as representative of the nasopharyngeal matrices for the subsequent analysis. the simili sars-cov- contaminated swabs contained a fixed amount of swab peptidome, plus a precise amount of viral peptidome corresponding to the expected quantities extracted from ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), and . ng ( pfu) of sars-cov- to cover the range of the viral load kinetics of sars-cov- infection. it is important to note that the virus produced in vero e cells and purified on sucrose gradient is only partially infectious, and thus, the data are also presented in quantities of viral proteins. the real number of viral particles could be much higher in these samples and could be roughly estimated as the molecular weights of each viral protein are known and if the numbers of molecules per virus particle were documented for sars-cov- . here, we refer to the infectious dose as this is the most important parameter in terms of health concern, but the ratio of infectious particles in the nasopharyngeal swabs of patients may drastically differ from the purified virus fraction used here and could even fluctuate during the course of the pathology. the strategy proposed for the analysis of these simili swabs consisted in a shotgun ms analysis based on a short acquisition of min with a short lc gradient. for the clinical samples, we added an inclusion list of viral peptides in the ms method. the inclusion list allowed forcing the fragmentation of candidate viral peptide ions contained in the background matrix, even when they were not included in the top from the data dependent acquisition method. the shotgun strategy resulted in the detection of viral peptides in six out of the seven conditions tested for the simili swab experiment. from the five peptides of interest, gfyaqgsr and adetqalpqr proved to be the most detectable and most sensitive in this background matrix, allowing proteotyping the virus up to the condition of ng of viral material ( pfu). one of the most interesting results was the omnipresence of peptide adetqalpqr and its two misscleaved versions kadetqalpqr and kkadetqalpqr. these peptides were consistently detected in out of identifications in the six most abundant conditions from the simili swab experiment. peptide kadetqalpqr was identified in all simili swabs from figure . these results clearly show that adetqalpqr, despite being prone to missed-cleavages, is one the most abundant and ionizable peptides and should be the main target for proteotyping sars-cov- . this result was confirmed from the analysis of the clinical swab samples since peptides adetqalpqr, kadetqalpqr, and kkadetqalpqr were undoubtedly the most abundant in samples from covid- patients ( figure and table ). in our previous work, we showed that this peptide sequence is also specific to sars-cov- but presented several variants among the available sars-cov- genomes. therefore, when targeting this peptide for viral detection with ms/ms, we can also take into account both its missed-cleaved versions and its different variants. surprisingly, the high intensity peptide eitvatsr was only identified in simili swabs with high concentration of viral proteic material (figure ). by analyzing the ms and ms/ms spectra from these samples, we confirmed that this peptide coeluted with another intense precursor from the background matrix that was fragmented simultaneously. the low mascot ion score attributed to these spectra hindered the confident identification of this peptide. this coelution effect is most likely due to the use of the short chromatographic gradient, and one way to tackle it would be to use smaller isolation windows for fragmentation. this parameter was tested for the analysis of clinical swabs, but little or no improvement was observed. no ms/ms spectra were validated at fdr % for this peptide in swabs t and t , even with the presence of a ms peak corresponding to this peptide in swab t . this peptide is therefore problematic in this type of matrix and probably not suited for tracking sars-cov- in nasal swab samples with our specific experimental setup. the distribution of the peptides along with the chromatogram from figure shows that the two most detectable peptides gfyaqgsr and adetqalpqr eluted in a narrow window of retention time between − min in simili swab samples. for the clinical swab samples, we observed that the retention time for these two peptides was . ± . min for peptide adetqalpqr, and . ± . min for peptide gfyaqgsr as established with skyline (table s ). in light of these new results, we argue that targeting peptides adetqalpqr and gfyaqgsr with an extra short lc gradient of min coupled to the enrichment of these hydrophilic peptides prior the lc injection could be one way to develop quick and robust assays for detection of the virus in clinical samples and gain in signal/noise ratio. besides their high intensity, these peptides provide the needed specificity for a confident assay: peptide gfyaegsr is highly conserved among different sars-cov- genomes, and peptide adetqalpqr is specific to sars-cov- . the simultaneous detection of these two peptides therefore could provide unequivocal evidence for the presence of the virus. interestingly, a recent not yet published study showed the high potential of the same two peptides by using a targeted proteomics assay. the authors report limits of detection in journal of proteome research pubs.acs.org/jpr article the midattomole range corresponding to theoretically sars-cov- particles in their specific experimental setup. besides shortening the lc gradient to less than three min, sample preparation can also be optimized to develop more rapid peptidome preparation assays and remove too hydrophilic and too hydrophobic peptides that could saturate the chromatography column. here, we performed a sds-page gel and in-gel proteolysis with trypsin to denature proteins and to remove any mass spectrometry-chromatography deleterious compounds that could be present in the nasal swab. this procedure is known to not be optimal as only % of the peptide material deposited on the gel is recovered. the literature is becoming rich in alternative sample preparation protocols for ms-based proteomics. for example, we recently proposed a proteotyping assay based on sp magnetic beads for protein purification and digestion in roughly min. being easily adapted to -well plates and robotization, sp based digestion is the method of choice for quick, highthroughput, and highly reproducible proteome digestions, as recently demonstrated. , pathogen proteotyping by mass spectrometry has emerged in recent years as an interesting alternative to molecular biology assays because of its high specificity and speed. numerous strategies based on tandem mass spectrometry have been already proposed for the detection of pathogens from a large source of samples including environmental and clinical samples. as recently highlighted, faster protocols can now be developed for routine mass spectrometry diagnostics for covid- patients. , however, efforts at automatizing sample preparation and diminishing the costs of tandem mass spectrometry are urgently required to deploy this technology in routine diagnostic laboratory if superior performances or throughput can be achieved. a simplified sample preparation protocol, an associated robust instrument, and low operating costs made the success of whole-cell maldi-tof mass spectrometry. here, the clinical setting of the proposed methodology would require a tandem mass spectrometry instrument coupled to chromatography and an automatized sample preparation. we have recently shown that a sp automated preparation of samples for proteotyping can be performed in a -well plate format within half an hour. the high-throughput of such sample preparation protocol is perfectly adapted to the min tandem mass spectrometry measurement established in the present study. in this proof-of-concept study, we did not evaluate extensively the limit of detection and false positive rate of the methodology. however, we could document on medical samples its feasibility. we have shown that nasal swabs with relatively low viral loads (ct and ct ) can be detected positively with the proposed tandem mass spectrometry method. samples with a trace amount of virus (ct ) were negative. as recently demonstrated, patients with ct above are not contagious and thus can be discharged from hospital care or strict confinement for nonhospitalized patients. therefore, the limit of detection of the method should be further documented once sample preparation, chromatography, and mass spectrometry parameters are further optimized. indeed, sample preparation based on sp capture, as well as the use of functionalized swabs, may further significantly increase the sensitivity of the tandem mass spectrometry proteotyping proposed in the present work. furthermore, more sensitive instrument and ms acquisition modes could be tested to gain further sensitivity. importantly, because test sensitivity is considered as secondary to frequency and turnaround time for covid- surveillance, quick approaches such as the mass spectrometry methodology developed in this proof-ofconcept may have direct application in clinical settings. in this proof-of-concept study, we tested the potential of lc− ms/ms based methods for proteotyping sars-cov- in nasopharyngeal swabs. with a min ms-acquisition window, we were able to identify and quantify several virus-specific peptides that allowed proteotyping the virus in simulated swabs and clinical swabs from covid- patients. we argue that peptides adetqalpqr (and its variant forms) and gfyaqgsr from the nucleocapsid protein are of utmost interest to develop quick and robust targeted assays for proteotyping the virus in nasopharyngeal swab samples. a bigger number of clinical specimens must be tested to validate the usefulness and limits of detection of these peptides to calculate the percentage of confirmation rate of positive pcr results and to develop the shortest possible pipeline to ultimately increase the throughput of the method. given the success of the measures adopted to limit the spread of sars-cov- , a large scale test of our strategy is currently not foreseeable in our country. as a result, while further studies in this context will certainly be of great benefit, results described here offer insight into potential opportunities for the development of new types of clinical diagnostics and significantly facilitate future studies. author contributions ¶ dg and gm contributed equally and should both be considered as first coauthor. ja conceived the study with help from dg, gm, lg, and op. gm and jcg performed the mass spectrometry experimental work. jpl and as contributed the medical covid- swab samples. fg, sd, and lb contributed the sars-cov- biological material. dg, lg, gm, op, and ja analyzed the data. dg and ja wrote the manuscript with help from lg. the authors declare no competing financial interest. the mass spectrometry and proteomics data acquired on simili swabs have been deposited to the proteomexchange consortium via the pride partner repository with the data set identifiers pxd and . /pxd . the authors are indebted to dr silvia meschi (national institute for infectious diseases "lazzaro spallanzani" irccs, via portuense , rome, italia) for making the human -ncov strain -ncov/italy-inmi ( n- ) available. this publication was supported by the european virus archive goes global (evag) project that has received funding from the european union's horizon research and innovation programme under grant agreement no. . the authors are also grateful to the french alternative energies and atomic energy commission (cea) and the anr program "phylopeptidomics" (anr- -ce - - ) that supported part of this study. a pneumonia outbreak associated with a new coronavirus of probable bat origin shortlisting sars-cov- peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data mass spectrometric identifcation of sars-cov- proteins from gargle solution samples of covid- patients development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis rapid detection of covid- causative virus (sars-cov- ) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor detection of sars-cov- in different types of clinical specimens overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for covid- detection correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases chest ct for 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can optimize whole viral particle antgen producton for vaccines coalition, c.-m. the covid- ms coalition-accelerating diagnostics, prognostics, and treatment fast and low-cost detection of sars-cov- peptides by tandem mass spectrometry in clinical samples rna-binding proteins are a major target of silica nanoparticles in cell extracts the unipept metaproteomics analysis pipeline skyline: an open source document editor for creating and analyzing targeted proteomics experiments evaluating the impact of different sequence databases on metaproteome analysis: insights from a lab-assembled microbial mixture the human nasal microbiota and staphylococcus aureus carriage evaluation of sample preparation methods for fast proteotyping of microorganisms by tandem mass spectrometry mass spectrometry: a revolution in clinical microbiology? high-throughput proteotyping of bacterial isolates by double barrel chromatography-tandem mass spectrometry based on microplate paramagnetic beads and phylopeptidomics viral rna load as determined by cell culture as a management tool for discharge of sars-cov- patients from infectious disease wards detection of influenza a virus in swine nasal swab samples with a wash-free magnetic bioassay and a handheld giant magnetoresistance sensing system test sensitivity is secondary to frequency and turnaround time for covid- surveillance key: cord- -w sqy authors: crone, niek s. a.; kros, alexander; boyle, aimee l. title: modulation of coiled-coil binding strength and fusogenicity through peptide stapling date: - - journal: bioconjug chem doi: . /acs.bioconjchem. c sha: doc_id: cord_uid: w sqy [image: see text] peptide stapling is a technique which has been widely employed to constrain the conformation of peptides. one of the effects of such a constraint can be to modulate the interaction of the peptide with a binding partner. here, a cysteine bis-alkylation stapling technique was applied to generate structurally isomeric peptide variants of a heterodimeric coiled-coil forming peptide. these stapled variants differed in the position and size of the formed macrocycle. c-terminal stapling showed the most significant changes in peptide structure and stability, with calorimetric binding analysis showing a significant reduction of binding entropy for stapled variants. this entropy reduction was dependent on cross-linker size and was accompanied by a change in binding enthalpy, illustrating the effects of preorganization. the stapled peptide, along with its binding partner, were subsequently employed as fusogens in a liposome model system. an increase in both lipid- and content-mixing was observed for one of the stapled peptide variants: this increased fusogenicity was attributed to increased coiled-coil binding but not to membrane affinity, an interaction theorized to be a primary driving force in this fusion system. intramolecular cross-linking of peptides, commonly referred to as peptide stapling, is often employed to change or constrain the secondary structure of small peptides and to induce unstructured peptides to mimic complex protein folds and protein−protein interactions (ppis). − stapling also contributes to an increased resistance to denaturation and proteolytic degradation, making it a useful technique for the modification of peptide-based therapeutics. hydrocarbon stapling, a technique which is based on catalyzed olefin metathesis, has seen widespread application with multiple compounds being investigated in academic, preclinical, and clinical studies. − peptide stapling techniques can be broadly divided into two categories: single-and two-component strategies. singlecomponent strategies incorporate amino acids that can be cross-linked selectively, or protection strategies are chosen that allow selective cross-linking. common single-component stapling strategies include disulfide bonding, lactam bridges, , and olefin metathesis. two-component stapling adds a bifunctional cross-linker to bridge two amino-acid side chains; the most common techniques are based on cysteine cross-linking and triazole linkages. − two-component strategies are in principle more complex than singlecomponent strategies, but they allow for a more flexible cross-linker design, as it does not need to be compatible with solid-phase peptide synthesis. although most stapling techniques are used to increase or constrain peptide helicity, systems that compare different methods are often based around short peptide sequences, and although multiple comparisons have been made, , the ideal cross-linking technique is still open to debate. the α-helix secondary structure motif has been mimicked using stapled peptides due to its common occurrence in proteins and therefore its potential as a ppi mimic. coiled coils, which are protein-folding motifs comprising two or more α-helices, are intrinsically helical, and therefore, techniques commonly used for the stapling of helices should permit modulation of coiled-coil interactions. indeed, rao et al. have shown lactam bridges can be used to generate short, helical, cfos binding peptides, and haney and horne have used oxime cross-linking to generate stapled variants of the gcn -p coiled-coil domain. more recently, wu et al. used a bistriazole stapling technique to increase peptide binding to the polymerase α accessory factor ctf , and lathbridge and mason showed that lactam-bridged heptapeptides can be used for the de novo design of a coiled-coil binding peptide. together, these studies provide methods for the crosslinking of coiled-coil or coiled-coil-binding peptides, but it is unclear which method would prove to be the most effective when applied to a different coiled-coil system. the size of the macrocycle formed varies significantly between the different cross-linking techniques, as do the polarity and hydrophobicity of the cross-linkers in question. interactions of the asymmetric oxime moiety with different amino acid side chains resulted in different binding strengths when the cross-linker was reversed in haney and horne's method. this necessitated the preparation and evaluation of both variants, and indicates oxime cross-linking effectiveness is dependent on amino acid composition. our lab has developed a model system for membrane fusion, inspired by naturally occurring snare (soluble nsf attachment protein receptors) proteins. this system consists of a pair of complementary peptides dubbed e and k, which form a heterodimeric coiled-coil that can be attached to lipid membranes via a peg spacer and lipid anchor. like snare proteins, this model system promotes the fusion of lipid membranes, and it can be facilely modified to study the process of membrane fusion via structure−activity relationships. it has recently been discovered that these two peptides play different roles in the fusion process. the interactions of the k peptide with lipid membranes have been hypothesized as an important factor in membrane fusion efficiency. membrane interactions can occur simultaneously with the formation of the coiled-coil domain in a membrane fusion interface (as visualized in scheme c); therefore, a fine balance between the two must be achieved. in addition, both membrane binding and coiled-coil formation depend on the peptides adopting a helical structure; we believe stapling should allow for the generation of peptides with varied helical structures, which will in turn affect coiled-coil formation and membrane binding interactions. studying the effects of modulating the membrane interactions and coiled-coil binding affinity will generate insights into the importance of both factors in membrane fusion. when attempting to modulate the behavior of the heterodimeric coiled coil used in our group, the choice of cross-linking technique was not obvious, due to the differences observed in previously employed cross-linking techniques (vide supra). the position of the cross-linker and the macrocycle size were deemed the most influential characteristics in the previously mentioned cross-linking strategies; therefore, we wanted to evaluate both of these criteria independently for our system. the most favorable candidates could then be used to test the effect of structural changes on coiled-coil-based membrane fusion. one stapling strategy that attracted our attention was developed by the degrado lab, and it is based on the alkylation of cysteine using dibromoxylenes. the advantage of this system lies in the rigidity provided by the aromatic ring, allowing precise spacing between the two thiol moieties by selecting one of the three different structural isomers of dibromoxylene: ortho, meta, and para; scheme . in the original study, meta-xylene showed the most promise as a cross-linker, and further investigations in the same group have therefore focused on this variant. , other recent investigations have also predominantly used the meta derivative, , and when a comparison was made between the isomers, only short or unstructured peptides were used. this means the question of whether, for a helical or coiled-coil peptide, metaxylene is indeed the best cross-linking moiety is unanswered. therefore, to probe the effect of stapling on coiled-coil peptides, we elected to investigate dibromoxylene cross-linking of cysteines, employing all three structural isomers in order to elucidate the role of cross-linker size and its effect on structure and activity. in this study, a library of nine stapled peptides was prepared by modifying peptide k via cysteine alkylation. these stapled k-peptide derivatives exhibited systematic variations in helicity and thermal stability, as observed by circular dichroism (cd) spectroscopy. the coiled-coil binding thermodynamics were studied using isothermal titration calorimetry (itc), and it was discovered that increased coiled-coil binding is based on a preorganization effect. these observed changes in structure and binding dynamics were heavily dependent on the location of the staple and the choice of cross-linker. in lipid-and content-mixing experiments, a significant change in fusogenicity was measured for selected stapled peptides, which was attributed to the altered coiled-coil interactions. stapled peptide design. the starting point for structural modification is one peptide of a three-heptad heterodimeric coiled-coil pair first reported by litowski and hodges. the two peptides are named after the abundance of either glutamic acid (glu, e) or lysine (lys, k), and each peptide contains a c-terminal glycine and either tyrosine or tryptophan as a fluorescent reporter, giving rise to e gy and k gw. to facilitate stapling, two amino acids in peptide k gw were modified to cysteine, spaced i to i + to best match a single αhelical turn. amino acids that are involved in electrostatic (positions e and g) or hydrophobic (positions a or d) interactions were not varied to ensure the stapled peptides retained the same stabilizing coiled-coil interactions as the parent peptides. three different variants were generated each with the cysteines and therefore the staple, in a different heptad, table . each of these positional variants was stapled with ortho-, meta-, and para-dibromoxylene, generating a library of nine stapled peptides. when referring to these stapled peptide variants, a notation which reflects the position and type of cross-linker is used, for example, k gw- m signifies the cross-linker is in the first heptad and the meta variant has been employed. secondary structure analysis. cd spectroscopy was employed to determine the secondary structure of the stapled peptide variants; the effects of both the stapling location and the size of the cross-linker can be clearly observed, figures and s . peptide stapling close to the c-terminus (k gw- variants) showed the largest increase in α-helicity for all three xylenes, whereas modification in the second heptad (k gw- variants) showed the lowest increase. notably, when paraxylene was used as the cross-linker in the second heptad, the overall peptide helicity was reduced, figure b , showing paraxylene is too large to form an ideal α-helix. the n-terminal positions (k gw- variants) all show a moderate increase in helicity, largely independent of staple size, confirming the previously observed trend for hydrocarbon stapling to be most effective at peptide termini. using temperature-dependent cd spectroscopy, an increase in melting temperature (t m ) could be determined for the stapled peptide variants, as shown in figures and s , with the change in t m closely following the observed changes in helicity. c-terminal modification showed the largest increase in melting temperature, with the ortho-xylene cross-linker yielding the most stable peptides over all three peptide variants, followed by the meta-xylene crosslinker. all stapled peptides interacted with e gy, showing typical coiled-coil spectra as is evident in figures c and s . c-terminal stapling showed the highest helicity, while the nterminal stapled peptides did not have increased coiled-coil helicity compared to the staples located in the central heptad. in contrast to the stapled peptides in isolation, meta-xylenemodified peptides show the most α-helical structure as a coiled coil. ortho-xylene stapled peptides had the largest increase in t m for all three positions (figure ), and the trends in coiledcoil stability are similar to those observed for the single peptides, with an average increase in t m of . °c for the stapled peptides (table s ) and . °c for their coiled coils (table s ) . meta-xylene was previously shown to have the largest increase in helicity in small unstructured peptides, but in the e/k system ortho-xylene stapled variants yielded the highest single-peptide helicity and largest increase in t m for both the peptides and their respective coiled-coils. because it is possible that stapling affects coiled-coil interactions without changing peptide helicity as observed via the thermal unfolding experiments, the effect of peptide stapling on coiled-coil binding was further investigated using isothermal titration calorimetry (itc). binding thermodynamics of stapled coiled-coils. direct determination of the dissociation constant (k d ) and enthalpy of binding (Δh b ) and therefore calculation of the free energy (Δg b ) and entropy of binding (Δs b ) is possible using itc ( figure s ), allowing investigation of peptide interactions independent of peptide structure. the results shown in figure and table s show that, in general, coiled-coil binding of peptides k gw and e gy is strongly enthalpically favored but entropically unfavored. the effect of enthalpy can be explained via the formation of amide hydrogen bonds and electrostatic interactions upon folding of the peptide. when the c-terminally stapled variants of peptide k gw are analyzed, the k d is decreased from to and nm for the o and m variants, respectively, and to nm for the p variant. a large decrease in Δs b was observed and was directly related to the size of the implemented staple. ortho-xylene stapling at the c-terminus reduced the effect of entropy upon binding from to kj/mol, a reduction of %. at the same time, an increase in the Δh b from − to − kj/mol was observed, counteracting the observed entropic effects and leading to the conclusion that the mechanism of peptide stapling relies on a preorganization effect: through conformational restriction, the peptide is preorganized in a helical conformation which reduces the entropic effects of binding, but some of the energy that is gained upon formation of an αhelix is also lost. although the k d for the c-terminal ortho-and meta-xylene stapled peptides is comparable, the Δs b is more favorable for the ortho variant, explaining the large differences in t m observed for these two peptides. at all three stapling positions, the ortho variants show a reduced effect of entropy upon binding compared to the meta variants, which is likely caused by the smaller size of the ortho cross-linker. a smaller cross-linker restricts the maximum distance between the two helical turns and therefore limits the number of possible conformations that the peptide can assume. recently, miles et al. screened hydrocarbon-stapled peptides as protein−protein interaction (ppi) mimics against bcl-x l / mcl- and observed similar changes in the Δh b and Δs b for their stapled peptides; however, they observed an overall increase in Δg b . binding kinetics determined via a surface plasmon resonance (spr) assay showed that the binding of their ppi mimic could best be explained via an induced fit mechanism, where the ppi can interact via multiple binding modes. restricting the potential conformations of the peptide through the introduction of a staple reduced the number of possible binding modes and therefore increased the overall k d bioconjugate chemistry pubs.acs.org/bc article of the system. the e/k peptides used in this paper are designed and experimentally confirmed to form heterodimeric coiled-coils exclusively. as there is only one binding mode, the observed changes in structure and stability, as determined via cd, show a direct correlation with the binding thermodynamics in itc: c-terminal stapling using orthoand meta-xylene is the most effective way to increase the binding strength of coiled-coil peptides. membrane interactions of peptide k gw are perturbed by peptide stapling. the effectiveness of e/ k-based membrane fusion is partially attributed to the membrane interactions of peptide k, which are theorized to induce membrane curvature and therefore accelerate the transition from membrane docking to hemifusion. the interactions of peptide k with lipid membranes are based on a lysine snorkeling mechanism, which describes the hydrophobic amino acids in the "a" and "d" position inserting in a lipid membrane, helped by the favorable electrostatic interactions between lysines and the phosphate groups of the lipid membrane. this is a reversible process that can only happen when the peptide folds into an amphipathic helix and all the hydrophobic amino acids are positioned on the same face. peptide stapling, which changes the overall peptide conformation, is therefore theorized to have an effect on membrane binding. the membrane partition coefficient (k p ) of the stapled k variants was assayed via tryptophan fluorescence titration experiments, and the results are shown in figure . membrane binding was either comparable to that of unmodified k gw or was increased up to a factor of and did not show any correlation to the location of the staple or to the overall helicity of the peptide ( figure s ). the difference in partition coefficient between k gw- o and k gw- m is striking, as the value is almost half for the ortho variant despite the helicity of the two being very similar. this shows that the and are shown in figure s . bioconjugate chemistry pubs.acs.org/bc article addition of a hydrophobic cross-linking moiety between the "b" and "f" position does not increase the membrane affinity of amphiphilic α-helical peptides in a structure-dependent manner and leads to the hypothesis that peptide k gw does not bind to liposomal membranes as a highly structured αhelix. cd experiments were performed with the c-terminal stapled peptides in the presence of liposomes, and this data showed a reduced ellipticity at nm and a high / nm ratio (see figure s ). this indicates that the peptides are less α-helical in the presence of liposomes, which supports this hypothesis. if partitioning from the aqueous phase into the membrane is assumed to require partial unfolding of the peptide helix, the difference in binding strength between the ortho and meta variants can also be explained by the smaller size of the ortho cross-linker, which restricts the ability of the peptide to unfold. lipid-and content-mixing is increased for c-terminal stapled peptides. complete fusion of two lipidmembrane-enclosed spaces will result in homogeneous mixing of the lipids in the inner and outer leaflets, as well as mixing of the inner contents. in a liposomal system, this process can be studied via the incorporation of chromophores into the lipid bilayer or on the inside of the liposomes. fusion of these liposomes with nonlabeled liposomes will result in a fluorescence change which can be quantified to compare the peptide fusogenicity. lipopeptides were prepared which contained cholesterol and a polyethyleneglycol (peg ) spacer at the n-terminus, facilitating membrane anchoring. stapled peptides k gw- o and k gw- m were selected for fusion studies because these gave rise to the largest structural and thermodynamic changes. moreover, their binding strength is comparable, but their partition coefficient differs by a factor of ; therefore, by testing both and comparing them to unmodified k gw, the effect of both coiled-coil binding strength and membrane binding on fusogenicity can be determined. the lipopeptides were prepared using a novel on-resin stapling technique enabled by the use of methoxytrityl (mtt) protected cysteine; full details are available in the experimental section. these peptides were tested for fusogenicity together with the lipidated variant of e gy (structures can be found in scheme s ). lipid mixing was quantified using a forster resonance energy transfer (fret) pair incorporated in the lipid membrane, and the results are shown in figure a . the amount of lipid mixing observed was comparable for k gw and k gw- m at % peptide concentration, while the k gw- o variant showed increased lipid mixing min after the start of the experiment. this indicates that docking of the liposomes occurs at the same speed but more lipid mixing occurs for the k gw- o variant. as the absolute amount of lipid mixing was low, the same experiment was also performed with % of the lipopeptides, which doubled the amount of lipid mixing observed while retaining the same trends ( figure s ). content mixing experiments when performed properly are the best measure for complete fusion of two lipid membranes. the membrane-impermeable sulforhodamine b (srb) dye was employed as a fluorescent reporter and showed significant increases in fusion for both stapled peptide variants, figure b , with the k gw- m variant doubling the amount of content mixing compared to k gw (from . % to . %). the k gw- o variant produced an even larger increase; up to % content mixing was observed after min, with an average of . % . table s and figure s . bioconjugate chemistry pubs.acs.org/bc article this is surprising, since there was no observed difference between k gw and the k gw- m variant during lipid mixing experiments. an immediate difference between the three peptides is observed at the start of the experiment, which is not the case for lipid mixing, raising the concern that the stapled peptide variants might be destabilizing the liposomes and causing leakage of srb across the lipid membranes. plain liposomes and liposomes modified with % lipidated e gy were tested for leakage but did not show significant differences ( figure s ), indicating that the stapled peptides do not destabilize the liposomal membranes. insights into the mechanism of coiled-coil based membrane fusion. membrane fusion occurs in multiple stages, starting with the docking of two membranes to create a membrane fusion interface, followed by hemifusion which results in the mixing of the outer lipid leaflets, and proceeding via the formation of a fusion pore to complete fusion of the two liposomes, meaning their contents are exchanged. both lipid-and content-mixing experiments showed increased fusiogenicity for the lipidated k gw- o peptide, with increased content mixing also observed for the k gw- m variant. differences in the lipid-mixing amount are obvious after min, indicating that the rates of initial docking and outer leaflet mixing are comparable for the three peptides. because complete fusion of the liposomes, as judged by content mixing, is increased significantly for the k gw- o variant, the observed difference in lipid mixing is most likely caused by an increased mixing of the inner leaflet lipids. the increased coiled-coil binding strength observed via itc,could explain the increase in fusion except for the fact that k gw- o and k gw- m are dissimilar in their fusogenicity, yet they have a comparable k d . the k gw- o and k gw- m stapled peptides differ in their effects of entropy on coiled-coil binding and the strength of their membrane interactions, which are both increased for k gw- m. the k d of coiled-coil formation is dependent on the association and dissociation rate constants, which show different behavior in temperature dependent stopped-flow experiments of coiled-coil peptides. the dissociation rate was shown to be more dependent on temperature and therefore had a much larger entropic component then the rate of association. the stapled peptide variants tested have a decreased entropic binding component and should therefore also show a lower rate of dissociation. at a membrane fusion interface, dissociation of the coiled-coil is most likely followed either by another peptide binding event or by the insertion of peptide k into the lipid membrane. a decrease in the dissociation rate should therefore result in an increase in the rate of fusion, although the total amount of fusion observed is not expected to change. for snare-mediated membrane fusion, it is known that multiple protein complexes are required to drive fusion of a single vesicle, and the likelihood of fusion occurring is dependent on the number of protein complexes at the fusion interface. , this cooperativity is likely also necessary for our coiled-coil based system, and any interactions that influence the amount of coiled-coils that can be coassembled around a fusion interface will influence the amount of fusion observed. in this case, both k gw- o and k gw- m show increased binding and lowered binding entropy and therefore increased fusion via a lower dissociation rate. for k gw- m, this difference is less significant and is likely to be partially counteracted by the increased membrane affinity of the peptide. this is a competitive interaction in the formation of the coiled-coil complex, and an interaction which can provide a pathway for dissipation of the free peptide after dissociation of the coiled coil. in this manner, the total number of peptide complexes that are formed around a membrane fusion interface is reduced, and no increase in membrane fusion is observed. this reasoning can also be applied to homomeric peptide interactions, which could provide a pathway for dissipation of the lipopeptide away from the fusion interface. cd titration was performed with k gw and the k gw- o and k gw- m analogues to test for homodimerization ( figure s and table s ), but the dimerization constant was found to be comparable for all variants and weak enough that this should not be bioconjugate chemistry pubs.acs.org/bc article considered an important part of the fusion mechanism. this mechanistic understanding derived from the observed differences between the two stapled peptide variants will require further confirmation in different systems and experiments. we have employed a cysteine bisalkylation stapling technique to generate of a series of nine structurally isomeric α-helical peptides that can form a heterodimeric coiled-coil when mixed with their binding partner. cd and itc experiments showed that both stapling location and choice of staple affected the properties of the resulting peptides and coiled-coil complexes, with the largest increase in structure, binding, and stability observed for peptides stapled close to the c-terminus with ortho-xylene. binding strength is increased via a preorganization mechanism, which consists of a large reduction of the unfavored entropic binding component, combined with a negative change in binding enthalpy. ortho-and meta-xylene cross-linkers resulted in similar coiled-coil binding strengths, although ortho-xylene reduced the effect of entropy the most. this effect was true for all three stapling sites and is due to the smaller size of the ortho-xylene cross-linker. although there may be some dependence on amino acid composition, we conclude that ortho-xylene is the best cross-linker to stabilize helical peptides, despite meta-xylene being more widely employed to date. the effect of stapling on peptide-membrane partitioning was determined and showed a -fold difference between stapled peptide variants, although no direct correlation to location or staple type could be made. lipopeptides of k gw- m and k gw- o were prepared via a novel on-resin stapling method. these peptides were tested in lipid-and content-mixing experiments, and large increases in fusogenicity for the k gw- o variant were observed. k gw- m also showed significantly increased content mixing, but it exhibited a similar amount of lipid mixing to the parent peptide. we theorize that these differences in fusogenicity can be explained via reduced dissociation; increasing coiled-coil interactions without increasing lipid membrane interactions allows accumulation of more coiled-coil pairs at the fusion interface and therefore increases membrane fusion. tentagel resin was purchased from rapp polymere. dimethylformamide (dmf), piperidine, pyridine, acetic anhydride, trifluoroacetic acid (tfa), and acetonitrile (mecn) were supplied from biosolve. n,n-diisopropylethylamine (dipea) and oxyma were purchased from carl roth. dichloromethane (dcm) and diethyl ether were supplied by honeywell. hbtu and all protected amino acids except fmoc-cys(mtt)−oh were purchased from novabiochem. all other chemicals were purchased from sigma alrdrich. ultrapure water was obtained from a milli-q water purification system. peptide concentration was established via absorption at nm, determined using a cary- uv−vis spectrophotometer. peptide synthesis and purification. all peptides were synthesized on solid phase using a cem liberty blue automated, microwave-assisted, peptide synthesizer. peptides were prepared on a . mmol scale using tentagel hl ram resin with a loading of . mmol/g. fmoc deprotection was performed using % piperidine in dmf at °c for s. amide coupling was achieved using equiv of protected amino acid, equiv of dic as the activator, and equiv of oxyma as the activator base, heated at °c for s. acetylation of the peptide n-terminus after automated synthesis was performed using an excess of acetic anhydride and pyridine in dmf. lipidated peptides were made on resin via the coupling of . equiv of n -peg -cooh (see supporting information methods for synthesis details), with . equiv of hbtu, and equiv of dipea in dmf for h at room temperature. after washing the resin with dmf, the azide was reduced using equiv of pme ( m in toluene), with : dioxane/water as solvent for . h. after the reaction was finished, the resin was washed thoroughly with : dioxane/water, meoh, and dmf. lipidation was achieved using equiv of cholesteryl hemisuccinate, equiv of hbtu, and equiv of dipea in : dmf/dcm, and this lipidation step was performed twice to achieve complete conversion. after the final coupling, the resin was washed with dmf, meoh, and dcm and dried under vacuum, and the peptide was cleaved using a . : . : . : . mixture of tfa/tips/eddt/water for h, after which the peptide was precipitated in cold diethyl ether, collected via centrifugation, and lyophilized. all peptides were purified by hplc on a shimadzu system consisting of two kc- ar pumps and an spd- a or spd-m a detector equipped with a kinetix evo c column. eluents consisted of . % tfa in water (a) and . % tfa in mecn (b), with all peptides eluted using a gradient of − % b over min, with a flow rate of ml/min. collected fractions were checked for purity via lcms, with the pure fractions being pooled and lyophilized. lc/ms spectra were recorded using a thermo scientific tsq quantum access max mass detector connected to a ultimate liquid chromatography system fitted with a × . mm phenomenex gemini μm c column. lc/ms spectra of the purified peptides can be found in the supporting information. peptide stapling. intramolecular cross-linking was achieved by dissolving the peptide in a : mixture of mecn/h o containing mm nh hco up to a peptide concentration of μm. tcep, equiv, was added as a mm stock solution, and the reaction was stirred for h, followed by addition of . equiv of the dibromoxylene crosslinker ( mm in dmf) and reacted for h. the reaction was quenched by the addition of % acetic acid and purified using preparative hplc. for the lipidated peptides, the cross-linking was performed on the solid phase. in short, cysteines protected with mtt were incorporated into the peptide, and after automated synthesis these protecting groups were removed by incubating the resin with % tfa, % tis in dcm for min, followed by washing the resin with dcm twice. this was repeated until no more color appeared when a small amount of the resin was mixed with tfa. cross-linking was achieved by addition of . equiv of the cross-linker and . equiv of dipea in : dmf/tfe and incubating this reaction for h. on-resin stapling was usually performed before lipidation. circular dichroism measurements. cd spectra were recorded on a jasco j- cd spectrometer fitted with a peltier temperature controller. unless otherwise specified, samples were measured at °c in a quartz cuvette with a mm path length. spectra were recorded from to at nm intervals, with a bandwidth of nm, with the final spectrum consisting of the average of sequentially recorded ( ) with [θ] obs representing the observed ellipticity in mdeg, c being the peptide concentration in mm, n being the number of peptide bonds, and l being the path length of the cuvette in cm. the fraction of the α-helical peptide could be calculated from the mean residue molar ellipticity using eq : tryptophan fluorescence titration. fluorescence was measured in -well plates using a tecan infinite m pro microplate reader. liposomes of the composition : : dopc/dope/cholesterol were prepared at a mm concentration via extrusion in pbs buffer, using an avanti mini extruder with nm polycarbonate membranes. titration series of liposomes in pbs buffer were prepared with concentrations between and μm, with the peptide concentration held constant at . μm. samples were prepared in -well plates, and after min of incubation a fluorescence spectrum was taken between and nm. the maximum fluorescence of each sample was plotted as a fold increase of the fluorescence of the peptide without liposomes present and fitted against eq to determine the partition constant: where the normalized fluorescence, f, is dependent on the maximum fluorescence when all peptide is bound to the membrane f max , the molar partition coefficient k p , the lipid concentration x, and the concentration of water which is assumed to be constant at . m. experimental data representing three separate experiments was fitted to eq using the least-squares method to yield the partition coefficient and the standard error of fitting. isothermal titration calorimetry. itc measurements were performed on a malvern microcal peaq-itc automated calorimeter. in a standard experiment, the measurement cell contained μl of μm peptide k and the syringe was filled with e gy at μm concentration, with both peptides dissolved in pbs. the syringe content was added in injections of . μl at s intervals, except the first injection which was . μl. the reference power was set at . μcal/s, and experiments were performed at °c. the data was analyzed with the microcal peaq-itc analysis software and fitted to a single binding site model to generate the thermodynamic binding parameters. the experiment was repeated on three separate occasions, and the experimental results with the lowest reduced χ value are represented in this paper. lipid and content mixing experiments. liposomes with the lipid composition : : dopc/dope/cholesterol were used at a μm concentration, where % of the lipids was substituted with the respective lipopeptide. lipid films were prepared via evaporation of lipid and lipopeptide stock solutions in : chcl /meoh under a stream of nitrogen, followed by high vacuum for at least h. the lipid films were rehydrated via vortex mixing with pbs buffer and sonication for min at °c in a branson bath sonicator. the liposomes were checked for size and polydispersity (pdi) via dynamic light scattering (malvern zetaszier nano s) and then sonicated for a second time if the pdi was larger than . . lipid mixing was assayed via the incorporation of . % dope-nbd ( , -dioleoyl-sn-glycero- -phosphoethanolamine-n-( nitro- - , -benzoxadiazol- -yl)) and . % dope-lr ( , dioleoyl-sn-glycero- -phosphoethanolamine-n-(lissamine rhodamine b sulfonyl)) in the lipid membranes of the cpkcontaining liposomes. a volume of μl of fluorescent cpkcontaining liposomes was mixed with μl of nonfluorescent cpe-decorated liposomes, and the emission of nbd at nm was followed over time. each experiment included a positive control consisting of liposomes at a μm concentration and . % of both dope-lr and dope-nbd, and a negative control where the fluorescent liposomes were combined with liposomes without cpe. the standard deviation was calculated on the average of four separate measurement samples, and the experiment was repeated at least three times. content mixing was assayed via the incorporation of mm sulforhodamine b in the hydration buffer of cpe-decorated liposomes. after sonication, the unincorporated rhodamine was removed using an illustra nap- size-exclusion column. for each experiment, μl of sulforhodamine-containing cpe-liposomes was mixed with μl of cpk-containing liposomes, and the fluorescence of sulforhodamine followed over time at nm. the value was normalized via referencing a positive control consisting of liposomes containing mm sulforhodamine b prepared in the same manner and a negative control where the fluorescent cpe liposomes were combined with plain liposomes. the standard deviation was calculated on the average of four separate measurement samples, and the experiment was repeated at least three times. change in fluorescence was measured in -well plates using a tecan infinite m pro microplate reader. the percentage of lipid and content mixing was calculated using the following formula (eq ): where f t is the fluorescence at time t and f and f max are the fluorescence of the negative and positive controls at the same time point, respectively. processing of fluorescence data and one-way anova analysis were performed in graphpad prism . structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes stabilized helical peptides: overview of the technologies and its impact on drug discovery constraining cyclic peptides to mimic protein structure motifs analysis of loops that mediate protein-protein interactions and translation into submicromolar inhibitors intramolecular thioether crosslinking to increase the proteolytic stability of affibody molecules activation of apoptosis in vivo by a hydrocarbon-stapled bh helix hydrocarbon-stapled peptides: principles, practice, and progress discovery of hydrocarbon-stapled short alpha-helical peptides as promising middle east respiratory syndrome coronavirus dual inhibition of mdmx and mdm as a therapeutic strategy in leukemia multifaceted roles of disulfide bonds. peptides as therapeutics modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus lactam-stabilized helical analogues of the analgesic mu-conotoxin kiiia design of triazole-stapled bcl α-helical peptides to target the β-catenin/b-cell cll/lymphoma (bcl ) protein− protein interaction targeting the genome-stability hub ctf by stapled-peptide design diversity-oriented stapling yields intrinsically cell-penetrant inducers of autophagy stapling peptides using cysteine crosslinking comparative alpha-helicity of cyclic pentapeptides in water effect of stapling architecture on physiochemical properties and cell permeability of stapled alpha-helical peptides: a comparative study peptide stapling techniques based on different macrocyclisation chemistries contemporary strategies for the stabilization of peptides in the alphahelical conformation oxime side-chain cross-links in an -helical coiled-coil protein: structure, thermodynamics, and folding-templated synthesis of bicyclic species combining constrained heptapeptide cassettes with computational design to create coiled-coil targeting helical peptides membrane fusion: grappling with snare and sm proteins a reduced snare model for membrane fusion distinct roles of snare-mimicking bioconjugate chemistry pubs lipopeptides during initial steps of membrane fusion interplay between lipid interaction and homo-coiling of membrane-tethered coiled-coil peptides controlled liposome fusion mediated by snare protein mimics development of alpha-helical calpain probes by mimicking a natural protein-protein interaction exposing the nucleation site in alpha-helix folding: a joint experimental and simulation study design of a short thermally stable -helix embedded in a macrocycle a new strategy for the in vitro selection of stapled peptide inhibitors by mrna display phage selection of chemically stabilized alpha-helical peptide ligands designing heterodimeric two-stranded alpha-helical coiled-coils -effects of hydrophobicity and alpha-helical propensity on protein folding, stability, and specificity cyclic analogs of galanin and neuropeptide y by hydrocarbon stapling isothermal titration calorimetry of protein-protein interactions hydrocarbon constrained peptides -understanding preorganisation and binding affinity nmr solution structure of a highly stable de novo heterodimeric coiled-coil a coiled-coil peptide shaping lipid bilayers upon fusion spontaneous adsorption of coiled-coil model peptides k and e to a mixed lipid bilayer situ modification of plain liposomes with lipidated coiled coil forming peptides induces membrane fusion energetics of coiled coil folding: the nature of the transition states snare-mediated membrane fusion is a two-stage process driven by entropic forces entropic forces drive self-organization and membrane fusion by snare proteins the authors declare no competing financial interest. the authors gratefully acknowledge professor nathaniel i. martin and ioli kotsogianni from the institute of biology (ibl) at leiden university for access to, and technical assistance with, the itc measurements. key: cord- -bvtchcbt authors: domingo-espín, joan; unzueta, ugutz; saccardo, paolo; rodríguez-carmona, escarlata; corchero, josé luís; vázquez, esther; ferrer-miralles, neus title: engineered biological entities for drug delivery and gene therapy: protein nanoparticles date: - - journal: prog mol biol transl sci doi: . /b - - - - . - sha: doc_id: cord_uid: bvtchcbt the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the design of new chemical entities (nce) for diagnosis and treatment of human diseases has relied on the discovery of active chemical drugs from a diverse library of compounds or from naturally occurring molecules. , further chemical modifications improve pharmacokinetic properties to obtain a final product with a known mechanism of action and decreased toxicity. nonetheless, using such approaches, the final products present low specificity for their target molecules, interacting with many other molecules and accumulating in some tissues, disturbing the correct homeostasis of the system. in some cases, the adverse effects of drug administration exceed pharmacological effect and despite the concise mechanism of action of the drug over the target molecule representing an improvement in the patient's state, the treatment has to be prevented or discontinued. in fact, although a maintained steady increase in the number of launched nce has been observed in the last years, the question arises whether this classical approach has already exhausted the discovery of innovative molecules. on the other hand, macromolecular new biological entities (nbe) have been used to supplement cellular deficiencies or to inhibit cellular pathways exploiting their relatively specific mode of action. proteins and peptides have been obtained first from their natural source or produced as recombinant versions after the development of genetic engineering techniques in the late s. however, the delivery of biological entities is sometimes hampered by its low half-life in the bloodstream by unspecific degradation, resulting in an expensive and ineffective process. nevertheless, some solutions have already been explored for biopharmaceuticals to increase solubility and stability and to reduce immunogenicity including postranslational modifications such as glycosylation and covalent conjugation of polyethylene glycol. thus, one of the main objectives in the use of drugs (for either nce or nbe) is the need to optimize the delivery system to reduce the pharmacological dose which would consequently represent a concomitant reduction in toxicity and cost. in that scenario, new delivery approaches have been implemented using biological interactions such as antigen-antibody binding (immunoliposomes) or more sophisticated interactions including the binding between nutrient concentrator sparc (secreted protein acidic and rich in cysteine) and albumin in the treatment of some types of cancer (abraxane ). , proteins can be then used for their targeting qualities as molecular delivery vehicles both for the specific delivery of drugs or nucleic acids in gene therapy approaches and by themselves as therapeutic molecules. one of the interesting characteristics of proteins is their ability to form intermolecular driven complexes as sophisticated and structurally perfect as in the case of viral capsids. in addition, through the use of genetic engineering, recombinant proteins can be tuned to include additional properties to optimize drug delivery and nucleic acid delivery in gene therapy. in this chapter, the main available strategies to develop protein-based nanovehicles or biopharmaceuticals will be described. in this context, several parameters will be defined such as proper formulation, stability, immunogenicity, and delivery to the correct cell type and cell compartment. modular protein engineering, virus-like particles (vlps), and other self-assembling entities are envisioned as modulatable novel protein nanoparticles able to include many desirable properties in the correct delivery of drugs and nucleic acids. finally, some successful examples of protein nanoparticles on the market will be described in addition to protein products currently in clinical trials and under preclinical research in order to envision which type of protein nanoparticles will be available soon on the market. with the therapeutic molecule to generate a vehicle capable of being transported in the blood if a systemic administration is needed and retaining a significant stability before reaching the target cell. , in addition, the biological system poses specific barriers that have to be overcome such as membranes (cytoplasmic, endocytic, and nuclear), degradation (protease degradation induced by acid denaturalization in lysosomes, cytosolic proteosomes, and nucleases), cytosolic transport, and nuclear entry if necessary. , for central nervous system therapies, the blood-brain barrier (bbb) represents the main bottleneck, and for that, a specific strategy has to be designed. furthermore, the therapeutic complex has to be flexible enough in order to release the therapeutic molecule in the specific cell compartment. thus, several protein motifs have been described to overcome each and every process described earlier so that a modular multifunctional protein can be generated including those modules that are necessary to achieve its goal. in order to get a rational construction of the multifunctional vector, each step has to be carefully taken into account so as to overcome every step which is needed to achieve its final goal (table i) . the dna/rna condensation or drug interaction with the protein vector is a critical step in the formulation of protein nanoparticles for gene therapy. they have to remain attached to the vector during the whole transport process through the body and the cell until it can be released in the desired localization within the target cell. highly positively charged peptides containing a large number of arginines or polylysines have been used to promote electrostatic interactions since nucleic acids are highly negatively charged molecules. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] natural dna-condensing proteins as nuclear histidines or protamines can also be used to bind nucleic acids. [ ] [ ] [ ] [ ] protamine, which is the protein that replaces histidines during the spermatogenesis process, is a sperm chromatin component and just as the histidines do, it has very high dna condensation ability to protect nucleic acids form cytosolic endonucleases. , in addition, as soon as the complex reaches the cellular nucleus, protamine is degraded by chromatinremodeling proteins, releasing the transported dna allowing its expression. , in contrast, polycationic dna condensation modules such as polylysines and polyarginines-even they can present higher dna condensation ability depending on the polycationic chain length-usually present lower dna-releasing ability, interfering negatively with the accessibility of cellular transcription factors and dna expression capacity. all these dna condensation modules described above interact with any dna that is incubated in an unspecific way. however, there are proteins such as gal that are able to recognize specific dna sequences [ ] [ ] [ ] and that permit to bind and condensate specific dna sequences in the final vector. in many cases, the multifunctional protein vector is in vivo administrated by the systemic route in order to travel in the blood and reach the target cells. that exposes the vector to all blood components, making it susceptible to be degraded. thus, it is completely necessary that the vector remain in the blood long enough to be able to reach the target cells. it has also been described that naked dna has an estimated half-life in blood of minutes ; so protein nanovehicles in gene therapy, among other properties, are intended to protect nucleic acids from degradation. one important factor when the vector is exposed to the blood is that it can be recognized by the immune system components and produces an immune response against the vector. thus, it is also very important to try to make the vector as less antigenic as possible in order to avoid being degraded or even being toxic to the organism. peptide uptake or internalization involves a step before the protein binding to the cell surface. this attachment can be either specific or unspecific but in all cases the promotion of its internalization is required. positively charged peptides usually bind the cellular surface by unspecific electrostatic interactions with the negatively charged cell surface proteoglicans. this kind of peptides can be used in the multifunctional protein if specific targeting is not required. cell-penetrating peptides (cpps) have been widely described as unspecific cell-binding and internalization peptides [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (see also the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume). however, specific interactions can be obtained by incorporating cell receptor ligands if cell or tissue targeting is required for the therapeutic action. moreover, some of those ligand-receptor interactions promote the ligand-receptor complex internalization. many peptides have been described in the literature as receptor-specific ligands so any of them can be added to the multifunctional proteins in order to confer them cell specificity. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the most natural specific ligands that can also be used for cell targeting are monoclonal antibodies. , [ ] [ ] [ ] in addition, if no specific peptides are available for an intended target, new specific binding peptides can be found by using phage display or combinatorial chemistry. . endosomal escape several internalization pathways are possible depending on the vector properties, , including endocytosis (clathrin/caveolae-mediated, clathrin/ caveolae-independent), macropinocytosis, and non-endocytic pathways. it is known that more than one internalization pathway can be performed at the same time but usually the peptide-based vector uses endocytic pathways. moreover, it seems that proteins that interact with a specific cellular receptor are internalized by the clathrin-mediated endocytic pathway. most of the generated endosomal vesicles will converge to late endosomes that eventually will fuse with cellular lysosomes. , remaining in the cellular endosomes, the multifunctional protein will be degraded, so it is strictly necessary that the internalized multifunctional proteins be released into the cellular cytoplasm escaping from degradation. several peptides have been described that are able to promote endosomal escape and can be classified into two types depending on their escape mechanism: fusiogenic peptides and histidine-rich peptides. the fusiogenic peptides are small peptides that have hydrophobic amino acids (aa-s) interspersed at constant intervals with negatively charged aa-s. , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] thus, when early endosomes become late endosomes, their low ph induces a conformational change in the peptide, which adopts a alpha-helix structure, in an amphipathic structure able to fuse with the endosomal membrane, leading to pore formation and releasing all the endosomal content into the cell cytoplasm. the histidine-rich peptides are small peptides with a high histidine content whose endosmolytic activity is mediated by a mechanism called ''proton sponge''. , , [ ] [ ] [ ] [ ] when the endosomal ph becomes low in late stages, the imidazole groups of the histidines are protonated and attract endosomal cl À ions, buffering against the proton pump. thus, the endosomes collapse by an osmolytic swelling process and the endosomal content is released to the cell cytoplasm. further details are given in the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume. once the protein has achieved the cellular cytosol, it can be degraded by cellular proteases or by the cellular proteosome system. it is important to avoid this process, especially if the protein has to reach the cellular nucleus. if the final target of the nanoparticle is the cellular cytoplasm, it is necessary that it remain there at least long enough to perform its therapeutical action. several peptide proteosome inhibitors have been described that are able to avoid this type of protein degradation. by adding these peptides to the final protein vector it is possible to protect it and enhance cytoplasmatic stability. epstein-barr virus nuclear antigen (ebna ) contains a proteosome inhibitor consisting of glycine-alanine repeats able to prevent proteosomal proteolysis. it has been shown that a minimum of aa-s gly-ala repeats are necessary to achieve such protective activity. [ ] [ ] [ ] if the protein vector is carrying nucleic acids (dna or rna), degradation by the cytosolic endonucleases has to be taken into account, so it is also very important to protect this nucleic acid in order to maintain its integrity. some dna/rna condensing peptides as protamines also protect the dna against cytoplasmic endonucleases and enhance its stability as has been described above. the cellular cytoplasm is a very crowded and compartmentalized environment where cellular organelles and cytoskeleton make the free diffusion of macromolecules such as protein vectors difficult. however, cytoskeleton elements such as microtubules are used by endosomes and other cytosolic macromolecules for intracytosolic mobility. dyneins have been described as being capable of carrying those macromolecules and endosomes along the microtubules in a retrograde transport toward the nucleus. some small peptides that are able to bind dyneins have been identified. they can be added to the multifunctional protein vector in order to mediate an intracytosolic mobility toward the cellular nucleus. several dynein-binding proteins have been identified in viruses that are able to use this transport system. comparing those protein sequences, a consensus peptide sequence (kstqt) that is able to bind to the dynein lc light chain has been identified. molecules lower than kda/ - nm are able to enter in the cellular nucleus by passive diffusion. however, macromolecules higher than kda/ - nm generally require an active transport system through the nuclear pore system. this transport mechanism generally requires a specific targeting signal peptide named nuclear localization signal (nls). these signaling peptides are usually rich in basic aa-s, which are recognized by the cellular importines and actively transported through the nuclear pore. , monopartite or bipartite nls sequences which are nls peptides that have one or two nls recognized sequences respectively have been described. thus, these peptidic sequences can be added into the final multifunctional protein if nuclear localization is required in order to express a carried dna. it has been reported that a single nls sequence is sufficient to transport the vector to the nucleus and that a large number of nls sequences can result in inhibition of its activity. one of the most used nls signal peptides are fragments derived from the - aa-s of the simian virus sv large tumor antigen (t-ag). other nls sequences can be found in gal , protamines, or tat. , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it is important that when the transported dna reaches the cellular nucleus, it has to be released in order to be accessible to the nuclear transcription factors and achieve the desired expression level. thus, while designing the multifunctional protein vector, this aspect has to be taken into account. once the dna has been released in the cell nucleus, it will be necessary to control its expression level depending on which therapeutic action is being promoted. when the goal is to kill a cell as in cancer therapies, the uncontrolled dna expression levels would not be a problem. however, when a specific protein expression level is required, achieving good control is very important. some expression systems have been developed that can be pharmacologically regulated by oral drug formulation. cell-specific promoters and enhancers can be also used in order to confer high cell specificity to the therapy. , d. ways to get over the bbb the bbb is a hermetic barrier that only allows nonlipophilic molecules smaller than da to cross it. however, some human proteins such as insulin, transferrin, insulin-like growth factor, or leptins are able to go across it by receptor-mediated transporters. thus, the most important factor limiting central nervous system-targeting therapeutics is the presence of the bbb. finding the way to cross it will be the main challenge. some peptides have been described that are able to reach the brain crossing the bbb. moreover, it has been seen that they can be associated with another molecule and transported through the barrier. thus, they could be interesting candidates to be included in the multifunctional vectors if central nervous system targeting is required. , , , antibodies have also been described that bind transferrin and insulin receptors and that are able to cross the bbb efficiently. they can be conjugated with large molecules, allowing its translocation to the central nervous system. , , [ ] [ ] [ ] synthesis, and rational design the development of genetic engineering techniques has increased the natural repertoire of proteins for the design of useful and/or valuable proteins with the aim to obtain new proteins with desired functions. there are three main strategies leading to the construction of engineered proteins: (a) direct evolution, (b) de novo protein design, and (c) rational design. directed evolution has developed quickly to become a method of choice for protein engineers in order to create enzymes having desired properties for all kind of processes. over the past decade, this technique has become a daily part of the molecular toolbox of every biochemist. this is emphasized by the increasing number of publications about the subject. in nature, evolution and creation of new functionalities is achieved by mutagenesis, recombination, and survival of the fittest. directed evolution mimics this and is a process of iterative cycles of producing mutants and finding the mutant with the desired properties. mutations can be introduced at specific places using site-directed mutagenesis or throughout the gene by random mutagenesis. several mutagenesis techniques have been developed in order to avoid codon bias. , the first technique used to mimic evolution was dna shuffling. this method is based on the mixing and subsequent joining of different related small dna fragments in order to form a complete new gene. in the process of shuffling, the recombination frequency is dependent on the degree of homology. a high level of recombination is important to get all possible combinations of mutations. since recombination can be biased, several methods to overcome problems arising from the use of shuffling in the early years were tackled by novel strategies, all having their own advantages and disadvantages. the products obtained by these methods have to be screened for desired qualities and not all of them can be easily screened. de novo protein design offers the broadest possibility for new structures. it is based on searches for amino acid sequences that are compatible with a three-dimensional protein backbone template using in silico techniques. several research groups in the field have applied in silico methods to design the hydrophobic cores of proteins, with the novel sequences being validated with experimental data. in silico protein design has allowed novel functions on templates originally lacking those properties, modifying existing functions, and increasing protein stability or specificity. beyond any doubt, intense research activities are ongoing in the field, the potential of which is simply enormous. so far there have been numerous examples of full sequences designed ''from scratch'' that were confirmed to fold into the target three-dimensional structures by experimental data. the zinc-finger protein designed by dahiyat and mayo was the first one to appear by this method. rational design of proteins is based on the modification or insertion of selected amino acids or domains in a polypeptide chain backbone to obtain proteins with new or altered biological functions. when using that strategy, a detailed knowledge of the structure and function of the backbone protein is needed to make desired changes. this generally has the advantage of being inexpensive and technically feasible. however, a major drawback of this approach is that detailed structural knowledge of a protein is often unavailable or it can be extremely difficult to predict the effects of various mutations. modular engineering enables, by using simple dna recombinant techniques, the construction of chimerical polypeptides in which selected domains, potentially from different origins, provide the required activities. an equilibrate combination and spatial distribution of such partner elements has generated promising prototypes, able to deliver expressible dna or molecules to tissue culture but also to specific cell types in whole organisms. modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of dna or drugs present an attractive approach for the development of self-assembling vectors for targeted gene or drug delivery. one of the first examples was described by the group of uherek et al. they combined a cell-specific target module (antibody fragment specific for the tumor-associated erbb antigen), a dna-binding domain (gal ), and a translocation domain for endosomal escape. in this context, many strategies for the construction of safer vehicles are being explored and the number of nonviral prototype vectors for gene and drug delivery is noticeably increasing. here, the common steps that an approach like this might explore are presented ( fig. ) . when designing a new protein for drug or gene delivery there are many critical aspects, namely (a) design of the vehicle itself, required functions, stability, etc.; (b) production of the protein, suitable expression system, purification procedure, scaling up process, etc.; (c) characterization of the vehicle by physicochemical and functional tests; and finally (d) the administration route and regulatory guidance for biological products. although all these aspects belong to different disciplines, they have to be overviewed together. here, the major needs of a modular protein for gene and drug delivery are presented. to enhance the physicochemical stability of the cargo molecules and their resistance to nuclease/protease-mediated degradation, protein vehicles should ideally exhibit, like their natural counterparts (viruses), nucleic-acid binding and condensing properties. such abilities are, in general, conferred by cationic segments of the main scaffold molecules that interact with nucleic acids, mainly through electrostatic interactions. in addition, such complexes need to efficiently release the nucleic acid in the nucleus (if the cargo is a therapeutic gene), for which endosomal escape is required. such functions have been found in some peptides in many natural molecules and they are suitable for functionalizing protein vehicles. the ability to bind a particular cell type with high specificity is especially significant in a systemic delivery in which appropriate biodistribution and tissue targeting are essential. for nuclear targeting, only naked short nucleic acids can freely enter the nucleus of nondividing cells via free diffusion through the nuclear pore. large molecules require active transport mediated by nlss that are often found in viral proteins. because the molecular mass of plasmidic dna varies from to to mda, dna that is to be expressed, and essentially any macromolecular complex for nucleic acid delivery, requires nlss. the role and types of functional modules peptides used for all these purposes will be discussed in depth in the following sections. in vivo experiments finally, which protein or peptide is better for a given cargo is to be determined empirically and only few rules can be taken literally. , c. production of protein nanoparticles some steps in the production of a protein-based vehicle after molecular cloning such as protein production and protein purification might be experimentally labor intense with a variable success rate. for that reason, when small proteins are needed, solid-phase peptide synthesis guarantees the process. however, the classical procedure of biological production allows scaling up the process in most of the cases and the production of larger polypeptides and fulllength proteins. generally, in protein nanoparticle approaches, the protein is composed by different modules of natural sources such as the cell-penetrating peptide transactivator of transcription (tat) derived from the tat of the human immunodeficiency virus (hiv) or artificial sequences not present in any organism such as the polylysine dna-condensing sequence. once it has been defined which modules will be part of the protein, it is important to define the order they will have in the final construct. it has been demonstrated by boekle and coworkers using melittin conjugated to polyethylenimine (pei) that depending on the side of the linkage (c-or n-terminus), the lytic activity could be changed. some other modules have the need to be in a determined position for its correct function. when producing a protein for gene or drug delivery, it is important to know the origin of its domains to choose the most suitable expression system for its production. for instance, if any module naturally carries a posttranslational modification that is essential for its biological function, the expression system chosen will have to be able to reproduce the same crucial modification. the main biological production systems for protein drugs are described below. escherichia coli is the most widely used prokaryotic organism for the expression of recombinant proteins. the use of this host is relatively simple and inexpensive. added advantages include its short duplication time, growth to high cell densities, ease of cultivation, and high yields of the recombinant product. however, since it lacks fundamental prerequisites for efficient secretion, recombinant proteins manufactured by e. coli systems are mainly produced as inclusion bodies. , moreover, posttranscriptional modifications are not achieved with this system. there are many examples of proteins for gene delivery produced in e. coli with probed efficiency. , like e. coli, yeasts can be grown cheaply and rapidly and are amenable to high-cell-density fermentations. besides possessing complex posttranslational modification pathways, they offer the advantage of being neither pyrogenic nor pathogenic and are able to secrete more efficiently. species established in industrial production procedures are saccharomyces cerevisiae, kluyveromyces lactis, pichia pastoris, and hansenulapolymorpha. s. cerevisiae is the best genetically characterized eukaryotic organism among them all and is still the prevalent yeast species in pharmaceutical production processes. in spite of their physiological advantageous properties and natively high expression and secretion capacity, the employability of yeasts in some cases, however, might reach a limit, particularly when the pharmacological activity of the product is impaired by the glycosylation pattern. in such cases, either a postsynthetic chemical modification has to be considered or the employment of more highly developed organisms. most examples of nanoparticles produced in yeast are for vlps. animal cell expression systems show the highest similarity to human cells regarding the pattern and capacity of posttranslational modifications and the codon bias. however, their culture is more complicated and costlier and usually yields lower product titers. among the known systems, insect cells infected by baculovirus vectors have reached popularity since they are considered to be more stress-resistant, easier to handle, and more productive compared with mammalian systems and are thus frequently employed for high-throughput protein expression. for commercial application, scale-up related questions have to be solved. [ ] [ ] [ ] preferably applied in pharmaceutical production processes are mammalian systems like chinese hamster ovary (cho) cells and baby hamster kidney (bhk) cells. these systems are genetically more stable and easier to transform and handle in scale-up processes, to grow faster in adherent and submerged cultures, and to be more similar to human cells and more consistent in their complete spectrum of modification. in some cases, mammalian cell systems can be the only choice for the preparation of correctly modified proteins. peptides, being complex and unique complex molecules with regard to its chemical and physical properties, can be produced synthetically by the solidphase method. , this technology can be used to avoid problems related to biological production. general advantages of synthetic peptides are that they are very stable compounds, solid-phase chemistry produces highly standardized peptides, and the crucial polycation component is provided by a ''natural'' polycation, thus minimizing toxicity. however, some disadvantages related to synthetic peptides have been reported such as the difficulty to synthesize long and well-folded oligopeptides, peptides with multiple cysteine, methionine, arginine, and tryptophan residues due to technical limitations or production cost. when working with protein nanoparticles, it is very important to characterize them physically and functionally in order to understand their behavior. the size and charge of protein/cargo particles are crucial properties which influence rates of diffusion, binding to polyanionic components of connective tissues, transversal of anatomical barriers, binding of serum proteins, attachment to cells, and mechanisms of endocytosis, among other factors. stability in physiological salt solutions is a key issue for in vivo delivery, as salt is found everywhere in the body. mixing a multivalent polycation and dna results in electrostatic binding of both molecules, with charge neutralization of dna and a particle formation named conjugate. charge neutralization can be easily seen by retardation gel assays and particle formation by dynamic light scattering (dls). dls is a good method to see particle formation but not to quantify relative number of particles of different sizes. to visualize particles, many groups have used transmission electron microscopy (tem) , with good results while others have used fluid particle image analyzer (fpia) to photograph individual particles in physiological solutions. the net charge of protein/cargo particles is an important variable. generally, optimal gene delivery for cell lines requires a net positive charge but, as stated previously, it has to be determined empirically. one of the best techniques to determine the net charge is by calculating the zeta potential that measures the electrophoretic mobility of particles. despite the fact that physical characterization is a key element, understanding and testing the functionality and pharmacokinetics of a gene or drug is the most important part of its development process. most of the initial tests are done using cell lines in in vitro experiments using reporter genes, rna, or drugs. , quantifying the percentage of transfected cells or drug-induced changes is a very valuable tool to evaluate nanoparticle performance in both nuclear and cytoplasmic delivery, respectively. in addition, in vitro experiments may be designed to select a candidate for the in vivo experiments from a group of possible therapy vectors. the quantitative kinetics of particle binding, the molecular basis of particle interactions with target cell membranes, the efficiency of particle internalization, and endosomal escape are all poorly understood. interaction of particles with plasma membranes prior to protein internalization can be either unspecific or specific. untargeted delivery normally is the consequence of electrostatic interactions between anionic ligands in the cell surface and cationic components of the vehicle. on the other hand, targeted delivery to specific membrane molecules is a more sophisticated approach. it aims to improve cell specificity and efficiency, by directing to molecules, only expressed or overexpressed in a particular cell type, that initiate internalization by endocytosis. targeting moieties include many types of molecules and is discussed afterwards. internalization of particles, its mechanisms, and kinetics are not well known and most studies about nanoparticle delivery do not focus on this aspect. there are several endocytic pathways each initiated by different ligands. enhancing the delivery by addition of chloroquine, a synthetic molecule used primarily for the prophylaxis and treatment of malaria that disrupts endosomes, is an accepted parameter to demonstrate endosomal localization of particles. endosomal escape is the area most intensively investigated but is poorly understood. an important practical point to note is that some reagents that are used can be toxic. to enhance this step, anionic fusogenic peptides can be used. these peptides fuse to membranes in an acidic-dependent manner causing its disruption. in gene delivery approaches, translocation of dna expression plasmids into the cell nucleus involves an active, energy-dependent process through the nuclear pore complex. directly injected dna into the cytosol is usually, but not always, poorly transferred to the nucleus , and because of that, the use of proteins carrying cationic nuclear-localizing sequences (such as that of sv large t antigen) has been widely used to overcome this step. iv. natural self-assembling protein nanoparticles: vlps ideal drug delivery and gene therapy vehicles must accomplish some desired features such as appropriate packaging size for its cargo, target cellspecificity, safe and efficient cargo delivery, and protection against immune recognition, or capability to escape immune recognition. moreover, these vehicles must avoid inflammatory toxicity and rapid clearance. in this context, viral vectors have been exploited as one of the vehicles of choice. viruses are nano-sized ( - nm) supramolecular nucleoproteinbased entities, covered or not with a lipid bilayer (enveloped/nonenveloped viruses) that satisfy, into relatively simple structures, outstanding properties and functions that are relevant to drug and gene delivery. viruses are able to recognize and interact specifically with cells by receptor-mediated binding, internalize, escape from endosomes, and uncoat and release nucleic acids in different cellular compartments. they are also capable of transcribing and translating their viral proteins to self-assemble into new infectious virus particles and exit the host cell. , [ ] [ ] [ ] despite all these relevant properties of viral vectors or some other rising vehicles in drug and gene delivery such as cationic liposomes, their therapeutic use presents some limitations and risks because of the complexity of production, limited packaging capacity, insertional mutagenesis and gene inactivation, low probability of integration, reduced efficacy of repeat administration or reduced expression overtime, unfavorable immunological recognition or strong immune response against vehicle and transgene, inflammatory toxicity, and rapid clearance. , in this context, virus capsids or vlps, produced by recombinant capsid proteins but lacking the viral genome, have noticeably emerged as a safer alternative to viral vectors. a. structure of protein self-assembled nanovehicles vlps are classically described as self-assembling, nonreplicative and nonpathogenic, highly organized supramolecular multiprotein nanoparticles (coats) (ranging from to nm) that can be formed from the minimal spontaneous self-assembling of one or more viral structural capsid proteins. it has been described that the self-assembling process of the structural viral proteins for vlp formation involves both spontaneous assembly, under favorable experimental conditions, and the requirement of scaffold proteins as catalysts. , therefore, vlps are considered protein ''coats'', ''shells'', or ''boxes'' that lack the viral genome, still conserve the structure, morphology, and some properties of viruses. some of these properties such as cellular tropism and uptake, intracellular trafficking, membrane translocation, and transfer of nucleic acids or molecules across the cytoplasmic, endosomal, and nuclear membranes are important for drug delivery and gene therapy. , , , [ ] [ ] [ ] usually, the degree of similarity of vlps and their viruses depends on the number of proteins incorporated into the constructs. , since the first description in of the viral dna packaging into mouse polyomavirus (mpyv) vlps and its transduction in vitro, vlps of different viruses such as papillomaviruses, [ ] [ ] [ ] hepatitis b, c, and e viruses, [ ] [ ] [ ] polyomaviruses, vlps offer some structure, dynamics, characteristic features, and functions that make them appealing bionanomaterials to be exploited in the biomedicine arena as drug and gene delivery vehicles and are discussed in detail afterward. on the one hand, viral coat proteins have the ability to spontaneously selfassemble, which ensures the formation of highly organized, regular, repetitive structurally stable, and very low morphological polydisperse particles that provide useful properties to be used as scaffolds for bioimaging, synthesis of bionanomaterials, and as nanocarriers in drug and gene therapy. in addition, homogeneity of particle size and composition is a desired production factor when developing therapeutic molecules. the overexpression of structural viral proteins in a convenient expression system renders recombinant proteins capable of being folded and assembled in discrete organized nanoparticles with a defined size corresponding to the natural capsid geometry. [ ] [ ] [ ] moreover, even though vlps are structurally stable particles, some biochemical and structural studies have observed that viral capsids and bacteriophages may show some structurally dynamic properties varying in shape, size, or rearrangements of the coat proteins, in response to different factors such as ph. [ ] [ ] [ ] [ ] on the other hand, vlps are considered biologically safe nanostructures since they are not infectious (lack of viral genome) and do not replicate, representing a safer alternative to viral vectors. , [ ] [ ] [ ] [ ] however, they can elicit immune and inflammatory responses, especially when repeated administration is needed. it has to be also noted that when used in vaccination, vlps could show excellent adjuvant properties and the majority of vlps stimulate strong cellular and humoral immune responses as direct immunogens. it has been suggested that recombinant vlps derived from infection of insect cells with baculovirus or even those derived from prokaryotic systems could be contaminated with different residual components of these host cells, contributing those impurities to the adjuvant properties. one interesting property of vlps is that coat viral proteins present an enormous elasticity and adaptability to be modified chemically and/or by protein genetic engineering , , to incorporate multiple directed functionalities, in order to be addressed in biomedical applications such as drug delivery or gene therapy. it has been recently reviewed that chemically and/or genetically modified vlps, including cpmv, ccmv, ms , m bacteriophages, and other virus-based nanoparticles, , could maintain their structural integrity and improve their physical stability and, moreover, these modifications could also confer desired cell-targeting properties to the nanovehicle. [ ] [ ] [ ] , , vlps can be successfully engineered with spatial precision to incorporate (attached or genetically displayed on the surface) targeting tissue-specific ligands such as epidermal growth factor (egfr) and antibodies, or other molecules such as oligonucleotides, peptides, gold, and other metals, target proteins, carbohydrates, polymers, fluorophores, quantum dots, drugs, or small molecules. , , moreover, one of the potential benefits of such modifications is that the specific geometric rearrangement confers precise recognition patterns. , furthermore, accessibility of the materials carried within the particle and the ability of inclusion and separation of nucleic acids, small molecules, and unusual cargoes with appropriate charge is another outstanding feature and key advantage of vlps that has also made them excellent vessels for gene and drug delivery. , as described above, vlps can be used as empty nanocarriers to transport molecules chemically attached on their surface or can be loaded ex vivo with therapeutic small molecules such as drugs, dnas, mrnas, sirnas, oligonucleotides, quantum dots, magnetic nanoparticles, or proteins. , , vlps of different papillomavirus and polyomavirus have been widely characterized and used for directed delivery in biomedical applications. , , , , , osmotic shock and in vitro self-assembling of vlp subunits in the presence of the cargo have been the two main strategies used to packaged nucleic acid or other small molecules. it has to be taken into account that some attachment of the cargo on the vlp surface can occur. besides, diversity of natural tropism including liver for hepatitis b vlps, spleen for some papillomavirus and polyomavirus vlps, antigen-presenting cells for certain papillomavirus vlps, and glial cells for human polyomavirus jc (jcv) vlps, among others is one of the key advantages offered by vlps providing a wide spectrum of specific targeting and distribution profiles depending on the directed application. although each vlp has its own characteristic receptors, entry pathway, and intracellular trafficking, it has been demonstrated that tropism of vlps could be customized, modifying the residues identified as ligands of the cellular receptor on vlps' surface or even varying the delivery routes. , , another key advantage of vlps is that they can be easily produced by using a wide range of hosts and expression systems, each of them with its own conditionings. in the past years, there has been an increasing need to improve and optimize efficient large-scale production systems, process control and monitoring, and up-and down-streaming processes. , , , production of vlps usually involves transfection of the cell host expression system of choice with a plasmid encoding one or more viral structural proteins, further and rigorous purification for the removal of immunogenic cellular contaminants, and quality control of the produced vlp and encapsulation of the cargo ex vivo before administration. , the most frequent and convenient expression systems, adaptable to large-scale processes are ( ) yeast cells , , ( ) bacteria , , ( ) green plants infected with modified viruses , , and ( ) cell-free systems. , the preparative and large-scale manufacture of vlps in some of these hosts has been reviewed by pattenden et al. and can be classified into two main methods of bioprocessing: in vivo and in vitro systems. in addition, the capability of in vitro dissociation and reassociation of vlps contribute to the application of easy and more accurate purification methods than those of viral vectors. , furthermore, depending on the expression system, the resulting vlp might be significantly different even though expressing the same viral proteins. thus, a broad spectrum of vlps could be customized depending on the vlp type, the number of proteins needed for vlp assembling, and the targeted final application. , as described above, vlps have great potential as nanocarriers in drug and gene delivery. at the same time, although there is an increasing flow of developments in this area, these vehicles also present some limitations that should be addressed and taken into account, such as residual cellular components, variable yield of functional vlps after disassembly/reassembly process, immunostimulation and unsuitability for repeated administration, tolerance to the transgene, ineffective therapeutic molecule loading, and low transfection rates. protein nanoparticles engineered for drug delivery and gene therapy due to their versatile nanoparticulate structure and morphology, and nonreplicative and noninfecting nature combined with their natural immunogenic properties and ease production, vlps have principally emerged as an excellent alternative tool to attenuate viruses for vaccination. and ebola virus. , although vlp-based vaccines have been primarily developed for their use against the corresponding virus, in the last decades genetic engineering or chemical modifications have been applied in order to generate chimeric vlps. thus, on the one hand, commonly short heterologous peptide epitopes or full proteins that are unable to form vlps or that are unsafe for vaccination have been presented on surface-exposed loops or fused to n-or c-exposed termini of structural viral capsid proteins on vlps. , , different hpv, - hbv, , parvovirus, , and chimeric polyoma vlps have been engineered , and tested for different applications including vaccination against viral or bacterial diseases, against virus-induced tumors, and more recently, for immunotherapy of nonviral cancer. , on the other hand, chemical bioconjugation for covalent coupling of protein epitopes and small molecules to lysines, cysteines, or tyrosine residues of vlp surfaces has been applied in viral or cancer vaccines. chackerian et al. have demonstrated the efficient induction of protective autoantibodies using self-antigen conjugation to hpv vlps. it is important to point out that vlps can also be engineered to incorporate heterologous cell-specific ligands to cell receptors, thus altering their cellular tropism. , , , this great convertibility and flexibility of vlps to be modified (chemically and/or genetically), their high stability, natural and diverse tropism, their nanocontainer properties, and their ability to enter in the cell and incorporate, bind, and deliver nucleic acids and small molecules have positioned vlps as appealing entities not only for vaccination applications but also for a broad spectrum of other diverse and emerging applications in nanomedicine and nanotechnology such as immunotherapy against cancer, , gene therapy delivery of therapeutic genes into specific cells, , , , , , and targeted delivery of drugs and small molecules using vlps as nanocarriers. , domingo-espÍn et al. although there is no commercial vlp as vector in gene therapy, since the initial work in of uncoating polyoma pseudovirus in mouse embryo cells as gene delivery vector and the establishment in of the viral dna packaging into mpyv vlps and its transduction in vitro, different vlps such as hbv and hepatitis e virus, hpv and polyomavirus nanoparticles , , have been modified toward the specific delivery of therapeutic genes and proteins in different target cells, organs, and tissues in vitro and in vivo by systemic injection or oral administration. for example, recombinant vp -based polyomavirus vlps can encapsulate in vitro exogenous dna, and deliver it by cell surface sialic acid residues to human brain cells and fetal kidney epithelial cells. furthermore, vlps have recently emerged as novel nanocarriers or nanocontainers to store unnatural cargos, deliver modified oligonucleotides, synthetic small interfering rnas, and plasmids expressing short hairpin rnas as therapy to downregulate gene expression. , in this context, chou et al. have recently described the use of jcv vlps as an efficient vector for delivering rnai in vitro using murine macrophage raw . cells and in vivo using balb/c mice in silencing the cytokine gene of il- without significant cytotoxicity for systemic lupus erythematosus gene therapy. one of the key aspects in targeted gene and drug delivery is cell-specific delivery. it is important to point out that vlps are tunable nanoparticles that can also be chemically or genetically engineered to modify their natural cellular tropism in order to diversify the range of therapeutic applications in targeted gene or drug delivery. , some effective approaches to modify the natural cellular tropism include: ( ) genetic engineering of vlp chimeras incorporating heterologous cellspecific short peptides that contain recognition sites of target cell receptors. in this context, polyoma and papillomavirus, with solved atomic structures of their major structural capsid proteins, have been extensively used to obtain chimeric vlps as delivery vector systems. , however, this approach has some bioprocessing limitations such as low production levels as a consequence of vlp modification, alterations of size and properties of the vlps that could affect the structural interactions and conformations for vlp assembly, disassembly and packaging, and low transduction efficiencies. ( ) chemical bioconjugation of purified vlps with epitope-containing peptides , or a wide range of small molecules conferring cell-specific targeting such as transferrins, folic acid, or other targeting molecules. as an example, cmpv vlps have been successfully conjugated with tfn using ''click'' chemistry and with nhs-ester-derivatized folic acid, demonstrating both as internalized into hela cells and kb cells, respectively. , ( ) high-throughput library and directed evolution method is a rational approach that has been recently used to engineer viral vectors with the desired tropism properties. ( ) pseudotyping, which consists of replacing the envelope protein of one virus species by the envelope protein of another virus species. ( ) modification of the delivery route of the vlps. it has been shown that the levels of expression of b-galactosidase in heart, lung, kidney, spleen, liver, and brain are different depending on the delivery route of polyomavirus vp vlps. the great accessibility and reactivity showed by vlps, as well as their ability to serve as nanocarriers, which made them suitable to be exploited in gene therapy, have also been applied to targeted drug delivery. genetic modification and/or chemical functionalization of exposed amino acid residues on the capsid surface in order to attach small molecules, such as markers or bioactives molecules, is one of the most common approaches applied to target drug delivery. , as an example, canine parvovirus (cpv) vlps produced in a baculovirus expression system and exhibiting natural tropism to transferrin receptors (tfrs) were chemically modified on accessible lysines of the capsid surface with fluorescent dye molecules and delivered to tumor cells. derivatization of cpv-vlps did not interfere with the binding and internalization into tumor cells. , one limitation of vlps in gene therapy is the low efficiency of gene transduction due to inefficient dna packaging. however, a recent study presented a novel in vivo dna packaging of jcv vlps in e. coli that effectively reduced human colon carcinoma volume in a nude mouse model. in this study, the exogenous plasmid dna was transformed into the jcv vp expressing e. coli. the packaging of the second plasmid occurs simultaneously as the in vivo assembly of the jcv vlp. even though it is still not clear how the plasmid dna molecules are encapsidated in the vlp, the authors showed that gene transduction efficiency by their in vivo package system was about % in contrast to the - % of gene transduction efficiency achieved by the in vitro osmotic shock system. in addition, the administration of exogenous proteins may induce the immune system response, reducing therapy effectiveness or causing undesirable secondary effects, albeit immunological response of protein nanoparticles can be modulated. spontaneous protein self-assembly to form ordered oligomers is a common event in biology. it can prove advantageous in terms of genome-size minimization, formation of large structures, stabilization of complexes, and inclusion of functional features. it has been widely documented that cellular oligomer proteins as well as viral capsids are stabilized by several weak noncovalent interactions as hydrophobic interaction, electrostatic energy, and van der waals forces. [ ] [ ] [ ] these interactions result in a complex quaternary structure described by three symmetry point groups named cyclic (cn), dihedral (dm), and cubic (t, o, i). , the development of computational techniques to predict protein-protein interactions using solved d protein structures makes it possible to predict and/or strengthen experimental data performing in in silico approaches. furthermore, its use opens up the possibility to design proteins not only displaying specific biological functions but also interesting intermolecular interactions to obtain increased multivalency in the resulting complexes. moreover, it should be considered that not only whole proteins can self-assemble in smart nanoparticles; oligopeptides are also capable of forming organized structures. many applications are possible due to the enormous quantity of different combinations and features that can be exploited with peptides. , furthermore, protein-protein interactions are not the unique parameters involved in particle formation, nucleic acid-peptide interactions, salt concentration, order of mix, and ratio between nucleic acid and protein can also strongly influence the condensation process. , due to their natural tendency to self-assemble forming highly ordered structures, viruses provide a wide variety of scaffold proteins which are used as gene/drug carriers. among them, vlps have been reviewed in the previous section. however, simple bacterial proteins can be also utilized as carriers for gene delivery. for example, heat shock proteins (hsp) from hyperthermophilic archeaon methanococcus jannaschii can assemble in a small structure of subunits having an octahedral symmetry. these nm structures are stable at high temperature, up to c, and wide range of ph. residue modifications are allowed to elicit specific attachment of small molecules. , in bacteria, bacterial microcompartments (bmc) which are intracellular organelles consisting of enzymes encapsulated within polyhedral, protein-only shells, somewhat similar to viral capsids, have been described. bmcs are composed of a few thousand copies of a few repeated protein species (including one or more enzymes involved in specific metabolic pathways), and with sizes of around - nm in cross section. the general role of bmcs is to confine toxic or volatile metabolic intermediates, while allowing enzyme substrates, products, and cofactors to pass. the first described bmc, the carboxysome, was isolated in the early s , and has been found to contain both co -fixing ribulose bisphosphate carboxylase/oxygenase (rubisco) , and carbonic anhydrase [ ] [ ] [ ] enzymes. carboxysomes' function is to enhance autotrophic co fixation at low co levels. other bmcs were later identified in cyanobacteria and some chemoautotroph bacteria. among them, bmc proteins have been later found to be encoded in the propanediol utilization operon (pdu) of the heterotroph salmonella and by an operon for metabolizing ethanolamine (eut) in enteric bacterial species, including salmonella and escherichia. salmonella enterica forms a polyhedral organelle during growth on , -propanediol ( , -pd) as a sole carbon and energy source, but not during growth on other carbon sources. , the pdu organelles' function is to minimize the harmful effects of a toxic intermediate of , -pd degradation (propionaldehyde). [ ] [ ] [ ] other studies have shown that a polyhedral organelle is involved in ethanolamine utilization (eut) by s. enterica. the function of the eut microcompartment is to metabolize ethanolamine without allowing the release of acetaldehyde into the cytosol, therefore minimizing the potentially toxic effects of excess aldehyde in the bacterial cytosol [ ] [ ] [ ] and also preventing volatile acetaldehyde from diffusing across cell membrane. so far, about proteins containing bmc domains have been identified, covering at least different bacterial phyla. the typical bmc protein consists of approximately amino acids, with an alpha/beta fold pattern. , some individual bmc proteins self-assemble to form hexamers, which further assemble side by side to form the flat facets of the shell. , , the formation of icosahedral, closed shells from such flat layers was elucidated in part by structural studies in carboxysomes: some bmc proteins assemble to form pentamers, which are located at and form the vertices of the icosahedral shell. mechanisms directing enzyme encapsulation within protein-based bmcs have been studied during the last years. it has been described that, in some carboxysomes, protein ccmm is used as a scaffold to form interactions between both shell proteins and enzymes, , through a ccmm c-terminal region with homology to the small subunit of rubisco. other studies revealed that pdu shells can self-assemble without needing interior enzymes and that carboxysomes can self-assemble in vivo when rubisco has been deleted. regarding properties of the encapsulated enzymes, in the pdu bmc some of the internal enzymes are encapsulated by specific n-terminal targeting sequences. , in this line, sutter and colleagues described a conserved c-terminal amino acid sequence that mediates the physical interaction of an iron-dependent peroxidase (dyp) or a protein closely related to ferritin (flp) with a specific type of bmc (encapsulins). in another example, an icosahedral enzyme complex, lumazine synthase (aals) from bacillus subtilis and aquifex aeolicus, was engineered to encapsulate target molecules by means of charge complementarity and can also be modified to give different characteristics to the assembled structure. moreover, enzymatic subunits, like e of pyruvate dehydrogenase from bacillus stearotermophilus, can be modified to be used in gene delivery. e peptides naturally form a dodecahedron of subunits of nm in diameter allowing modification for drug-like accommodation. the assembling/disassembling of these structures can be modulated by changing the operative ph in the experimental environment. these nanoparticles can also be functionalized with antigens for vaccine development. , according to these results, specific targeting sequences could be of use in biotechnological applications to package proteins inside the stable selfassembled icosahedral shell of bmcs, offering appealing opportunities to manipulate in the laboratory such nanocages to fill them with therapeutic molecules. the simplicity of this system makes it very attractive for engineering studies to design, mimicking nature, new applications in biotechnology, providing a new, intriguing platform of microbial origin for drug delivery. bovine serum albumin (bsa) is able to form microspheres after sonochemical treatment in aqueous medium. chemical effects of ultrasound radiation and coupling with an anticancer drug such as taxol (paclitaxel) led to the assembling of a spherical carrier with an average diameter of nm. bsa particles resulting from s-s bonds, due to ho radical formation, are able to release the encapsulated taxol in cancer tissue with best results if compared with mere taxol treatment. this drug for breast cancer treatment is commercially available. , also little cationic peptides can lead to self-assembling particles. among others, arginine-rich cationic peptides are widely known as good tools for gene delivery. for example, purified r -tailored gfp in solution is described to form nanodisk particles nm in diameter. this structure is proved to be induced by the arg tails and is able to bind and condense dna. these nanodisks are also able to deliver dna toward the nucleus where the reporter gene is expressed. on the other hand, the expression of recombinant proteins over physiological rates can cause a bad functioning of cellular quality control system, leading to self-organizing, pseudo-spherical, protein aggregates known as inclusion bodies. these mechanically stable nanoparticles, ranging from to nm in diameter, were considered for a long time as undesired bio-products. recently, it became clearer that they are suitable for medical approaches when utilized as scaffold surface to promote cellular proliferation. [ ] [ ] [ ] one of the most difficult goals for a foreign gene delivery is to reach the nucleus. an approach to overpass this obstacle is by fusing an nls in a nonessential position of a dna-binding protein. such type of modification has been described for a tetracycline repressor protein (tetr) fused with an sv nls. the tetr-nls affinity and specificity to teto dna sequence is exploited to form spontaneous protein-dna complexes which allow an enhancing of dna transportation into the nucleus and subsequent expression of foreign genes, combining the two peculiar characteristics of each fusion component. there is still a tremendous gap between progresses made in protein-based nanoparticle research for drug delivery and clinical reality. hundreds of publications in basic research describe the combination of two or more functional elements in a single protein nanoparticle, by which the delivery of a carried drug is enhanced. these agents act by improving critical steps in the drug delivery process, such as increasing the systemic stability or tissue specificity, favoring internalization, endosomal escape, and entry into the nucleus, or transporting therapeutic material through the bbb, in in vitro and in vivo studies. besides the human recombinant therapeutic proteins currently on the market (or functional segments of them), there are also some fusion proteins approved for clinical use (most by incorporating an antibody fragment or a ligand to enhance cell specificity). sadly no gene therapy trials have so far used full protein carriers in vivo, but rather peptide-functionalized vehicles. bottlenecking the gap between research and clinical application, the us fda/european medicines agency (emea) only approves human proteins, to avoid the risk of an immune response that could affect not only the effectiveness of the nanoparticle but also challenge patients' health. another critical factor is the administration route, where the protein is degraded before arriving at the target; this problem could be solved or minimized by the use of protein d-isomers, pegylation, or the design of protecting groups for labile sites. despite the current situation mentioned above, there are many good examples of multifunctional modular proteins that, when carrying therapeutic material, can improve the prognosis in vivo in animal models for different diseases. these examples are reviewed below, along with those few protein nanoparticles that are currently on the market or in clinical trials. albumin is a natural protein transporter of hydrophobic molecules throughout plasma that has been approved by the fda to reversibly bind water-insoluble anticancer agents, as is the case of albumin-bound (nab) paclitaxel, abraxane . this albumin-nab technology-based drug is in use in patients with metastatic breast cancer who have failed combination therapy, and it is the first protein nanoparticle approved by the fda. albumin potentiates paclitaxel concentration within the tumor by increasing paclytaxel endothelial transcytosis through caveolae formation. it also contributes to the fact that tumors secrete an albumin-binding protein sparc (also called bm- ) to attract and keep albumin-bound nutrients inside the tumor cell. the albuminpaclitaxel complex was not formally considered a nanoparticle in the united states (due to an average size of nm) but only so in europe. apart from whole recombinant therapeutic proteins being currently commercialized, there are also some examples of vehicles formed by chimerical proteins with target ligands already in the market. dab il- (denileukin diftitox or ontak) is a fusion of diphtheria toxin catalytic and translocation domains for lethal effect and interleukin- (il- ) to gain cell specificity in the treatment of persistent or recurrent t-cell lymphoma. belatacept (bms- ) is a ctla -ig fusion protein formed by the cytotoxic t-lymphocyteassociated antigen joined to an immunoglobulin g fc fragment fusion protein, developed by bristol-miers-squibb. etanercept (enbrel) fusion tumor necrosis factor receptor (tnfr), which binds and inhibits specifically tnf activity, to an immune globulin g fc, to prevent inflammation mediated by tnf in autoimmune diseases like arthritis and psoriasis. on the other hand, fusion proteins which include an antihuman epidermal growth factor receptor (her ) monoclonal antibody that binds tumor cell surfaces, among them the so-called ''trastuzumab'' (commercialized as herceptin by roche), associated to dm- , an antimitotic drug, aimed at improving the treatment of breast cancer. finally, vlps, that is, empty viral entities formed by the self-assembly of a viral capsid protein, are the only truly protein nanoparticles (architectonically speaking) which are currently used in clinical practice. hbsag recombinant protein of hbv expressed in yeast and the capsid l recombinant protein of hpv (types , , , and ) administered currently as vaccines tend to form spontaneously vlps that elicit t and b immune response. recently, there have been preclinical and clinical trials to test the security and efficacy of vlp vaccines against chikungunya and seasonal influenza virus (http://www. medpagetoday.com/meetingcoverage//icaac/ ), respectively. influenza vlp vaccines have proven to provide complete protection against h n flu pandemics, within a record preparation time when compared to months for traditional vaccines. the use of vlps as a delivery system for drugs or nucleic acids in gene therapy is still under investigation. drugs and proteins may be transformed through pegylation, a process that can assist them in overcoming some of the potential problems that delay the adoption of protein nanoparticles for clinical use. the covalent attachment of peg can reduce immunogenicity and antigenicity by hiding the particle from the immune system, can increase the circulating time by reducing renal clearance, and can also improve the water solubility of a hydrophobic particle. the use of pegylation has been approved for commercial use by the fda and emea, and some examples of pegylated protein products are adagen (peg-bovine adenosine deaminase), the first pegylated protein approved by the fda in , pegasys (peg-interferon alpha), and oncaspar (peg-l-asparaginase). the majority of protein nanoparticles studied in clinical trials (http://clinicaltrias.gov) are fusion proteins composed of a therapeutic protein/peptide and a target cell-specific ligand. an example is alt- , a biologic compound composed of il- genetically fused to a humanized soluble t-cell receptor directed against the p -derived antigen. the clinical trials evaluated whether directing il- activity using alt- to the patient's tumor sites that overexpress p results in clinical benefits (nct , nct ). another ligand joined to il- is l , a tumor-targeted immunocytokine constituted of a single chain fragment variable (scfv) directed against the ed-b domain of fibronectin, one of the most important markers for neoangiogenesis. l -il- is in a phase i/ii study for patients with solid tumors and renal cell carcinoma (rcc) (nct ). l has also been fused to tnfa with the intention to target tnfa directly to tumor tissues resulting in high and sustained intralesional bioactive tnfa concentrations. the l tnfa is under clinical trial using isolated inferior limb perfusion (ilp) with the standard treatment with melphalan mg/l limb volume in subjects affected by stage iii/iv limb melanoma (nct ). ngr-htnf is another bifunctional protein which combines a tumor-homing peptide (ngr) that selectively binds to amino peptidase n/cd highly expressed on tumor blood vessels, thus affecting tumor vascular permeability, and htnf, with direct anticancer activity. ngr-htnf is undergoing clinical trials as a single agent to treat different cancers, as well as in combination with chemotherapy agents. another strategy to direct a therapeutic protein to the target cell is through fusion to a growth factor receptor ligand. an example is tp- , a recombinant chimerical protein composed of the egfr binding ligand (tgf-a) and a genetically engineered form of the pseudomonas exotoxin, pe- , to treat recurrent grade iv malignant brain tumors (nct ). many clinical trials are based on a therapeutic protein fused to a targeting antibody, as is the case of apc . this drug stimulates the immune system and stops cancer cells from growing by the combination of biological therapies with bevacizumab , an already approved monoclonal antibody that locates tumor cells and kills them in a specific way (nct ). there are also many putative protein drugs against cancer which include antibodies antiintegrins (e.g., cilengitide and imgn ), sometimes in combination with classical therapies. a recently developed tool, the nanobodies or single domain antibodies, have several advantages: small size (only - kda), which lowers the possibility of triggering immune response, safety in clinical trials (nct ), and is easy to be joined to different kinds of compounds. all these features make nanobodies competent drugs against different diseases, and have been tested in vivo as bifunctional proteins associated to a prodrug, very efficient in mice cancer xenografts. even though cpps are very useful tools to deliver drugs and in gene therapy (see the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume), their toxicity and endosomal entrapment slows their inclusion for systemic delivery in clinical trials. nevertheless, there are a few examples of use to prevent undesirable cell proliferation in coronary artery bypass grafts, as is the case of a cpp (r-ahx-r) ahxb-pmo conjugate targeted to human c-myc to be applied ex vivo. the trial, in phase ii, has been completed in (nct ). another case is psorban , a product patented for the treatment of psoriasis based on a cyclosporine-polyarginine conjugate of local application, which circumvents the specificity problem of intravenous (i.v.) application. it is in clinical trial phase iii, but not yet in the market. finally, kai- , a pkcd inhibitor peptide conjugated to tat to function as an intravenous drug for the treatment of acute myocardial infarction, is currently in phase b clinical trial (nct , kai pharmaceuticals). there are many proteins, often organized as nanoparticles, that when associated to a drug, therapeutic protein, peptide, or nucleic acid increase the therapeutic efficacy of a cargo alone in the treatment of various diseases. some of them proved effective in animal models, which are discussed in more detail in this section, with relevant examples listed in table ii . these nanoparticles may simply be (a) a cpp to promote nonspecific internalization, - (b) a peptide to confer cargo specificity by joining a receptor distinctive of a cell type, including scfvs or peptides obtained by phage display, and (c) a mixture of both, since as observed in several studies the cpp does not reduce ligand specificity and increases nanoparticle potency. [ ] [ ] [ ] complex and multifunctional vehicles including endosomal escape peptides enhance the therapeutic potency of the complex, or other domains that allow their selective activation in certain contexts. , apart from the cases listed in table ii , the spectrum of additional examples of multidomain protein nanoparticles tested in vivo is wide, and a considerable proportion of them include cpps, mainly tat and polyarginines. a classical tat fusion protein is the transducible d-isomer ri-tatp c ' fusion protein that activates p protein in cancer cells, but not in normal cells. ri-tatp c treatment in terminal peritoneal carcinomatosis and peritoneal lymphoma preclinical models results in significant increases in life span (higher than sixfold) and full recovery from the disease. there are also several studies in vivo using tat-fused therapeutic proteins which have proven effective in treating tumors [ ] [ ] [ ] and cerebral ischemia , when applied intraperitoneally (i.p.). regarding polyarginines, kumar and colleagues have presented two different models in which a bifunctional peptide formed by nine arginines ( r) and a specific ligand constitute an effective sirna vehicle. in the first model, a chimerical peptide derived from rabies virus glycoprotein (to confer neuronal specificity) fused to d-arginines (rvg- r), was able to transport si-rna across the bbb and silence specific gene expression in the brain when applied intravenously. in the second model, a cd -specific single-chain antibody was conjugated to oligo- -arginine peptide (scfvcd - r) for t cell-specific antiviral sirna delivery in humanized mice reconstituted with human lymphocytes. in hiv-infected humanized mice, this treatment controlled viral replication and prevented the disease-associated cd t cell loss. moreover, it effectively suppressed viremia in infected mice. some other examples of polyarginines in tumor models are -d-arginines fused to a tumor-suppressor peptide, which stopped tumor growth in hepatocellular carcinoma-bearing mice when applied intraperitoneally, and also colesteryl oligoarginines carrying vegf sirna, which inhibited tumor growth in colon adenocarcinoma after local application. another bbb-crossing peptide is g , which is able to transport nanoparticles loaded with loperamide. in general, the partner fusion peptide can confer specificity instead of penetrability, as is the case of egfr fab fragment associated to liposomes that contain anticancer drug, which increases efficiency of anticancer effect in egf overexpressing xenograft tumors ; in addition, rgd- c-doxorubicin in human breast xenografts increases efficacy and diminishes toxicity. in many conjugates, the therapeutic peptide of the chimerical proteins is a toxin. anthrax lethal toxin has been modified to be activated by methaloproteases, and it has probed to be effective for human xenografted tumors such as melanoma, lung, and colorectal cancer. anthrax toxin has also been associated to antibodies or growth factors for lethal effects specifically on cancer cells. the specific cytotoxicity desired to treat a tumor might derive from a tissue factor, which promotes clotting to restrict blood supply in tumor vessels, fused to peptides that provide specificity, like v-cam antibodies, fibronectin, and integrin ligands. eventually, drug activity may decrease when conjugated to a carrier protein, although if the entry of the drug is favored, the overall balance of activity can be much more efficient. on the other hand, the use of noncovalent bond drug carrier could avoid interfering with the activity of the drug. an important issue in a preclinical study to be considered for a clinical trial is the administration route. in in vivo experiments, most of the protein nanoparticles are administered by local or intraperitoneal injection, avoiding systemic spreading and clearance in the vascular system, in a way very similar to in vitro experiments. the fda and emea, on the other hand, will preferentially approve i.v. and oral administrations rather than intraperitoneal or local injections except for very accessible tissues. another relevant issue is the number of active domains to be included in a therapeutic protein carrier, an issue that seems to be relevant for the functionality of the construct. for example, the cpp neutralization of a ligand may depend on the cpp/ligand ratio that is in the vehicle. it has also been observed that the integrin binding power of rgd-containing motives increases with the number of rgd domains over the monomer until a maxim of four moieties. another example is tat activity empowerment when attached to molecules that form tetramers, such as beta-galactosidase and p- . some multidomain protein carriers allow the drug entrance only in selected target cells by tailored smart selective mechanisms. for instance, cpps neutralized by polyanions are activated and enter the cells when they are released by metalloproteases or by lowering the ph, both situations being very common in tumors. cpp-morpholino oligomer (pmo) nanoparticles have also shown their effectiveness in treating viral infections by inhibiting viral replication, as demonstrated with the carrier (r-ahx-r) ahxb-pmo administered i.v. in animal models infected with picornaviruses, i.p. in mice infected with coronaviruses and flaviviruses, and the carrier r f c-pmo administered also i.p. in mice infected with ebola virus. furthermore, it has also been shown in some of these studies that the efficacy of the treatment is dependent on the incorporation of arginine-rich peptides in the nanoparticle. a good example of how a cpp can improve the internalization of a therapeutic protein is the case of insulin. the instability and low absorption in the digestive tract of insulin prevents its oral administration, even though it would be very convenient for a daily administrated drug. in recent studies, noncovalent conjugation of insulin to different cpps enhances its absorption without toxic intestinal effect, l-penetratin being the most efficient as insulin carrier. among the protein nanoparticles tested in vivo, it is worth making special mention of trojan horses generated in pardridge's laboratory to cross the bbb, through a strategy of fusing within a chimerical peptide the therapeutic protein which has to reach the cns to a monoclonal antibody against the human insulin receptor (hirmab). this trojan horse is very potent for humans and primates, and has proven effective to transport b-glucuronidase, a-l-iduronidase, gdnf, abeta amyloid peptides, paroxonase, etc., with potential benefits in diseases like mucopolysaccharidosis type vii, hurler syndrome, parkinson, alzheimer, and organophosphates toxicity, respectively. there are also promising results when protein nanoparticles have been tested as carriers for gene therapy in vivo, some examples being listed in table ii . in this regard, the use of modular proteins generated by insertional mutagenesis of b-galactosidase condensing the sod gene are able to protect neurons against ischemic injury ; a bifunctional galactosylated polylysine is able to conjugate plasmid dna and to differentially promote expression in hepatocytes that display asialoglycoprotein receptor ; a suicide multidomain protein particle formed by herpes simplex virus thymidine kinase (hsv-tk) conjugated to transferrin (tf) by a biotin-streptavidin bridging, which, administered i.v. in k massively metastasized nude mice, was able to reduce tumor size and to increase mouse survival. in this chapter, proteins and peptides have been envisioned as potent biotechnological tools for the development of new biocompatible biological entities that can be used as therapeutic agents by themselves or as nanovehicles for the delivery of associated drugs. proteins are nanostructures that can form complex high-order entities such as vlps, resulting in appropriate cages for the internalization of therapeutic molecules. in addition, the design of modular proteins displaying selected functions has been possible by using in silico approximations to the feasibility of recombinant protein production. this approach has demonstrated the versatility of such molecules in the generation of novel delivery nanovehicles opening up the possibility of new functional combinations to enhance the specific interaction with the target tissue. such tunable specificity in the delivery of drugs, nucleic acids, or other proteins is one of the main properties that make multifunctional proteins appealing as more rational delivery vehicles. the presence on the market of such complex entities, which started with the approval of insulin for the treatment of diabetes, has been increasing over the past years, and this tendency is expected to continue. in fact, there are some products in clinical trials that will probably end up being approved and some more are being explored in preclinical experiments which might enter in clinical 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involved in coenzyme b -dependent degradation of , -propanediol in salmonella enterica serovar typhimurium lt pdup is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in b -dependent , -propanediol degradation by salmonella enterica serovar typhimurium lt dna polymerase i function is required for the utilization of ethanolamine, , -propanediol, and propionate by salmonella typhimurium lt glutathione is required for maximal transcription of the cobalamin biosynthetic and , -propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, , -propanediol, and propionate in salmonella typhimurium lt microcompartments for b -dependent , -propanediol degradation provide protection from dna and cellular damage by a reactive metabolic intermediate conserving a volatile metabolite: a role for carboxysome-like organelles in salmonella enterica protein structures forming the shell of primitive bacterial organelles bacterial microcompartment organelles: protein shell structure and evolution atomic-level models of the bacterial carboxysome shell structure and mechanisms of a protein-based organelle in escherichia coli a multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria analysis of carboxysomes from synechococcus pcc reveals multiple rubisco complexes with carboxysomal proteins ccmm and ccaa analysis of a genomic dna region from the cyanobacterium synechococcus sp. strain pcc involved in carboxysome assembly and function synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric rubisco species short n-terminal sequences package proteins into bacterial microcompartments structural basis of enzyme encapsulation into a bacterial nanocompartment a simple tagging system for protein encapsulation multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly thermostability and molecular encapsulation within an engineered caged protein scaffold ph-triggered disassembly in a caged protein complex characterization and activity of sonochemically-prepared bsa microspheres containing taxol-an anticancer drug paclitaxel-clusters coated with hyaluronan as selective tumor-targeted nanovectors protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (r ) peptide the nanoscale properties of bacterial inclusion bodies and their effect on mammalian cell proliferation surface cell growth engineering assisted by a novel bacterial nanomaterial nanostructured bacterial materials for innovative medicines development of a selfassembling nuclear targeting vector system based on the tetracycline repressor protein unraveling mysteries of the multifunctional protein sparc a virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection recombinant h n viruslike particle vaccine elicits protective immunity in ferrets against the pandemic h n influenza virus properties, production, and applications of camelid singledomain antibody fragments efficient cancer therapy with a nanobody-based conjugate effect of cell-based intercellular delivery of transcription factor gata on ischemic cardiomyopathy morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters in vivo delivery of the caveolin- scaffolding domain inhibits nitric oxide synthesis and reduces inflammation a non-covalent peptide-based carrier for in vivo delivery of dna mimics targeting cyclin b through peptide-based delivery of sirna prevents tumour growth antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors selective inhibition of erbb -overexpressing breast cancer in vivo by a novel tat-based erbb -targeting signal transducers and activators of transcription -blocking peptide design of a tumor-homing cell-penetrating peptide a tumor-homing peptide with a targeting specificity related to lymphatic vessels penetratin improves tumor retention of single-chain antibodies: a novel step toward optimization of radioimmunotherapy of solid tumors killing hiv-infected cells by transduction with an hiv protease-activated caspase- protein development of elastin-like polypeptide for thermally targeted delivery of doxorubicin treatment of terminal peritoneal carcinomatosis by a transducible p -activating peptide antitumor effect of tat-oxygen-dependent degradation-caspase- fusion protein specifically stabilized and activated in hypoxic tumor cells the - amino acid sequence of the beta-domain of von hippel-lindau gene product is sufficient to inhibit renal tumor growth and invasion dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity protein kinase c delta mediates cerebral reperfusion injury in vivo in vivo delivery of a bcl-xl fusion protein containing the tat protein transduction domain protects against ischemic brain injury and neuronal apoptosis t cell-specific sirna delivery suppresses hiv- infection in humanized mice cholesteryl oligoarginine delivering vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo cancer treatment by targeted drug delivery to tumor vasculature in a mouse model matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature anthrax fusion protein therapy of cancer comparison of three different targeted tissue factor fusion proteins for inducing tumor vessel thrombosis overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cellpenetrating peptide tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide improved targeting of the alpha(v)beta ( ) integrin by multimerisation of rgd peptides probing the impact of valency on the routing of arginine-rich peptides into eukaryotic cells cell-penetrating and cell-targeting peptides in drug delivery tumor imaging by means of proteolytic activation of cell-penetrating peptides tat peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors cell penetrating peptide conjugates of steric block oligonucleotides usefulness of cell-penetrating peptides to improve intestinal insulin absorption biopharmaceutical drug targeting to the brain gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake in vivo gene delivery to tumor cells by transferrin-streptavidin-dna conjugate the authors appreciate the financial support received through grants bfu - from micinn, ps from fiss, and sgr- from agaur. the authors also acknowledge the support of the ciber de bioingeniería, biomateriales y nanomedicina (ciber-bbn), an initiative funded by the vi national r&d&i plan - , iniciativa ingenio , consolider program, ciber actions and financed by the instituto de salud carlos iii with assistance from the european regional development fund. protein nanoparticles engineered for drug delivery and gene therapy key: cord- -gsp dozg authors: franci, gianluigi; falanga, annarita; zannella, carla; folliero, veronica; martora, francesca; galdiero, marilena; galdiero, stefania; morelli, giancarlo; galdiero, massimiliano title: infectivity inhibition by overlapping synthetic peptides derived from the gh/gl heterodimer of herpes simplex virus type date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: gsp dozg herpes simplex virus (hsv) is a human pathogen that infects epithelial cells. the cutaneous lesions, caused by the virus, spread to the nervous system creating several complications. fusion of host membranes with the viral envelope is mandatory and mediated by a group of glycoproteins conserved in all herpesviridae subfamilies, such as the glycoproteins b (gb), h (gh), l (gl) and d (gd). we investigated the inhibitory activity mediated by synthetic overlapping peptides spanning the entire ectodomains of gh and gl glycoproteins. we have performed a brute analysis of the complete gh/gl heterodimer in order to explore the inhibitory activity of peptides modelled on these glycoproteins against hsv‐ infection. twenty‐four of the gh peptides at a concentration of μm reached the % of inhibition cut‐off. interestingly, they are mainly located in the gh carboxy‐terminal domain. none of the gl peptides had a clear inhibiting effect. no peptide toxicity was observed by lactate dehydrogenase assay at the concentrations used in our experimental conditions. hsv‐ therapy is based on acyclovir treatment, but some resistant strains are emerging. in this scenario, innovative approaches for hsv‐ treatment are necessary. our data support the direct involvement of the described domains in the process of virus penetration; therefore, these results are of relevance to the potential development of novel therapeutic compounds to prevent hsv‐ infections. copyright © european peptide society and john wiley & sons, ltd. herpes simplex viruses (hsv) are human pathogens that infect epithelial cells [ ] . they are dsdna viruses divided in three sub-families: alfaherpesvirinae, betaherpesvirinae and gamma herpesvirinae. the alfaherpesvirinae are grouped together for their relative fast replicative cell cycle, their host range and the ability to generate latency in the sensitive ganglial nerves [ ] . among this sub-family, the important species for human infections are represented by hsv- , hsv- and varicella-zoster virus. the common hsv treatment is based on acyclovir, valacyclovir and farmcyclovir. resistant strains are emerging; therefore, new strategies are needed to deal with these infections [ ] . therapeutic peptides have become an attractive tool in drug discovery, and the best characterized therapeutic antiviral peptide inhibitor is enfuvirtide (fusion inhibitor) which mimics the cterminal repeat coil region of human immunodeficiency virus (hiv) fusion protein, gp [ ] [ ] [ ] [ ] . specific functional domains of the proteins involved in the mechanism of cell penetration of a number of viruses have been found able to inhibit infectivity [ ] [ ] [ ] [ ] [ ] [ ] . hsv- infection is initiated by the binding of glycoprotein c (gc) to cell surface heparan sulfate [ , ] . the hsv- gb protein (gb) can also bind to heparan sulfate proteoglycans, but the binding is less efficient [ ] . following attachment, gd binds to one of a number of co-receptors on the host cell membrane, including herpesvirus entry mediator, nectin , nectin or -o-sulfated heparan sulfate, resulting in a conformational change in gd [ ] [ ] [ ] [ ] [ ] [ ] . the conformational change in gd is believed to trigger the formation of a fusion complex, which enables fusion to ensue. the coexpression of gd, gb and gh-gl heterodimer in the same cell results in cell-cell fusion, indicating that these four proteins constitute the minimal fusion apparatus [ ] [ ] [ ] [ ] . although the entry pathways of other enveloped viruses share similar patterns, most systems for which molecular details have been gathered rely on a single fusion protein [ ] . hsv- is singular because it uses both gb and gh/gl as its core fusion machinery [ , ] . the gh/gl complex and gb only interact with each other in response to receptor binding by gd [ , ] and this interaction is required for allowing fusion to occur [ ] . the tridimensional structure of gh/gl of hsv- showed that the gh/gl heterodimer does not resemble any known viral fusogen, which may support different roles for gh including that of being a fusion regulator through its interaction with gb [ ] . a broad literature describes gh as involved in the entry process, for example, certain mutations in the transmembrane (tm) region and cytoplasmic tail affect fusion [ ] [ ] [ ] [ ] , as do mutations in the region preceding the tm [ ] . furthermore, peptides matching a number of regions of the gh ectodomain have been shown to interact with membranes and proposed to play a role in the fusion process, probably through regulation of gb activity and/or direct membrane interactions at the fusion site [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in hsv-infected cells and on mature virions, gh and gl are always found together as an heterodimer, in a stable : complex [ , ] and rely on one another for proper folding, post-translational processing and transport to the cell and virion surface [ ] [ ] [ ] [ ] . hsv- gh is a -residue type membrane glycoprotein. the ectodomain contains seven n-glycosylation sites and eight cysteine residues forming at least two disulphide bonds between cysteines and (residue and ) and cysteines and (residues and ) [ , , ] ; gl is a -residue protein with a signal peptide but no tm region. the crystal structure of the gh/gl complex has been solved [ ] and shows an overall shape of a boot. gh can be divided in three distinct domains: (i) the n-terminal domain, which binds gl (h ), (ii) the central helical domain (h ), and (iii) the c-terminal βsandwich domain (h ). the n-terminal domain is located in the upper part of the gh-gl boot and consists of two subdomains, which are connected by a linker (residues gly -pro ); the first subdomain (residues arg -leu ) contains a β-hairpin that forms a six-stranded mixed β-sheet with four strands coming from gl plus three short helices, while the second subdomain (residues ala -pro ) contains a six-stranded antiparallel β-sheet. it is interesting to note that the n-terminal domain does not have a folded core and is the most divergent in sequence. the central domain (residues asn -phe ) is globular and mostly helical. the c-terminal domain (residues val -pro ) is located at the toe end of the boot. it is a -stranded β-sandwich where each side is composed of a fivestranded β-sheet and leads to the tm region. the central and cterminal domains are more conserved and probably have the same fold in different gh proteins. similarly to the n-terminal domain of gh, gl does not have a stable core. only~ % of gl residues adopt regular secondary structure, which include three helices and two β-sheets. peptide-based strategies have recently been used to study herpesvirus glycoprotein function. several laboratories have analysed synthetic peptides homologous to regions from gc, gb and gh of different herpesviruses in order to inhibit infection [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we have previously shown that peptides modelled on gh domains with high interfacial hydrophobicity were able to inhibit hsv infectivity [ , ] , as well as peptides corresponding to helical domains [ ] . recently, several studies have analysed viral glycoproteins with generalized peptide-based approaches to dissect the whole glycoproteins in order to search for functionally important regions besides the predictable bioinformatic motifs. the mechanism that underlies the ability of some synthetic peptides to inhibit fusion is currently unclear, but some regions of these glycoproteins are exposed on the surface, and they may be involved in interactions with other proteins, or they may become exposed to the surface after the conformational rearrangements that probably take place during the fusion process; this could perhaps explain the ability of the corresponding synthetic peptides to inhibit cell fusion. to date, few peptide molecules outside the well-known inhibitory regions of class viral fusion proteins, the heptad repeats, should be as fusion; therefore, a brute force approach to the identification of peptide entry inhibitors may help in the dissection of hsv- glycoproteins domains. in the present study, we used a peptide scanning inhibition approach for the identification of functional domains and/or lead compounds as infectivity inhibitors. we generated overlapping peptide libraries of the complete sequences of gl and gh ectodomains. these peptides were screened for their ability to inhibit hsv- infection in vero cells at a concentration of μm. peptides were prepared by standard -fluorenylmethoxycarbonyl polyamine solid-phase synthesis, using a pssm multispecific peptide synthesizer (shimadzu corporation biotechnology instruments department kyoto japan). the tga resin (substitution . mmol g À ) was used as the solid-phase support, and syntheses were performed on a scale of μmol. all amino acids, equiv. relative to resin loading, were coupled according to the tbtu/hobt/diea method: equiv. of fmoc-amino acid, equiv. of tbtu, equiv. of hobt ( m hobt in dmf) and equiv. of diea ( m diea in dmf). the fmoc protecting group was removed with % piperidine in dmf (v/v). peptides were fully deprotected and cleaved from the resin by tfa solution ( % tfa, . % thioanisole, . % ethandithiol and . % anisole as scavengers); the crude peptides were precipitated with ice-cold ethyl ether, filtered, re-dissolved in water and lyophilized. the crude peptides were purified to homogeneity by preparative reverse-phase high-pressure liquid chromatography on a waters delta prep chromatographic system, equipped with an uv lambda max mod. detector. the samples were injected on a jupiter (phenomenex) c column ( . mm × cm, μm) eluted with a h o/ . % tfa (a) and ch cn/ . % tfa (b) solvent mixture. a linear gradient from to % of b over min at a flow rate of ml min À was employed. the collected fractions were lyophilized to dryness and analysed by analytical reverse-phase high-pressure liquid chromatography on a shimadzu class-lc equipped with a diode array detector spd-m av using a phenomenex c analytical column ( × mm, μm); a linear gradient from to % of b over min at a flow rate of ml min À was used. the identity of purified peptides was confirmed by maldi spectrometry. all purified peptides were obtained with high yields ( - %). african green monkey kidney cells (vero) (atcc ccl- ) were grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum. hsv- (strain sc ), carrying a lacz gene driven by the cmv ie- promoter to express βgalactosidase, was propagated on vero cells monolayers. peptides were dissolved in dmem without serum and used at final concentration of μm. experiments were conducted in parallel with scrambled peptides and no-peptide controls. to assess the effect of peptides on inhibition of hsv infectivity, a co-treatment assay was performed in triplicate. the statistical analysis was carried out via t-test and p-value. the cells were incubated with peptides at μm in the presence of serial dilutions of viral inoculum for min at °c. after inactivation of nonpenetrated viruses by citrate buffer (ph . ), cell monolayers were incubated for h at °c in dmem supplemented with carboxymethylcellulose. finally, monolayers were fixed and stained with x-gal ( -bromo- -chloro- -indolylβ-d-galactopyranoside), and plaque numbers were scored. experiments were performed in triplicate, and the percentage of inhibition was calculated with respect to no-peptide control experiments. the ic and ic values, peptide concentration that resulted in and % of hsv infectivity reduction in a co-treatment assay, respectively, were extrapolated via graphpad prism software from the resulting sigmoidal dose-response curve. peptide cytotoxicity was measured by a lactate dehydrogenase (ldh) assay [ ] and was carried out according to manufacturer's instructions using a cytotoxicity detection kit (roche diagnostic spa., milano, italy). release of ldh from the cytosol to culture is a marker of cell death. the increase of ldh activity in the supernatant was related with the percentage of necrotic cells. vero cells were cultured in a -well plate at a density of × cells/well for h, followed by treatment with investigated concentrations of each peptides for h. maximal ldh release was obtained after the treatment of control cells with % solution of triton x- (sigma chemical company) for min at room temperature. one hundred microlitres of supernatant from the top of all the wells was mixed with the prepared detection kit reagent. after -min incubation, the absorbance was measured at nm by tecan spectrophotometer plate. for each experiment, at least three replicate wells were examined. to test the potential of using peptides as an antiviral agent and to identify functionally important regions of hsv- membrane glycoproteins, two peptide libraries were constructed on the sequences of gh and gl ectodomains. considering the different size of the two glycoproteins under study, we decided to prepare libraries with different overlapping lengths: a library of -mer peptides extending from residues to (just at the beginning of the tm domain) of gh and a second library of -mer peptides from residues to corresponding to the whole sequence of gl. the first (gh) library was made of peptides overlapping each other by residues (supplementary table s ), while the latter was designed with a residue overlap (supplementary table s ), and both signal peptides were excluded from our analysis. in detail, we excluded the first aa from gh sequence (including the peptide signal - aa) and the first aa from gl sequence. the libraries were screened for peptides with inhibitory activity by using a plaque reduction assay in which both virus and cells were exposed to the peptides prior to infection and the peptides remained present throughout the assay. the peptide efficacy was measured by a plaque reduction assay as described in the materials and methods. the criterion for considering a peptide of potential interest was the inhibition of at least % of plaque formation at a concentration of μm. figure shows the gh-overlapping peptides and their antiviral activities in the screening. of the peptides in the gh-library, peptides showed an inhibition activity more than % (table ) , while the threshold was not reached by any of the gl peptides (supplementary table s h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h % of viral inhibition activity p-value < , figure . inhibitory activity of gh peptides. the relative activity was normalized and reported as % of inhibition. the threshold lane was fixed at % of infection activity. the statistical analysis was carried out via p-value < . . interesting to note that some of the best inhibitors were located at the domain of interaction between gh (aa - ) and gl. previous works using a physico-chemical algorithm, the wimley-white interfacial hydrophobicity scale (wwihs), in combination with other structural data allowed us to predict regions in gh potentially involved in membrane interactions during the entry and fusion process, and some of them were found to possess hsv antiviral activity in dose-dependent inhibition assays [ ] . in the present study, we have extended these studies applying a more generalized peptide-based approach to scan the entire gh ectodomain and its accompanying glycoprotein gl. the chosen threshold for including into the category of putative inhibitory peptides was dictated by the general high concentration needed for exerting an antiviral activity of hsv-derived peptides. previous works have shown that concentrations of peptide ranging from to over μm are needed to inhibit hsv infectivity. nevertheless, the results obtained prove that peptide-based strategies targeting regions of the proteins with predictable structures or motifs represent a valid option when searching functional regions of a protein; in fact, our whole protein scan confirmed as regions of interest several domains which were already found to posses some antiviral activity based on their structural characteristics. peptides h , h and h , spanning the region of gh from to , partially overlap the helical peptide previously named h-hr (aa - ) able to reduce infectivity in a consistent manner, albeit at the high concentration of μm [ , ] . peptides h , h , h and h , h , h , h and h overlap peptides gh - and gh - , which were found able to inhibit hsv entry with approximately % of inhibition at μm. hydropathy analysis based on the hydrophobicity-at-interface scale proposed by wimley and white [ ] enabled the detection of these two domains of hsv gh able to induce rapid membrane fusion and inhibit viral entry. a shorter peptide gh - , corresponding only to the n-terminus of gh - and almost completely overlapping h , h and h of the present work, was able to inhibit hsv entry even more effectively [ ] . finally, peptides h and h at the extreme c-terminus of the gh ectodomain (the tm helical domain starts at aa ) have been previously individuated as being part of a larger pre-tm domain with the ability of inhibiting virus infectivity, and a peptide previously named ptm [ ] exactly corresponds to peptide h of the present study. nevertheless, the remaining peptides have been newly identified and presented an inhibitory activity above the set threshold. these are peptides h -h , h -h , h , h and h , which were found to exhibit inhibitory effects against hsv- plaque formation (table ) . in order to define if chemical as well as conformational properties could represent common and shareable characteristics of active peptide, we performed bioinformatics analysis based on pepcalc (www.pepcalc.com) and pep-fold (http://bioserv.rpbs. univ-paris-diderot.fr/) databases, respectively (figures and ) . chemical and physical properties highlight shared characteristic for some peptides. in detail, we reported in figure the peptides subdivided for their isoelectric point grouped as acid (ph from to ), neutral (ph from to ) and basic (ph from to ). in second instance, the peptides were clustered based on their charge at ph . as shown in figure , we found that some peptides are represented in specific clusters like h , h and h for the first group as well as for h , h and h in the second. interestingly, most new inhibiting peptides are present in the acid (h , h , h , h ) and basic (h , h ) groups, while only one peptide (h ) is present in the neutral group. moreover, we performed a primary and secondary structure analysis on the inhibiting gh peptides (figure ). peptides clustered together by a sequence homology point of view ( figure a) , and the clusters are directly associated with inhibition activity ( figure b ). for instance, the cluster had a percentage of viral inhibition close to %, while the clusters and just above %. secondary structure bioinformatics peptide analysis is reported in figure c , where we observed that in the cluster , a different distribution of helical and coil structure with low presence of extended regions is present compared to the others. finally, to confirm that these peptides did not exert toxic effect on vero cells, monolayers were exposed to a range of concentrations ( , , , μm) of each inhibitory peptide for h, and cell viability was assayed by an ldh assay. no statistical difference was observed between the viability of control (untreated) cells and that of cells exposed to the peptides (data not shown); thus, none of the peptides exhibited cytotoxic effects at the concentrations tested. most of the viral peptide entry inhibitors described so far were intentionally designed in order to inhibit viral entry by competing with intermolecular interactions using amino acid specific sequences mostly derived from viral fusion proteins. however, an activity without a specific molecular target can be envisaged for many entry inhibitor peptides. these peptides, albeit being derived from viral surface proteins, do not belong to well-known hr domains, but are often scattered in exposed regions of surface proteins or in domains that may only become exposed following conformational modifications triggered by binding of viruses to receptors or by environmental changes inside endosomes. the current understanding of the mechanisms of action of these peptides finds a hallmark in their propensity for membrane binding due to their interfacial hydrophobicity or amphipaticity [ , , , ] . in view of this characteristic, peptides could hamper virus entry by establishing direct physical interactions at hydrophobic interfaces on membranes or fusion proteins. essentially, peptides can bind membranes, membrane proteins or virus particles, thereby exert a steric hindering effect on the early stages of viral penetration such as viral binding and/or membrane fusion. sometimes, they can interfere with fusion protein modifications by premature initiation of the fusion proteins, which become unable to promote fusion once the peptides are bound to the viral envelope membrane. in this study, we have described an alternative approach for identifying functionally important regions of hsv- gh and gl, showing for the first time the hsv- inhibition activity of unreported peptides derived from gh protein. the approach involved the synthesis of two libraries of overlapping peptides that encompassed the entire ectodomains of gh and gl. brute-force scanning approaches have proved to be of interest for analysing proteins of several viruses; in fact, potential antiviral peptides against hepatitis c virus (hcv) have been discovered by screening overlapping peptides ( -mers) covering the entire hcv polyprotein [ , ] . eleven peptides with strong antiviral activity were identified. the most active inhibitory peptide, called c a, showed significant hydrophobic and membrane binding propensity and was suggested to directly interact with the hcv viral envelope blocking infection when treating viruses during or prior to the initial entry stage. interestingly, c a was also able to inhibit hiv by disrupting the integrity of the viral envelope [ ] . following this line, four proteins considered to play a role for promoting hcv entry (namely: cd , scavenger receptor b , claudin and occluding) were analysed by an overlapping amino acid peptide scanning strategy, and out of peptides, two of them derived from claudin showed potent inhibitory activity against hcv infectivity [ ] . furthermore, hcv has also been recently analysed by screening a peptide library covering the full-length e and e amino acids sequences where a peptide from the e stem domain was found to block cell entry of hcv peseudoparticles at nanomolar concentrations. this peptide is of sure potential interest because it proved to be efficient against the major hcv genotypes from to [ ] . another study assessed the activity of -overlapping peptides ( -mers) covering the entire vp capsid protein of enterovirus , an important human pathogen which may cause severe neurological complications. in this case, the strategy proved to be also usable for searching potential peptide inhibitors in non-enveloped viruses, with the discovery of four peptides able to inhibit ev- infection by more than % [ ] . a further study described a -mer overlapped peptide library synthesized by combinatorial approach where a peptide derived from the s subunit of the spike glycoprotein of the severe acute respiratory syndrome associated coronavirus (sars-cov) could block the binding to the cellular receptor and, thus, the entry into target cells [ ] . bai et al. [ ] used a phage display methodology to identify a peptide (named p ) to inhibit west nile virus (wnv) infectivity, possibly by binding to the envelope glycoprotein (e protein) necessary for membrane fusion. a modified version of p , named p , showed a strongly increased inhibitory activity against wnv at micrometre concentrations, and, more interestingly, entered the brain by crossing the blood brain barrier to reduce infectivity in a mouse model of brain infection. moreover, an interesting study sparkling from the observation that hiv patients co-infected with the common and asymptomatic gb virus c (a virus related to hcv) present lower hiv viremia suggested the analysis of overlapping peptides from the e protein from the gb virus c and allowed the identification of two peptides able to inhibit hiv in vitro by their competitive binding at the gp -gp interface [ , ] . also, hsv glycoproteins have been analysed by a peptide scanning strategy. a library of overlapping -mer peptides encompassing the ectodomain of gb was synthesized and tested for hsv infectivity inhibition [ ] . seven of the peptides inhibited infectivity by % or more when tested at -μm concentrations. interestingly, many of the antiviral peptides identified by these brute force approaches overlap with many peptides discovered by analysing hydrophobicity at interface scales [ ] . the example of hsv gb is remarkable, because all the peptides (at least the most active) had also been previously described as membraneinteracting [ ] sequences using the wwihs . our results, scanning the entire sequences of hsv- gh and gl, confirm that the wwihs is a powerful mean to identify potential antiviral peptides, but some regions of potential interest can remain underscored; therefore, a systematic analysis of the whole sequence by overlapping peptide libraries can add more detailed information on the regions involved in inhibition. in fact, we were able to detect four areas where peptides could be grouped for their antiviral activity. some of the identified regions overlap with peptides already described which were discovered by bioinformatics tools, thereby strengthening this clear relationship between the function and the physiochemical character of peptides. in particular, the four identified inhibitory areas are mainly located in the regions of the glycoprotein named h , while only a small area (s ) is located in the h region. the h domain of gh is mainly characterized by a bundle of helices and a few extended regions. in gh derived from pseudorabies virus (prv), a synthaxin like bundle (slb) is present in this region [ ] , and this motif has proved to be of importance because its disruption can lead to impaired replication activity of the virus [ ] . interestingly, one of our inhibitory peptides is located in the hsv- gh corresponding region. surprisingly, we did not detect any activity in inhibition assays when testing gl derived peptides. this may likely account for the negligible role of gl in membrane interactions. nevertheless, also domain h of gh which is mainly devoted to interact with gl did not provide any inhibitory peptides. h sub-domains clamp gl like tongs and make extensive contacts between the interacting highly complementary surfaces. in fact, the two proteins need each other to fold correctly and gl is a powerful scaffolding protein for gh. the inability of gh peptides derived from the h domain to function as inhibitors of infectivity can be explained by the fact that the formation of the highly stable complex between the two glycoproteins happens during the maturation and egress from the infected cell; therefore, the structure is already definitive when the heterodimer becomes expressed on the mature virion envelope. disruption of the gh:gl interaction is not likely to happen because the whole h domain would result in an unfolded structure in absence of gl. the four areas of active peptides are depicted in figure where the filled surface of the protein is shown. it is of interest to note that inhibitory areas s , s and s are mainly occupying regions of gh domains h and h and are exposed to the external surface of the protein, while only area s is partially buried inside and only partially exposed to the outside. therefore, this observation is consistent with the minor inhibitory activity of peptides belonging to area s . furthermore, inhibitory area s precedes the c-terminal tm domain of gh and is part of the c-teminal h ectodomain mainly composed of β-sheets which constitute a large patch of hydrophobic residues able to interact with the cell membrane during the fusion process or modifying the curvature of the viral envelope. membrane fusion is an important step in enveloped virus entry into host cells. the present study on the antiviral activity of hsv- derived peptides provides a large set of data obtained with overlapping peptides covering the whole ectodomains of gh and gl. these peptides may be useful for probing gh activity during membrane fusion, but their major use could be as direct antiviral molecules. acting on early stages, the discovered peptides can be used as microbicide for topical administration following further analysis and appropriate modifications for improving their viability and efficiency. as a matter of fact, considering their molecular weight, these peptides can be categorized as mid-size drugs, which are, nowadays, receiving considerable attention in medicinal chemistry. the herpes simplex virus latencyassociated transcript gene is associated with a broader repertoire of virus-specific exhausted cd + t cells retained within the trigeminal ganglia of latently infected hla transgenic rabbits herpes simplex virus type latency and reactivation: an update resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management enfuvirtide: the first therapy to inhibit the entry of hiv- into host cd lymphocytes potent suppression of hiv- replication in humans by t- , a peptide inhibitor of gp -mediated virus entry a rationally engineered anti-hiv peptide fusion inhibitor with greatly reduced immunogenicity peptide inhibition of hiv- : current status and future potential release of dengue virus genome induced by a peptide inhibitor peptide inhibitors of dengue-virus entry target a late-stage fusion intermediate inhibition of hendra virus fusion inhibition of hiv entry by targeting the envelope transmembrane subunit gp a new class of synthetic peptide inhibitors blocks attachment and entry of human pathogenic viruses perspective use of antiviral peptides against influenza virus herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry three classes of cell surface receptors for alphaherpesvirus entry glycoprotein cindependent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein b the soluble ectodomain of herpes simplex virus gd contains a membrane-proximal pro-fusion domain and suffices to mediate virus entry entry of alphaherpesviruses mediated by poliovirus receptor-related protein and poliovirus receptor the target protein (gh) is rendered as a filled surface (light blue), and the locations of the four areas (from the n-terminal to the c-terminal of the protein) are shown. s is depicted in orange, s is in yellow, while s is in pink, and s is in green. gl is not shown, and the protein is rotated to show the best rendering for each of the inhibitory areas herpes simplex virus- entry into cells mediated by a novel member of the tnf/ngf receptor family structure of unliganded hsv gd reveals a mechanism for receptor-mediated activation of virus entry function of herpes simplex virus type gd mutants with different receptorbinding affinities in virus entry and fusion regulation of herpes simplex virus gb-induced cell-cell fusion by mutant forms of gh/gl in the absence of gd and cellular receptors glycoproteins gb, gd, and ghgl of herpes simplex virus type are necessary and sufficient to mediate membrane fusion in a cos cell transfection system characterization of cell-cell fusion mediated by herpes simplex virus glycoproteins gb, gd, gh and gl in transfected cells cell fusion induced by herpes simplex virus glycoproteins gb, gd, and gh-gl requires a gd receptor but not necessarily heparan sulfate substitution of herpes simplex virus entry glycoproteins with those of saimiriine herpesvirus reveals a gd-gh/gl functional interaction and a region within the gd profusion domain that is critical for fusion structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme fusing structure and function: a structural view of the herpesvirus entry machinery herpes virus fusion and entry: a story with many characters bimolecular complementation reveals that glycoproteins gb and gh/gl of herpes simplex virus interact with each other during cell fusion a functional interaction between herpes simplex virus glycoprotein gh/gl domains i and ii and gd is defined by using alphaherpesvirus gh and gl chimeras bimolecular complementation defines functional regions of herpes simplex virus gb that are involved with gh/gl as a necessary step leading to cell fusion crystal structure of the conserved herpesvirus fusion regulator complex gh-gl an endoplasmic reticulumretained herpes simplex virus glycoprotein h is absent from secreted virions: evidence for reenvelopment during egress the transmembrane domain and cytoplasmic tail of herpes simplex virus type glycoprotein h play a role in membrane fusion mutations in the cytoplasmic tail of herpes simplex virus glycoprotein h suppress cell fusion by a syncytial strain herpes simplex virus capsid-organelle association in the absence of the large tegument protein ul p sitedirected and linker insertion mutagenesis of herpes simplex virus type glycoprotein h fusogenic domains in herpes simplex virus type glycoprotein h the ectodomain of herpes simplex virus glycoprotein h contains a membrane alpha-helix with attributes of an internal fusion peptide, positionally conserved in the herpesviridae family structure and orientation of the gh - membrane interacting region of herpes simplex virus type in a membrane mimetic system the presence of a single n-terminal histidine residue enhances the fusogenic properties of a membranotropic peptide derived from herpes simplex virus type glycoprotein h analysis of a membrane interacting region of herpes simplex virus type glycoprotein h evidence for a role of the membrane-proximal region of herpes simplex virus type glycoprotein h in membrane fusion and virus inhibition peptides containing membraneinteracting motifs inhibit herpes simplex virus type infectivity role of membranotropic sequences from herpes simplex virus type i glycoproteins b and h in the fusion process a peptide derived from herpes simplex virus type glycoprotein h: membrane translocation and applications to the delivery of quantum dots structural and antigenic analysis of a truncated form of the herpes simplex virus glycoprotein gh-gl complex n-terminal mutants of herpes simplex virus type gh are transported without gl but require gl for function a novel herpes simplex virus glycoprotein, gl, forms a complex with glycoprotein h (gh) and affects normal folding and surface expression of gh a mutant herpes simplex virus type unable to express glycoprotein l cannot enter cells, and its particles lack glycoprotein h interplay between the herpes simplex virus gb cytodomain and the gh cytotail during cell-cell fusion contribution of cysteine residues to the structure and function of herpes simplex virus gh/gl stuck in the middle: structural insights into the role of the gh/gl heterodimer in herpesvirus entry hydrophobic alpha-helices and of herpes simplex virus gh interact with lipids, and their mimetic peptides enhance virus infection and fusion coiled-coil domains in glycoproteins b and h are involved in human cytomegalovirus membrane fusion a synthetic peptide from a heptad repeat region of herpesvirus glycoprotein b inhibits virus replication inhibition of viral-induced membrane fusion by peptides peptide inhibitors against herpes simplex virus infections structural and functional features of the polycationic peptide required for inhibition of herpes simplex virus invasion of cells structural characteristics and antiviral activity of multiple peptides derived from mdv glycoproteins b and h cell entry mechanisms of hsv: what we have learned in recent years identification of peptide inhibitors of enveloped viruses using support vector machine analysis of synthetic peptides from heptadrepeat domains of herpes simplex virus type glycoproteins h and b role of mitogen-activated protein kinases in the inos production and cytokine secretion by salmonella enterica serovar typhimurium porins experimentally determined hydrophobicity scale for proteins at membrane interfaces a heptad repeat in herpes simplex virus gh, located downstream of the alpha-helix with attributes of a fusion peptide, is critical for virus entry and fusion peptide entry inhibitors of enveloped viruses: the importance of interfacial hydrophobicity a virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis c virus infection in vitro antiviral activity of virocidal peptide derived from ns a against two different hcv genotypes: an in vitro study rational design of peptides with anti-hcv/hiv activities and enhanced specificity a human claudin- -derived peptide inhibits hepatitis c virus entry identification of a potent and broad-spectrum hepatitis c virus fusion inhibitory peptide from the e stem domain inhibition of enterovirus (ev- ) infections by a novel antiviral peptide derived from ev- capsid protein vp screening and identification of linear b-cell epitopes and entryblocking peptide of severe acute respiratory syndrome (sars)-associated coronavirus using synthetic overlapping peptide library antiviral peptides targeting the west nile virus envelope protein peptides derived from a distinct region of gb virus c glycoprotein e mediate strainspecific hiv- entry inhibition hiv- fusion is blocked through binding of gb virus c e -derived peptides to the hiv- gp disulfide loop multiple peptides homologous to herpes simplex virus type glycoprotein b inhibit viral infection the identification and characterization of fusogenic domains in herpes virus glycoprotein b molecules identification of a pheromone regulating caste differentiation in termites a replication defect of pseudorabies virus induced by targeted alphahelix distortion in the syntaxin-like bundle of glycoprotein h (v p) is corrected by an adjacent compensatory mutation (v a) additional supporting information may be found in the online version of this article at the publisher's web site. table s . gh peptide names with the respective sequences. table s . gl peptide names with the respective sequences. key: cord- - k ll authors: bastos, paulo; trindade, fábio; da costa, joão; ferreira, rita; vitorino, rui title: human antimicrobial peptides in bodily fluids: current knowledge and therapeutic perspectives in the postantibiotic era date: - - journal: med res rev doi: . /med. sha: doc_id: cord_uid: k ll antimicrobial peptides (amps) are an integral part of the innate immune defense mechanism of many organisms. due to the alarming increase of resistance to antimicrobial therapeutics, a growing interest in alternative antimicrobial agents has led to the exploitation of amps, both synthetic and isolated from natural sources. thus, many peptide‐based drugs have been the focus of increasing attention by many researchers not only in identifying novel amps, but in defining mechanisms of antimicrobial peptide activity as well. herein, we review the available strategies for the identification of amps in human body fluids and their mechanism(s) of action. in addition, an overview of the distribution of amps across different human body fluids is provided, as well as its relation with microorganisms and infectious conditions. human antimicrobial peptides (amps) represent approximately % of all curated amps catalogued to date. human host defense peptides are an intrinsic part of the innate immune system and exhibit a broad activity spectrum against bacteria, fungi, viruses, and parasites while amps can be antibacterial (abps), antifungal, antiprotist, antiviral, anticancer, antiparasitic, insecticidal, spermicidal, chemotactic, antioxidant, protease inhibitors, or even exhibit wound healing properties (supporting information table s ), their scope of action overlaps considerably and some peptides show activity at several levels (fig. ). [ ] [ ] [ ] [ ] [ ] for instance, on the one hand, human plasma adrenomedullin-derived peptides are active against multiple bacteria species, human urinary and gingival fluid calcitonin gene-related peptide displays antimicrobial activity against several bacteria and fungi species, and human neutrophil peptides (hnps)/defensins isolated from most biological fluids display activity against several bacterial, fungal, and viral species (fig. ) . on the other hand, dozens of human amps known to date display antimicrobial activity against the same human colonizers or pathogens, including escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus (figs. and ) . also, it is intriguing to observe that even though some amps display broad activity spectra, others seem to be anywhere from species-to kingdom-specific (fig. ) . while likely to be confounded global network depicting the distribution of human antimicrobial peptides across biological fluids and their microbial targets. rectangular nodes correspond to biofluids, circular nodes to target species, and each edge correspond to a given gene encoding one or more antimicrobial peptides. the thicker the edge, the stronger is the association of a biofluid to a pathogen, representing increased number of antimicrobial peptides defending against such pathogen in such biofluid. also, pathogens represented by bigger nodes (e.g., escherichia coli, candida albicans, staphylococcus aureus) represent those that are targeted by more antimicrobial peptides across different biofluids. by observational bias (whereby some species, e.g., e. coli, tend to be tested more frequently than others), this observation also reflects that the activity spectra of amps depend not only on their physicochemical properties (which group them according to unique families) but also on target properties and on the environment/biological fluid in which they are found. as the large majority of amps identified to date are abps (ß %, figs. - ) , most concepts and examples provided in this review will be based on results from studies focusing on abps, closely followed by those derived from studies on antiviral and antifungal peptides. however, the same principles tend to apply across all amps. amps are very potent cationic molecules displaying minimum inhibitory concentrations (the minimum concentration that prevents bacterial growth) as low as - μg/ml, which highlights their promise as broad-spectrum antimicrobial agents. furthermore, amps act more rapidly than conventional antibiotics and are not affected by the typical resistance mechanisms involving conventional drugs, making them very attractive for therapeutic purposes. in addition, amps are not mere microbicide agents, as their scope of action may encompass other functions, such as cellular development and immune system modulation. for instance, fetal keratinocytes express significantly more amps compared to neonatal and adult keratinocytes, despite the lower degree of exposition to the environment or to pathogenic agents. these a rough estimate of all amps catalogued to date (supporting information table s ) indicates . % to be α-helices, followed by β-sheets ( . %), and peptides presenting with both helices and β-sheets ( . %). however, approximately % of all amps currently identified do not have a known d structure, while only . % have a known structure that is neither a helix nor a β-sheet. excluding those that have unknown structures, . % of all amps are either α-helices and/or β-sheets, suggesting a highly conserved structure for a highly primordial biological function. secondary structures of human amps may include α-helices (e.g., ll- ), β-sheets (e.g., defensin ), and extended peptides (e.g., indolicidin). considering that β-sheets and α-helices in polypeptide chains typically contain three to ten amino acids per strand and . residues per turn, respectively, and that amino acid sequences can be virtually unlimited, one can easily envision that a given peptide can display a multitude of secondary structures and, thus, many diverse conformations (see for protein secondary structures predictive approaches). among secondary structures, α-helices (in addition to being the most frequently encountered in nature) are the most regular and predictable, and, consequently, the most extensively studied. amps with linear α-helices are notably positively charged and amphipathic, but only adopt such secondary conformation upon binding to bacterial membranes. therefore, their antimicrobial activity is directly dependent on the secondary structure. in contrast, β-sheet amps possess well-defined conformations due to the presence of disulfide bridges formed between thiol groups in cysteines. in these peptides, such bridges are often required for antimicrobial activity. contrariwise, bovine lactoferricin was shown to be effective against e. coli even when such bridges were blocked by pyridylethylation. other human amps, such as human β-defensin (hbd- ), present both α-helices and β-sheets. in the specific case of hbd- , disrupting the three stabilizing disulfide bonds does not compromise its antimicrobial activity, but it does diminish its chemotactic properties. furthermore, the correlation of the conformation with peptides' antimicrobial activity is not always as straightforward as one could imagine. for example, disruption of hbd- 's disulfide bonds results in peptides with free cysteines at the carboxy terminal that show increased antimicrobial activity against the pathogenic candida albicans and the gram-positive commensals bifidobacterium and lactobacillus. moreover, linear extended peptides do not keep a fixed conformation in solution, and are enriched in one or more amino acids, particularly arginine, proline, and tryptophan. for instance, the amp histatin found in human saliva is enriched in histidine residues, while prophenin is very rich in proline and phenylalanine residues. when it comes to loop rich peptides, the presence of many prolines makes an amp much less likely to form amphipathic structures, adopting what is frequently referred to as polyproline helical type-ii (ppii) structures. these structures are specifically bound by sh domains, which are usually found in proteins that interact with other proteins and mediate assembly of specific protein complexes (upon proline-rich peptides binding). human antibacterial peptides (abps), which compose the largest group of human amps, exhibit a weighted average net charge of + . per peptide, which is considerably above the average net charge of all amps identified to date. also, despite being intuitive that the more positively charged an amp is, the more potency it will present, human amps do not show greater potency or efficacy when compared to those of other species, suggesting that the environment and the presence of different amino acids (d and rare amino acids) also play a significant role. notwithstanding the large diversity of amps across human biologic fluids (figs. and , table i ), it should be noted that amps are much more diverse across all species, than those identified in humans, which present rather conserved properties. in fact, some amps from other species are characterized by more intriguing sequences, structures, and physicochemical growth-regulated alpha protein properties. case in point, bacillus subtilis is known to produce several extremely potent lipopeptides (active at concentrations as low as . - . μm) with antimicrobial activity against several species. one of these, gageotetrin a is a very unique anionic amp that consists of only leucine and glutamic acid residues conjugated with a unique fatty acid, -hydroxy- -methyltridecanoic acid. another example is that of sonorensin from bacillus sonorensis, a broad-spectrum amp from the heterocycloanthracin subfamily, active against both gram-negative and gram-positive bacteria, including listeria monocytogenes and s. aureus. sonorensin displays an amino acid sequence with multiple copies of the same motif, making this peptide rather unique. baceridin is a circular peptide from a plant-associated bacillus that is synthesized by ribosome-independent machinery and bears only six amino acids, % in the d configuration. moreover, baceridin is a proteasome inhibitor, compromising cell cycle progression and inducing apoptosis in tumor cells by a p -independent pathway. copsin from coprinopsis cinerea is bactericidal against enterococcus faecium and l. monocytogenes by interacting with the peptidoglycan precursor lipid ii and interfering with the cell wall biosynthesis. curiously, it is modified with a pyroglutamate, which confers a higher degree of thermal stability and resistance toward protease digestion. even though some display fungicidal and virucidal properties, the chief activity of human amps is against gram-positive and gram-negative bacteria (figs. - ). furthermore, while amps represent an extremely diverse group of biological active molecules, most share common properties that contribute for their membranolytic or otherwise antimicrobial activity: positive charge, hydrophobicity, and amphiphilicity. therefore, the mechanism of action of most amps involves membrane disruption (fig. ) , a process that is also required when their translocation is warranted and the respective targets are localized intracellularly. the interaction of amps with bacterial membranes depends on the establishment of attractive electrostatic forces between these. bacterial species are known to carry negatively charged outer membranes due to phosphate groups of the lipopolysaccharides in gram-negative bacteria and to the lipoteichoic acids in gram-positive bacteria. the fact that all gramnegative and gram-positive bacteria display these type of negatively charged lipids accounts for the lack of specificity of most abps, and promotes the attraction between amps and bacterial membranes while preventing their binding to most host cells membranes. moreover, the inherent hydrophobicity and amphiphilicity allow abps to form clusters at the membrane's surface once attached. , at low concentrations (low peptide-to-lipid ratio), abps tend to adsorb onto the surface, adopting an orientation parallel to the membrane bilayer. however, clustering increases the peptide-to-lipid ratio, which subsequently promotes additional clustering. once a certain peptide-to-lipid ratio threshold is reached, abps adopt a perpendicular orientation relative to the bacterial membrane, which allows them to insert themselves into the membrane, as will be further discussed (fig. ) . peptides can adopt many different secondary structures and conformations, which depend on their amino acids sequence, as well as on the environment. consequently, different peptides and different environments favor the formation of unique structures, such as barrel staves, carpets, and toroidal pores. , , when the peptides attach and are inserted into the membrane bilayer so that the hydrophobic regions align with the phospholipids' acyl groups and the hydrophilic regions create the central region of a pore, a barrel stave is formed. in addition, these structures act as a primer for the aggregation of new peptides, thus expanding the pore's diameter. due to abps' cationic sites, membrane crossover would be very energetically unfavorable in their original parallel orientation. however, this orientation changes to a ( ) cluster at the cell surface and cause membrane disruption by several different mechanisms (e.g., barrel staves, carpets, toroidal pores), ( ) translocate into cells and impair intracellular organelle machineries, ( ) impair protein-protein interactions, enzymatic cascades, and cytosolic signaling pathways, ( ) interact with nucleic (not in the case of bacteria) acids, trap replication forks, and compromise nucleic acids as well as protein synthesis, ( ) preclude several steps of viral replication, ( ) inhibit genetic material trafficking, reverse transcriptase, and viral proteases, ( ) block the interaction between virus and host cells (e.g., viral envelope glycoproteins gp and gp or coreceptor cxcr ), compromising virus binding and entry, and ( ) cause membrane lysis on enveloped viruses. see text for detailed description. designed using servier medical art. perpendicular one upon abps' clustering, and the peptides are reoriented with their hydrophobic portions facing the membrane acyl groups and their cationic sites facing the interior of the pores. thus, clustering and pore formation provides a means for membrane disruption and the leakage of intracellular molecules from bacterial cells. in contrast, if peptides remain parallel to the membrane surface with their hydrophobic residues facing the membrane while their hydrophilic residues face the surrounding milieu, a "carpet"-like structure is achieved. with such organization, membrane's rigidity weakens progressively as new peptides are added, culminating in its dissolution and breakup. mechanistically, this mode of action is less stringent because peptides do not need to adopt a specific structure. however, the necessary concentration of abps appears be much higher than that required for the formation of barrel staves. [ ] [ ] [ ] for toroidal pores to form, peptides need to aggregate onto the membrane's surface and insert themselves in a perpendicular fashion. , subsequently, abps are reoriented parallel to the membrane so that polar residues interact with the polar heads of the membrane lipids, thus causing membranes to bend. this induced bending of the bilayer causes the upper and lower leaflets to meet, and allows a mixture of peptides and lipid head groups to line with the interior of the pore, forming toroidal or wormhole structures. , , abps may also inhibit bacterial growth and have a bactericidal effect by promoting the clustering of anionic lipids, such as phosphatidylglycerols (pgs) and cardiolipin. for instance, for ll- , initially believed to act according to the "carpet" model, there is evidence that its fragments, namely kr- , act as a magnet that competes for the negatively charged pgs, promoting their redistribution into "pg-rich domains." such phospholipidic reorganization may disturb cell signaling and compromise the activity of membrane-bound proteins, namely, the voltage-dependent potassium channels, which can seriously jeopardize bacteria survival. likewise, n-acylated peptides derived from lactoferricin were shown to induce defects in e. coli cell division by rearranging the distribution of inner membrane phospholipids, essentially due to the clustering of cardiolipin and other anionic lipids. the bias toward positive charges confers amps the ability to easily interact not only with biological membranes, but also with other negatively charged molecules (fig. ) such as nucleic acids and those bearing phosphate groups. , in addition, because this type of interaction is largely unspecific, amps can exhibit not only a broad-spectrum activity, but also multiple mechanisms of action (fig. ) . , for instance, lactoferricin is known to inhibit atp-dependent multidrug efflux pumps as well as dna, rna, and protein synthesis. ten human amps derived from lactoferricin and lysozyme are thought (by sequence homology) to act by trapping the replication fork and preventing base recombination and dna repair. furthermore, the presence of many prolines in some peptides (also known as ppii structures as aforementioned) hampers the organization of amphipathic structures. these structures favor interactions between peptidic structures and between these and nucleic acids, thus playing a major role in signal transduction and complexes assembly. however, ppii structures do not necessarily contain prolines, and abps rich in arginine residues are also able to translocate across membranes and to interact with nucleic acids and proteins. hence, both proline and/or arginine-rich abps may be capable of inhibiting prosynthetic signaling pathways and enzymatic activity (fig. ) , shifting the conformation of cellular structures and activating autolysins. however, this is a speculation that warrants further studying. in addition to targeting bacterial membranes, human defensins also bind to the peptidoglycan precursor lipid ii and inhibit cell wall biosynthetic enzymes in staphylococci species. moreover, when stabilizing disulfide bridges between conserved cysteine residues in human amps with β-hairpin or β-sheet conformations are disrupted, the resulting linear peptides still maintain their antimicrobial properties despite losing membranolytic activity. , the antifungal histatins act at multiple levels by mechanisms of action conserved across the histatin family of amps, with histatin the most potent amp in this family. this amp binds the yeast transmembrane receptor heat-shock protein ssa / p, is internalized, and then targets primarily yeast mitochondria. , by doing so, it leads to the formation of reactive oxygen species (ros), causes atp efflux, and inhibits oxidative phosphorylation. , simultaneously, histatin also interacts with the potassium transporter trk p, causing release of k + ions and further atp efflux. when the functional domain of histatin was synthetically multiplied, its antifungal activities were significantly potentiated and it became significantly faster-acting compared to the normal histatin . such an observation might prove itself very useful, because it indicates that modulating the number of functional domains per amp may enhance its antimicrobial activity without requiring the administration of increased quantities of amp. histatins may also exert their antimicrobial activities by inhibiting host and microorganismal proteolytic enzymes. histatin is a strong inhibitor of human matrix metalloproteases / and metalloproteases from porphyromonas gingivalis. thus, its inhibitory activity over proteolytic enzymes may attenuate tissue damage and microbial propagation during the onset of periodontal inflammatory disease. also, by inhibiting the cysteine protease clostripain from clostridium histolyticum, histatin mitigates the virulence factors produced by this clostridium species and may therefore alleviate the complications associated with gas gangrene syndrome. so far, the above-mentioned mechanisms of action have been discussed in the context of abps or antifungal peptides, and sometimes the same peptide is active against both bacteria and fungi. in turn, the action of antiviral peptides can diverge from these mechanisms and should thus be independently commented (fig. ) , although some overlap does exist. extracellularly, most native antiviral peptides often present as α-helical structures, have primarily lytic activity on enveloped viruses, and can directly inactivate cell-free virions (fig. ) . human lysozyme fragments are this group's prototype, capable of aggregating on the viral surface and causing disruption of membrane envelopes. , similarly, cap peptide appears to exert its antiviral activity by destructing the virus envelope of type i herpes simplex virus (hsv) or even by disrupting the capsids of adenoviruses. however, because membrane lipids of enveloped viruses derive from host cell membranes, special attention is required when considering the exploitation of this feature, as antiviral peptides may disrupt host cell membranes indiscriminately. alternatively, antiviral peptides may block the interaction between the virus and the host cell (fig. ) . that is believed to be, at least in part, the mechanism of action of human defensins, θ -defensin, human α -antitrypsin virip, cyclic lactoferricin, and serine protease inhibitor elafin. for instance, it has been demonstrated that all α-defensins [hnp- , hnp- , hnp- , and hnp- and human α-defensin (hd)- and hd- ] and hbd- prevented hsv infection by blocking virus binding and entry. such antiviral activity was attributed to hnp- - , hd- , and hbd- 's high affinity to hsv's glycoprotein b and hnp- , hd- , and hbd- 's avidity toward endogenous heparan sulfate. similarly, protection against hiv is conferred by peptides with lectin properties that can bind viral envelope glycoproteins gp and gp , crucial for viral attachment, fusion of envelopes with host membranes, and subsequent entry into host cells. hnp- that binds and blocks the action of gp , and θ -defensins (e.g., the pseudogene retrocyclin) together with the human α -antitrypsin-derived -residue viral-inhibitory peptide both targeting gp represent examples of such peptides. [ ] [ ] [ ] likewise, hsv- infection can also be blocked by retrocyclin, which binds tightly to glycoprotein b . the cyclic peptide lactoferricin seems to compete for the ligation to extracellular heparan sulfate or chondroitin sulfate, thus preventing the very first event in cytomegalovirus, hsv, and papillomavirus infection. [ ] [ ] [ ] finally, elafin is thought to act indirectly in host cells by hampering viral attachment and probably by modulating host's antiviral and inflammatory responses toward hsv- infection. synergistically with this mechanism, antiviral peptides can also bind and inhibit the activation of viral co-receptors, leading to further inhibition of glycoproteins-mediated viral attachment. not only that, hbd- and - inhibit viral infection by promoting the internalization of its co-receptor cxcr . , virucidal peptides may also preclude several steps of viral replication by interacting with intracellular targets (fig. ) . this is the case of hnp- and hd- , which may prevent hsv replication by binding directly to its dna. also, hnp- arrests influenza virus replication by inhibiting protein kinase c phosphorylation, an essential step for nuclear trafficking events. human cathelicidin (ll- ) derived peptides, in turn, inhibit reverse transcriptase and viral proteases, thus preventing integration of the viral genetic load into human dna. , the few resistance mechanisms against amps demonstrated so far among microorganisms have been observed almost exclusively in bacteria. with very few exceptions, bacteria do not seem to develop complete resistance against cationic abps, though some strategies do exist among bacterial species for diminishing their potency and efficacy. the difficulty in acquiring resistance may stem mainly from the fact that abps target an essential cellular component for bacteria survival, which cannot be easily modulated by the cell without causing significant adverse effects. therefore, resistance mechanisms are not likely to be a limitation in the therapeutic utilization of abps. in light of this scarce resistance mechanisms de novo acquisition, what mechanisms do bacteria naturally develop/possess against abps? first, one should consider that the primary target of abps consists of bacterial cell membranes and bear in mind how abps depend on the electrostatic interactions between membrane negative charges and positive peptidic residues. accordingly, bacteria have learned how to modulate cell membrane charges and composition. gram-positive bacteria can partially modify the peptidoglycan's teichoic acid and gram-negative bacteria can partly change its lipopolysaccharide lipid a moiety, thus compromising this interaction. for instance, d-alanine-activating enzyme and d-alanine transfer protein are known to transport positively charged d-alanines to the surface anionic teichoic acids in gram-positive s. aureus, reducing the negative net charge of its outer membrane. also in s. aureus, the five-component system graxsr-vrafg increases the expression of mprf and dltabcd genes upon exposure to cationic amps. when s. aureus strains are mutated so that the regulation of these genes is disturbed, these strains become more susceptible to daptomycin, polymyxin b, hnp, and platelet factor -derived peptide, and less infectious in vivo in a nonclinical endocarditis model. [ ] [ ] [ ] the gene product mprf attaches lysine residues, which bear a positively charged ε-amino group, to the anionic phophatidylglycerols, making these less negatively charged and thus less susceptible to interactions with cationic amps. , the gram-negative vibrio cholerae can increase its resistance to polymyxins more than -fold by modifying its lipopolysaccharides in a process mediated by almf. the lpta gene product found in gonococcal species, such as the gram-negative bacteria neisseria gonorrhoeae, protects lipopolysaccharide lipid a moieties by attaching phosphoethanolamine, making these bacteria less susceptible to amps and increasing their capacity to modulate host's immune response by evading complement-mediated cellular death. , moreover, in n. gonorrhoeae and neisseria meningitides, the presence of phosphoryl and phosphoethanolamine in lipid a moieties enhances the activation of the nf-κb pathway and the production of proinflammatory cytokines in human cells, which can amplify their virulence. despite this modification-dependent virulence, the absence of such modifications in commensal neisseriae species allow these to colonize and coexist with the human host without inducing bactericidal host immune responses. in addition, the adaptation of the composition and, hence, the electrostatic properties of the membrane in order to circumvent the host immune system is not an exclusive phenomenon of pathogenic bacteria. commensal bacteria of the intestinal tract can remove phosphate groups from their membrane's lipopolysaccharides, decreasing their negative charge and allowing greater survival and proliferation in the gut. similarly to the above-mentioned paradoxical modification-dependent virulence, mutated commensal gut microbiota bacteroides that fail to remove phosphate groups from their lipopolysaccharides are killed during inflammation while normal strains keep their resilience. therefore, there appears to be an inverse relationship between acquired resistance and bacterial virulence, so that induced/acquired modifications render cells less virulent while simultaneously allowing these to more easily colonize and coexist within the human host environment. second, bacteria are also able to adapt the fluidity of the membrane by increasing the hydrophobic interactions within their outer membrane, thus hindering pore formation. third, bacteria can express proteases against abps or transporters and efflux pumps like atp-binding cassette (abc) transporters to circumvent such innate defense barriers. , glu-c protease produced by s. aureus induces the loss of antibacterial activity of neuroendocrine peptides, and chromofungin, procatestatin, and human catestatin are enzymatically degraded when treated with bacterial supernatants. in e. faecalis, bacitracin binds the abc transporter ef - , which interacts with the regulatory domain of the two-component system ef - , increasing its expression and conferring resistance against bacitracin. two-component systems regulate the expression of abc transporters and are constituted by a transport permease and a sensor kinase. however, at least in b. subtilis, the abc transporter itself is required for both sensing of and resistance to bacitracin, suggesting these transporters to also function as environmental sensors. lastly, bacteria may also synthesize molecules capable of directly neutralizing abps, which can provide a way for bacteria to bypass amps. however, such resistance mechanisms are very limited and resistance to amps does not take place to a large extent. r bastos et al. in humans, endogenous amps have been found in several fluids throughout the body, as depicted in figures and as well as in table i . as noted, multiple amps have been found in tears, saliva, milk, blood, urine, sweat (and others not-so-well-explored biofluids), reflecting not only a remarkable versatility but also a huge conservation that reinforces its essential role. the robust knowledge of amps on some fluids (e.g., tears, urine, saliva) over others (e.g., amniotic fluid, cerebrospinal fluid) most likely reflects their availability, but different exposure to pathogens from the environment (figs. and ) should also account for these discrepancies to a large extent. additionally, saliva and urine screening is of special interest, since both the oral cavity and the urinary tract represent the main doorways for microbial invasion and colonization, and are thus enriched with host defense peptides. likewise, milk screening is of high relevance due to the presence of particular proteins and amps that will boost neonate's immunity. despite underrepresented, amps from other fluids are equally relevant (figs. and ). for instance, amps collected from airway secretions, bronchoalveloar lavages, and nasopharyngeal aspirates can be valuable sources of information regarding the physiological response to pathogens of the respiratory tract and may, themselves, constitute raw models for new therapeutic drugs. also, other than circulating immune cells derived, blood has not been regarded as a rich source of amps, but this view can be questioned (figs. and ), most likely due to tissue-derived amps' passage to the blood circulation. however, it should be noted that human fluid reservoirs are not independent/isolated, and, consequently, peptides may be present throughout and cross between, possibly undergoing modifications between such reservoirs. amps may be present in fluids as a result of many different processes, including exocytosis, in situ proteolysis, tissue necrosis, and cellular lysis. therefore, in this section, we will characterize naturally occurring amps based on structural and functional similarities rather than their source of collection. defensins are cationic peptides rich in cysteine residues, presenting as β-sheet structures stabilized by disulfide bridges. according to the alignment of these stabilizing disulfide bounds, mammalian defensins are classified into three subclasses, α-defensins, β-defensins, and θdefensins. disulfide bounds of α-defensins occur between cysteines - , - , and - . these peptides consist of - amino acids and adopt a triple-stranded β-sheet conformation. human neutrophils express four distinct α-defensins, known as hnp- to hnp- . despite having been initially isolated from peripheral blood leukocytes, hnp- to hnp- can also be found in bone marrow, spleen, and thymus. the remaining two already identified human α-defensins, hd- and hd- , are found only in paneth cells of the small intestine and in epithelial cells, thus being tissue specific. expressed in the aforementioned cell types, amps can also be found in bodily fluids in direct contact with these cells. among the most recent discoveries, hd- has been found to be active against bifidobacterium adolescentis and other anaerobic gut commensals, but not (directly) bacteriostatic or bactericidal against most pathogenic bacteria, and may therefore play an important role in maintaining a balance among the microbiota of the gut. similarly, human hd- is particularly potent against the gram-positive bacillus clostridium difficile, one of the most common pathogens responsible for nosocomial infections, whose highly virulent strains often attack small intestine mucosa. in addition, α-defensins may show both cellular and regional specificity throughout the gastrointestinal tract, either secreted or active inside intracellular granules and constitutively expressed or amenable to induction. , , it should also be emphasized that the activity spectrum of α-defensins is not limited to bacteria and fungi, exhibiting activity against several virus, including hiv, hsv, adenovirus, and human papillomavirus (hpv), and displaying several unique mechanisms of action (please refer to section ). it should be noted that the antimicrobial activity of α-defensins is influenced even by the difference in one amino acid, such as for hnp- to hnp- . in fact, an additional acidic residue in hnp- lowers its effectiveness (most accurately, its potency) against s. aureus, p. aeruginosa, and e. coli. this is explained by the easily donated proton, which makes this defensin less positively charged (at least when considering interaction domains only), thus reinforcing the importance of a positive net charge for the antimicrobial activity of amps, , as evidenced in section . in terms of disease, α-defensins are known to have a paramount role, particularly when the demands for defense mechanisms are increased. accordingly, a remarkable -to fold increase in the concentration of hnp- and hnp- (and hnp- to a smaller extent) in human tears as been reported after ocular surgery, an increase that allowed these peptides to reach the required concentrations for antimicrobial activity but that is followed by a decrease once healing has been completed and the demands for immune defenses have returned to baseline. nonetheless, the goal of achieving this increased α-defensin concentration in tears is not restricted to the enhancement of the immune defenses, as it is also known to promote local cellular proliferation and tissue repair. in parallel, salivary α-defensins are active against streptococcus mutans, but their secretion has been found deregulated in caries-positive human subjects. in contrast, prolonged physical exercise has been shown to significantly increase the levels of salivary α-defensins, showing that lifestyles may have a considerable impact on our amp-based defenses against microorganisms and it is possible to modulated the activity thereof. disulfide bounds of β-defensins occur between cysteines - , - , and - . these defensins are longer than α-defensins, spanning from to amino acids, but also share a triple-stranded βsheet conformation. the first hbd was isolated from plasma samples and kidneys constitute the major source of hbd- . since then, peptides derived from human hbd- have been identified in tears, urine, vaginal lavage, sweat, and blood plasma, and these are effective primarily against e. coli. [ ] [ ] [ ] [ ] in turn, hbd- was isolated from psoriatic skin samples, displaying bactericidal activity toward gram-negative e. coli, p. aeruginosa, and c. albicans, and bacteriostatic activity toward gram-positive s. aureus. despite very effective in vitro, both hbd- and hbd- present attenuated activities in vivo as a result of their sensitivity to physiological salinity. in contrast, hbd- is active against s. aureus and vancomycin-resistant e. faecium even at physiological salt concentrations. the hbd- defensin has been found in seminal plasma, and it has been reported as potential efficacious against e. coli, s. aureus, and c. albicans. , furthermore, hbd- has been identified in epididymal fluid and has been shown to regulate lipopolysaccharide-mediated inflammation toward e. coli, s. aureus, and c. albicans, protecting sperm from motility loss. in disease conditions, increased concentrations of hbds in plasma and bronchoalveolar lavage fluid were reported in patients with diffuse panbronchiolitis. in particular, β-defensin a was suggested to play a role against p. aeruginosa. also, while β-defensins are expressed throughout the gastrointestinal tract, these seem to have evolved together with changes in human gut bacteria, displaying characteristic expression patterns in response to certain microorganisms. their expression may be significantly altered in human diseases, most notably in inflammatory bowel diseases (for insightful reviews on this topic, please refer to , and enhanced in inflammatory conditions of the ocular surface ). with α-defensins, the role of the ph for amps' activity is considered. here, β-defensins serve to illustrate the role of salt concentration. in fact, tears contain a remarkable concentration of sodium chloride, which inhibits the activity of human amps (by ß % in the case of hbd- , an inhibition that increases up to % in the presence whole tear fluid, suggesting the presence of other inhibiting factors). therefore, whenever there is an increase in the demand of amps activity, (e.g., upon infection or tissue damage), their concentrations must surge to reach the required protective levels. however, an excessive increase in the concentration of any amp may lead to tissue damage and local functions may be compromised. for this reason, a complex interplay of amps is required at surfaces like the ocular epithelia, which is achieved by a qualitative enrichment, in addition to the expected quantitative increase (for a depiction of the multitude of amps found in human tears, please refer to ). similarly to α-defensins, the activity spectrum of β-defensins is not limited to bacteria and fungi, and they are active against several virus, namely, hiv, influenza a virus (iav), respiratory syncytial virus, and vaccinia virus, and display several unique mechanisms of action, depending on their target (see above section on mechanisms of action). lastly, genomic studies have predicted additional β-defensins to be expressed in human cells, but their presence in human bodily fluids and their antimicrobial activity in vivo is yet to be demonstrated. while αand β-defensins adopt triple-stranded β-sheet conformations, θ -defensins have shorter sequences and adopt a complete circular conformation, with stabilizing disulfide bounds between cysteines - , - , and - . the peptide rhesus θ -defensin is the prototype of this more recently discovered family of amps. like αand β-defensins, θ -defensins possess broadspectrum microbial activity against bacteria and fungi, while also being capable of protecting mononuclear cells from infection by hiv- . , moreover, these peptides display antiviral activity against hsv, hiv, iav, and severe acute respiratory syndrome (sars) coronavirus, acting by several mechanisms, depending on their target. structurally, θ -defensin peptides are very interesting and very odd, representing the first cyclic peptide family discovered in animals, albeit not in humans. moreover, their circular structure appears to be required for antimicrobial activity. these peptides were first isolated as antimicrobial octadecapeptides expressed in leukocytes of rhesus monkeys, and their formation results from the head-to-tail joining of two independent nine-amino acid peptides derived from truncated pro-α-defensins. in contrast to the previously described classes of defensins, human θ -defensins are thought to be completely inactivated, despite the existence of mrnas encoding at least two θ -defensins expressed in the bone marrow. while genes encoding θ -defensins in humans also encode premature stop codons (thus hampering θ -defensin expression), synthetic human θ -defensins (called retrocyclins) have been shown as promising antiviral agents. in fact, retrocyclin- inhibits the entry of hiv, hsv, and iav into host cells. in other mammals, retrocyclin- also protects from infection by bacillus anthracis spores and the rhesus θ -defensin protects mice from sars coronavirus infection. cathelicidins are a very diverse group of cationic α-helical and amphipathic amps that exhibit a broad-spectrum activity against bacteria, fungi, and virus. the term "cathelicidin" originally referred only to the entire precursor protein, though currently is commonly used for the whole family, including the resulting peptides with antimicrobial activity. in contrast to the mammalian trend in which cathelicidins and defensins are the main amps, there is only one human gene encoding multiple cathelicidin-related peptides, , located on chromosome and expressed in the airways, mouth, tongue, esophagus, epididymis, and small intestine. , despite this tissue expression pattern, ll- is constitutively formed in spleen, liver, stomach, intestine, and bone marrow. ll- is highly positively charged (+ charge at physiological ph . ), due to the high content of arginine and lysine amino acids, and adopts an α-helical structure in solutions with ionic composition similar to that of human plasma. while defensins share common structural features, cathelicidin-related peptides are highly heterogeneous despite deriving from the same precursor. cathelicidins are characterized by a highly conserved n-terminal signal peptide (the so-called "cathelin domain") and a highly variable c-terminal antimicrobial domain that can be released after cleavage by proteinases. , cathelicidin is cleaved into the antimicrobial peptide ll- by both kallikrein and kallikrein serine proteases, it is upregulated by vitamin d, and has been shown to significantly reduce the risk of death from infection dialysis patients. ll- was shown to disrupt bacterial membranes through the formation of toroidal pores and carpet structures, and to exhibit chemotactic properties, attracting leukocytes and activating secretion of chemokines. in addition, ll- is internalized by cells, acidified in endosomes, and activates the signaling pathway downstream to toll-like receptor by interacting with double-stranded rna. profiling of airway fluids collected from premature infants' tracheal aspirates during infection evidenced the production of ll- even at an early stage of development. in these samples, ll- was found in significantly increased concentrations during pulmonary or systemic infections, suggesting this peptide to be as important to avoid infectious processes as to fight already established infections. the concentration of ll- has been measured in seminal plasma samples from healthy donors and found to be up to -fold higher than in blood plasma. furthermore, it was found to be attached to spermatozoa, eliciting a possible role in human fertilization or its precursor to be extensively cleaved by the high concentration of serine proteases present in seminal plasma. ll- , an alternative form of human cathelicidin peptides, contains one more alanine at the n-terminus than ll- and was initially found to be produced in female vaginal secretions due to the action of gastricsin on sperm precursor cathelicidin. both ll- and ll- are active against bacteria such as e. coli, s. aureus, p. aeruginosa, and bacillus megaterium, and synergistically with peptides from seminal plasma play an essential defense role protecting the human reproductive system. , interestingly, both citrullination and adp (adenosine diphosphate)-ribosylation of arginines can compromise the ability of ll- to prevent endotoxin-induced sepsis, perhaps because the attachment of these negatively charged molecules reduces the peptide's cationicity or by raising steric hindrance. , histatins are a normal component of human saliva and part of the innate immune system, protecting against a broad array of infectious agents, especially against candida species, saccharomyces cerevisiae, cryptococcus neoformans, and neurospora crassa. [ ] [ ] [ ] there are only two genes enconding histatins (both located on chromosome ), and they are exclusively expressed in human salivary glands. however, other active amps resulting from the cleavage of histatins and also exist. histatins and are encoded by genes htn and htn , respectively. histatins , , and - are the products of posttranslational proteolytic cleavage of histatins and , though histatin can also result from posttranscriptional modification of histatin mrna. , peptides derived from histatin bear distinct domains associated with specific functions, namely an n-terminal domain with antimicrobial activity and a c-terminal domain with wound-healing properties. , histatin has two important metal-binding motifs, consisting of an amino-terminal copper (ii)/nickel (ii)-binding motif and a zinc (ii)-binding motif, although they can also bind to cobalt (ii) ions. , the ability to coordinate metal ions appears to be important for the stabilization of the secondary structure, particularly in the presence of negatively charged membranes. nevertheless, histatins lack any defined secondary structure and are unordered when in aqueous solutions, assuming α-helical structures only in organic solutions. , in addition, histatin binding to copper (ii) and nickel (ii) ions induces the formation of ros, which contributes for its fungicidal activity. , histatins can bind to a receptor on fungal cell membranes and induce cell death by disrupting the cell cycle and causing nonlytic loss of atp, as discussed above. while histatins are effective, potent, and fast-acting amps against several antimicrobial species, these peptides are nontoxic to human cells and human microflora, which may be due to the fact that their translocation across the membrane is site and species specific. , both parotid, submandibular, and sublingual glands secret histatins, and their concentration and secretion have been shown to decrease with age, even when accounted for total protein concentration. moreover, oral candidal infections increase with age, suggesting a link between age-associated deficiency of human histatins and an increased risk of oral infections. dermcidins are broad-spectrum anionic amps with no apparent homology to other known amps. peptides in this group comprise amino acid residues, which are subsequently processed into smaller but still active ones. dermcidins are constitutively expressed in human eccrine sweat glands and secreted in sweat, exhibiting antimicrobial activity against common human pathogenic microorganisms such as s. aureus, staphylococcus epidermidis, e. coli, e. faecalis, and c. albicans. [ ] [ ] [ ] this constitutive production contrasts with human defensins and cathelicidins, which are induced during inflammatory and stressful conditions. a small percentage of human breast cancer cells displays rna encoding dermcidin, and after oxidative stress induction different types of tumor cells leads to the production of diverse and biologically active proteolytically processed dermcidin peptides. , therefore, the upregulation of dermicidins may confer human breast cancer cells a selective advantage compared to nonmalignant cells. in contrast, reduced expression of dermcidins in sweat from atopic dermatitis patients contributes to skin infections and altered skin colonization. hence, the downregulation of dermicidins in atopic dermatitis patients may represents a suppression of an innate and fundamental defense mechanism, which, in turn, could confer an advantage to infectious microorganisms. unlike histatins, which are nontoxic to human cells and human microflora (see above), dermicidins can have serious deleterious consequences to the human host. dermcidin isoform is known as a stress-induced oxidative protein capable of inhibiting the synthesis of nitric oxide in endothelial cells, insulin in pancreatic islet β-cells, and hepatic hormone synthesis. also, hypertensive patients show significantly higher levels of plasma dermcidin, but salicylic acid has been shown to reduce dermcidin levels while simultaneously attenuating dermcidin-mediated insulin inhibition in patients with acute myocardial infarction. therefore, the exploitation of dermcidin isoform for antimicrobial therapeutic purposes is most likely not possible due to severe side effects. despite this, it may be a promising therapeutic target for hypertension and diabetes management. in animal models, dermcidin has been shown to induce platelet aggregation and coronary artery disease by activating platelet cyclooxygenase and inhibiting the constitutive form of nitric oxide synthase, respectively, with an efficiency times higher than that of adp at activating cyclooxygenase. notably, dermcidin was able to stimulate the development of coronary artery disease in one animal model, even at suboptimal concentrations of adp within min. together, these observations suggest that the ability of amps to fight infections depends on their induction by infectious microorganisms, the particular peptides generated, and the local environment/tissue in which they act. also, biological roles performed by human amps are more diverse than immediately expected, playing essential functions other than fighting infectious microorganism(s). in fact, dermcidin-mediated inhibition of insulin is unique in the sense that no other protein is known to arrest pancreatic insulin synthesis in such way, and similar observations might result in novel and unexpected applications of human amps. amps expressed by human liver (leap and ) were initially discovered in human blood, and subsequently found in urine. these peptides are known as hepcidins and are characterized by a high content in cysteine residues (approximately % of all residues, with eight disulfide bounds). leap- or hepcidin- is essential for maintaining iron homeostasis, and, when mutated, leads to severe iron overload and juvenile hemochromatosis. hepcidins are the main regulators of plasma iron concentration and its secretion is promoted during inflammatory responses, particularly by the action of interleukin , which is one of the main mediators of the acute phase response. as iron bioavailability is a limiting factor for bacterial growth, hepcidins represent an organic mechanism for sequestering iron from invasive pathogens. however, when several iron sources are available, as it happens within the human host, eliminating a single reservoir may not be sufficient to attenuate microorganisms' virulence. moreover, bacteria are known to exploit almost every host iron-binding protein and reservoir and, consequently, human laeps/hepcidins are a rather limited source of antimicrobial activity. for these reasons, the antimicrobial role of iron sequestration depends not only on hepcidins, but also on several other mediators, such as transferrin, lactoferrin, ceruloplasmin, haptoglobin, and hemopexin. mutations responsible for decreasing the levels of hepcidins or compromising the function of other regulators of iron homeostasis, such as the major histocompatibility complex class i like hereditary hemochromatosis protein (hfe), transferrin receptor , and ferroportin, lead to increased iron blood levels. when levels are high enough to produce hemochromatosis, individuals are at an increased risk of infectious complications, including bacteremia and meningitis caused by e. coli, bacteremic cellulitis, and hemorrhagic bullous skin lesions caused by v. cholera, multiple pyogenic abscesses in the liver parenchyma as a result of yersinia enterocolitica infection, meningitis, endocarditis, and pericarditis resulting from infection with l. monocytogenes, and fatal septicemia during infection with vibrio vulnificus. in contrast, hemochromatosis can also be associated with decreased macrophage iron storages in the case of hfe-associated hemochromatosis. this depletion makes these cells more efficient in fighting salmonella enterica subsp. enterica, serovar typhimurium, and mycobacterium tuberculosis infections. , however, these protective effects of macrophages iron storage depletion have only been demonstrated in animal models and appear to be mediated not by hepcidins, but rather by lipocalin- , which reduces the availability of iron for salmonella, and transferrin and lactoferrin, both of which compromise iron acquisition by m. tuberculosis. , neuropeptides and neuroendocrine peptides may exert mitogenic actions through which innate barriers are reinforced and may display neurotransmitter, immunoregulatory, and direct antimicrobial activity. vasostatin- was the first natural antifungal n-terminal chromogranin a derived fragment peptide, having been first isolated from chromaffin secretory granules from bovine adrenal medulla. however, its sequence is highly conserved in humans ( % identity homology) and its microbial activity also overlaps, to some extent, with that of bovine origin. irrespectively, human vasostatin- displays antimicrobial activities against gram-positive bacteria (micrococcus luteus, b. megaterium) and a large variety of filamentous fungi (n. crassa, aspergillus fumigatus, alternaria brassicola, nectria haematococca, fusarium culmorum, fusarium oxysporum, trichophyton mentagrophytes) and yeast cells (s. cerevisiae, c. albicans). moreover, many autocrine and paracrine biological activities of chromogranin a, its precursor protein, have been attributed to peptides located along its sequence, which are co-released with catecholamines by chromaffin cells upon stimulation and can be found in human bodily fluids. , therefore, the antimicrobial activity spectrum of vasostatin- may be extended by that of co-released peptides also formed by chromogranin a proteolytic cleavage, and some of the coproduced peptides are even more potent than vasostatin- itself. in addition to the structural requirements for the antimicrobial activity of chromogranin a derived peptides, posttranslational modifications may also modulate their activity. for instance, s-pyridylethylation (an alkylating modification consisting of the addition of pyridylethyl moieties to the -sh functional groups of cysteine residues that disrupts disulfide bridges) renders it selectively active against b. megaterium but not m. luteus, while oxidation seems to render it inactive against bacteria but still active against fungi. in addition, chromogranin a derived peptides also undergo phosphorylation and glycosylation, which can modulate their physicochemical properties. currently, vasostatin- is known to be stored in endocrine, neuroendocrine, and neuronal cells, and to be released from stimulated chromaffin and immune cells upon stress. moreover, the stimulation of polymorphonuclear immune cells induces the processing of chromogranin a and the secretion of vasostatin- and other chromogranin a derived peptides, which may account for local inflammation. lastly, recombinant and synthetic human vasostatinderived fragments have been shown to inhibit vascular contraction induced by endothelin- , parathyroid hormonal secretion, neuronal survival, expression of neurofilaments, and neuronal gaba uptake. , adrenomedullin was first isolated from a human adrenal pheochromocytoma and its plasma concentration is raised under specific physiological conditions, namely, renal failure, hypertension, and sepsis. this peptide has antibacterial activity against several grampositive and gram-negative bacteria, including propionibacterium acnes, s. aureus, m. luteus, p. gingivalis, actinomyces naeslundii, s. mutans, c. albicans, eikenella corrodens, actinobacillus actinomycetemcomitans, streptococcus pneumoniae, haemophilus influenzae, streptococcus pyogenes, bacteroides fragilis, e. coli, and helicobacter pylori. human neuropeptide y displays antimicrobial activity against bacteria and fungi, but truncated fragments are tenfold more potent than the intact precursor neuropeptide y, perhaps because the net charge of the fragments is more positive than that of the intact neuropeptide. encephalins and their derived peptides may either enhance or inhibit the immune response and the corresponding circulating immune cells/mediators in both humans and animal models. , as well as their subsequent antimicrobial effects, may thus take place indirectly in immune system modulation. simultaneously, antibacterial assays have revealed that peptide b/enkelytin (a c-terminal fragment of proenkephalin a) specifically targets gram-positive bacteria, including m. luteus, b. megaterium, m. luteus, and s. aureus, while having no effect over gram-negative bacteria. , classically, proenkephalin a has been ascribed to the secretory granules from adrenal medullary chromaffin cells and various brain regions, such as the striatum and the pituitary, according to some animal studies. , despite this, it has also been demonstrated to be expressed in and secreted by polymorphonuclear neutrophils (accounting for its particular enrichment in wound fluids), t and b lymphocytes, macrophages, and mast cells. , however, it should be noted that the antimicrobial effects of encephalins and their derived peptides result mostly from animal studies and have not been adequately studied in human secretions, despite the high conservation of their sequences across species, which most likely contribute for the similar activity spectrum. even so, phosphorylation and glycosylation are characteristic of these peptides, , and it is possible to envision that differences at the level of posttranslational modifications or as a result of gene divergence may result in distinct activity spectra. interestingly, peptide b/enkelytin is metabolized in vivo to opioid peptides, thus revealing an intricate relationship between innate immunity and pain modulation, and its plasma concentration is increased after coronary artery bypass grafting in humans. because it is expressed in both immune and neuronal cells, it is possible that the same stimuli may act on both cells populations, thereby accounting for immune system modulation at the periphery and alterations in central nervous system signaling. however, the exact roles and mechanisms of this interplay are not clear. in addition, other amps found in epithelial secretions from the gut and the skin, for example, include peptides derived from alpha-melanocyte-stimulating hormone, which is active against s. aureus and c. albicans. accordingly, evidences suggest that amps like these act by inhibiting mrna and protein synthesis rather than inducing direct membrane disruption, as is the case for most amps. several cationic proteins display nonenzymatic antimicrobial activity, which is sustained by the resulting cleavage peptides after protein fragmentation. for example, lactoferrin is a glycoprotein enriched in milk and neutrophilic granules, which has the ability to sequester iron and thus prevent bacterial overgrowth. when digested by pepsin, lactoferrin yields lactoferricin h (human), an amp able to neutralize bacterial toxins, regulate gene transcription, inhibit complement activation, viral infection, and tumor overgrowth, thus further extending the antimicrobial properties of its precursor. human chemokines are secreted by macrophages and polymorphonuclear immune cells and, therefore, found in the blood circulation. moreover, during allergic inflammatory responses to bacterial infections of the airways, the eosinophil-recruiting chemokines eotaxin- /ccl , eotaxin- /ccl , and eotaxin- /ccl are generated by mast cell proteases, active against several airway pathogens, including s. pneumoniae, s. aureus, h. aeruginosa. peptides resulting from the cleavage of pepsinogen a and c prosequences have also been shown to exert antimicrobial activity. although these were initially identified from the stomach of bullfrogs, synthesized human pepsinogen c prosequences with similar structural characteristics (amphipathic and helical structures) to these cleavage peptides have also exhibited antimicrobial activity. calcitermin is an amp that results from the cleavage of a amino acids long sequence from the c-terminal region of protein calgranulin c, isolated from human airway secretions. by adopting an alpha-helical conformation in bacterial membranes, calcitermin is able to target gram-negative bacteria (e.g., e. coli and p. aeruginosa) and fungi (e.g., c. albicans). furthermore, the precursor protein and fragment peptide may act synergistically to protect the human host against foreign microorganisms present in the airways. on the one hand, the cleavage peptide calcitermin inserts into membranes of microorganisms. on the other hand, the precursor protein calgranulin c has a proinflammatory activity. by acting as a dangerassociated molecular pattern molecule and binding to the receptor for advanced glycation end products in innate immune cells, it activates the map-kinase and nf-κb signaling pathways, leading to the production of proinflammatory cytokines and histamine, upregulation of cell adhesion molecules, recruitment of leukocytes, and degranulation of granulocytes. , dermcidin-derived peptides such as dcds, ssls, and leks (the letters correspond to the first three amino acids of the corresponding precursor dermcidin protein) are also formed in eccrine sweat glands (but not in apocrine sweat glands) at these body sites with high probability for contact with pathogens (e.g., palms, face, arms) and exhibit antimicrobial activity against a large number of microorganisms, including s. aureus, e. faecalis, e. coli, s. epidermidis, pseudomonas putida, methicillin-resistant s. aureus, and rifampicin/isoniazid-resistant m. tuberculosis. [ ] [ ] [ ] such dermcidin-derived amps are regulated by proteolytic processing at several levels (see section ) and are differentially expressed during inflammatory conditions. these peptides are resistant to proteolytic degradation in sweat up to at least hr and, interestingly, most dermcidin-derived peptides are anionic in nature. if such distinct properties may lead to different antimicrobial activity spectrum is unclear, though not surprising, as skin represents a harsh and particularly unique environment. as of specific interest, it should be noted that the consensual average skin surface ph has been decreasing in the last few years and is now generally accepted to be below . while showering and cosmetic products decrease skin's surface ph, plain tap water can increase the skin ph up to hr. noticeably, negative charges seem to be important for dermcidin-derived peptides' antimicrobial activity, in contrast with what would be expected for amps. in fact, most dermcidin-derived peptides are naturally anionic, but low ph (high h + concentration) leads to an increase in their net charge (toward positivity). therefore, while an acidic skin actually keeps the resident bacterial flora attached to it, a more alkaline skin compromises such attachment, suggesting that the more negatively charged such skin peptides are (alkaline skin), the more efficient these become. despite the average length of . residues and net charge of + . for the close to amps already identified, human amps tend to be longer and more positively charged. amps with more than amino acids have been identified across the six kingdoms of life. curiously, most (approximately half) appear to be of human origin, presenting as amino acid sequences of residues on average and suggesting large amps to be a human feature. moreover, compared to all the remaining amps, large human amps tend to be biased toward leucine ( . %) and lysine ( . %) residues, displaying nonpolar aliphatic and positively charged side groups, respectively (table i) . therefore, large human amps are enriched with amino acids that promote distinguishing features of amps: high overall positive charge, hydrophobicity domains, and amphiphilicity. nevertheless, this bias toward lysine residues is neither unique to human amps nor more pronounced among these. for instance, the amphibian cathelicidin rc- is composed by % lysine residues, and its homologous snake crotalicidin contains % lysine residues, showing high potency against s. pyogenes, acinetobacter baumannii, e. faecalis, s. aureus, e. coli, klebsiella pneumoniae, and p. aeruginosa. as evidenced in table i , large human amps have been found in a variety of bodily fluids, including tears, saliva, urine, airways secretions, breast milk, blood, sweat, and epididymal fluids. in addition, other large amps are also expected to exist despite not constitutively found in biologic fluids, due to their inducible nature. for instance, paneth cells in the small intestine release amps-rich granules upon stimulation by cholinergic or bacterial stimuli, and granulocytes are known to contain amps in granules destined for extracellular secretion. , therefore, as the synthesis and secretion of large amps may not be constitutive but amenable to induction by microbial macromolecules and inflammatory cytokine stimuli, several other large amps may yet to be catalogued. hence, exploration of novel large antimicrobial human peptides is an ongoing work that may yield novel therapeutic agents. peptidomics is the complete study or global analysis of the peptides present in a biological sample, at a given time, under a particular condition. , the identification of naturally occurring amps in human biological fluids is a laborious, daunting, and sometimes unsuccessful task. such naturally occurring peptides can directly result from gene encoding, from passage to bodily fluids as a result of cellular damage and renewal, or from extracellular proteolytic processing of precursor proteins. the latter, considered to be the most prevalent, hampers the identification and interpretation of biologically active amps because precursor proteins may themselves display antimicrobial activity, but may also be cleaved into multiple functional and nonfunctional peptides. , moreover, owing to the large dependency of amps on their sequence and d structure, these peptides should not be subject to mass spectrometry (ms) based approaches employing enzymatic or chemical digestion, neither to any in vitro step that may cause their fragmentation. instead, top-down proteomic strategies are preferred. classically, different peptides fractions have been separated and tested for their biological activity, often without ever addressing the peptides' biological/antimicrobial activity, their sequence, target, or hypothetical mechanism(s) of action. however, current strategies are more focused, precise, and informative, capable of addressing all these parameters, sometimes in a single study (see below). , , human biological fluids consist of complex samples, containing lipids, salts, proteins, carbohydrates, and other molecules that make the study of endogenous peptides extremely difficult. consequently, enrichment and separation steps are almost always required. of these biomolecules, proteins and larger peptides as well as salts are the most complicated to separate from the target peptides. luckily, salts can sometimes be removed in the same procedures applied for protein removal. first and foremost, proteases/peptidases have to be inactivated, which can be done by denaturing procedures (e.g., microwaving, boiling) or by adding protease/peptidase inhibitors. , however, both of these can modify the peptide structure and, thus, these steps can be avoided when samples are resistant enough to further protease/peptidase activity. this is the case for urine samples, for example, as, when collected, have long been subject to proteolytic processing in the bladder. proteins are frequently removed by selective precipitation, most commonly by organic solvent (e.g., acetone) or acidic (e.g., trichloroacetic acid) precipitation, which also removes salts (left in the supernatant). , however, precipitation-based proteins removal is not complete and may lead to the formation of peptide aggregates, which are consequently lost in the precipitate. since peptides can present a great variety of sizes, sequences, structures, hydrophobicity properties, net charges, and other characteristics, their isolation is complex. still, such complexity allows for isolation steps to take place at multiple dimensions, increasing peptides isolation efficiency and accuracy. for instance, peptides can first be isolated based on their size and charge, , bias toward specific amino acids (e.g., cysteines, tryptophans, methionines), [ ] [ ] [ ] or on the presence of post-translational modifications (ptms) (e.g., phosphopeptides, glycopeptides). , still, because of the aforementioned limitations, the search for endogenous amps in human bodily fluids has mostly relied on the identification of candidate amps by chromatographic techniques (e.g., high-performance liquid chromatography, hplc) and sequencing (e.g., edman sequencing), followed by their de novo chemical synthesis and in vitro antimicrobial activity testing. for these reasons, the identification and in vivo testing of novel amps is often time consuming and requires multiple studies/phases and resources. in order to mitigate this problem, candidate human amps have come to be first predicted in multiple ways, including mining of the entire human genome for particular motifs known to confer antimicrobial activity, and screening of human peptidomes (e.g., salivary, urinary) in order to find homologous peptides to other known amps. accordingly, several libraries and databases are currently available for data comparison, and some search programs can even combine both previously obtained data and de novo information. even so, the identification of amps benefits the most from combinations of chromatographic and immunological techniques, genomics and proteomics. while genomics and proteomics both allow the prediction of novel amps and have the potential for contributing with a larger number of putative amps, peptides with proven in vivo antimicrobial activity have to be more accurately identified by combinations of chromatographic and immunological techniques, chemical sequencing and antimicrobial assays. though immunoassays can detect the intended target in a variety of complex biological samples, these techniques do not provide an integrative view of the peptidome, can suffer from cross-reactivity or lack of specificity issues and require previous knowledge of the target structure. , moreover, this issue becomes a particular problem when considering that shorter or longer peptides resulting from the same precursor protein may all contain the same epitopes and thus be recognized as the same peptide fragment despite exhibiting distinct biological activities. electrospray ionization (esi) and matrix-assisted laser desorption ionization (maldi) have been the most frequently and successfully applied ionization techniques in the field of bodily fluids peptidomics. in line with these, peptides are most frequently previously separated by lc-based and capillary electrophoresis (ce) based approaches, allowing different peptide fractions to be independently separated. from this point onwards, almost any mass analyzer can be used, although in practice quadrupole time-of-flight (q-tof) and ion traps (it) instruments are the most frequently utilized ones in peptidomics, especially in exploratory studies. [ ] [ ] [ ] in turn, fourier transform-ion cyclotron resonance-ms is more suitable for targeted/confirmatory analysis when dealing with fewer peptides. because these apparatuses allow tandem ms to be performed, peptides can be readily sequenced in addition to their identification, circumventing the above-mentioned limitations concerning immunological assays and largely simplifying peptidomics workflows. in summary, when aiming at the isolation of a particular peptide, with predicted or known charge and molecular weight, an ms-based procedure can theoretically be very straightforward. first, fluid samples require (sometimes multiple) centrifugation steps to remove cells, cellular debris, wastes, and possible contaminants. then, proteins and peptides in the supernatant are separated and fractionated by multidimensional chromatographic techniques. within these procedures, proteins and peptides can be initially fractionated by gel filtration (size dimension), followed by cation-exchange (charge dimension) fractionation. cation-exchange chromatography uses a negatively charged ion exchange resin with affinity for molecules with net positive charge. a salt gradient is used to separate the peptides of interest from other bound peptides, based on their predicted isoelectric point. finally, peptides can be subject to ms analysis. they are typically ionized by esi or maldi and subsequently analyzed in a q-tof, it, or hybrid ltq. as amps tend to be highly basic peptides due to a bias toward arginine and lysine residues, these have a propensity to be particularly enriched when applying cation exchange chromatography based techniques. however, because biological fluids are complex matrices, these sometimes require multiple fractionation, isolation, and extraction steps. these are laborious optimization techniques for the detection and quantification of amps and, despite their inherent complexity, they are often based in not always accurate functional and structural predictions. a recent approach has proved itself capable for the isolation of peptides present in bodily fluids that can bind directly to certain bacterial membrane components, such as lipopolysaccharide, and induce an inflammatory response against these. , after obtaining a bacterial homogenate and extracting membrane components with an organic solvent such as n-butanol, functionalized beads with n-hydroxysuccinimidyl-sepharose (an agarose used in affinity and protein chromatography) are conjugated with these membrane bacterial components and incubated with a human biological fluid (e.g., saliva, urine). then, peptides bound to the conjugated beads are eluted and analyzed in a ltq-orbitrap hybrid fourier transform apparatus. moreover, functionalized beads conjugated with bacterial membrane components can be tested in vivo for their capacity to induce the production of inflammatory mediators. thus, this exploratory approach could result in the identification of potential human amps with selective properties capable of binding bacterial membranes and inducing an inflammatory response. recently, dallas and colleagues have attained the most complete and prolific analysis of naturally occurring peptides with potential antimicrobial activity in human milk by nano-lc quadrupole-tof tandem mass spectrometry (ms/ms), while searching against libraries of human milk peptides and known amps. in order to remove milk peptides produced by in vitro proteolysis, fresh milk derived peptides were compared to control samples of the same origin but immediately subject to boiling. as boiling denatures proteases, in vitro proteolysis does not take place and peptides present in these controls could then be confidently assigned in the original analysis. moreover, masses fragmented in the first round of ms/ms were excluded from subsequent rounds of ms. the authors realized that more than % of the + unique naturally occurring peptides identified with % confidence have yet unknown functions, which serves to illustrate how much there is still to discover regarding the biological roles of human endogenous peptides in biological fluids. nevertheless, the antimicrobial activity of the milk peptides ( predicted amps) was confirmed in vivo by radial diffusion assays against the gram-negative e. coli and the gram-positive s. aureus. furthermore, this study reinforced the observation that the background against which mass spectra are searched seems to be a more critical factor than the mass analyzer or the ionization technique used, when it comes to both the number of peptides identified and the confidence by which these can be assigned. in the same study, authors have emphasized and demonstrated the importance of avoiding in vitro hydrolysis of amps in human bodily fluids, which can be attained by adding protease inhibitors after sample collection or by adequately accounting for this phenomenon, as the authors did by using an appropriate control. in contrast, not all bodily fluids seem to be susceptible to this degree of hydrolysis, at least not to the same extent. urine, for instance, is thought to be more stable and not to require treatment with protease inhibitors. however, by the time urine is collected it has already remained stagnant for considerably longer periods of time, and, hence, probably already extensively exposed to the action of proteases/peptidases inherently present in urine. therefore, how intact urinary amps actually are should always be questionable and these should ideally be interpreted considering the background of hydrolytic enzymes present in the same urine sample. also questionable is the inhibition of proteolysis in seminal plasma samples. in fact, the liquefaction process of human semen performed by seminin and other proteolytic enzymes is physiological, and, therefore, naturally occurring amps should be analyzed in samples that were allowed to liquefy without the addition of protease inhibitors. curiously, seminin, which is the primary agent responsible for liquefaction of the seminal coagulum, is partially destroyed by freezing at − °c, reinforcing the vitality of analyzing fresh samples. similarly, the degradation of peptide fragments derived from semenogelins in seminal plasma samples is time dependent and responsible for the decline in hiv-enhancing activity of sperm, once again underscoring the importance of timely performing analyses of peptides in bodily fluids. as previously stated, amps activity depends largely on their d structures, and their correct characterization becomes of paramount importance. the best experimental techniques have been and currently are x-ray crystallography and nuclear magnetic resonance, both of which allow for the study of structure-function relationships, though very expensive. therefore, currently, these are commonly replaced or complemented by bioinformatics prediction tools whenever possible. using such tools, the sequence of amino acids of a peptide allows for the accurate and immediate prediction of the presence of secondary structures (e.g., αhelices, β-sheets). then, the overall d structure is devised by algorithms employing energy minimization principles and molecular dynamics, sometimes resulting in multiple and not mutually exclusive d conformations, which is in accordance with peptides dynamic structure in biological fluids. , the most common identification software referenced in the literature for peptide/precursor protein identification are sequest and mascot. , currently, however, there is a considerable need for algorithms and software capable of correctly identifying amps and predicting their structure-function relationship. amps are a promising replacement for conventional antibiotics owing to their effectiveness against multiresistant bacteria and over multiple bacterial species simultaneously, invoking a diverse set of mechanisms that cannot be easily shortcut by bacteria, fungi, and/or virus. an ideal amp would be (i) highly selective against its target while leaving human host cells unaffected, (ii) not prone to resistance mechanisms, (iii) relatively easy to produce at low costs, and (iv) stable during storage or upon administration. drug design of such amps focuses on peptidic and nonpeptidic mimetic drugs, modulating the hydrophobicity, positivity, and amphiphilicity of endogenous amps to increase their antimicrobial potency. however, exploiting amps for therapeutic purposes is actually more complex than initially envisioned. while linear amps present a simpler design and a more predictable structure-function relationship, circular peptides may allow the design of longer acting and more resistant amps. such peptides have been designed by inserting the intended amp into other circular peptide (cyclotides) and have been proven efficacious against hiv and inflammatory diseases by circumventing their instability and poor bioavailability. , similarly, while humans produce amps with l-amino acids and their antimicrobial activity may depend on the action of endogenous enzymes, which can only act upon l-type amino acids, producing amps with d-amino acids may render these more resistant to hydrolysis. rather than administering exogenous amps, it is possible and perhaps more secure and controllable to pharmacologically stimulate the production of endogenous amps. for instance, , -dihydroxycalcypherol (active vitamin d ) has been successfully used to induce the expression of defb (defensin, beta ) and camp (cationic antimicrobial peptide) genes, the production of both ll- and human β-defensin in cell cultures of keratinocytes from diabetic foot ulcers and to promote wound healing. accordingly, the persistence of functional milk human κ-caseinoglycopeptides in plasma of human infants after human milk feeding serves the purpose of exogenously administering amps without the requirement for their de novo synthesis. amps can help preventing biofilm formation and microorganisms' growth. that is the case of ll- and its fragments, which were already shown to have antibiofilm formation properties against several methicillin-resistant s. aureus strains and p. aeruginosa, common pathogens of hospital-acquired infections, as well as against the melioidoisis' etiological agent burkholderia pseudomallei and the uropathogenic e. coli. [ ] [ ] [ ] [ ] another amp with antibiofilm formation activity is hbd- , found to prevent the development of methicillin-resistant s. epidermidis and s. aureus biofilms, largely responsible for orthopedic implants associated infections. likewise, amps may also inhibit the formation of fungal biofilms. for instance, histatin has been demonstrated to successfully inhibit the growth of c. albicans biofilm on denture acrylic. these examples may broaden the applications of amps in the clinical and biomedical fields by allowing functionalization of catheters. alternatively, implants and other medical devices can also reduce bacterial colonization, biofilm formation, and infection rates, presenting long lasting functionality, broad-spectrum activity, and minimal cytotoxicity against human cells. , human amps could, theoretically, also be used as biosensors immobilized onto microchips for identification purposes, allowing the identification of microorganisms. however, such applications have only been exploited with animal or synthetic amps. these biosensors on electrical impedance alterations resulting from the presence of bacterial or fungal surface elements, or on the specificity of peptide nucleic acid probes against their targets, presenting higher versatility, lower costs, and lower sensitivity limits compared to other conventional methodologies. [ ] [ ] [ ] alternatively, amps could be used as drug delivery systems in the form of conjugates in order to accurately target drugs and other agents to tumor sites or intended organs, as typified by monodisperse "endosomolytic" nanoparticles for in vivo gene delivery using intravenous injection, or by functionalized nanoparticles with higher membrane penetration targeted against gliomas. , such functionalization may allow for deeper tissue penetration, increased antimicrobial potency, sustained release, and novel routes of administration to be achieved. ever since the isolation of the first peptide from the frog skin in , several breakthroughs were made in the isolation, synthesis, and application of amps. still, many challenges are yet to overcome in the field of amps peptidomics, reflecting mainly their huge diversity and how little is known about their behavior in vivo and their structure-function relationships. in terms of costs, producing amps can be several hundred times more expensive than the production of conventional antibiotics. moreover, amps' design can be very complex and the quest for the perfect amp a never-ending search. in fact, considering the possibility of working with only amino acids, a small peptide with ten amino acids could have up to = . × different sequences, a larger peptide with amino acids could present with up to = . × different sequences, and one with amino acids could adopt any one of = . × different sequences. such possibilities do not even account for the presence of posttranslational modifications. then, each one of these peptides could shape into several different secondary structures and fall into a wide spectrum of activity, depending on the milieu conditions. furthermore, in a way to overcome the challenge of peptide stability, one has to consider the possibility to synthesize and to administer amps containing d-amino acids, thus decreasing their susceptibility to hydrolysis by human enzymes. to deal with such large amount of amino acid combinations one should mind using peptide libraries and computational models (such as by quantitative structure-activity relationship analysis) in order to narrow large collections to few surrogate amps (for a review please see blondelle and lohner's paper. another challenge to overcome is the lower amp's activity in vivo when compared to that observed in vitro. this has largely hampered the development of amps as therapeutic agents, as this decrease in activity stems from differences in ph and salt concentrations, the presence of proteases and corresponding inhibitors, and other interacting molecules hindering antimicrobial activity. accordingly, several amps (e.g., pexiganan, iseganan, neuprex have reached phase ii clinical trials only to fail after approval for marketing because they did not evidence superior activity over already marketed conventional antibiotics. in early , clinicaltrials.gov (a registry and result database of publicly and privately supported clinical studies of human participants conducted around the world) listed few clinical trials involving amps. for instance, ll is currently being studied for its efficacy in melanoma patients due to its ability to stimulate the immune system, while c g and chromogranin a derived peptides are under scrutiny for their therapeutic potential against dental diseases. simultaneously, therapeutic strategies are also being studied to augment amps' expression in amp-deficient patients. this deficiency is believed to be due to an increase in th lymphocytes derived cytokines, il- , il- , and il- , and a decrease in tnf-α, il- , il- , and interferonγ . one example of such strategy is pimecrolimus, a calcineurin inhibitor that binds with high affinity to macrophilin- , preventing the translocation of nuclear factor of activated t cells to the nucleus and the transcription and release of such inhibitory cytokines (for more up-to-date trials, readers can access clinicaltrials.gov). another possible caveat concerns the cytotoxicity to mammalian cells when in concentrations above naturally occurring values. when bacteria produce amps, they also develop means to avoid their targeting by such peptides, such as efflux pumps and the sequestering of enzymes. however, eukaryotic cells are equally prone to damage if the administration of exogenous amps is carried out in high enough concentrations. therefore, administering amps not constitutively produced or at concentrations above those normally found in the human host environment can be toxic and very detrimental to human cells. furthermore, rapid metabolism and low bioavailability are characteristic of amps because these peptides can suffer extensive proteolysis in vivo, thereby accounting for their inactivation and short half-life. , , similarly, for peptides to be available at sites distant from their origin, these would have to cross many cellular membranes. however, due to amps' polarity, membrane hydrophobicity may prevent them from doing so. moreover, in contrast to other highly polar mediators (e.g., ionic salts and a few metabolites), endogenous peptides tend not to have a transporter, further compromising their bioavailability in different compartments and the subsequent impossibility to be absorbed through the gastrointestinal tract. therefore, the number of possibilities rapidly escalades, and each amp-based therapy can be further optimized based on the modulation of chemical properties and engineering of delivery systems. even acknowledging all these factors, the human host is most certainly an unpredictable environment and the same peptide may behave differently, displaying unique individual pharmacokinetics and pharmacodynamics. on a final note, due to amps' interaction with and dependence on other immune mediators, variability will most likely result from immune status alterations as those frequently found in human diseases, including infectious ones. taken together, the above discussed results support amps as constituting a paramount group of defense molecules in human biological fluids, being present in most fluids, contributing for the sterility of some of these, but being most notably enriched in biological fluids bathing tissues with the largest load of microorganisms (fig. ) , such as saliva and urine. also, while some amps seem the display activity spectra against only a handful of microorganisms, others display broad-spectrum effects. thus, the expression and activity patterns of human amps enriched in biofluids may have suffered selective pressures resulting from exposers to microorganisms specifically present in each fluid (fig. ) . this work was financed by national funding from fct (fundação para a ciência e a tecnologia) through the project uid/bim/ / , uid/ic/ / . rv thanks fct for if/ / fellowship. the authors report no declarations of interest. with saliva proteomics research. since then, he extended the application of proteomics to the characterization of other biofluids' proteome and peptidome in order to identify the biological pathways modulated by diseases such as diabetes mellitus and bladder cancer. currently, he works to a large extent with bioinformatics tools for the identification of enzymes involved in the regulation of proteome remodeling. additional supporting information may be found in the online version of this article at the publisher's web site: apd : the antimicrobial peptide database as a tool for research and education anticancer efficacy of magainin and analogue peptides structure-activity analysis of thanatin, a -residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides cationic host defence peptides: potential as antiviral therapeutics the n-and c-terminal fragments of ubiquitin are important for the antimicrobial activities cationic peptides: effectors in innate immunity and novel antimicrobials fetal human keratinocytes produce large amounts of antimicrobial peptides: 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antimicrobial peptide (hcap ) predicts increased infectious disease mortality in patients undergoing hemodialysis ll- peptide enhancement of signal transduction by toll-like receptor is regulated by ph identification of a peptide antagonist of ll- increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection identification of novel semenogelin iderived antimicrobial peptide from liquefied human seminal plasma processing of seminal plasma hcap- to all- by gastricsin. a novel mechanism of generating antimicrobial peptides in vagina citrullination alters immunomodulatory function of ll- essential for prevention of endotoxin-induced sepsis nad-dependent adp-ribosylation of the human antimicrobial and immune-modulatory peptide ll- by adpribosyltransferase- human salivary histatin- exerts potent fungicidal activity against cryptococcus neoformans the effect of histatin , adsorbed on pmma and hydroxyapatite, on candida albicans colonization anticandidal activity of major human salivary histatins localization of the genes for histatins to human chromosome q and tissue distribution of the mrnas histatins, a novel family of histidine-rich proteins in human parotid secretion. isolation, characterization, primary structure, and fungistatic effects on candida albicans histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (his and his ) structural relationship between human salivary histatins zinc and copper bind to unique sites of histatin is salivary histatin a metallopeptide? amino terminal cu(ii)-and ni(ii)-binding (atcun) motif of proteins and peptides: metal binding, dna cleavage, and other properties † zn( +) ions selectively induce antimicrobial salivary peptide histatin- to fuse negatively charged vesicles. identification and characterization of a zinc-binding motif present in the functional domain nmr studies of the antimicrobial salivary peptides histatin and histatin in aqueous and nonaqueous solutions salivary histatin : dependence of sequence, chain length, and helical conformation for candidacidal activity antimicrobial peptides with therapeutic potential the antimicrobial peptide histatin- causes a spatially restricted disruption on the candida albicans surface, allowing rapid entry of the peptide into the cytoplasm effect of donor age on the concentrations of histatins in human parotid and submandibular/sublingual saliva functional and structural characterization of recombinant dermcidin- l, a human antimicrobial peptide dermcidin: a novel human antibiotic peptide secreted by sweat glands polysaccharide intercellular adhesin (pia) protects staphylococcus epidermidis against major components of the human innate immune system proteolysis-inducing factor is expressed in tumours of patients with gastrointestinal cancers and correlates with weight loss identification of a survival-promoting peptide in medium conditioned by oxidatively stressed cell lines of nervous system origin deficiency of dermcidin-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo the role of dermcidin isoform : a two-faceted atherosclerotic risk factor for coronary artery disease and the effect of acetyl salicylic acid on it the appearance of dermcidin isoform , a novel platelet aggregating agent in the circulation in acute myocardial infarction that inhibits insulin synthesis and the restoration by acetyl salicylic acid of its effects hepcidin revisited, disulfide connectivity, dynamics, and structure mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis the gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation sequestration and scavenging of iron in infection escherichia coli bacteremia, meningitis, and hemochromatosis bacteremic cellulitis caused by non- , non- vibrio cholerae: report of a case in a patient with hemochromatosis yersinia enterocolitica infection with multiple liver abscesses uncovering a primary hemochromatosis fatal listeria meningitis, endocarditis and pericarditis in a patient with haemochromatosis hemochromatosis, iron and septicemia caused by vibrio vulnificus absence of functional hfe protects mice from invasive salmonella enterica serovar typhimurium infection via induction of lipocalin- hereditary hemochromatosis results in decreased iron acquisition and growth by mycobacterium tuberculosis within human macrophages antibacterial and antifungal activities of vasostatin- , the n-terminal fragment of chromogranin a the primary structure of human chromogranin a and pancreastatin phosphorylation and o-glycosylation sites of human chromogranin a (cga - ) from urine of patients with carcinoid tumors intracellular and extracellular processing of chromogranin a. determination of cleavage sites the vasoinhibitory activity of bovine chromogranin a fragment (vasostatin) and its independence of extracellular calcium in isolated segments of human blood vessels chromogranin a induces a neurotoxic phenotype in brain microglial cells matsuoka h. plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure elevation of circulating and ventricular adrenomedullin in human congestive heart failure increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis an investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora enhancement of antimicrobial activity of neuropeptide y by n-terminal truncation inhibition of pulmonary metastases and enhancement of natural killer cell activity by methionine-enkephalin methionine-enkephalin secreted by human colorectal cancer cells suppresses t lymphocytes characterization of antibacterial cooh-terminal proenkephalin-a-derived peptides (peap) in infectious fluids: importance of enkelytin, the antibacterial peap - secreted by stimulated chromaffin cells two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin quantitation of proenkephalin a messenger rna in bovine brain, pituitary and adrenal medulla: correlation between mrna and peptide levels differential expression of preproenkephalin and preprodynorphin mrnas in striatal neurons: high levels of preproenkephalin expression depend on cerebral cortical afferents preproenkephalin mrna in t-cells, macrophages, and mast cells regulated expression of proenkephalin a in normal lymphocytes polymorphism and absence of leu-enkephalin sequences in proenkephalin genes in xenopus laevis the presence of antibacterial and opioid peptides in human plasma during coronary artery bypass surgery antimicrobial effects of alpha-msh peptides effect of cyclic amp on rna and protein synthesis in candida albicans human lactoferrin and peptides derived from its n terminus are highly effective against infections with antibiotic-resistant bacteria many chemokines including ccl /mip- alpha display antimicrobial activity eotaxin- (ccl ) exerts innate host defense activities that are modulated by mast cell proteases antimicrobial peptides derived from pepsinogens in the stomach of the bullfrog, rana catesbeiana calcitermin, a novel antimicrobial peptide isolated from human airway secretions mast cell and monocyte recruitment by s a and its hinge domain s a provokes mast cell activation: a potential amplification pathway in asthma and innate immunity generation of multiple stable dermcidin-derived antimicrobial peptides in sweat of different body sites natural skin surface ph is on average below , which is beneficial for its resident flora vipericidins: a novel family of cathelicidin-related peptides from the venom gland of south american pit vipers paneth cell defensins: endogenous peptide components of intestinal host defense defensin-rich dense granules of human neutrophils the human antibacterial cathelicidin, hcap- , is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils peptides in body fluids and tissues as markers of disease peptidomics-based discovery of novel neuropeptides the glucagon-like peptides human cathelicidin, hcap- , is processed to the antimicrobial peptide ll- by extracellular cleavage with proteinase alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of aggregatibacter actinomycetemcomitans extensive in vivo human milk peptidomics reveals specific proteolysis yielding protective antimicrobial peptides proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation antioxidant peptidomics reveals novel skin antioxidant system targeted protein degradation by salmonella under phagosome-mimicking culture conditions investigated using comparative peptidomics optimisation of protein precipitation based upon effectiveness of protein removal and ionisation effect in liquid chromatography-tandem mass spectrometry peptidomics technologies for human body fluids human cerebrospinal fluid peptidomics selective enrichment of tryptophan-containing peptides from protein digests employing a reversible derivatization with malondialdehyde and solid-phase capture on hydrazide beads selective solid-phase isolation of methioninecontaining peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine-and of methionine-sulfoxide-containing peptides porous polymer monolithic column with surface-bound gold nanoparticles for the capture and separation of cysteine-containing peptides mass spectrometric identification of n-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging highly selective enrichment of phosphorylated peptides using titanium dioxide identification of protein modifications using ms/ms de novo sequencing and the opensea alignment algorithm immunoassays for the incretin hormones gip and glp- mass-spectrometric identification of a novel angiotensin peptide in human plasma salivary peptidomic as a tool to disclose new potential antimicrobial peptides capillary electrophoresis-electrospray-mass spectrometry in peptide analysis and peptidomics the endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by lc-esi-paul ion trap and qq-tof endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption/ionization and electrospray ionization hybrid quadrupole time-offlight matrixassisted laser desorption/ionization quadrupole time-of-flight mass spectrometry: an elegant tool for peptidomics the differential diagnostic model for serous peptidomics in hbv carriers established by maldi-tof-ms analysis peptidomics-based discovery of an antimicrobial peptide derived from insulin-like growth factor-binding protein naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease biochemical aspects of the coagulation and liquefaction of human semen liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances hiv infection x-ray crystallography of peptides: the contributions of the italian laboratories chapter nmr of antimicrobial peptides pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides prediction of peptide structure: how far are we? comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations large-scale database searching using tandem mass spectra: looking up the answer in the back of the book molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis -dihydroxyvitamin d induces ll- and hbd- production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion uropathogenic escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide ll- antimicrobial and antibiofilm activity of ll- and its truncated variants against burkholderia pseudomallei anti-staphylococcal biofilm effects of human cathelicidin peptides human host defense peptide ll- prevents bacterial biofilm formation human β-defensin inhibits antibiotic-resistant staphylococcus biofilm formation sensitivity of candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine biocompatibility of antimicrobial melimine lenses: rabbit and human studies development of a catheter functionalized by a polydopamine peptide coating with antimicrobial and antibiofilm properties impedimetric detection of pathogenic gram-positive bacteria using an antimicrobial peptide from class iia bacteriocins design and evaluation of peptide nucleic acid probes for specific identification of candida albicans electrical impedance detection of bacterial pathogens using immobilized antimicrobial peptides novel endosomolytic peptides for enhancing gene delivery in nanoparticles silicon microfluidic flow focusing devices for the production of size-controlled plga based drug loaded microparticles a novel peptide designated pyla and its precursor as predicted from cloned mrna of xenopus laevis skin therapeutic peptides under the spotlight antibacterial peptides for therapeutic use: obstacles and realistic outlook optimization and high-throughput screening of antimicrobial peptides pexiganan acetate a multinational, randomized phase iii trial of iseganan hcl oral solution for reducing the severity of oral mucositis in patients receiving radiotherapy for head-and-neck malignancy prolyl peptidases: a serine protease subfamily with high potential for drug discovery bioavailability and transport of peptides and peptide drugs into the brain key: cord- - code oh authors: benincasa, monica; skerlavaj, barbara; gennaro, renato; pellegrini, antonio; zanetti, margherita title: in vitro and in vivo antimicrobial activity of two α-helical cathelicidin peptides and of their synthetic analogs date: - - journal: peptides doi: . /j.peptides. . . sha: doc_id: cord_uid: code oh two α-helical antimicrobial peptides (bmap- and - ) and four synthetic analogs were compared for in vitro and in vivo antimicrobial efficacy. all peptides proved active in vitro at micromolar concentrations against a range of clinical isolates, including antibiotic-resistant strains. bmap- and two analogs were more effective towards gram-negative, and bmap- towards gram-positive organisms. in addition, bmap- provided some protection in vitro against human herpes simplex virus type (hsv- ). the parent peptides and mbmap- analog protected mice from lethal i.p. infections in an acute peritonitis model at peptide doses significantly lower than those toxic to the animals, suggesting a satisfactory therapeutic index. the emergence of clinical bacterial strains exhibiting resistance against conventional antibiotics has urged the search for novel anti-infective agents. among the compounds that are currently under investigation for their therapeutic potential are a number of cationic antimicrobial peptides of the innate immune system, and their synthetic derivatives [ , ] . cationic antimicrobial peptides are present in all living organisms as a first-line host-defense mechanism against invading microbes. most of these peptides rapidly inactivate bacterial, fungal or viral pathogens by a mechanism which in most cases is mediated by disruption of integrity of the microbial membranes [ ] . this relatively non-specific mechanism is difficult to evade by susceptible bacteria, as indicated by the relatively rare selection of bacterial strains resistant to cationic peptides [ ] and can be considered an interesting feature of these peptides in view of their development as anti-infective drugs. in this study, we investigated the therapeutic potential of two natural peptides belonging to the mammalian cathelicidin peptide family [ , , ] . these molecules were denoted bmap- and - (bmap is an acronym for 'bovine myeloid antimicrobial peptide', to indicate that both are antimicrobial components of the bovine neutrophils). the two peptides are and amino acid residues long, respectively, and are c-terminally amidated. they share a . % sequence identity and an additional % similarity [ ] . prior structural studies suggested that both molecules include a - n-terminal region that undergoes an ␣-helical conformation in membrane mimicking environments, and a non-helical, hydrophobic c-terminal region. both bmaps kill gram-negative and -positive bacteria and fungal species at micromolar concentrations, and retain strong and broad spectrum antimicrobial activity in the presence of physiologic salt concentrations [ ] . in addition, in vitro antibacterial and anti-parasite efficacy against leptospira interrogans serovars and against cryptosporidium parvum has recently been reported for bmap- [ , ] . the mechanism of action of these peptides is based on the ability to rapidly bind to and permeabilize the membranes of target microorganisms. however, bmap- and - prove toxic to mammalian cells in a concentration range above m, suggesting poor discrimination between prokaryotic and eukaryotic cell membranes. structure-activity relationship (sar) studies aimed to increase selectivity towards microbial organisms led to the design of truncated bmap derivatives comprising the - n-terminal sequence and lacking the hydrophobic c-terminal region [ ] . these compounds, denoted bmap- ( - ) and bmap- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , were virtually devoid of cytotoxic and haemolytic activities and displayed antimicrobial potency comparable to, or only slightly lower than that of the parent bmap peptides. similar properties were also shared by an analog of bmap- with modified c-terminal region, named mbmap- . collectively, these sar studies suggested that the hydrophobic c-terminal tail is a major determinant of the toxicity of bmap molecules to mammalian cells [ ] . as an important step to investigate their therapeutic potential, we extended the functional analysis of bmaps and determined their in vitro and in vivo efficacy. specifically, we analysed the in vitro activity of bmap- and - and of their analogs against a wide panel of gramnegative and -positive clinical isolates, also including many antibiotic-resistant strains, and against the human herpes simplex virus type (hsv- ). additionally, an -residue peptide comprising the c-terminal region and lacking the - n-terminal residues, has also been tested. their potential to protect in vivo from infections by gram-negative and -positive organisms was assessed using a mouse acute peritonitis model. fmoc-pal-peg-ps resin, coupling reagents for peptide synthesis and fmoc-amino acids were purchased from applied biosystems (foster city, usa), novabiochem (laufelfingel, switzerland) and chemimpex (wood dale, il, usa). peptide synthesis-grade n,n-dimethylformamide, n-methyl- -pyrrolidone, dichloromethane, dimethylsulfoxide, piperidine and hplc-grade acetonitrile were from biosolve (valkenswaard, the netherlands). trifluoroacetic acid, n-methylmorpholine and trifluoroethanol (tfe) were obtained from acros chimica (beerse, belgium), , -diaza bicyclo ( . . )-undec- -ene (dbu) and n-acetylimidazole from sigma-aldrich (st. louis, mo, usa). the peptides bmap- and - and their analogs bmap- ( - ), bmap- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , bmap- ( - ), and mbmap- were synthesized as previously described [ ] on a milligen automated synthesizer (applied biosystems, foster city, usa) using the fmoc chemistry. side chain protecting groups were as follows: trityl (trt) for his, t-butyl (tbu) for tyr and ser, t-butyloxycarbonyl (boc) for lys, and trp and , , , , pentamethyldihydrobenzofuran- -sulfonyl (pbf) for arg. to obtain c-terminally amidated peptides, the fmoc-pal-peg-ps resin ( . meq/g) was used. the synthesis was carried out at • c in n,n-dimethylformamide (dmf) with a six-fold excess of fmoc-amino acids, activated in situ by an equivalent amount of -( h-benzotriazole- yl)- , , , -tetramethyluronium tetrafluoroborate (tbtu), in the presence of . n n-methylmorpholine. to improve the yield, difficult couplings were performed with an eight-fold amino acid excess, using an equimolar amount of o-( -azabenzotriazole- -yl)- , , , -tetramethyluronium hexafluorophosphate (hatu) as coupling agent. after cleavage and deprotection with a mixture of trifluoroacetic acid/water/thioanisole/phenol/ethandithiol/triisopropylsilane ( . : : : : . : , v/v) for h at room temperature, the crude peptides were repeatedly extracted with methyl butyl ether and purified by reversed phase high performance liquid chromatography (rp-hplc) on a preparative ( mm× mm) c delta-pak column (waters, bedford, ma, usa) using an appropriate - % water/acetonitrile gradient in the presence of . % trifluoroacetic acid. molecular masses were determined by electrospray mass spectrometry (es-ms), using an api i instrument (pe sciex, toronto, canada). purified peptides were dissolved in . % tfa at approximately mm concentration, divided in small aliquots and kept frozen at − • c until use. the concentration of bmap- and bmap- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was determined at nm considering a molar extinction coefficient of . for each phe residue [ ] , bmap- peptides were measured at nm, using a molar extinction coefficient of for trp [ ] and of for tyr [ ] . a total of gram-positive ( staphylococcus aureus, enterococcus faecalis, enterococcus faecium, streptococcus agalactiae), and gram-negative ( acinetobacter baumanni, pseudomonas aeruginosa, and serratia marcescens) bacterial strains were tested in this study. these clinical isolates were routinely characterized for antibioticresistance and were kindly provided by prof. e. tonin from the department of biomedical sciences of the university of trieste. the escherichia coli o :k :h bort, used in the acute peritonitis animal model, was a generous gift from prof. p. abraham from the academic medical center, university of amsterdam. all the strains were stored at − • c and subcultured in luria-bertani (lb) broth prior to use. the in vitro antibacterial activity of purified bmap peptides and of their analogs was determined as the minimum inhibitory concentration (mic) by a microdilution susceptibility test in -well microtiter plates, according to the guidelines of the national committee for clinical laboratory standards (nccls). the activity was measured in mueller-hinton broth (difco) with logarithmic-phase microorganisms as previously reported [ ] . the hsv- and the porcine respiratory corona virus (prcv) (kindly provided by dr. wunderli, institute for medical virology, and dr. engels, institute of virology, faculty of veterinary medicine, university of zürich) were titrated by inoculation of vero cells with hsv- and swine testicular cells with prcv (cells kindly provided by dr. engels,) with -fold dilutions using the endpoint dilution method of reed and muench [ ] . cells were grown in minimal essential medium (mem) with earle's salt and l-glutamin (gibco brl) and supplemented with % foetal calf serum (fcs, gibco brl), u/ml penicillin, and g/ml streptomycin (gibco brl). swine testicular cells were cultured in iscove's modified dulbecco's medium without l-glutamine, containing mm hepes, % non-essential amino acids and mm na-pyruvate. neutral red uptake assay was performed following a procedure based on borenfreund and puerner [ ] . briefly, h after infection, the maintenance medium was removed and the cells were incubated for h with mem containing g/ml neutral red (fluka) ( l per well). cells were then washed and fixed with l of % formaldehyde and % cacl . the dye taken up by the cells was extracted using l of % acetic acid in % ethanol and the absorbance was measured at nm in a mr dynatech microplate ® -reader (dynatech laboratories inc., chantilly, va, usa). each sample was tested in triplicate and the percent protection achieved by the compounds in the infected cells was calculated as described by pauwels et al. [ ] . increasing concentrations of peptides (from . to m) were dissolved in mem and incubated with confluent cell monolayers, infected with tcdi , in -well tissue culture plates for min at • c and % co . after removal of the medium, the cell monolayer was washed with phosphate-buffered saline (pbs) and further incubated in mem for h. the inhibition of the cytopathic effect was assessed by light microscopy and measured by the neutral red uptake assay using infected vero cells as control. peptide cytotoxicity was evaluated by using the same assay, incubating uninfected cells with serial dilutions of each peptide for min at • c and % co . confluent cell monolayers were infected and treated with the peptides as described in the previous paragraph. after h incubation at • c and % co , the plates were frozen and thawed three times. ten-fold dilutions of the supernatants were used to infect confluent cell monolayers, grown in -well plates, to quantify hsv- inhibition. after h at • c and % co , the inoculum was removed, cell monolayers were washed once with pbs and overlaid with mem supplemented with . % carboxymethyl cellulose, % fcs, u/ml penicillin and g/ml streptomycin and incubated at • c and % co for days. after removal of the medium and fixation with methanol, monolayers were stained with . % crystal violet and the plaque number was counted. results of repeated experiments performed in triplicate were expressed as percentage of plaque inhibition by comparison with untreated control cell monolayers. for the in vivo experiments, male balb/c mice of approximately g and weeks of age were obtained from harlan nossan (harlan nossan s.r.l., correzzana, milan, italy). the in vivo toxicity of the peptides was investigated by injecting mice with increasing amounts of each bmap peptide dissolved in sterile pbs via i.p. ( . ml per mouse) or i.v. ( . ml per mouse). the controls received the vehicle alone. animal behaviour and survival were monitored over a -day period and the ld was calculated according to litchfield and wilcoxon [ ] . for these experiments, an encapsulated strain of e. coli o :k :h bort, a methicillin-resistant s. aureus (mrsa) and a reference strain of p. aeruginosa, atcc , were grown in mueller-hinton broth. inocula containing an amount of bacteria expected to result in - % mortality were prepared by diluting log-phase cultures in pbs. depending on the test organism, inocula ranged from to × cfu for e. coli o :k :h bort, from to × cfu for p. aeruginosa atcc and from to × cfu per mouse for mrsa. at least mice per dose level were infected and monitored for survival over a -day period after infection. mic values are presented as the mean of at least four independent experiments. statistical significance of the differences between mortalities in the in vivo experiments was determined by fisher's exact test. a p value ≤ . was accepted as indicating significance. the sequences of all bmap peptides used are shown in table . the antibacterial activity of these peptides was tested by a microdilution susceptibility assay towards bacterial isolates. these included a variety of clinically relevant, antibiotic-resistant species: mrsa, vancomycin-resistant e. faecalis (vref) (fig. ) and multi-resistant strains of p. aeruginosa and a. baumannii (fig. ) . bmap- and - displayed a broad-spectrum activity with mic values ranging from to m in most cases. these values confirm and extend those previously obtained against a limited panel of atcc strains [ ] . despite structural similarity, the two peptides revealed different and somewhat complementary spectra of activity, i.e. bmap- was more effective against gram-negative species (mic values of - m, fig. and table ), whereas bmap- showed a better activity against gram-positive strains (mic values of - m, fig. and table ). importantly, similar or even lower mic values were observed when the activity towards antibiotic-susceptible versus antibiotic-resistant strains (e.g. mssa versus mrsa strains and vsef versus vref strains) was compared (fig. ). table ). conversely, bmap- ( - ) was slightly less effective than bmap- against gram-positive strains but showed significantly improved activity against gram-negative strains. thus, based on these in vitro data, removal of the c-terminal hydrophobic sequence not only results in improved selectivity towards bacterial versus mammalian cells, as already reported [ ] , but also towards gram-negative versus gram-positive microorganisms. the bmap- ( - ) fragment, which lacks part of the n-terminal ␣-helix and maintains the c-terminal hydrophobic region of bmap- , showed comparable activity against both gram-positive and -negative microorganisms, and was only slightly less potent than bmap- against p. aeruginosa (mic values increased from -to -fold, fig. ). the modified mbmap- analog, characterized by a more hydrophilic c-terminal region than bmap- , was highly potent against gram-negative strains, with mic values significantly lower than those of bmap- (mics of . - m) towards a. baumannii and p. aeruginosa (fig. ) . by contrast, this analog showed a marked decrease in the activity against gram-positive strains (most mics are > m), with the remarkable exception of three highly susceptible s. agalactiae strains. the ability of bmap- and - and of their synthetic - fragments to inhibit the cytopathic effect of hsv- was determined with the neutral red uptake assay, using a broad range of peptide concentrations. vero cell monolayers were incubated with tcdi of hsv- particles and . - m peptide for min, then washed and incubated in the absence of the peptide for h to observe cell growth. bmap- was poorly cytotoxic at m, with only % of the cells taking up the neutral red dye. at this concentration, the peptide provided % protection from hsv- and inhibited viral replication to the same extent, as assessed by the virus yield inhibition assay (table ) . bmap- ( - ) was also not toxic to vero cells at m, but unlike bmap- , this peptide did not exert any protection from viral infection at any concentration tested (not shown). despite structural resemblance to bmap- , bmap- was cytotoxic even at the lowest peptide concentration tested (not shown), thereby precluding the evaluation of its antiviral activity. both peptides were also tested against porcine respiratory corona virus using swine testicular (st) cells. however, none of the peptides proved active against this virus at . - m (not shown), further supporting poor antiviral activity of these peptides. not tested a confluent vero cells were incubated with bmap- for min at • c, then washed and resuspended in growth medium. cell survival was determined by measuring the uptake of neutral red after incubation for h at • c. b cells were incubated with peptide and viral particles ( tcdi ) for min at • c, then washed and resuspended in growth medium. cell protection was determined by measuring the uptake of neutral red after h incubation at • c. c quantification of hsv- inhibition was performed by virus yield inhibition assay, as described in section . the results are the mean of two independent experiments performed in triplicate. as a first step to evaluate the therapeutic potential of bmap peptides, the in vivo toxicity of the parent peptides and of their - fragments was determined in mice injected intraperitoneally and/or intravenously with increasing single peptide doses. no toxicity was observed when any of the peptides were administered i.p. up to mg/kg, whereas doses of mg/kg resulted in - % mortality within days from injection, and the ld values were determined to be in the range - mg/kg. the toxicity of bmap- and - after intravenous administration proved much higher, with ld values of and mg/kg, respectively. these results indicate that the compounds are not safe via i.v., and therefore, their therapeutic potential is restricted to topical applications. the potential of bmaps to protect mice from a bacterial challenge was tested in a model of acute peritonitis induced by i.p. injection of a lethal dose of p. aeruginosa atcc ( - × cfu/ml), e. coli o :k :h bort ( - × cfu/ml), an encapsulated strain isolated from a neonatal meningitis patient, or mrsa ( - × cfu/ml). all these strains were susceptible to bmaps in vitro in the micromolar concentration range, as shown in table . adult male balb/c mice were infected i.p. with a lethal dose of the test microorganism, immediately followed by a single dose of peptide. the number of survivors was monitored for weeks and compared to that of control mice that only received the lethal bacterial challenge or the vehicle. the results of these experiments are summarized in table . as little as . mg/kg of bmap- resulted in full protection of e. coli-infected mice. a higher dose ( . mg/kg) of this peptide was required to obtain % protection from p. aeruginosa. conversely, bmap- was poorly effective against s. aureus, even at . mg/kg peptide concentration. bmap- provided a % protection against s. aureus and e. coli-infected mice, respectively, at . and . mg/kg. this compound was somewhat less effective against p. aeruginosa, with % protection at a dose of . mg/kg. the in vivo results thus reflect the in vitro behaviour of the two peptides, bmap- being more effective against gram-negative, and bmap- against gram-positive bacterial strains. the effects of the - fragments were tested in mice i.p. infected with p. aeruginosa and mrsa. the compounds were administered i.p. at or mg/kg immediately after bacterial challenge. only bmap- ( - ) showed % protection of p. aeruginosa-infected mice at mg/kg, and neither peptide was effective against s. aureus challenge (data not shown). finally, the protective effect of mbmap- against a lethal dose of i.p. injected p. aeruginosa was tested at . , . and . mg/kg. the former dose was ineffective, while the latter two doses protected and % of the infected mice, respectively (data not shown). the mbmap- analog was not tested in the mrsa-induced peritonitis model, since it proved inactive in vitro against gram-positive microorganisms. in this study, we investigated the in vitro and in vivo activity of two cathelicidin peptides, bmap- and - , and of fragments and analogs derived from them. the in vitro activity was tested against a wide panel of clinical isolates including mrsa and vref strains, as well as a. baumannii and p. aeruginosa multi-resistant strains. the results indicate a broad spectrum activity for the parent bmap- and - , which are comparably effective against antibiotic-resistant and antibiotic-susceptible isolates. this further confirms that the mechanism of action of these peptides, based on their ability to permeabilize bacterial membranes [ ] , is different from that of the antibiotics to which the strains tested are resistant. the truncated and substitution analogs, designed to probe the importance of the hydrophobic c-terminal region, showed significant differences in activity when compared with the parent bmaps. the lack of the c-terminal region, or replacement of hydrophobic residues in this region with more hydrophilic ones, had no effect towards gram-negative microorganisms and in some cases led to improved activity, as specially observed with mbmap- . conversely, these modifications resulted in decreased activity against gram-positive strains, with the notable exception of s. agalactiae, which remained highly susceptible. it is interesting to note that the decreased activity against gram-positive bacteria parallels the decreased cytotoxicity towards mammalian cells observed upon removal or modification of the c-terminal tail [ ] . understanding of these differences in activity will require deeper investigations of bacterial surface components and of their interaction with the peptides. while indicating potent and wide spectrum in vitro antibacterial activity, the results of this study do not support antiviral efficacy of bmap peptides. in fact, only bmap- provides some protection in vitro against hsv- , whereas all the other peptides are ineffective at non-cytotoxic concentrations. the results obtained with in vitro assays prompted us to exploit the in vivo potential of these peptides against lethal bacterial challenge in animals. the use of an acute peritonitis model in mice was suggested by the results of in vivo toxicity tests, that indicated a much better ld for i.p. rather than i.v. administration of these peptides. the in vivo results using the parent bmaps are encouraging and parallel those obtained in vitro. bmap- was highly protective against e. coli and p. aeruginosa, with % protection at respectively . and . mg/kg, and was considerably less effective against i.p. challenge with s. aureus (table ). conversely, administration of bmap- at . mg/kg resulted in % protection against i.p. injection of an mrsa strain and was less effective against e. coli and p. aeruginosa. importantly, protection is achieved at peptide doses that are significantly lower than those which are toxic to animals (i.e. no i.p. toxicity up to mg/kg), suggesting a satisfactory therapeutic index for these compounds. in keeping with the in vitro results, mbmap- also protects mice from lethal i.p. dose of p. aeruginosa, whereas the - fragments are more effective in vitro than in vivo. these peptides are poorly or no protective in the mouse model used, suggesting that the hydrophobic c-terminal region is important for the in vivo activity. despite a wealth of published in vitro activity studies of antimicrobial peptides against antibiotic-resistant clinical isolates, there are only a few published reports of their in vivo activity. pg- , a -residue cathelicidin peptide with a ␤-sheet structure, was tested in a mouse model of acute peritonitis induced by s. aureus and p. aeruginosa [ ] . the effective doses of pg- compare well with those of bmap- and - . a highly potent pg- analog proved effective in reducing oral microflora in humans, suggesting that antimicrobial peptides may be useful for treatment of oral infections [ ] . other in vivo studies examined the effect exerted by the sheep cathelicidin peptide smap- in an ovine model of acute pulmonary infection. this peptide is highly similar to bmap- ( % identity) and proved effective after pulmonary deposition in a lamb pneumonia model induced by mannheimia haemolytica. smap- reduced the bacterial concentration in bronchoalveolar fluid and in consolidated pulmonary tissues, as well as the severity of the lesions in the lungs, suggesting it may find an application in the treatment of respiratory tract infections [ ] . further in vivo studies were performed with indolicidin, a bovine cathelicidin peptide, using an animal model of systemic fungal infection caused by intravenous injection of aspergillus fumigatus spores [ ] . free indolicidin was highly toxic to mice when injected i.v. (all treated animals died at mg/kg peptide), whereas administration of the peptide encapsulated in lipo-somes significantly reduced toxicity. liposomal entrapped indolicidin at mg/kg considerably increased animal survival, suggesting that liposome carriers may improve the therapeutic index of toxic peptides [ ] . finally, indolicidin and other antimicrobial peptides were investigated in three rat models of septic shock. treatment with all the peptides used in this study resulted in a significant reduction in plasma endotoxin and tnf-␣ concentrations and reduced the rates of animal deaths as compared to controls [ ] . in addition to the above reported in vivo studies, several clinical trials have been performed or are currently underway on analogs derived from natural antimicrobial peptides. the results so far obtained indicate that, despite failure in a few cases, these peptides still have therapeutic potential, particularly for topical applications [ , ] . liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity toxicity determined in vitro by morphological alterations and neutral red adsorption the ovine cathelicidin smap kills ovine respiratory pathogens in vitro and in an ovine model of pulmonary infection protein estimation by the product of integrated peak area and flow rate development of protegrins for the treatment and prevention of oral mucositis: structure-activity relationships of synthetic protegrin analogues cationic peptides: distribution and mechanisms of resistance spectroscopic determination of tryptophan and tyrosine in proteins structural features and biological activities of the cathelicidin-derived antimicrobial peptides potential therapeutic role of cationic peptides in three experimental models of septic shock in vitro effect on cryptosporidium parvum of short-term exposure to cathelicidin peptides clinical development of cationic antimicrobial peptides: from natural to novel antibiotics cathelicidins: a family of endogenous antimicrobial peptides simplified method of evaluating dose-effect experiments ib- , a protegrin peptide with in vitro and in vivo activities against the microflora associated with oral mucositis rapid and automated tetrazolium-based colorimetric assay for the detection of anti-hiv compounds a simple method of estimating fifty per cent endpoints comparative in vitro activity of five cathelicidin-derived synthetic peptides against leptospira from innate immunity to de-novo designed antimicrobial peptides biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities rapid membrane permeabilization and inhibition of vital functions of gram-negative bacteria by bactenecins protegrin- : a broad-spectrum, rapidly microbicidal peptide with in vivo activity cathelicidins: a novel protein family with a common proregion and a variable c-terminal antimicrobial domain cathelicidin peptides as candidates for a novel class of antimicrobials we thank dr. marco stebel for assistance with the animal model experiments. this work was supported by grants from the istituto superiore di sanita', programma nazionale di key: cord- - f g m authors: herzig, volker; cristofori-armstrong, ben; israel, mathilde r.; nixon, samantha a.; vetter, irina; king, glenn f. title: animal toxins — nature’s evolutionary-refined toolkit for basic research and drug discovery date: - - journal: biochem pharmacol doi: . /j.bcp. . sha: doc_id: cord_uid: f g m venomous animals have evolved toxins that interfere with specific components of their victim’s core physiological systems, thereby causing biological dysfunction that aids in prey capture, defense against predators, or other roles such as intraspecific competition. many animal lineages evolved venom systems independently, highlighting the success of this strategy. over the course of evolution, toxins with exceptional specificity and high potency for their intended molecular targets have prevailed, making venoms an invaluable and almost inexhaustible source of bioactive molecules, some of which have found use as pharmacological tools, human therapeutics, and bioinsecticides. current biomedically-focused research on venoms is directed towards their use in delineating the physiological role of toxin molecular targets such as ion channels and receptors, studying or treating human diseases, targeting vectors of human diseases, and treating microbial and parasitic infections. we provide examples of each of these areas of venom research, highlighting the potential that venom molecules hold for basic research and drug development. venomous animals have taken advantage of the inherent biological complexity of their victims by developing toxins that target one or all of the nervous, musculoskeletal, or cardiovascular systems in order to facilitate prey capture or defense against predators. for example, many predatory venoms contain neurotoxins that disrupt neuromuscular transmission in order to rapidly paralyse envenomated prey [ ] . due to the inherent specificity and potency of most venom molecules, venoms have become an invaluable source of modulators to study a wide range of molecular targets, especially ligand-activated [ ] [ ] [ ] and voltage-gated ion channels [ ] [ ] [ ] . while research on many potential drug targets is hampered by the lack of selective modulators, our increasing knowledge of venom pharmacology is beginning to shed light on some of these poorly studied targets (e.g. [ ] ). here we provide examples of how venom compounds have been used to study diverse molecular targets and aid our understanding of their role in human disease. we use knowledge gained from approved venom-derived drugs [ , ] to illustrate how basic research can lead all the way from venom to a human therapeutic. finally, we touch on other potential uses of toxins for human health, such as targeting vectors of human diseases, and development of antimicrobial and antiparasitic drugs (fig. ). during the course of evolution, venom has evolved independently at least times across eight different phyla [ ] . it has been estimated that there are more than , venomous species on the planet, representing ~ % of extant animal biodiversity [ ] , spanning invertebrates such as annelids, arthropods, cnidarians, molluscs, nematodes, sea urchins, star fish, and vertebrates such as snakes, lizards, fish, shrews, and platypus [ , [ ] [ ] [ ] . chemically, venoms are complex mixtures of salts, small molecules, peptides, and proteins [ , ] , with an estimated million compounds in spider venoms alone [ ] . most of these compounds are toxins, which are defined as substances that cause dose-dependent pathophysiological injury to a living organism, thereby reducing their viability [ ] . the high metabolic cost of venom production [ ] has ensured that over the course of evolution only the most potent toxins have prevailed. venoms can therefore be considered natural libraries of highly optimised bioactive compounds [ , ] . given the vastly different phyletic origins of venomous species and the even greater diversity of their target organisms, it is expected that venom toxins modulate an extremely diverse range of molecular targets (fig. ) . the dominant components of most venoms are small peptides. the high potency, selectivity and biological stability of many venom-derived peptides has made them particularly attractive from a drug discovery perspective. there are already two fda-approved venom-derived peptides in clinical use -ziconotide, an analgesic drug from the venom of a marine cone snail [ ] , and exenatide, an anti-diabetic drug from a venomous lizard [ ] -plus numerous others in clinical trials [ , ] or preclinical development [ , ] . the larger size of peptides compared to small molecules provides a larger binding surface with their molecular target, typically yielding higher potency and greater target specificity, thereby minimising unwanted side-effects. however, the larger size and polar nature of peptides translates into poor oral bioavailability. other disadvantages of peptides can be poor membrane permeability and low metabolic stability [ ] , although these disadvantages can often be overcome by chemical modification [ ] [ ] [ ] . in addition, progress in the chemical synthesis [ ] [ ] [ ] and recombinant production [ , ] of complex peptides has facilitated cost-effective venom-peptide manufacture. venomous animals have evolved peptide toxins with a huge diversity of molecular frameworks, most of them with tertiary structures stabilised by disulfide bonds [ ] . the most well-known of these disulfide-rich frameworks is the ubiquitous inhibitor cystine knot (ick) or knottin motif [ , ] , which is defined as an antiparallel -sheet stabilised by a cystine knot [ ] . a major advantage of ick peptides for drug development is their resistance to proteases, high temperature, and harsh chemicals, which has been attributed to their knotted structure [ ] [ ] [ ] . however, despite our extensive knowledge about a limited number of disulfide frameworks present in animal venoms, there are many families of disulfide-rich venom peptides that remain completely unexplored (e.g. [ ] [ ] [ ] [ ] ). further research on these novel disulfide-rich frameworks is therefore required to unleash any potential they might hold for drug development. recent advances in proteomics and transcriptomics have now made it possible to rapidly elucidate the venom proteome of even miniature venomous animals [ ] . while these developments have greatly accelerated our understanding of the structure, function and evolution of venom proteins, they have led to a general neglect of non-proteinaceous venom molecules. a wide variety of small molecules have been found in venoms including salts, amino acids, hormones, nucleosides, neurotransmitters, and polyamines [ ] [ ] [ ] . in addition to playing important roles in envenomation [ ] , some of these molecules are valuable pharmacological tools; for example, the polyamine toxins found in spider and wasp venoms are potent modulators of glutamate and nicotinic acetylcholine receptors [ , ] . early toxinological research focused on study of venoms that adversely affect humans, with the aim of understanding their mechanism of action. the molecular targets of these venoms were typically delineated using low-throughput approaches such as in vivo and ex vivo tissue studies, manual patch-clamp recording, or biochemical approaches such as radioligand binding studies [ ] . these bioassays can be highly informative because they provide information on integrated tissue or cellular responses that can be used to provide target information [ ] . however, they require a priori knowledge about well-defined receptor populations and signalling pathways in target tissues, as well as the availability of selective agonists or antagonists. in addition to providing fundamental insights into the molecular targets and mode of action of venoms and toxins, these traditional approaches yielded key insights into mammalian physiology, as exemplified below. however, these approaches neglected toxins that are of no consequence to human health, those that are present in low abundance in venom, and toxins that act on novel targets that are not represented in conventional bioassays. these neglected toxins may nonetheless offer fundamental insights into human disease, and some may even have therapeutic benefit. accordingly, high-throughput screening approaches have been developed over the past decade to exploit the exceptional biodiversity of animal toxins for drug discovery. since many venoms and toxins exert these biological effects through actions on cell membranes, receptors and ion channels, high-throughput techniques assessing changes in cellular signalling have proven particularly insightful. these include electrophysiological techniques [ ] , fluorescent imaging of na + , ca + , k + or clflux [ , ] , enzyme-linked immunosorbent assays (elisas), as well as assays based on detection of bioluminescence, fluorescence polarisation, fluorescence-resonance energy transfer (fret), bioluminescence resonance energy transfer (bret), and scintillation proximity [ ] . invariably, these assays are designed to determine pharmacological activity at specific targets of interest, although each technique has limitations with regard to assay flexibility, throughput, amount of toxin required, and specificity (for a review see [ ] ). thus, the choice of assay will depend on whether the intention is to identify the biological target of venom components that exert effects on biological processes either at the level of the cell or organism, or whether the screen is aimed at discovery of novel bioactivity at specific molecular targets or processes of interest. it has been known since the early s that the specific and virtually irreversible blocking action of -bungarotoxin, a venom peptide from the banded krait bungarus multicinctus, at the vertebrate neuromuscular junction is due to functional antagonism of the depolarising effects of acetylcholine [ ] . however, the identity of the presumed target of -bungarotoxin, the cholinergic receptor protein, was unclear until a single toxin-bound membrane protein was isolated from electric tissues of the marbled electric ray torpedo marmorata [ ] and the electric eel electrophorus electricus [ ] . it is now known that acetylcholine receptors (achrs) can be classified into muscarinic (machr) and nicotinic (nachr) subtypes, with the latter subtype being the target of bungarotoxin and numerous other venom peptides. machrs are metabotropic receptors whereas nachrs are pentameric ligand-gated ion channels. the profound impact of -neurotoxins on various biological processes highlighted the importance of nachrs in both physiology and pathology. early medical applications included the surgical use of tubocurarine, a plant-derived toxin that potently inhibits muscle nachrs and is famous for its use as a paralysing agent in poison arrows employed by south american indians [ ] . the use of as a tubocurarine as a muscle relaxant allowed patients undergoing surgery to be paralysed without using dangerously high doses of general anaesthetics [ ] . more recently, nachrs have gained attention as putative therapeutic targets for a range of human disorders. for example, a number of nachr subtypes are thought to play key roles in chronic pain and inflammation [ ] , and several α-conotoxins with good selectivity for these subtypes are being developed as drug leads [ , ] . the high affinity interaction of α-neurotoxins with nachrs also provided key insights into the structure and mode of action of these receptors [ ] , and they remain important pharmacological tools due to their exquisite selectivity for specific achr subtypes. for example, fluorescent derivatives of α-bungarotoxin are excellent probes for identifying α nachrs [ ] , while subtypeselective α-conotoxins from the venom of marine cone snails can distinguish between nachr subtypes containing common subunits [ , ] . since ablation of nachr subunits using gene editing approaches would lead to complete loss of all nachr isoforms containing the deleted subunit, α-neurotoxins remain invaluable tools for dissecting the physiological roles of specific nachr subtypes and their contribution to human disease. in addition to being a valuable source of molecules that modulate known drug targets, venoms can also be used to discover new therapeutic targets. for example, the centre of excellence for new target discovery in brazil, which is co-funded by glaxosmithkline, is focused on using venoms to identify and validate new drug targets. one therapeutic area in which venom peptides have been successfully used to identify new drug targets is chronic pain [ , ] . although the primary role of venom for most animals that possess a venom system is predation, most venomous animals also use venom for defense. some animals, such as bees, caterpillars, stonefish, and stingrays, use venom exclusively for this purpose. in contrast to the diverse mechanisms by which predatory venoms debilitate prey, defensive venoms are largely algogenic, that is they are designed to cause intense pain (as most people that have been stung by a bee or wasp can testify). unlike death, pain induces a lasting memory that can be conveyed to conspecific predators. since pronociceptive venom toxins have evolved to target vertebrate sensory neurons they have the potential to uncover new components of mammalian pain signalling pathways and to serve as pharmacological tools for studying these components [ , ] . from a screen of venoms that activate mouse sensory neurons, osteen et al. recently isolated two homologous peptides (hm a and hm b) from venom of the african tarantula heteroscodra maculata that activated only a subset of sensory neurons [ ] . the effect of these peptides was blocked by tetrodotoxin (ttx), a non-selective inhibitor of voltage-gated sodium (na v ) channels. this ultimately led to the discovery that these peptides are highly potent and selective agonists of na v . channels [ ] , which were not previously thought to be involved in pain signalling. na v . was shown to regulate the excitability of a nerve fibres that transmit mechanical pain signals to the spinal cord, thereby highlighting this ion channel for the first time as a potential analgesic target [ ] . moreover, na v . was shown to be present in sensory neurons that innervate the gut, and its expression was upregulated in a mouse model of irritable bowel syndrome (ibs) [ ] . this suggests that compounds that specifically inhibit na v . might be useful for treatment of ibsrelated gut pain, and indeed na v . inhibitors were recently shown to reduce mechanical hypersensitivity in several models of chronic visceral pain [ ] . an unexpected consequence of the discovery of hm a and hm b was their application to dravet syndrome (ds) epilepsy [ , ] , which is caused by heterozygous loss-of-function mutations in the gene encoding na v . . ds is a pharmacoresistant epileptic encephalopathy characterised by childhood-onset polymorphic seizures, cognitive impairment, psychomotor regression, autistic traits, and increased risk of sudden death [ , ] . in the central nervous system, na v . is found in the axon initial segments of gabaergic inhibitory interneurons, and consequently epileptic seizures in ds are thought to result from the reduced inhibitory activity of these neurons [ ] . remarkably, in a mouse model of ds, intracerebroventricular infusion of hm a restored the function of inhibitory interneurons and led to almost complete abolition of seizures within days, which rescued the mice from premature death [ ] . in a similar approach to that described above, a screen of snake venoms on somatosensory neurons revealed robust activation of trigeminal ganglia by texas coral snake venom [ ] . this activity could only be reproduced when two toxin components were combined to form the high affinity heteromeric mittx complex. subsequent patch-clamp studies revealed the target of mittx to be acid-sensing ion channels (asics). asics are unusual homo-or heterotrimeric channels that are activated by protons and expressed throughout the nervous system [ , ] . although mittx promotes activation of several asic subtypes, it is ~ -fold more potent at the splice-isoforms asic a and asic b compared with other subtypes. texas coral snake envenomation causes intense pain in humans, and indeed mittx elicited robust nocifensive behaviour when injected into the hindpaw of mice. this response was absent in pan-asic knockout mice, but present in an asic knockout, revealing the contribution of asic in peripheral pain sensation. a subsequent study further delineated the specific roles of asic a and asic b in different pain pathways using mambalgin toxins isolated from black mamba venom [ ] . mambalgins were found to block asics expressed in the central (asic a, asic a/ a, and asic a/ b) and peripheral (asic b and asic a/ b) nervous system. intrathecal and intracerebroventricular injection of mambalgins in mice produced potent analgesia that was naloxone resistant and dependent on the presence of asic a, suggesting an opioid independent mode of action proposed to be via central inhibition of asic a/ a channels. strikingly, peripheral intraplantar injection of mambalgins in mice produced analgesic effects in wild-type and asic a knockout mice. this demonstrated for the first time that asic b-containing channels in rodent nociceptors, as opposed to the splice isoform asic a, are important for peripheral analgesia. more recent work using an asic b knockout animal confirmed the role of this subtype in peripheral pain sensing [ ] . in this model, peripheral injection of mambalgin had no effect on acid-induced hyperalgesia in asic b knockout mice. thus, the combined use of genetic techniques and venom toxins (mittx and mambalgins) enabled dissection of the roles of asic splice isoforms in pain signalling. in addition to venom peptides, non-peptidic marine toxins have proven to be invaluable tools for elucidating diverse pain pathways. ciguatoxins (ctx) are temperature-stable polyethers produced by various species of gambierdiscus, a benthic dinoflagellate [ ] . the consumption of fish with high levels of ciguatoxins present in their flesh and viscera, a result of bioaccumulation, causes ciguatera fish poisoning [ ] . these effects are due the action of ctx on ion channels including na v channels [ ] . the most potent congener p-ctx- has been used to pharmacologically probe neuropathic pain (fig. ). ciguatera is characterised by both gastrointestinal and neurological symptoms including paresthesia, pruritus and cold allodynia, and it can even lead to death in severe cases [ ] . the pathognomonic cold allodynia observed in ciguatera patients is also reported in a broad range of clinical indications including in chemotherapy patients, complex regional pain syndrome, and fibromyalgia [ ] [ ] [ ] . defined by sensitivity to innocuous cooling temperatures, cold allodynia is a poorly understood phenomenon. subcutaneous injection of p-ctx- into the rodent hindpaw causes robust and concentration-dependent cold allodynia [ ] , and therefore it is uniquely positioned to elucidate the molecular basis of cold allodynia. the large family of thermosensitive transient receptor potential (trp) channels plays a critical role in temperature sensation [ ] . interestingly, trpm , the canonical cold sensing channel that is sensitive to menthol, is not associated with p-ctx- -induced cold allodynia, as trpm null mice administered p-ctx- display robust pain behaviours in response to innocuous cooling [ ] . rather, the combined findings from ex vivo skin-saphenous nerve recordings, in vivo studies and in vitro assays highlighted a role for trpa in p-ctx- -induced cold allodynia [ ] . however, trpa is not directly activated by p-ctx- . both ttx-sensitive and ttx-resistant na v channels were responsible for the altered neuronal excitability, which drives a trpa -dependent change in cold sensing [ ] . although the precise role of different trp channel isoforms in cold sensing remains contentious, the use of this marine toxin has proved valuable in delineating these complex mechanisms. p-ctx- was also utilised to assess a range of analgesic treatments for cold allodynia. using a suite of selective pharmacological agents including venom-derived peptides (such as analgesic conotoxins; recently reviewed in [ ] ), p-ctx- -induced cold allodynia was found to be dependent on na v . and na v . [ ] . oxaliplatin-induced cold allodynia was completely blocked by -conotoxin giiia, a na v . inhibitor from cone snail venom [ ] , although intraplantar administration of cn , a na v . -selective agonist from scorpion venom [ ] , elicited spontaneous pain and mechanical allodynia but did not enhance cold sensitivity. the mechanisms underlying neuronal cold sensitivity were further delineated using a class of presynaptic neurotoxins produced by mamba snakes (dendroaspis sp.) (fig. ) . specifically, in trigeminal neurons, application of αdendrotoxin-k, a blocker of kv . channels, elicited a reversible, concentration-dependent shift in the cold threshold [ ] . subtype-selective na v channel toxins have confirmed a crucial role for both na v . and na v . in pain signalling (fig. ) . the na v . subtype is highly expressed in the central and peripheral nervous system, in particular at the nodes of ranvier [ ] . due to the expression of na v . on motor neurons, na v . null mice are not viable beyond post-natal day (p ) [ ] . this greatly impeded the study of na v . in peripheral sensory neurons, where its role in excitability was less well defined. the subtype-selective scorpion toxin cn was therefore used to probe the role of this channel in nociception. cn causes na v . to open at hyperpolarised (more negative) potentials, and it enhances resurgent currents in large diameter sensory neurons in vitro and increases excitability of medium-diameter a fibres ex vivo [ , ] . therefore, it follows that in vivo nocifensive behaviours elicited by cn administration are primarily mediated by medium-diameter rather than smaller sensory neurons. critically, this demonstrates how toxins can be used to pharmacologically isolate a target of interest (in this case na v . ) in the presence of highly similar homologs of the target (na v . , na v . , na v . and na v . ). the toxin od , isolated from venom of the iranian yellow scorpion odonthobuthus doriae, potently enhances the activity of na v . by inducing a hyperpolarising shift in the voltagedependence of activation, inhibiting channel fast inactivation, and increasing peak current [ ] . injection of od into the footpad of mice elicits pain behaviours including licking, lifting and flinching of the injected paw that can be reversed by local or systemic treatment with selective na v . inhibitors. thus, the analgesic efficacy of na v . inhibitors can be rapidly profiled in vivo using od -induced pain behaviour, demonstrating that even toxins with no therapeutic potential can be effective tools for understanding the complexities of peripheral pain sensing [ ] (fig. ). snakes are the most well-studied venomous animal, and hundreds of snake-venom proteins have been isolated over several decades. these venom components have improved our understanding of venom toxicity, significantly advanced our the knowledge of hemostatic disease mechanisms, and led to the development of diagnostic agents and life-saving drugs [ ] [ ] [ ] . due to the immense amount of research on snake venoms, we focus here on just a few examples where studies of snake venom have advanced our understanding of hemostasis and associated diseases. studies into the lethal effects of envenomation by the burrowing asp atractaspis engaddensis led to the discovery of a novel peptide named sarafotoxin (sftx). sftx is a potent vasoconstrictor of coronary blood vessels that can induce cardiac arrest [ ] [ ] [ ] . several sftx isoforms were soon isolated from other stiletto snakes [ , ] , and it was subsequently discovered that the sftxs are in fact orthologs of mammalian vasoconstrictor peptides known as endothelins (ets) [ ] . however, a major difference between these peptides is that a protease cleavage site involved in et inactivation is not present in the sftxs [ , ] , making them more stable in vivo. this allowed the sftxs to be used as molecular tools to study vasoconstriction and expanded the available pharmacological toolbox beyond the ets. the sftxs and ets have both been used to characterise et receptors and downstream signalling pathways [ , ] . von willebrand factor (vwf) is important for platelet adhesion, and its deficiency in humans leads to a common blood coagulation disorder known as von willebrand disease (vwd). botrocetin is a heterodimeric protein found in venom of the south american pit viper bothrops jararaca and it functions as a platelet-aggregating protein. it was initially named venom coagglutinin [ ] as it leads to agglutination and aggregation of platelets by forming a complex with vwf and enhancing its affinity for the platelet receptor glycoprotein ib (gpib). this effect makes botrocetin an excellent diagnostic tool to assay for vwf binding to gpib in diseases such as vwd, or thrombopathies such as bernard-souler disease [ , ] . botrocetin has also been used as a tool to study the vwf-platelet interaction and its role in disease [ , ] . unlike other vwf binding proteins such as the antibiotic ristocetin, botrocetin-induced platelet agglutination activity is independent of the vwf multimer size. this is advantageous as it facilitates quantitation of vwf and allows assessment of vwd severity. the combination of botrocetin and ristocetin has allowed identification of missense mutations that cause of type b vwd [ ] . a fortuitous observation using viper venom to study neurite outgrowth in healthy and tumorous cells led to the nobel prize in . in studies of the regulation of cell growth, a fraction from mouse sarcoma extracts was shown to induce nerve cell growth in chick embryos [ ] . this fraction was termed nerve growth factor (ngf) [ ] , and it was shown to be composed of both nucleic acid and protein. snake venom from the cottonmouth viper agkistrodon piscivorus was known to contain phosphodiesterase activity, and thus it was proposed that this venom could degrade the nucleic acid component (which was thought to be a contaminant) and reveal the active ingredient. surprisingly, minute amounts of crude snake venom strongly promoted nerve growth, approximately - times more potently than the ngf-containing sarcoma fraction [ ] . comparison of the sarcoma extract and venom contents ultimately led to isolation and characterisation of the proteinaceous ngf. ngf has since been found in several snake venoms from the viperidae, crotalidae, and elapidae families and it has been used widely to study cell growth regulation [ , ] . the use of snake venom in this case not only led to a serendipitous finding with widespread importance to basic science, but ngf has also been important for elucidating the etiology of diseases such as developmental/growth deformations, tumours, and delayed wound healing. cobra venom factor (cvf) from the venom of cobra (naja sp.) snakes has the intriguing property of disrupting activation of the complement component of the innate immune system. the complement system is comprised of a set of interacting proteins, and it can be activated in three different ways for it to play a role in pathogen clearance. the alternative pathway of activation involves spontaneous hydrolysis of complement factor c to c b, which allows the plasma protein complement factors b and d to bind to c b and transform it to c -convertase on the external surface of an invading pathogen membrane. subsequent recruitment of additional components of the complement pathway system leads to assembly of the membrane attack complex, which forms pores in the target cell membrane and that kill or damage the pathogen [ , ] . cvf is a structural and functional analogue of the human complement factor c , and it acts in a similar manner to human factors c b and c c to activate and deplete the complement cascade [ , ] . these properties of cvf have made it possible to independently study the role of complement in host defense, immune responses, and pathogenesis of disease. cvf was in fact pivotal to the discovery of complement factor b itself, which facilitated further characterisation of this complex pathway [ , [ ] [ ] [ ] [ ] . cvf is not only an immensely useful research tool, but it has recently been used as an immunosuppressant in tissue and organ transplants and cancer therapies [ , ] , and cvf derivatives are being designed to activate the complement system in disease states where it has been depleted [ , ] . stroke is the second leading cause of death worldwide [ ] , yet therapeutic options are limited to dissolution of blood clots with tissue plasminogen activator (tpa). unfortunately, due to the risk of inducing an intracranial haemorrhage, tpa is used in < % of stroke patients [ ] , and consequently there is an unmet need for compounds that can protect the brain from stroke-induced injury. clinical trials of numerous agents, especially glutamate receptor antagonists, have failed to provide effective neuroprotection in stroke patients, which has spurred interest in better understanding the etiology of ischemia-induced brain injury [ ] . spider-venom peptides have been crucial for uncovering the key role of asics in stroke-induced brain damage, and validating these channels as a target for neuroprotective drugs [ ] [ ] [ ] [ ] . during ischemic conditions the brain switches from oxidative metabolism of glucose, its primary fuel, to anaerobic glycolysis, which results in lactic acidosis. the brain extracellular ph can fall from ~ . to as low as . in the ischemic core [ , , ] , which is sufficiently acidic to robustly activate proton-gated asics. the asic a isoform is the dominant subtype in rodent and human brain [ , ] and it primarily conducts sodium ions, although it is the only asic subtype that is also permeable to calcium (p na /p ca ~ ) [ ] . it has been proposed that asic a activation contributes to toxic intracellular calcium overload during stroke via both direct calcium permeability and downstream activation of voltage-gated calcium (ca v ) channels and nmda receptor-gated channels via asic-induced membrane depolarisation [ ] . however, the primary mechanism by which asic a activation leads to neuronal cell death appears to be recruitment and activation of ripk [ , ] , a master regulator of necroptosis. although several small molecules and endogenous ligands of asic a have been discovered to date, their lack of potency and selectivity limits their use as pharmacological tools for dissecting the role of asics in disease. the first potent and moderately selective asic a ligand discovered is pctx , a -residue peptide from venom of the trinidad chevron tarantula psalmopoeus cambridgei [ ] . pctx inhibits homomeric asic a (ic ~ nm), asic a/ b heteromers (ic ~ nm), and asic a/ a ( - % inhibition at nm) channels, and potentiates proton-evoked asic b currents by over -fold control (ec ~ nm) [ ] [ ] [ ] [ ] [ ] . the key role of asic a in stroke-induced brain damage was first highlighted in a seminal study which showed that genetic ablation of the asic a-encoding gene reduced brain infarct size by ~ % in a mouse model of ischemic stroke [ ] . however, since genetic ablation can give rise to compensatory upregulation of related channels with unknown consequences [ , ] , these researchers also used "pctx venom" (i.e., crude p. cambridgei venom) as a pharmacological tool to validate the genetic studies. they showed that "pctx venom" administered min before and after stroke onset reduced infarct size by the same magnitude as genetic ablation of asic a [ ] . however, spider venoms are complex molecular cocktails that modulate an array of ion channels, and the venom of p. cambridgei is no exception. pctx constitutes only ~ . % of total p. cambridgei venom [ ] , and other components in this venom are known to target trpv and voltage-gated potassium (k v ) channels [ , ] . this left open the question of whether the observed neuroprotective effects were induced by pctx , other components in the venom, or a combination of both. to address this question a more recent study used pure recombinant pctx to confirm the involvement of asic a in stroke [ ] . structure-activity relationship (sar) studies of pctx [ , ] facilitated design of a weakly active pctx mutant, which was not neuroprotective in the same rodent stroke model, thereby strengthening the evidence that asic a inhibition reduces stroke-induced brain damage. hadronyche infensa proved to be highly neuroprotective in a focal model of ischemic stroke, even when administered h after the stroke onset [ ] . in comparison to pctx , hi a is a slightly more potent inhibitor of asic a (ic . nm) and it is more selective, with > -fold higher potency for asic a than homomeric asic b, asic a, and asic channels. hi a inhibition of asic a is much less reversible than pctx [ ] , and these venom peptides have a different mechanism of action [ , ] . the different properties of pctx and hi a broaden the pharmacological toolkit for studying asic a and asic a-related pathologies. together these two venom peptides have provided striking pharmacological evidence that asic a is involved in ischemic injuries of not just the brain, but also the heart [ ] and kidney [ ] . the evolution of drug resistance across bacteria, fungi and parasites has created an unmet need for novel anti-infectives. the united nations report on antimicrobial resistance estimated that > , people die annually due to drug-resistant infections [ ] . while most focus has been on human antibiotic resistance, control of parasitic nematodes in both livestock [ , ] and companion animals [ , ] is also seriously threatened by resistance. despite the clear need for new anti-infectives, there is a diminishing pipeline of anti-infective drug candidates in both the human and veterinary spaces [ ] [ ] [ ] , which has created interest in venoms as a potential source of novel anti-infectives. antimicrobial peptides (amps) are found in many animal venoms, and hundreds of examples have been reported with varying activity against fungi and both gram-positive and gram-negative bacteria [ ] [ ] [ ] [ ] [ ] [ ] . venom-derived amps (vamps) are typically small, linear peptides that are rich in cationic residues (lys, his and arg), which facilitates their interaction with anionic membranes [ , ] . they are often unstructured in aqueous solution but form ordered -helices upon interaction with lipid membranes [ , ] . many vamps exhibit broad-spectrum cytolytic effects. this can be a double-edged sword for antimicrobial development as it often confers desirable broad-spectrum activity against a wide range of bacterial and fungal pathogens but also cytotoxicity against host cells. for example, the ponericin peptides isolated from the amazonian stinging ant neoponera goeldii have broadspectrum activity against bacteria and fungi, but they are potently hemolytic [ ] . likewise, cupiennin a from venom of the wandering spider cupiennius salei exhibits non-specific cytolytic activity against bacteria, insects, protozoan parasites, and human cells [ ] . mammalian cytotoxicity is typically assessed by screens against red blood cells or mammalian cell lines, but an often overlooked property of cytolytic vamps is that they can also cause pain. for example, venom-peptide ∆-myrtoxin-mp a from the australian ant myrmecia pilosula has submicromolar activity against bacterial pathogens but it causes mechanical allodynia when injected into mice [ ] . melittin, the primary component of honeybee venom, is potently antimicrobial but it induces pain through direct and indirect activation of sensory neurons [ ] . hence, future research on vamps should place greater emphasis on screening across a broad range of cell lines and phenotypic readouts, particularly for cytolytic peptides. a small number of vamps show some degree of inherent taxonomic selectivity. for example, bicarinalin from the ant tetramorium bicarinatum is highly active against bacterial and fungal pathogens (minimal inhibitory concentration (mic) of . - µm, which is comparable to commercial anti-infectives), with at least -fold selectivity over human lymphocytes [ ] . it also has similar potency to commercial antibiotics against clinical isolates of the helicobacter pylori, a pathogen that causes stomach ulcers [ ] . however, most vamps will require further engineering of their selectivity in order for them to be therapeutically useful [ ] , and several strategies have been employed successfully for this purpose. for example, shorter analogues of the scorpion-venom peptides hadrurin and vejovine were engineered with -fold improvement in selectivity for escherichia coli over red blood cells [ ] . modification of the hydrophobicity and net charge of the wasp-venom peptide mastoparan through selective amino acid substitutions [ ] and n-terminal acylation [ ] increased its affinity for negatively-charged bacterial membranes and decreased its cytotoxicity towards mammalian cells. although most vamps have not progressed beyond basic in vitro studies, some have proven effective in in vivo infection models. zy is a -residue cyclised derivative of cathelicidin-bf from venom of the snake bungarus fasciatus with improved potency, selectivity and stability over the parent toxin [ ] . in vitro, zy kills multidrug-resistant isolates of the human pathogens pseudomonas aeruginosa and acinetobacter baumannii at lower concentrations than commercial antibiotics, and it reduces bacterial load and inflammation in the lungs of mice in a p. aeruginosa infection model, with no reported side-effects [ ] . lyetxi-b, an n-terminal acetylated derivative of lyetxi from venom of the wolf spider lycosa erythrognatha, has improved potency and selectivity against e. coli and also reduces both bacteraemia and inflammation in a mouse model of septic arthritis when administered intraarticularly [ ] . route of administration is a key issue that must be considered for therapeutic deployment of vamps; traditionally, peptides have poor oral bioavailability [ ] , and hence alternative methods of vamp administration such as topical application should be considered. the effectiveness of this approach was demonstrated for kn - , a derivative of the peptide bmkn from venom of the scorpion mesobuthus martensii, which protected the skin of mice in a staphylococcus aureus in vivo infection model [ ] . topical administration can also circumvent or alleviate off-target effects that are evident when the vamp is present systemically. finally, it might be possible to achieve additive or synergistic effects by combining vamps with existing antibiotics by utilising the pore-forming activity of the vamp to enhance antibiotic delivery. for example, garcia et al. found an additive effect when pathogen-specific antibiotics were combined with vamps isolated from the venom of arachnids [ ] . viruses continue to pose an enormous threat to global health, as exemplified by the current global pandemic caused by sars-cov- [ ] . despite intensive intervention efforts, an estimated , people continue to die annually from human immunodeficiency virus acquired immunodeficiency syndrome (hiv-aids) [ ] . many serious viral diseases such as severe acute respiratory syndrome (sars), middle eastern respiratory syndrome (mers), dengue, zika, and ebola lack vaccines or efficacious therapeutic treatments, highlighting the need for novel antivirals. most reported venom toxins with antiviral activity are vamps with broad-spectrum activity; examples include melittin, snake venom pla and l-amino oxidases, and mastoparan derivatives [ , ] . thus, a key challenge will be engineering these compounds to have selectivity for either the viral particles or the process by which they infect host cells. mastoparan- , derived from wasp-venom mastoparan, has broad-spectrum activity against lipid-enveloped viruses (including influenza and flaviviruses) through direct insertion and disruption of the envelope, but it remains highly cytotoxic to host cells in vitro [ ] . peptides with activity on specific viral protein targets rather than lipids may provide better selectivity. lactarcin- from the central asian spider lachesana tarabaevi inhibits replication of dengue virus- (ic ~ µm) by virtue of its ability to inhibit the viral protease ns b-ns , but it has low selectivity over mammalian cells [ ] . the antibacterial activity of mucroporin, a vamp from the scorpion lychas mucronatus, was improved through histidine and arginine substitutions to create the analog mucroporin-m with increased net charge. mucroporin-m has improved activity against both gram-positive and gram-negative bacteria [ ] , and these modifications also conferred micromolar antiviral activity in vitro (ec - µm) against measles virus and pseudovirus models of the highly pathogenic sars-cov and avian influenza h n viruses [ ] . a key limitation of current research on venom-derived antiviral toxins is that few studies have progressed to in vivo testing. one example found that pre-treating vesicular stomatitis virus, a serious pathogen of cattle, with mastoparan- significantly improved clinical outcomes in a mouse challenge model through presumed inactivation of viral particles [ ] . however, this needs to be confirmed in a more clinically relevant challenge model of viral infection followed by toxin administration to probe efficacy. overall, further research is required to ascertain the potential of venom-derived peptides as viable antiviral drugs. parasites remain one of the most serious threats to human and veterinary medicine, with current treatment options limited by drug resistance, side-effects and limited efficacy [ ] . this has stimulated interest in venoms as a source of novel antiparasitic compounds (for a review see [ ] ). most such studies have focused on protozoan parasites, which are responsible for a range of human diseases. the most problematic parasitic disease is malaria, which is caused by plasmodium parasites; despite intensive intervention efforts, malaria kills an estimated , people each year and control is threatened by drug resistance [ ] . a further million people worldwide are infected by parasitic protozoans of the genera leishmania and trypanosoma, with approximately million new infections per year [ ] . most antiparasitic compounds isolated from animal venoms are small cationic vamps, which again raises the issue of taxonomic selectivity. the vamps mastoparan [ ] and melittin [ ] inhibit development of trypanosoma cruzi (the causative agent of chagas disease), but selectivity over host cells is low. however, bicarinalin potently inhibits growth of leishmania infantum (mic . µm), the causative agent of infantile visceral leishmaniasis, with good selectivity over human lymphocytes [ ] . remarkably, crotamine, a peptide from venom of the south american rattlesnake crotalus durissus terrificus, crosses infected red blood cell membranes to lyse the plasmodium parasitophorous vacuole, thereby killing the parasite [ ] . this may present an opportunity for combination therapy with commercial antimalarials, facilitating drug transport to the parasite. further examples of venom components active against protozoan parasites include trimorphin, a pla from venom of the north american rear-fanged snake trimorphodon biscutatus lambda that inhibits growth of leishmania major [ ] , and crovirin, a cysteine-rich secretory protein (crisp) from the rattlesnake crotalus viridis viridis that is active against protozoan parasites at concentrations that are not toxic to host cells [ ] . the protozoan toxoplasma gondii, which causes the key zoonotic disease toxoplasmosis, is susceptible to a venom peptide from the marine cone snail conus californicus [ ] , while a c-type lectin and a pla from b. pauloensis snake venom impair toxoplasma adhesion and replication [ , ] . the greatest burden of disability-adjusted life years in the neglected tropical diseases comes from helminth infections, including roundworms (nematoda) and flatworms (platyhelminthes) [ ] . these worms cause important parasitic diseases in humans, including onchocerciasis, lymphatic filariasis, and schistosomiasis. about million people worldwide suffer from schistosomiasis, and it is considered the second most socioeconomically devastating parasitic disease after malaria [ ] . the burden of parasitic disease is also a key concern in animal health and production, with the worldwide cost of controlling livestock parasites amounting to tens of billions of dollars [ ] . despite this clear need for novel anthelmintics, it is an underexplored area of venom research with very few reports of anthelmintic venom toxins. crotamine [ ] and ε-latroinsectotoxin from venom of the black widow spider latrodectus mactans tedecimgutattatus [ ] both impair growth of the non-parasitic nematode caenorhabditis elegans, but anthelmintic activity has not been reported against parasitic species. more recently, the spider-venom peptide hi a was shown to inhibit larval development of the barber's pole worm haemonchus contortus, a parasite of sheep production [ ] . gomesin, a peptide found in the venom [ ] and hemocytes [ ] of spiders, has broad-spectrum activity against bacteria, fungi, parasites and tumor cells [ , ] , and a cyclized version was recently shown to have improved stability and activity against plasmodium in vitro [ ] . to our knowledge, no venom-derived antiparasitics have yet been demonstrated to have in vivo efficacy. as for antibacterials, antifungals and antivirals, most venom-derived antiparasitics will likely require engineering to enhance their selectivity over host cells. an alternative strategy to developing venom compounds as anti-parasitic drugs is to instead impair plasmodium development and transmission via transgenic mosquitoes engineered to express the anti-parasitic compound in the mosquito midgut, as recently demonstrated with scorpion-venom peptides [ ] . as most research on venom compounds with antibacterial, antiparasitic, or antifungal activity has been performed in the last decade, we expect that more toxins with such activities will be discovered in the near future. most work to date has focussed on human pathogens, but there is a clear need for novel veterinary anti-infectives which could be further explored in future. due to the commonly found cytolytic nature of these peptides, a key challenge across all of these diseases will be engineering analogs with selectivity for the targeted pathogen. arthropods, and in particular hematophagous (blood-sucking) species, are vectors for a wide-range of organisms that can cause human disease [ ] . the most pernicious vectors are undoubtedly mosquitoes, which transmit protozoans (e.g. plasmodium species that cause malaria), parasitic nematodes (e.g. brugia species that cause filariasis), parasitic platyhelminths (e.g. trematodes that cause schistosomiases), and viruses (e.g. flaviviruses that cause yellow fever, west nile fever, and dengue) [ , , ] . other important hematophagous disease vectors include triatomine bugs which transmit trypanosomes that cause chagas disease, fleas which transmit the bacterium yersinia pestis that causes bubonic plague, and ticks which transmit a wide variety of viral, bacterial and protozoan pathogens [ , ] . rather than directly targeting disease-causing organisms, venom toxins could be used instead to target the vectors of these diseases. insects are the dominant vectors of human diseases, and chemical insecticides are the primary means of controlling these insect pests [ , ] . unfortunately, all chemical insecticides target one of only a handful of molecular targets, and consequently their indiscriminate use has led to widespread resistance [ ] . thus, new insecticides with novel modes of action are much sought after. spiders are professional insect killers, and they are the most speciose venomous animal, and consequently their venoms have been intensively studied as a potential source of novel bioinsecticides [ , [ ] [ ] [ ] [ ] [ ] . as a result, many insecticidal toxins have been isolated from spider venoms, including peptides, proteins and polyamines, but they mostly lack the necessary taxonomic selectivity to be useful as insecticides. an ideal insecticide should be inactive on humans and other vertebrates, as well as beneficial insects such as pollinators and natural predators of the targeted insect pest [ ] . this narrow target-organism profile makes insecticide development particularly difficult. however, spidervenom peptides have been isolated with desirable taxonomic selectivity. for example, by carefully counter-screening insect toxins from the venom of australian funnel-web spiders in vertebrate assays, a number of insect-selective neurotoxins were isolated [ ] . one of these peptide toxins (gs-/-hexatoxin-hv a), which has complex polymodal pharmacology, has been developed commercially as a bioinsecticide by vestaron corporation; this peptide is active against a broad range of insect pests but is inactive against bees and vertebrates [ ] . in order to target anopheline mosquitoes that transmit malaria, a gene encoding gs-/-hexatoxin-hv a was engineered into the entomopathogenic fungus metarhizium pingshaense [ ] , and this weaponised entomopathogen proved highly successful in controlling malaria vectors in a field trial in burkina faso [ ] . an advantage of this approach is that the range of susceptible species is determined by the entomopathogen rather than the venom peptide, thereby allowing deployment of insecticidal venom toxins that would otherwise lack the desired taxonomic selectivity [ , ] . some insecticidal venom peptides have exceptional taxonomic selectivity. for example, -diguetoxin-dc a (dc a), a -residue knottin from venom of the desert bush spider diguetia canities [ ] , and -theraphotoxin-ae a, a -residue knottin from venom of african tarantula augacephalus ezendami [ ] , have potent insecticidal effects on german cockroaches (blattella germanica), without significantly affecting american cockroaches (periplaneta americana). both knottins target insect na v channels, and mutational analyses revealed that the susceptibility of insects to these toxins is determined by small sequence differences in their proposed binding site on the extracellular surface of the domain ii voltage sensor (vsd ii ) [ , ] . a high-resolution structure of dc a bound to a cockroach nav channel was recently determined using cryoelectron microscopy [ ] , and it not only confirmed that vsd ii is the binding site for dc a but revealed that the toxin's inactivity against vertebrates [ ] can be explained by sequence variations in the vsd ii region of each of the human na v channels. the dc a-na v channel structure allows predictions to be made about which insects will be susceptible to dc a based on their na v channel vsd ii sequences, and it should allow use of structure-guided protein engineering to develop toxin analogs with improved potency and/or altered taxonomic selectivity. in addition to highlighting how venom compounds can be used to elucidate disease mechanisms and uncover new drug targets, we want to provide examples of venom compounds that have progressed all the way from basic research to marketed drugs. there are currently six venomderived drugs approved by the fda, plus a snake-venom-derived serine protease (batroxobin; marketed as defibrase®) that is approved for clinical use outside of the usa [ , ] . two further venom-derived drugs -dalazatide for treatment of autoimmune diseases [ ] and tozuleristide for intraoperative imaging of brain tumours [ ] -are currently undergoing clinical trials. the examples below demonstrate the potential for developing venom compounds into drugs [ ] but, like any class of therapeutics, there are many challenges and the success rate is low [ ] . the first venom-derived drug was captopril, an angiotensin-converting enzyme (ace) inhibitor that is used to treat hypertension. the observation that patients envenomated by the lethal brazilian viper bothrops jararaca suffered from hypotension and severe intestinal contractions led to the discovery that the venom inhibits ace [ ] [ ] [ ] . inhibition of ace lowers blood pressure by preventing conversion of angiotensin i to the vasoconstrictive hormone angiotensin ii, and by decreasing degradation of the vasodilatory peptide bradykinin. researchers at squibb isolated six ace inhibitory peptides from b. jararaca venom, one of which (teprotide, residues) was both potent and stable in vivo [ ] . detailed sar studies of teprotide ultimately led to a smaller, orally active peptidomimetic known as captopril (d- -methyl- -mercaptopropanoyl-l-proline) [ , ] . captopril was approved by the fda for treatment of hypertension in , and by the mid s it had become a blockbuster drug, reaching peak annual sales of more than usd $ . billion [ ] . another example of a reptile peptide that was developed into a human drug is exendin- , a residue peptide isolated from venom of the gila monster heloderma suspectum. in striking contrast to captopril, the marketed drug (exenatide) is identical to the peptide found in the lizard's venom. exenatide mirrors the effects of human hormone glucagon-like peptide- (glp- ), an incretin that stimulates glucose-dependent insulin secretion, suppresses glucagon secretion, slows gastric emptying, and promotes satiety [ ] , but it is much more stable in vivo than human glp- with a plasma half-life of . h [ ] . byetta®, a formulation of exenatide that requires twice daily subcutaneous injections, was approved by the fda in for control of type diabetes, and it achieved worldwide sales of usd $ million in fiscal year [ ] . in order to improve patient acceptance and compliance, a new formulation was developed in which the peptide is embedded in biodegradable polymeric microspheres that extends the duration of exenatide release to such an extent that therapeutic levels can be maintained with weekly injections [ ] . this new formulation (bydureon®) was approved by the fda in , and annual sales have remained steady at usd $ - million despite the introduction of competing incretin mimetics. another drug that is an exact copy of a venom peptide is ziconotide, a synthetic version of ωconotoxin mviia, a -residue knottin peptide found in venom of the marine cone snail conus magus. ziconotide is used to selectively inhibit ca v . channels in the spinal cord, thereby preventing the propagation of pain signals from the spinal cord to processing centres in the brain [ ] . it was approved by the fda in for treating patients with chronic pain. ziconotide is administered by intrathecal infusion, which limits its use to patients with intractable chronic pain that have failed on frontline analgesic drugs such as morphine. while ziconotide set a landmark as the first venom-derived drug with a neuronal target, its limited market acceptance due to its mode of administration and neuropsychiatric side-effects [ ] has caused venom researchers to focus attention on analgesic targets in peripheral nociceptors rather than central neurons, as outlined in section . . the integration of advanced venom proteomics with next-generation sequencing of venom-gland transcriptomes has made it possible to rapidly determine, at only moderate cost, the complete venom proteome of even miniature venomous animals [ ] . as a result, novel venom peptides and proteins, often with unique three-dimensional folds, are being discovered at an unprecedented rate. the coupling of these omics workflows with high-throughput screening technologies [ , ] is allowing the pharmacology of these compounds to be explored with unprecedented efficiency, often leading to the discovery of toxins with novel pharmacology [ , , , , ] . all of these advances have combined to create tremendous interest in venoms as a potential source of drugs and bioinsecticides [ , ] . while there have been some notable successes -there are seven venom-derived drugs in clinical use and several in phase / clinical trials -the triumphs are scarcer than many predicted. no novel venom-derived drug has been approved by the fda since [ , ] , and only one venomderived compound has been commercialised as a bioinsecticide [ ] . the reasons for this are manifold. drug development is inherently difficult and the overall success rate is low [ ] ; venomderived drug candidates are no different than any other class of therapeutic in this respect, although the overall approval rate for venom-derived drugs entering clinical trials is higher than other classes of therapeutics. most venom researchers are not trained in the nuances of drug development, and this has led to over-hyped claims about the therapeutic potential of venom compounds, often in the absence of in vivo data. we encourage collaboration between venom researchers and those with experience in drug development so that, at a minimum, in vivo efficacy and toxicity can be assessed in validated disease models, using clinically viable routes of administration, before making claims about therapeutic potential. renewed interest from pharmaceutical companies in venoms as source of drug leads and pharmacological tools [ ] [ ] [ ] [ ] will help to drive these collaborations. notwithstanding these caveats, there is little doubt that the field of venoms-based drug discovery has rapidly matured over the past decade and is now producing drug leads at an unprecedented rate. advances in cryoelectron microscopy have facilitated determination of the three-dimensional structure of venom compounds bound to large membrane receptors [ , , [ ] [ ] [ ] [ ] , and this should facilitate the rational design of drug candidates with improved potency and selectivity for their cognate molecular target. we predict that these advances this will lead to significant impact in some disease areas. for example, ion channels are now the third most common human drug target after kinases and g-protein-coupled receptors [ ] ; venoms are unquestionably the best natural source of potent ion channel modulators and venom-derived ion channel modulators have already attracted significant interest from pharmaceutical companies [ , [ ] [ ] [ ] [ ] . the authors declare that they have no conflicts of interest. figure : fields of application in basic research and human health to which venom compounds have been applied. venom compounds have the potential to be used as pharmacological tools, therapeutics, insecticides, or for targeting vectors of human disease or disease-inducing organisms. botrocetin bound von willebrand factor (vwf) and platelet glycoprotein ib (gpib) ( u n) [ ] ; cobra venom factor (cvf) bound complement factor b ( hrz) [ ] ; captopril bound angiotensin converting enzyme (ace) ( uzf) [ ] ; triflin bound small serum protein (ssp ) ( imf) [ ] ; mittx-α and -β bound acid sensing ion channel (asic ) ( ntw) [ ] . (b) cone snail toxin complexes: α-conotoxin imi bound acetylcholine binding protein (achbp) ( byp) [ ] ; μconotoxin kiiia bound voltage-gated sodium channel (na v ) . /auxiliary β subunit ( j e) [ ] . (c) spider toxin complexes: pctx bound asic ( fz ) [ ] ; dc a and tetrodotoxin (ttx) bound na v pas ( a ) [ ] ; dktx and resiniferatoxin (rtx) bound transient receptor potential cation channel subfamily v member (trpv ) ( irx) [ ] ; protx-ii bound na v . voltage sensor domain ii/na v ab chimera ( n i) [ ] . (d) gila monster toxin exendin- bound glucagon-like peptide receptor (glp- r) ( c t) [ ] . (e) scorpion toxin complexes: charybdotoxin (ctx) bound voltage-gated potassium channel (k v ) . - . paddle chimera ( jta) [ ] ; α-mammal toxin aah bound human-cockroach hybrid na v channel ( nt ) [ ] . structures are shown as a cartoon representation and from two angles ( o rotation). toxin components are shown in purple and with a transparent surface representation where possible (note: ttx and rtx are shown as red sticks). where applicable for the target structure, protein subunits or domains are colored differently. structures are not to scale. toxins providing insight into the pathophysiological role of specific ion channels in sensory neurons. subtype-selective na v channel activators, including δ-trtx-hm a and the scorpion toxins cn and od , have highlighted modality-specific nociceptive roles for na v . , na v . and na v . , respectively, in sensory neurons. activation of na v . by the scorpion toxin od leads to spontaneous action potential firing in myelinated a-fibres and unmyelinated cfibres, while activation of na v . leads to symptoms of mechanical allodynia and increased mechanical sensitivity. cn , a selective na v . activator, elicits pain behaviour and mechanical allodynia after local administration consistent with effects on myelinated a-fibres. similarly, cold pain induced by ciguatoxin is elicited by selective activation of unmyelinated peripheral sensory neurons expressing the cold-sensitive trpa and na v . channels, while α-dendrotoxin revealed a crucial role for k v . in setting the activation threshold of cold-sensitive c-fibre neurons. the toxicogenomic multiverse: convergent recruitment of proteins into animal venoms venom toxins in the exploration of molecular, physiological and pathophysiological functions of acid-sensing ion channels conotoxins targeting nicotinic acetylcholine receptors: an overview nicotinic acetylcholine receptor inhibitors derived from snake and snail venoms calcium channel diversity and neurotransmitter release: the ω-conotoxins and ω-agatoxins peptide toxins that selectively target insect na v and ca v channels from foe to friend: using animal toxins to investigate ion channel function selective spider toxins reveal a role for na v . channel in mechanical pain venoms as a platform for human drugs: translating toxins into therapheutics peptide therapeutics from venom: current status and potential the diversity of venom: the importance of behavior and venom system morphology in understanding its ecology and evolution venoms to the rescue arthropod assassins: crawling biochemists with diverse toxin pharmacopeias entomo-venomics: the evolution, biology and biochemistry of insect venoms pharmacology and biochemistry of spider venoms venom composition and strategies in spiders: is everything possible? advances in insect physiology spider-venom peptides: structure, pharmacology, and potential for control of insect pests poisons, toxungens, and venoms: redefining and classifying toxic biological secretions and the organisms that employ them the venom optimization hypothesis revisited combinatorial peptide libraries in drug design: lessons from venomous cone snails were arachnids the first to use combinatorial peptide libraries? intrathecal ziconotide: a review of its use in patients with chronic pain refractory to other systemic or intrathecal analgesics exenatide: from the gila monster to the pharmacy safety and pharmacodynamics of dalazatide, a kv . channel inhibitor, in the treatment of plaque psoriasis: a randomized phase b trial phase safety, pharmacokinetics, and fluorescence imaging study of tozuleristide (blz- ) in adults with newly diagnosed or recurrent gliomas the future of peptide-based drugs fusion proteins containing neuropeptides as novel insect contol agents: snowdrop lectin delivers fused allatostatin to insect haemolymph following oral ingestion engineering stable peptide toxins by means of backbone cyclization: stabilization of the α-conotoxin mii optimization of physicochemical and pharmacological properties of peptide drugs by glycosylation recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins venoms to drugs: venoms as a source for the development of human therapeutics modern tools for the chemical ligation and synthesis of modified peptides and proteins production of recombinant disulfiderich venom peptides for structural and functional analysis via expression in the periplasm of e. coli high-throughput expression of animal venom toxins in escherichia coli to generate a large library of oxidized disulphidereticulated peptides for drug discovery the structural universe of disulfide-rich venom peptides toxin structures as evolutionary tools: using conserved d folds to study the evolution of rapidly evolving peptides knottin: the database of inhibitor cystine knot scaffold after years, toward a systematic structure modeling a common structural motif incorporating a cystine knot and a triple-stranded β-sheet in toxic and inhibitory polypeptides thermal, chemical, and enzymatic stability of the cyclotide kalata b : the importance of the cyclic cystine knot spider-venom peptides as therapeutics the cystine knot is responsible for the exceptional stability of the insecticidal spider toxin ω-hexatoxin-hv a a bioinformatics survey for conotoxin-like sequences in three turrid snail venom duct transcriptomes centipede venom: recent discoveries and current state of knowledge a dipteran's novel sucker punch: evolution of arthropod atypical venom with a neurotoxic component in robber flies venom peptide repertoire of the european myrmicine ant manica rubida: identification of insecticidal toxins venom collection and analysis in the pseudoscorpion chelifer cancroides (pseudoscorpiones: cheliferidae) one scorpion, two venoms: prevenom of parabuthus transvaalicus acts as an alternative type of venom with distinct mechanism of action nmr-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species giant fish-killing water bug reveals ancient and dynamic venom evolution in heteroptera targeting ionotropic receptors with polyamine-containing toxins polyamine toxins: development of selective ligands for ionotropic receptors venoms-based drug discovery: bioassays, electrophysiology, high-throughput screens and target identification ion channel screening development of a high-throughput fluorescent nowash sodium influx assay high-throughput fluorescence assays for ion channels and gpcrs pharmacological screening technologies for venom peptide discovery isolation of neurotoxins from the venom of bungarus multicinctus and their modes of neuromuscular blocking action isolation of the cholinergic receptor protein of torpedo electric tissue use of a snake venom toxin to characterize the cholinergic receptor protein from arrow poison to neuromuscular blockers toxins and drug discovery nicotinic acetylcholine receptors in neuropathic and inflammatory pain α -containing nicotinic acetylcholine receptors and the modulation of pain the α α nicotinic acetylcholine receptor antagonist αo-conotoxin gexiva[ , ] alleviates and reverses chemotherapy-induced neuropathic pain crystal structure of the extracellular domain of nachr α bound to α-bungarotoxin at . Å resolution moon is, carbofuran induces apoptosis of rat cortical neurons and downregulates surface ⍺ subunit of acetylcholine receptors conus peptides targeted to specific nicotinic acetylcholine receptor subtypes conus venom peptide pharmacology pain-causing venom peptides: insights into sensory neuron pharmacology receptor-targeting mechanisms of pain-causing toxins: how ow? nav . inhibition can reduce visceral hypersensitivity selective na v . activation rescues dravet syndrome mice from seizures and premature death a selective na v . activator with potential for treatment of dravet syndrome epilepsy a heteromeric texas coral snake toxin targets acid-sensing ion channels to produce pain structure, function, and pharmacology of acid-sensing ion channels (asics): focus on asic a acid-sensing ion channel (asic) structure and function: insights from spider, snake and sea anemone venoms black mamba venom peptides target acid-sensing ion channels to abolish pain involvement of acid-sensing ion channel b in the development of acid-induced chronic muscle pain differential toxin profiles of ciguatoxins in marine organisms: chemistry, fate and global distribution australian perspectives on a global problem clinical observations on , cases of ciguatera (fish poisoning) in the south pacific quantitative and qualitative perceptual analysis of cold dysesthesia and hyperalgesia in fibromyalgia chemotherapy-induced neuropathy reduction of allodynia in patients with complex regional pain syndrome: a double-blind placebo-controlled trial of topical ketamine ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling an introduction to trp channels conotoxins that could provide analgesia through voltage gated sodium channel inhibition analgesic treatment of ciguatoxin-induced cold allodynia μ-conotoxin giiia, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling, and receptor characterization resurgent current and voltage sensor trapping enhanced activation by a β-scorpion toxin solely in nav . channel. significance in mice purkinje neurons variable threshold of trigeminal cold-thermosensitive neurons is determined by a balance between trpm and kv potassium channels sodium channel na v . is localized at nodes of ranvier, dendrites, and synapses molecular and pathological effects of a modifier gene on deficiency of the sodium channel scn a (na v . ) na v . regulates excitability of mechanosensitive sensory neurons analgesic effects of gptx- , pf- and cnv in a mouse model of na v . -mediated pain toxins in thrombosis and haemostasis: potential beyond imagination from snake venom toxins to therapeutics-cardiovascular examples non-enzymatic proteins from snake venoms: a gold mine of pharmacological tools and drug leads a new type of toxin in the venom of snakes of the genus atractaspis (atractaspidinae) cardiotoxic effects of the venom of the burrowing asp coronary vasospasm as the primary cause of death due to the venom of the burrowing asp, atractaspis engaddensis sarafotoxins s : several isotoxins from atractaspis engaddensis (burrowing asp) venom that affect the heart a novel potent vasoconstrictor peptide produced by vascular endothelial cells endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance the hydrolysis of endothelins by neutral endopeptidase . (enkephalinase) three apparent receptor subtypes for the endothelin/sarafotoxin family endothelin-like peptides venom coagglutinin: an activator of platelet aggregation dependent on von willebrand factor structure and function of snake venom toxins interacting with human von willebrand factor role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von willebrand factor practical applications of snake venom toxins in haemostasis snake c-type lectin-like proteins and platelet receptors von willebrand disease type b: a missense mutation selectively abolishes ristocetin-induced von willebrand factor binding to platelet glycoprotein ib in vitro experiments on the effects of mouse sarcomas and on the spinal and sympathetic ganglia of the chick embryo nerve growth factors from snake venoms: chemical properties, mode of action and biological significance a nerve growth-stimulating factor isolated from snake venom nerve growth factor from cobra venom molecular diversity of snake venom nerve growth factors a molecular concept of immune cytolysis alternative pathway for the activation of complement in human serum. formation and composition of the complex with cobra venom factor that cleaves the third component of complement molecular characterization of the complement activating protein in the venom of the indian cobra (naja n. siamensis) cobra venom factor: structural homology with the third component of human complement the c -activator system: an alternate pathway of complement activation isolation of the anticomplementary protein from cobra venom and its mode of action on c alternate complement pathway: factors involved in cobra venom factor (covf) activation of the third component of complement (c ) the relationship of glycine-rich -glycoprotein to factor b in the properdin system and to the cobra factor-binding protein of huan serum inhibition of the membrane attack complex of complement for induction of accommodation in the hamster-to-rat heart transplant model snake venom: from fieldwork to the clinic functional characterization of human c /cobra venom factor hybrid proteins for therapeutic complement depletion cobra venom factor: structure, function, and humanization for therapeutic complement depletion arumugam tv, pathophysiology, treatment, and animal and cellular models of human ischemic stroke beyond nmda and ampa glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels prolonged activation of asic a and the time window for neuroprotection in cerebral ischaemia pctx affords neuroprotection in a conscious model of stroke in hypertensive rats via selective inhibition of asic a potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acidsensing ion channel a translational strategies for neuroprotection in ischemic strokefocusing on acid-sensing ion channel a identification of a calcium permeable human acid-sensing ion channel transcript variant acid-sensing ion channels in acidosis-induced injury of human brain neurons tissue acidosis induces neuronal necroptosis via asic a channel independent of its ionic conduction disruption of auto-inhibition underlies conformational signaling of asic a to induce neuronal necroptosis isolation of a tarantula toxin specific for a class of proton-gated na + channels interaction of acid-sensing ion channel (asic) with the tarantula toxin psalmotoxin is state dependent heteromeric acid-sensing ion channels (asics) composed of asic b and asic a display novel channel properties and contribute to acidosisinduced neuronal death functional and pharmacological characterization of two different asic a/ a heteromers reveals their sensitivity to the spider toxin pctx the modulation of acid-sensing ion channel by pctx is ph-, subtype-and species-dependent: importance of interactions at the channel subunit interface and potential for engineering selective analogues genetic compensation triggered by mutant mrna degradation ptc-bearing mrna elicits a genetic compensation response via upf a and compass components isolation and characterization of psalmopeotoxin i and ii: two novel antimalarial peptides from the venom of the tarantula psalmopoeus cambridgei spider toxins activate the capsaicin receptor to produce inflammatory pain a dynamic pharmacophore drives the interaction between psalmotoxin- and the putative drug target acidsensing ion channel a molecular dynamics and functional studies define a hot spot of crystal contacts essential for pctx inhibition of acid-sensing ion channel a the tarantula toxin psalmotoxin inhibits acid-sensing ion channel (asic) a by increasing its apparent h + affinity enhanced donor heart preservation by therapeutic inhibition of acid sensing ion channel a, biorxiv acid-sensing ion channel a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis no time to wait: securing the future from drug-resistant infections anthelmintic resistance to ivermectin and moxidectin in gastrointestinal nematodes of cattle in europe anthelmintic resistance in haemonchus contortus: history, mechanisms and diagnosis the emergence of macrocyclic lactone resistance in the canine heartworm, dirofilaria immitis multiple drug resistance in the canine hookworm ancylostoma caninum: an emerging threat? repurposing drugs for the treatment and control of helminth infections polishing the tarnished silver bullet: the quest for new antibiotics anthelmintic discovery and development in the animal health industry antimicrobial and cytolytic peptides of venomous arthropods antimicrobial peptides from scorpion venoms insects, arachnids and centipedes venom: a powerful weapon against bacteria. a literature review animal venoms as antimicrobial agents animal venom peptides: potential for new antimicrobial agents hitchhiking with nature: snake venom peptides to fight cancer and superbugs toxins and antimicrobial peptides: interactions with membranes latarcins: versatile spider venom peptides ponericins, new antibacterial and insecticidal peptides from the venom of the ant pachycondyla goeldii cupiennin a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells △-myrtoxin-mp a is a helical heterodimer from the venom of the jack jumper ant that has antimicrobial, membrane-disrupting, and nociceptive activities melittin, the major pain-producing substance of bee venom biochemical and biophysical combined study of bicarinalin, an ant venom antimicrobial peptide anti-helicobacter pylori properties of the ant-venom peptide bicarinalin enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin selective amino acid substitution reduces cytotoxicity of the antimicrobial peptide mastoparan selective acylation enhances membrane charge sensitivity of the antimicrobial peptide mastoparan-x the antimicrobial peptide zy combats multidrug-resistant pseudomonas aeruginosa and acinetobacter baumannii infection lyetxi-b, a synthetic peptide derived from lycosa erythrognatha spider venom, shows potent antibiotic activity in vitro and in vivo current challenges in peptide-based drug discovery antibacterial activity and mechanism of a scorpion venom peptide derivative in vitro and in vivo antimicrobial peptides from arachnid venoms and their microbicidal activity in the presence of commercial antibiotics the sars-cov- outbreak: what we know global, regional, and national incidence, prevalence, and years lived with disability for diseases and injuries for countries and territories, - : a systematic analysis for the global burden of disease study antiviral activity of animal venom peptides and related compounds antiviral peptides as promising therapeutic drugs a mastoparanderived peptide has broad-spectrum antiviral activity against enveloped viruses identification of natural antimicrobial agents to treat dengue infection: in vitro analysis of latarcin peptide activity against dengue virus mucroporin, the first cationic host defense peptide from the venom of lychas mucronatus virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses antiparasitic agents: new drugs on the horizon venoms as sources of novel anti-parasitic agents souto-padron t, melittin peptide kills trypanosoma cruzi parasites by inducing different cell death pathways trypanocidal activity of mastoparan from polybia paulista wasp venom by interaction with tcgapdh inhibition of malaria parasite plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom a comparative study of the effects of venoms from five rear-fanged snake species on the growth of leishmania major: identification of a protein with inhibitory activity against the parasite in vitro effect of the synthetic cal . a conotoxin, derived from conus californicus, on the human parasite toxoplasma gondii anti-parasitic effect on toxoplasma gondii induced by bnsp- , a lys -phospholipase a homologue from bothrops pauloensis venom insights into antiparasitism induced by a c-type lectin from bothrops pauloensis venom on toxoplasma gondii water-related diseases. world health organization impact of gastrointestinal parasitic nematodes of sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance -an australian perspective anthelmintic effects of a cationic toxin from a south american rattlesnake venom latrophilin is required for toxicity of black widow spider venom in caenorhabditis elegans the antitrypanosomal diarylamidines, diminazene and pentamidine, show anthelmintic activity against haemonchus contortus in vitro structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene isolation and characterization of gomesin, an -residue cysteine-rich defense peptide from the spider acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family the biological and biophysical properties of the spider peptide gomesin gomesin inhibits melanoma growth by manipulating key signaling cascades that control cell death and proliferation cyclization of the antimicrobial peptide gomesin with native chemical ligation: influences on stability and bioactivity from noxiustoxin to scorpine and possible transgenic mosquitoes resistant to malaria spider-venom peptides as bioinsecticides australian funnel-web spiders: master insecticide chemists the insecticidal potential of venom peptides versatile spider venom peptides and their medical and agricultural applications tying pest insects in knots: the deployment of spider-venom-derived knottins as bioinsecticides improved efficacy of an arthropod toxin expressing fungus against insecticide-resistant malaria-vector mosquitoes transgenic metarhizium rapidly kills mosquitoes in a malaria-endemic region of burkina faso construction of a hypervirulent and specific mycoinsecticide for locust control characterization and cloning of insecticidal peptides from the primitive weaving spider diguetia canities molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the african spider augacephalus ezendami a distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin dc a structural basis for the modulation of voltage-gated sodium channels by animal toxins venoms to drugs: venom as a source for the development of human therapeutics london history of the design of captopril and related inhibitors of angiotensin converting enzyme the discovery of captopril: from large animals to small molecules design of angiotensin converting enzyme inhibitors blockbuster drugs: the rise and decline of the pharmaceutical industry exenatide once weekly for the treatment of type diabetes ziconotide: neuronal calcium channel blocker for treating severe chronic pain do the potential benefits outweigh the risks? an update on the use of ziconotide in clinical practice venomics: a new paradigm for natural products-based drug discovery a distinct three-helix centipede toxin ssd inhibits i ks channels by interacting with the kcne auxiliary subunit novel venom-derived inhibitors of the human eag channel, a putative antiepileptic drug target potency optimization of huwentoxin-iv on hna v . : a neurotoxin ttx-s sodium-channel antagonist from the venom of the chinese bird-eating spider selenocosmia huwena engineering potent and selective analogues of gptx- , a tarantula venom peptide antagonist of the na v . sodium channel structural basis of na v . inhibition by a gating-modifier spider toxin comprehensive engineering of the tarantula venom peptide huwentoxin-iv to inhibit the human voltage-gated sodium channel hna v . mechanisms of channel block in calcium-permeable ampa receptors structural basis of ⍺-scorpion toxin action on na v channels structures of human na v . channel in complex with auxiliary subunits and animal toxins molecular basis for pore blockade of human na + channel na v . by the μ-conotoxin kiiia a comprehensive map of molecular drug targets the snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation insights into complement convertase formation based on the structure of the factor b-cobra venom factor complex structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin i-converting enzyme crystal structure of the complex between venom toxin and serum inhibitor from viperidae snake x-ray structure of acid-sensing ion channel -snake toxin complex reveals open state of a na + -selective channel structures of aplysia achbp complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations molecular basis for pore blockade of human na(+) channel nav . by the μ-conotoxin kiiia structural plasticity and dynamic selectivity of acid-sensing ion channelspider toxin complexes trpv structures in nanodiscs reveal mechanisms of ligand and lipid action crystal structure of the ligand-bound glucagonlike peptide- receptor extracellular domain structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent k + channel king: conceptualization, supervision king: writing, review & editing, funding acquisition key: cord- -q lgliph authors: zevenhoven-dobbe, jessika c.; wassenaar, alfred l. m.; van der meer, yvonne; snijder, eric j. title: production of monospecific rabbit antisera recognizing nidovirus proteins date: - - journal: sars- and other coronaviruses doi: . / - - - - _ sha: doc_id: cord_uid: q lgliph the importance of monospecific antisera for the experimental analysis of viral proteins is undisputed. they make it possible to identify and analyze the target protein against a background of a large number of other proteins, either in whole fixed cells or in cell lysates. this chapter describes our experience with the production of such rabbit antisera directed against proteins of coronaviruses and other nidoviruses. the use as antigens of either synthetic peptides (coupled to a carrier protein) or proteins expressed in escherichia coli is described, and detailed protocols for immunization and preparation of test bleeds are provided. for screening of the immune response following immunization, detailed protocols for three commonly used techniques are described, all of which are based on the use of infected cells or cells expressing the protein of interest, side by side with appropriate controls. the in situ immunodetection of the target in fixed cells by immunofluorescence microscopy is described, as are protocols for techniques that can be applied to cell lysates containing the target protein (western blotting and immunoprecipitation). the latter techniques are performed in combination with polyacrylamide gel electrophoresis, thus allowing confirmation of the molecular weight of the target that is recognized by the antiserum. coronaviruses, and other nidoviruses such as arteriviruses, produce one of the largest sets of viral polypeptide species among the rna viruses. this feature is related to the polycistronic nature of their genome, which contains up to open reading frames and, in particular, to the polyprotein strategy used to produce the large viral replicase/transcriptase, the complex set of nonstructural proteins that is responsible for replication and transcription of the nidovirus genome [see ( ) and the references therein]. in coronaviruses (fig. ) , the larger of the two replicase polyproteins (pp ab) can be more than amino acids long and is processed into or cleavage products, now commonly referred to as nonstructural protein (nsp) to ( , ) . in arteriviruses, despite their much smaller replicase size (pp ab is "only" ∼ to amino acids), the complexity is not very different and replicase processing yields or cleavage products. the regulated autoproteolysis of nidovirus replicase pp a and pp ab is driven by two to four proteinases that reside in the orf a-encoded part of the replicase. cleavage sites for coronavirus and arterivirus proteinases have been identified for several prototypic nidoviruses through a combination of theoretical and experimental methods and can now be confidently predicted for other members of these virus groups. since an additional to proteins are expressed from subgenomic mrnas produced from the -end of the viral genome ( fig. ) , including the structural polypeptides responsible for virion formation, the proteome of the average nidovirus consists of between and protein species. it should be noted that intermediates of replicase polyprotein processing, which have been described for various nidoviruses, are not taken into consideration in this count and are likely to even further increase the repertoire of viral polypeptides produced in infected cells. replicase precursors and processing intermediates in fact complicate certain types of analyses, such as those that rely on microscopy, since antibodies will usually not discriminate among precursors, intermediates, and processing end products. in the infected cell, many nidovirus proteins are targeted to specific locations (fig. ) , some of them even to multiple specific locations. over a period of about years, our laboratory has been involved in the production and characterization of polyclonal rabbit antisera directed against specific structural and, in particular, nonstructural proteins of nidoviruses ( - ). our experience in this area is summarized in this chapter. the usefulness and importance of monospecific antibodies for the experimental analysis of almost any protein in biology is undisputed. in particular, when depicted is the cytoplasmic replication cycle, which starts with virus entry and release of the genome into the cytoplasm. subsequently, the genome is translated to produce replicase polyproteins pp a and pp ab, which are cleaved to yield nonstructural proteins ( ). a complex for viral rna synthesis is assembled on cytoplasmic double-membrane vesicles (dmvs). this complex is involved in genome replication and the synthesis of a nested set of subgenomic mrnas, which are required to express the genes in the -proximal third of the genome. translation of the smallest subgenomic mrna yields the viral nucleocapsid protein (n) that assembles into new nucleocapsid (nc) structures together with newly generated genome rna. other subgenomic mrnas encode viral envelope proteins that are (largely co-translationally) inserted into membranes of the host cell ' the target protein has to be studied against a background of a large number of other proteins, either in whole fixed cells or in cell lysates, specific antibodies can provide a rapid and reliable method for discriminating signal from noise. three commonly used antibody-based detection techniques, which are also important for screening of the immune response during antiserum production, are discussed below: • in situ immunodetection of the target in fixed cells, either by immunofluorescence (if) microscopy or immunoelectron microscopy (iem). immunofluorescence microscopy images of vero-e cells infected with sars-coronavirus illustrating the expression of four important viral genes and the different subcellular localization of their products. nonstructural protein (nsp ) is one of the replicase subunits expressed from the genomic rna, revealing the localization of the membrane-bound viral replication complex. spike protein s localizes to different compartments of the exocytic pathway. membrane protein m accumulates in the golgi complex, in particular early in infection. nucleocapsid protein n is a largely cytoplasmic protein. • detection of the target by western blotting (wb), i.e., electrophoresis of a cell lysate containing the target and transfer of the (denatured) proteins to a solid carrier such as nitrocellulose or polyvinylidene fluoride (pvdf) membranes. • immunoprecipitation (ip) of the target from a cell lysate, often used in combination with radioactive labeling of protein synthesis in the cells for a specific time prior to cell lysis. although in several techniques experimental data obtained with monoclonal antibodies can be superior, in particular because of reduced background signal, the generation and screening of hybridoma cell lines requires a considerable investment, in terms of both labor and capital. furthermore, in the context of specific research questions, polyclonal antisera may sometimes even be preferred over monoclonal antibodies since they contain a mixture of immunoglobulin molecules, derived from different b-cell lines in the immunized animal, often recognizing multiple epitopes of the target protein. moreover, if desired, the chances of cross-reactivity of the antiserum with related proteins, e.g., from different strains of the same virus or from closely related other virus species, are considerably better for polyclonal antisera, particularly when they have been raised using a larger and/or conserved part of the target protein. obviously, the primary goal during polyclonal antiserum production in laboratory animals is to obtain a reasonable volume of a high-affinity antiserum. for rabbits, if necessary, bleeds of ∼ - ml can be obtained at -week intervals, and the final bleed can yield up to ml of serum from about ml of whole blood. since monoclonal antibodies are routinely produced in mice, the use of other species such as rabbits also creates the possibility of convenient in situ double-labeling experiments (see below). in such experiments, using speciesspecific secondary antibodies, a polyclonal rabbit antiserum against the target of choice and, for instance, a mouse monoclonal antibody recognizing a cellular protein can be used in combination to detect both proteins in the same specimen simultaneously. the (obvious) standard prerequisites for the production of a polyclonal rabbit antiserum are: (i) an antigen, (ii) a (pathogen-free) rabbit to be immunized, (iii) a test method to detect the response, and (iv) a permit for animal experiments. the last relates to institutional and/or governmental regulations controlling animal use, which differ from country to country and are not discussed here in detail. important considerations are the choice of adjuvant, method and site of administration of the antigen, sedation of animals, maximum volume of test bleeds, and various safety precautions regarding animals and personnel. two types of antigens have been used in our studies: synthetic peptides and proteins expressed in and purified from escherichia coli. the production of antigens in e. coli are not covered in any detail in this chapter and the reader is referred to the extensive literature on this topic published elsewhere (and in chapter in this volume). e. coli allows for the cheap production of large amounts of antigen, but clearly such antigens have to be purified prior to use for immunization. this can be facilitated, e.g., by the use of a variety of tags (such as fusion to glutathione-s-transferase, maltose-binding protein, or the commonly used hexahistidine tag) for which convenient affinity resins are available. reasonably pure antigens (> % pure) are required and in the case of larger tags one should consider removing the tag proteolytically to ensure that the immune response will be directed against the target rather than against its tag. if the e. coli expression product turns out to be insoluble, which will usually interfere with its straightforward affinity purification, it may still be possible to purify a reasonably pure protein sample from inclusion bodies by repeated extraction of this material with increasing concentrations of urea (or guanidium isothiocyanate). as long as small amounts of this material can be used (i.e., the protein concentration is sufficiently high), after washing of the protein pellets with pbs, such urea-containing samples can be used for mixing with the adjuvant and immunization without the need for dialysis. synthetic peptides offer the advantage of being pure, and with certainty they contain exclusively the amino acid sequence of the selected target. since they normally cover only to residues of this target, their sequence has to be selected with care (see below). it is not straightforward to confidently predict antigenic peptides from an amino acid sequence. several programs to support this activity can be found on the internet and algorithms for this purpose are included in most dna/protein analysis software ( - ). if the structure of the target is known, peptides located on its surface are to be preferred. entirely polar and helical peptides should be avoided. in our experience, peptides located at (or close to) the n-or cterminus of the target also have a high probability of being immunogenic. this was true in particular for peptides derived from the nidovirus replicase polyproteins, which were usually selected on the basis of known or predicted cleavage sites (although based on relatively small numbers; success rate with terminal peptides was around % and with internal peptides only around %). the peptides to be synthesized usually are - amino acids long. peptides that are very hydrophobic may be more difficult to handle (e.g., poor solubility prior to coupling). to protect peptides against host proteases and thus increase their stability, the c-terminal carboxyl group can be replaced with an amide group during synthesis. (this may not be advisable when using peptides that normally form the c-terminus of a protein.) to facilitate coupling to bsa used as the carrier protein (see below), one or two lysine residues can be added to one side of the peptide (to the n-terminus when targeting the c-terminus of a protein, and vice versa). synthetic peptides are available from a variety of commercial or in-house sources. in our institute, peptides are synthesized by solid-phase strategies on an automated multiple peptide synthesizer (syroii, multisyntech, witten, germany). the purity of the peptides is determined by analytical reversed-phase hplc and should be at least %. the identity and homogeneity of the peptides is confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and analytical reversed-phase chromatography. freeze-dried peptides, dissolved peptides, or coupled peptides in solution are best stored at - • c. synthetic peptides have to be coupled to a carrier protein to enhance their immunogenicity. bovine serum albumin (bsa) and keyhole limpet hemocyanin (klh) are the two most commonly used carrier proteins. we have always relied on bsa (a very soluble and stable plasma protein) and a coupling reaction using glutaraldehyde, which links the amino groups of carrier and synthetic peptide (in lysine residues and at the amino terminus of the peptide). for example, crosslinking at lysine residues can be represented as: note that the above calculations are based on a number of assumptions and should be considered as a rule of thumb. clearly, since the sequence is known, the mr of the peptide can be calculated more precisely. however, since we usually add extra lysines to the peptide there will be different ways in which it can be coupled to the carrier and a variety of complexes will be formed (bsa "trees" with a varying number of peptide "branches" scattered all over the backbone), presenting the epitope in many different ways. . to the peptide-bsa mixture, add pbs to give a final volume of l; then (carefully, slowly, and while mixing) add l of a freshly prepared . % glutaraldehyde solution (equaling approximately mol). . incubate the coupling reaction on a roller device at room temperature for h to allow cross-linking of peptide and carrier protein. . add l of -m glycine in order to quench the remaining free glutaraldehyde and continue rolling for h. . dialyze the coupling reaction overnight against pbs to remove small molecules such as uncoupled peptide, glycine, and glycine-glutaraldehyde. dialyze two or three times against at least volumes of pbs at • c. the final step can be overnight. usually, after dialysis, the concentration of peptide-bsa complex is around mg/ml and the solution is ready for direct use as an antigen in immunization. when using synthetic peptides or proteins purified from e. coli, adjuvants containing immunostimulatory molecules are applied to enhance the immune response to the antigen. freund's adjuvants have been used in our studies; freund's complete adjuvant (fca) for the initial immunization and freund's incomplete adjuvant (fica) for subsequent booster immunizations. fca (but not fica) contains heat-inactivated mycobacterium tuberculosis or mycobacterium butyricum (or extracts thereof), which stimulate both cellular and humoral immunity. the water-in-oil emulsion that is the basis for fca guarantees the slow release of the antigen from the site of immunization, but it should be noted that the mineral oil component is quite toxic and induces granulomatous reactions. mix fca/fica with the antigen-containing solution to form a "toothpaste-like" stable emulsion. see below for details. in our standard protocol, ∼ g of antigen (peptide-bsa complex) is used for the primary immunization, and the same amount for subsequent booster reactions. . before use, mix the fca, e.g., gently on a roller device to get the bacteria into suspension. for the primary immunization, the antigen ( - g) is diluted with pbs to give a final volume of . ml pbs, which is mixed with . ml of fca. transfer this mixture to a -ml syringe (luer-lock). use a p micropipette; put the tip in the opening of the syringe, and slowly pull the plunger back. in the same manner, fill a second syringe with . ml of fca and . ml of air (to promote the formation of the emulsion). . connect the two syringes with a three-way stopcock (fig. ) . mix the contents of the two syringes until a thick, white emulsion is obtained. this means that the suspension is converted into an oily mass that includes the water. this emulsion, when properly prepared, is stable and will not disperse when a droplet is put into pbs. the emulsion will ensure the slow release of the antigen into the animal. . disconnect the syringes and use the antigen suspension the same day. optionally, the syringes can be stored for later use, overnight at • c or for longer periods at - • c. . for booster immunizations fica is used instead of fca. material for two boosts can be prepared in one action by doubling the volumes above. filled syringes can be stored at - • c for prolonged periods of time. young adult new zealand white rabbits (preferably females, weighing between and kg) have been used routinely in our studies (fig. ) . the young age of the animals is thought to be particularly important to ensure a strong primary response to the antigen. although it is often advised to use two animals per antigen, in our experience-with just a few exceptions-both animals generally give the same (positive or negative) result, although titers of the antiserum and background signal(s) may vary between such duplicates. still, if the number of animals available or housing capacity is limited, and also for financial and ethical reasons, it may be wiser to use a single animal per antigen and-in the event of failure-return to such an antigen in a subsequent round of immunizations. the rabbits are injected subcutaneously at three or four places on their back (fig. ) . given the strong inflammatory response induced by fca, the animals are monitored closely following the primary immunization. the interval between the primary immunization and the first booster, and between subsequent boosters, is about weeks. it is essential to obtain a preimmunization bleed on (or before) the day of the primary immunization, which will be used to assess the response against the antigen (see below). the standard schedule for immunization and bleeding would look like this: bleeds should be limited to % of the animal's total blood volume and a -week recovery period should be allowed. as a "rule of thumb," the blood volume of rabbits is about ml/kg of body weight-assuming the animal is mature, healthy, and adequately nursed. in our experience, a (good) response is unlikely (but not impossible. . .) if there is no positive signal after two or three boosts. although there have been some exceptions to this rule, it is advisable-for financial reasons as well as ethical considerations-to terminate the experiment after ∼ days and, if necessary, try again with an additional animal or an alternative antigen later on. . collect - ml of blood (usually from an ear vein of the rabbit; fig. ) in a -ml centrifuge tube and allow complete clotting of the blood in a water bath at • c for h. . use a pasteur pipette with a melted tip to gently liberate the clot from the wall of the tube. be sure to cause minimal lysis of red blood cells, to ensure a clear serum sample later on. subsequently, leave the clot to shrink overnight at • c (fig. ) . . centrifuge the tube at × g for min to further reduce the volume of the clot. . carefully, with a pipette, remove as much serum as possible from the tube without touching/damaging the clot; in case of doubt, use a second collection tube for the last few ml (also an additional spin might help), which may be less clear and might be saved as a backup sample only. . prepare some smaller and some larger aliquots of the serum and store at - • c or - • c. sera can be kept for many years, but repeated freezing and thawing should be avoided. also, it is advisable to use tubes with a screw cap lid containing a rubber seal, which will minimize evaporation during prolonged storage. store a small sample ( . to ml) at • c for testing. . working stocks of antisera (in screw cap tubes) can be kept at • c for many months; if desirable, . % (final concentration) of sodium azide (highly toxic!) can be added as a preservative, but in our experience this is not necessary if sera are kept cool and handled with care. it is advisable to spin antisera (in particular the less clear ones) for min at full speed in a microcentrifuge prior to use. the native proteins that are the targets for antiserum production are usually highly structured molecules and obviously the reactivity of the antiserum will be influenced by the conformation of the antigen used for immunization. in particular, when synthetic peptides are used the structural resemblance between antigen and target may be limited. this probably explains why-in our experiencepeptides derived from the termini of the target protein, which are often less structured than the protein core, generally work better than internal peptides. although an elisa approach, based on the antigen used for immunization, can be employed for an initial screening of the immune response in the animal, this result may be relatively meaningless when it comes to the question of whether the antiserum will ultimately react with the viral protein target. one will in fact be measuring the response against the combination of peptide and carrier protein or, in the case of immunization with proteins expressed in and purified from e. coli, against the target and contaminants present in the antigen. all the immunizations we carried out with bsa-coupled peptides produced sera that reacted positively in an elisa using plates coated with the uncoupled peptide, but less than half of those turned out to be useful in if, wb, or ip. therefore, it is advisable to screen immediately using samples containing the native, full-length viral protein target. moreover, during this screening process, the conformation of the target is an issue. when screening formaldehyde-fixed whole cells by microscopy techniques the target is fixed but structured. during wb analysis proteins are subjected to denaturing conditions and fixed to the membrane used for blotting, whereas ips are done in solution and can be performed under both native and denaturing conditions. in fact, the level of denaturation in ip experiments can be easily influenced by varying the percentage of sds in the ip buffer. often, ips with antisera raised using synthetic peptides work better in the presence of relatively high ( . - . %) concentrations of sds, probably because the epitope in the denatured target resembles the antigen used for immunization more closely under these conditions. an additional significant advantage is the fact that the higher sds concentrations will strongly reduce the background ip signal and result in a much cleaner analysis (see below). in our experience, antisera that work well in if assays usually also work well in wb and ip. conversely, sera that are negative in if may still be highly reactive in wb and/or denaturing ip. consequently, it is important to test new antisera in at least two of these three assays. in our daily practice, we have usually relied on if for preliminary screening and wb for confirmation. there is an additional reason to confirm the if results by subsequent wb or ip analysis: in if assays antisera can sometimes show a strong reaction with cellular proteins (fig. ) , either-at low dilutions-because of the lack of a specific response against the target or-at higher dilutions-because of an unexpected cross-reactivity cov nsp ( ) , which was very specific in vero-e cells (see fig. ), resulting in a strong, punctate nuclear labeling in bhk- cells that expressed the ace- receptor for sars-cov. (d) sars-cov-infected cells labeled several weeks after fixation and embedded using prolong mounting fluid. an aspecific nucleolar background labeling was observed, especially in the green range of the fluorescence spectrum. the specific, much brighter signal is derived from an anti-nsp labeling using a rabbit antiserum that was directly coupled to alexa fluor- ( ). with cellular proteins. wb or ip analysis provides important information about the molecular mass of the target that is recognized, and may thus prevent premature conclusions about the specificity of the antiserum. the correct assessment of specificity is also aided by the inclusion of two important controls: the preimmunization serum (which should obviously not show the same signal) and mock-infected control cells (which might reveal that it is in fact a cellular target that is being recognized). when performing initial screening with if assays, it is practical to use cell cultures that have been infected at an moi of . - and therefore contain a mixture of virus-infected and mock-infected cells in the same specimen. semiconfluent cells seeded on glass cover slips ( -mm-diameter) are the preferred specimens for initial testing. the cells can either be infected with the target virus or transfected with a vector expressing the target protein. obviously, cells should be fixed at a time point when the target protein is convincingly expressed. the most reactive rabbit antisera that we have produced can be used in if assays at dilutions of between : and : . for initial testing, however, dilutions on the order of : to : are advised. . wash the cells once with pbs and fix the cells at room temperature with % paraformaldehyde in pbs for at least min (or overnight). . wash the cells once with pbs containing mm glycine (coverslips in sealed dishes can be stored at • c in pbs for many weeks). . using sharp tweezers, carefully transfer coverslips to the wells of a prelabeled well cluster containing pbs-glycine. be sure to remember, every time you handle the coverslips, on which side there are cells. while in the cluster, cells should always be facing upward. . permeabilize the cells at room temperature for min in pbs containing . % triton x- . . in min, wash the cells three times with pbs-glycine and leave them in the last wash step. . dilute the primary antiserum (initial dilutions, e.g., : and : ) in pbs containing % fcs; l per coverslip ( -mm-diameter) will be needed. . cut a large piece of parafilm, place it in a larger dish, label the position of the various samples, and place -l drops of the antiserum dilutions on the parafilm. one by one, take the coverslips from the -well cluster, remove excess pbs by touching a tissue to the side of the coverslips, and place the coverslips on the drops with the cells facing the antiserum. . incubate for - min at room temperature (or • c); make sure the samples do not dry out during this incubation (e.g., by placing a wet tissue in the dish). . return the coverslips to the -well cluster; in min wash three times with pbsglycine and leave them in the last wash step. . prepare a dilution of the fluorescently labeled secondary antibody. optimal dilutions of conjugates should be determined separately using a well-defined primary antiserum. again l per coverslip will be needed. optionally, g/ml of hoechst can be added to this dilution for staining of nuclear dna. . incubate and wash as described under steps to . . take the coverslips from the -well cluster. embed the specimens onto glass microscopy slides in a mounting fluid [e.g., mowiol or prolong (molecular probes)]. avoid air bubbles in the mounting fluid by slowly and carefully sliding the coverslip on a small drop (∼ l) of mounting fluid. . store the specimens in the dark at • c. the mounting fluid should harden at least overnight before high magnification lenses and immersion oil are used. . analyze the specimens in a fluorescence microscope using the filter sets required for the label attached to the secondary antibody. double-labeling experiments can be done to compare the localization of two (or more) proteins of interest in the same cell by combining a rabbit antiserum recognizing one protein and, e.g., a mouse monoclonal antibody recognizing a second target. obviously, the two primary antisera have to be detected with suitable conjugates carrying different fluorescent labels, preferably with wellseparated emission spectra. following initial optimization (testing of different dilutions, balancing of the two signals, and controls for specificity of primary and secondary antibodies), one can usually carry out the labeling using a twostep approach. first, specimens are simultaneously incubated in a combined dilution of the two primary antibodies and, subsequently, after extensive washing, in a combined dilution of the two conjugates. in case of background and/or specificity problems, however, it may be necessary to perform the labeling in four consecutive steps. in if assays, double labeling with two antisera from the same species is only possible when one of the two is directly coupled to a fluorescent group. we have recently been successful (fig. ) ( ) in purifying the ig fraction from small volumes ( - ml) of rabbit antisera using a commercially available protein a column and have subsequently conjugated these antibodies to alexa fluor- . the if assay then consisted of three incubation steps: (i) incubation with the uncoupled rabbit antiserum, (ii) incubation with the anti-rabbit conjugate recognizing the first antibody, and (iii) incubation with the alexa fluor- -coupled second rabbit antiserum. the order of these steps is very important to avoid binding of the anti-rabbit conjugate to the directly labeled second rabbit antiserum. during western blotting (wb) analysis samples containing the protein of interest are separated according to size in acrylamide gels and transferred to a membrane, which is subsequently incubated with the antiserum. protein bands reacting with the antiserum are detected using a secondary antibody and accompanying assay (a variety of enzyme-linked or fluorescent conjugates is available; here we use a peroxidase-coupled secondary antibody). as in the case of if assays, lysates to be tested can be derived from cells that are either infected or transfected with an appropriate expression vector. for infection lysates, we use a high multiplicity of infection to avoid a mixture of infected and uninfected cells in the sample. cells are lysed in protein lysis buffer, which leave the nuclei intact and allow their removal by centrifugation. for initial testing, an amount of lysate equaling ∼ × cells per cm gel width (or - slots) can be used. alternatively, purified protein (e.g., expressed in e. coli) can be used ( - ng is fig. . example of a standard test of a rabbit antiserum in western blot and immunoprecipitation. the antiserum used here was raised against a domain in the c-terminal region of pp a of the arterivirus eav ( ) and recognizes a large set of processing intermediates and end products, which are indicated by arrowheads: (a) western blot analysis: eav-and mock-infected cell lysates were prepared at h postinfection, run on an sds-polyacrylamide gel, blotted to pvdf membrane, and incubated with the postimmune serum at a : dilution. detection was with an anti-rabbit igg hrpo conjugate and a chemiluminescence assay. [reproduced from ( ).] (b) immunoprecipitation: eav-infected cells were labeled with s-methionine/cysteine for h, from - h postinfection and lysates were used for ip using l of antiserum per sample. following the ip, samples were run on an sds-polyacrylamide gel and signal was detected using autoradiography. as specificity controls, the preimmune serum was tested on eav-infected cells and the postimmune serum was tested on mock-infected cells. furthermore, for the left panel the binding of the antiserum to the antigen was done in the presence of . % sds, whereas . % sds was used for the right panel. the comparison illustrates how higher sds percentages can reduce the background signal and improve the overall picture. usually sufficient). most antisera can be used at a -fold dilution or higher, but we advise starting the testing with a : dilution (fig. a) . protein-antibody complexes are then purified by having them bind to beads carrying the ig-binding proteins a or g on their surface, which are subsequently spun down and washed repeatedly to remove unbound proteins. the simplest form of protein a carrying beads are formalin-fixed staphylococcus aureus cells (e.g., pansorbin; calbiochem), but alternatively protein a or g coupled to sepharose can be used. usually, metabolically labeled protein lysates (e.g., s-methionine-labeled) are used for immunoprecipitation studies and samples are run on sds-page gels that can then be used for autoradiography. in contrast to wb analysis, immunoprecipitation relies on recognition of the target in solution. depending on the conditions used, proteins can either be close to their native confirmation, partially denatured, or completely denatured (e.g., in the presence of high concentrations of sds). thus, the conformation of the antigen used for immunization may influence the results. in our experience, linear antigens such as synthetic peptides may yield antisera that work considerably better in immunoprecipitation assays carried out under stringent denaturing conditions (e.g., . - % sds), which will often also reduce the background in the assay (fig. b) . nevertheless, the optimal concentration of sds should be determined for every antigen-antiserum pair since the optimum may be as low as . % sds, e.g., when using antibodies recognizing mainly (or exclusively) conformational epitopes. furthermore, the amount of antiserum in the immunoprecipitation assay is important. for initial screening of rabbit antisera we usually test and l of the antiserum in a l total ip volume. coverslips with infected cells or cells expressing the protein of interest. . fixative: % paraformaldehyde in pbs pbs: see section . , step pbs with % fetal calf serum (fcs) pbs with mm glycine protein lysis buffer ( mm tris-hcl amersham biosciences, hybond p, #rpn f) x western blot transfer buffer (wtb): mm tris, . m glycine pbs: see section . , step pbs-tm with % bsa) anti-rabbit igg horseradish peroxidase conjugate, e.g., swine-anti-rabbit igg hrpo amersham biosciences, ecl plus western blotting detection kit ip buffer ( mm tris-hcl weak wash buffer a ( mm tris-hcl ph . ; mm nacl laemmli sample buffer (lsb): mm tris-hcl, ph . ; mm dtt; % glycerol formalin-fixed staphylococcus aureus cells; calbiochem # ) or protein a/g sepharose beads wash the cells with cold pbs and-to a -cm dish with - × cells-add l of cold protein lysis buffer transfer the lysate to a labeled microfuge tube and spin down the nuclei for min at full speed in a microcentrifuge transfer the supernatant to the new tube, leave the pellet (nuclei, often barely visible) behind and add / the lysate can now be used for wb or stored in the - • c/- • c freezer prepare an sds-page gel (or minigel) of a suitable acrylamide percentage (depending on the size of the protein of interest) and run: (i) the lysate containing the protein of interest, (ii) a control lysate (mock-infected or untransfected cells), and (iii) a molecular weight marker. several sets of these samples can be run on one gel to try different antiserum dilutions, or wider slots can be used and strips cut with the right samples after blotting with a pencil mark one side of the membrane; this is the side of the membrane that will face the gel during blotting and, subsequently, the solutions during incubation. prewet the pvdf membrane in methanol for min; never touch the pvdf membrane with bare fingers; always use gloves! dilute x wtb to give x wtb and incubate the membrane in x wtb for min take the gel from between the glass plates and briefly wash it in x wtb build the electroblot stack-from cathode to anode-with the following layers: three sheets of whatman paper presoaked in wtb (and optionally one sheet of whatman paper soaked in wtb with . % sds), the equilibrated gel, the equilibrated and marked membrane : ) and the preimmune serum as a control ( : ) in pbs-tmb and incubate the blots for h at room temperature while swirling dilute the peroxidase-conjugated secondary antibody (swine-anti-rabbit igg hrpo) in pbs-tmb and incubate the blots for h at room temperature while swirling prepare the solutions for the chemiluminescence assay according to the manufacturer's instructions gently "semidry" the blot with some whatman paper (let the fluid run off; do not really dry it!) put the chemiluminescence solution on the plastic foil and incubate the blot with the "protein side down" for min remove the excess of fluid with some whatman paper and wrap the blot in plastic foil expose an x-ray film to the blot for - min, develop the film, and check if a longer exposure is required. to avoid overexposure of the film, it may be wise to wait about min before making the first exposure take l of s-labeled cell lysate in protein lysis buffer ( ) (equaling about - × cells; obviously the expression level of the target protein is to be considered as well); add l of ip buffer and the antiserum ( or l). incubate these ∼ -l samples overnight wash the pansorbin cells ( l for each sample) by mixing with an equal volume of ip buffer, spin down for sec at full speed in a microcentrifuge, and resuspend the pansorbin cell pellet in the original volume (again add l of washed pansorbin cells to each immunoprecipitation and incubate at • c for more than h while swirling spin down for min at full speed in a microcentrifuge, remove the supernatant, and add l of weak wash buffer a; vortex until the pellet is completely resuspended. (optionally, this step can be repeated once to get a cleaner result spin down for min at full speed in a microcentrifuge again and repeat the same washing procedure with weak wash buffer b resuspend the pellet in l of lsb by pipetting up and down before loading the samples on an sds-page gel, denature at • c for min. (for some proteins, e.g., very hydrophobic ones, it may be better not to heat the samples and just leave them in lsb for min or longer spin the pansorbin cells down for min at full speed in a microcentrifuge; do not resuspend the pellet! transfer the supernatant to a new tube and load part of this sample onto an sds-page gel following sds-page, process the gel for autoradiography or phosphorimager analysis according to standard protocols and expose for - days not all bleeds of the same rabbit have the same concentration of antibody and thus the dilutions to be used can vary mounting solutions can sometimes have strange side effects, especially when the cells are not freshly fixed. for example, the use of prolong gold mounting fluid can generate a green nonspecific nucleolar signal when microscopy slides used for embedding are not clean, wipe them first with a tissue with % ethanol, next with water, and then with a dry paper towel/tissue. when ethanol is not removed, a (sometimes bright) orange background may result occasionally, antisera judged to be negative in if using paraformaldehyde-fixed cells can become positive using methanol-fixed cells (or the other way round) however, methanol fixation is less gentle and generally the morphology of the cell is less well preserved. fixation is performed for min at - • c (using ice-cold % methanol or % methanol/ % acetic acid) and cells are subsequently transferred to and kept in pbs-glycine. methanol immediately dissolves all cellular membranes topley and wilson's microbiology and microbial infections: virology volume virus-encoded proteinases and proteolytic processing in the nidovirales unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex nuclear localization of nonstructural protein and nucleocapsid protein of equine arteritis virus the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis processing of the equine arteritis virus replicase orf b protein: identification of cleavage products containing the putative viral polymerase and helicase domains proteolytic processing of the replicase orf a protein of equine arteritis virus prediction of protein antigenic determinants from amino-acid-sequences the antigenic index-a novel algorithm for predicting antigenic determinants semiempirical method for prediction of antigenic determinants on protein antigens structural proteins of equine arteritis virus proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp the authors would like to thank jan wouter drijfhout and willemien benckhuijsen (lumc department of immunohaematology) for advice and assistance on peptide design and synthesis. we are grateful to the staff of the lumc animal facility for almost years of pleasant collaboration and reliable housing and handling of the rabbits used for antiserum production. this work was supported (in part) by the european commission int the context of the activities of the euro-asian sars-dtv network (sp -ct- - ). key: cord- -e g la authors: lehrer, robert i.; bevins, charles l.; ganz, tomas title: defensins and other antimicrobial peptides and proteins date: - - journal: mucosal immunology doi: . /b - - / - sha: doc_id: cord_uid: e g la nan endogenous antimicrobial peptides are widely distributed among vertebrates. most are amphiphilic, polycationic molecules with an α-helical, cystine-stabilized β-sheet, or proline-rich structure. they represent elements of a robust, ancestral animal immune system that predates the advent of lymphocytes and immunoglobulins. secreted and cell-associated antimicrobial peptides enable their hosts to resist incursions by potential pathogens. from a pathogen's perspective, these peptides present a series of barriers to evade or overcome. in humans (and other mammals), defensins and cathelicidins are the principal antimicrobial peptides of neutrophils and epithelial cells. many mucosal surfaces are bathed by antimicrobial proteins, including lysozyme, lactoferrin, secretory leukoprotease inhibitor (slpi), and secretory phospholipase a . all defensins have a largely β-sheet structure and contain three intramolecular cystine disulfide bonds. the smallest, θ-defensins, are circular peptides that contain residues, α-defensins have between and residues, and β-defensins have up to (fig. . ) . to date, more than α-defensin, β-defensin, and θ-defensin molecules have been reported, and humans express six different α-defensins and at least four βdefensins. additional human defensin/defensinlike genes exist in five different chromosomal loci, but their expression and properties remain to be characterized. α-defensins occur in the neutrophils of humans, rats, rabbits, guinea pigs, and hamsters, but not in those of mice, horses, pigs, and sheep. although the small intestinal paneth cells of mice express at least selsted et al. ) and possibly as many as α-defensins (ouellette and selsted ) , only two very dissimilar defensin species are found in human paneth cells (mallow et al. ) . two atypical α-defensins exist in rabbit kidney, but their functions remain uncertain (bateman et al. ; wu et al. ) . thus far, with the exception of rabbit alveolar macrophages, mononuclear phagocytes are not known to produce appreciable amounts of α-defensins. although many human epithelial cells constitutively express human β-defensins (hbd- ), this peptide is particularly abundant in the distal renal tubules and in other parts of the genitourinary tract. hbd- , originally recovered from psoriatic skin (harder et al. ) , is induced in various epithelia by inflammatory signals. in the epidermis, it is present in very low amounts unless induced by interleukin- (il- ) .the hbd- gene was found by genomic searching (jia et al. ) and the corresponding peptide was isolated from psoriatic skin (harder et al. ) . hbd- is highly expressed in the testis (epididymis) and less abundantly in the gastric antrum (garcia et al. b) . since about different β-defensin genes have been identified in the mouse (scheetz et al. ) , sorting out their functions and relationships to human biology is unlikely to be a simple task. through its interaction with this chemokine receptor (yang et al. ) . the θ-defensins, initially discovered in rhesus macaque neutrophils, have a remarkable structure, so far unique among mammalian peptides. these -amino acid (aa) peptides with three intramolecular disulfide bonds have a cyclic amide backbone (tang et al. ; leonova et al. ) , formed from two nonapeptides by a novel posttranslational ligation reaction. the nonapeptides are encoded by two θ-defensin genes very similar to conventional α-defensins except that they are shortened by a "premature" stop codon, and contain only three cysteines. either homodimers or heterodimers can be joined to form a mature θ-defensin peptide. many different defensin genes have been sequenced. the myeloid α-defensin (defa) genes have similar layouts, with three exons that correspond approximately to the ′-untranslated region, signal sequence/propiece, and mature defensin region (linzmeier et al. ) .the genes for hd- and , αdefensins expressed predominantly within the human intestine, have only two exons as do the defb genes that encode β-defensins, suggesting that the evolution of α-defensins involved both deletional and insertional events. although dissimilar in their genomic sequence, the genes for hd- and hd- show considerable sequence similarity in their proximal ′-flanking regions, which presumably contain elements involved in their tissue-specific expression (mallow et al. ; salzman et al. ) . the defa genes encoding human myeloid and enteric α-defensins are clustered on human chromosome (linzmeier et al. ) . the human defb genes that encode hbd- , hdb- , and hdb- contain two exons and are all within a few hundred kilobases from the myeloid α-defensin locus on chromosome p (liu et al. ; peng et al. ) . this region is also home to a deft pseudogene that is highly homologous to the genes encoding the α-defensins of rhesus monkeys. θdefensin (deft) genes evidently arose in an old world monkey when the mutation of a preexisting α-defensin gene placed a premature stop codon within exon of a defa gene. additional defensin and defensinlike genes have been identified in four other chromosomal loci, but their tissue expression and biology remain to be characterized . a review of the antimicrobial activity of defensins against gram-positive and gram-negative bacteria is available (lehrer et al. ) . briefly, defensins are effective broadspectrum microbicides, especially when present in high local concentrations or acting in low-ionic strength media. high local concentrations occur within phagolysosomes, in the capillary-like lumen of small intestinal crypts, and at interfaces between effector and target cells. rabbit defensins np- and np- bind with high affinity to pseudomonas aeruginosa pao , forming small surface blebs and permeabilizing its outer membrane. this process, sometimes called "self-promoted uptake" (sawyer et al. ) , allows the polycationic peptide molecules to displace divalent cations that link adjacent lipopolysaccharide (lps) molecules. the consequent permeability changes allow otherwise excluded molecules, such as lysozyme, to enter the periplasmic space and attack the bacterium further. viljanen and coworkers (viljanen et al. ) noted that human defensins increased the outer membrane's permeability to hydrophobic probes (e.g., rifampin) in p. aeruginosa, escherichia coli, and salmonella typhimurium. bactericidal concentrations of hnp- permeabilized the outer and inner membranes of e. coli, causing immediate and simultaneous cessation of macromolecular synthesis and respiration (lehrer et al. a) . permeabilization of microbial cell membranes is a hallmark of defensin-mediated antimicrobial activity. especially with human defensins, such permeabilization is prevented by treatments that inhibit the target cell's metabolism, growth, or transmembrane protonmotive force (lehrer et al. a) . defensins permeabilize artificial phospholipid membranes only when a transmembrane electronmotive force of sufficient magnitude and correct polarity is applied (kagan et al. ). membrane conductance increases as the second to fourth power of the defensin concentration, suggesting that two to four defensin molecules interact to form a channel in this system. wimley and associates (wimley et al. ) examined interactions between human defensins and lipid bilayers. they found that hnp- (net charge, + ) bound to and permeabilized unilamellar vesicles composed of anionic phospholipids, but not those formed of electroneutral phospholipids. data from experiments with entrapped markers of chapter defensins and other antimicrobial peptides and proteins fig. . . a dimer of human α-defensin hnp- . the figure is based on coordinates obtained from x-ray crystallography (hill et al. ) . different sizes suggested that multimeric hnp- formed aqueous pores with a maximum diameter of ~ Å. if such pores formed in bacterial membranes, they should allow passage of defensin monomers and even dimers (~ × × Å) to more interior sites. certain bacteria are intrinsically resistant to defensins, including neisseria gonorrhoeae (qu et al. a) , burkholderia cepacia, burkholderia pseudomallei brucella spp. (martinez de tejada et al. ) . what can make bacteria resistant has been partially explained by identifying mutations that increase bacterial susceptibility to antimicrobial peptides. studies on the intracellular enteric pathogen s. typhimurium revealed the importance of the two-component regulatory system, phop/phoq. phoq is a magnesium-binding sensor kinase that responds to ionic and other environmental changes by phosphorylating phop, a dna-binding protein that can activate numerous genes, including some needed for resistance to antimicrobial peptides and intracellular survival (miller et al. ; chamnongpol et al. ) . these include important aspects of lps structure (ernst et al. ) , and expression of an outer membrane protease that cleaves, and presumably inactivates, certain antimicrobial peptides (guina et al. ) . studies with staphylococcus aureus associated its relative resistance to defensins and other cationic antimicrobial peptides to the modification of its membrane phospholipids with l-lysine (kristian et al. ) and of its lipoteichoic acids with d-alanine (collins et al. ) . both modifications reduce electrostatic binding of the peptides to the staphylococcal surface. n. gonorrhoeae may owe its defensin-resistance to an energy-dependent "mtr" efflux pump (shafer et al. ) . these findings suggest that interactions with cationic antimicrobial peptides may have profoundly influenced bacterial evolution. in summary, the effect of defensins on microbes can be envisioned to occur in stages. electrostatic adsorption to anionic sites on or near the target cell's membrane or (for certain defensins) to sugars in microbial glycoproteins or lps result in locally high concentrations that promote defensin aggregation and multimer formation. insertion of defensins into microbial membranes is assisted by their positive charge and by the target cell's transmembrane potential. the ensuing membrane disruption and channel formation permit intracytoplasmic entry of defensins and allow essential microbial components to leak out. unless repaired, these events lead to irreversible target cell injury. mammalian defensins can kill various fungal pathogens, including candida spp., cryptococcus neoformans, and hyphae and germinating spores of rhizopus oryzae and aspergillus fumigatus (reviewed in lehrer et al. ) . in recent years, the antifungal properties of defensins have been studied more by botanists and agriculturists than by immunologists. this reflects the importance of fungi as plant pathogens (thomma et al. ) and the likely utility of transgenic plants. plant defensins, like those of mammalian origin, probably owe their antifungal properties to an ability to induce membrane permeabilization (thevissen et al. ). both human and rabbit defensins show activity against mycobacteria, including m. tuberculosis (miyakawa et al. ; sharma et al. ) and m. avium-intracellulare (ogata et al. ) , even in the presence of divalent cations or physiologic salt concentrations. although the plasma membrane of m. tuberculosis is the principal target for hnp- action, its dna may be a secondary target (sharma and khuller ) . human neutrophils can ingest and kill both m. avium (hartmann et al. ) and m. tuberculosis, and the latter is apparently subdued by nonoxidative mechanisms (kisich et al. ) . in vivo, mycobacteria that evaded or survived the attentions of neutrophils would likely find a defensin-free sanctuary once resident within a macrophage, because the only mammalian macrophages known to contain significant quantities of defensins are alveolar macrophages of the rabbit. in a rabbit model of syphilis, large local amounts of defensins were seen during the first hours. this subsided, but reappeared on days to , when healing began (borenstein et al. ) .the nichols strain of treponema pallidum was neutralized by rabbit defensins in vitro and in vivo. borrelia burgdorferi, the causative agent of lyme disease, is also susceptible to defensins (lusitani et al. ) . the effect of defensins on protozoans has received little study, at least in vertebrates. human α-defensin hnp- , mouse paneth cell defensin- and defensin- (cryptdins), and rabbit α-defensin np- killed trophozoites of giardia lamblia, especially when the concentrations of sodium chloride and divalent cations were low (aley et al. ). both α-defensins (zhang et al. ) and θ-defensins (cole et al. ) can protect cells from infection by hiv- in vitro. persons infected with human immunodeficiency virus- (hiv- ) whose α,β cd t cells maintain the ability to produce and release α-defensins may exhibit relative resistance to developing acquired immune deficiency syndromedefining (aids-defining) symptoms, compared with hivinfected subjects whose t cells are defective in this regard (zhang et al. ) . θ-defensins act by blocking an early step in the uptake process, and their ability to do so correlates with their ability to bind gp with high affinity (cole et al. ) . α-defensins also bind gp with high affinity, but their mechanisms of action against hiv- has not yet been defined. α-defensins also protect cells from infection by herpes simplex virus (hsv) types and in tissue culture media (daher et al. ), by interfering with an early step in the infection process (sinha et al. ) . human and rabbit α-defensins also neutralized vesicular stomatitis and influenza a/wsn virus, but lacked significant activity against cytomegalovirus, reovirus, and echovirus (daher et al. ). among the nonenveloped viruses, only adenoviruses have so far been reported to be susceptible to defensins (gropp et al. ; bastian and schafer ) . purified human defensins kill various normal and tumor cell targets in a concentration and time-dependent fashion (reviewed in lehrer et al. ) and show synergistic activity when combined with sublytic concentrations of hydrogen peroxide. defensin molecules bound to mammalian target cells with biphasic kinetics, similar to those with candida albicans. k cells exposed to μg/ml of radiolabeled hnp- bound approximately . × defensin molecules per cell. this initial binding did not cause cytolysis and was comparable in defensin-resistant and defensin-sensitive target cells. after to minutes, sensitive targets became permeable to trypan blue (molecular weight = da) and manifested enhanced transmembrane ion flux. membranebound defensin molecules became progressively more difficult to dislodge by adding serum, and after to hours, the cells began releasing cr-labeled cytoplasmic components. strand breaks and adenosine triphosphate-ribosylation (adp-ribosylation) were first detected in k and raji targets - hours after incubation with hnp, and these increased to maximal levels by hours. dna was not degraded into nucleosome-sized fragments. other experiments suggested that the initial defensin-induced pores were voltage dependent and that defensin-mediated injury to mammalian cells depended on an energized target cell membrane. since defensins are bound by α -macroglobulin and other normal serum components, it is not surprising that defensins were minimally, if at all, cytotoxic in the presence of serum. whether and where defensins are cytotoxic in vivo is not known. although most studies of defensins have examined their antimicrobial properties, considerable evidence suggests that some defensins also play other roles, including immunomodulation, hormonal regulation, opsonization, and stimulating wound repair. human neutrophil defensins are chemotactic for human monocytes (territo et al. ) , t-cells (chertov et al. ) , and immature dendritic cells (yang et al. ) in vitro, so that their release from neutrophils could provide a signal to mobilize immunocompetent mononuclear cells. defensins also induce the synthesis of interleukin- (il- ), a c-x-c cytokine, by human airway epithelial cells, possibly providing a mechanism to recruit additional neutrophils to sites of inflammation (van wetering et al. ) .the α-defensins of human neutrophils were shown to act as adjuvants to enhance systemic igg antibody response (lillard et al. ) . murine β-defensin mbd- acted as an adjuvant for tumor immunization, by providing a costimulatory signal via the toll-like receptor (biragyn et al. ) . it is not yet clear whether these activities are shared by other defensins. rabbit np- a ("corticostatin") and certain other α-defensins bound reversibly to the adrenocorticotropic hormone (acth) receptor of rat adrenal cells in vitro and inhibited acth-stimulated steroidogenesis (zhu and solomon ) . rabbit defensins np- and np- greatly enhanced the ability of rabbit alveolar macrophages to ingest bacteria and fungi under serum-free conditions (fleischmann et al. ) . defensins were mitogenic for fibroblasts (murphy et al. ) , and small daily injections of rabbit defensins accelerated wound healing in rats (kudriashov et al. ). guinea pig defensins were reported to release histamine from rat mast cells in vitro (yamashita and saito ) . most normal mucosal surfaces contain considerably more epithelial cells than neutrophils or macrophages. notable exceptions include the gingival crevices that surround teeth, which are bathed in high concentrations of neutrophils and their components. neutrophil defensin concentrations as high as mg/ml have been detected in such fluids (t. ganz and k. miyasaki, unpublished) and could play an important role in controlling local microbial proliferation. in other locations, neutrophils and macrophages are rapidly recruited to epithelial surfaces when the primary mucosal defenses are overwhelmed by microbial invaders. the important function of these secondary defenses is illustrated by the particularly severe impact of neutropenia on the integrity of oral and gastrointestinal mucosae and the characteristic involvement of periodontal tissues in congenital or acquired neutrophil disorders. each human neutrophil contains several thousand cytoplasmic granules that are divided into three principal subtypes, called primary (or azurophil), secondary (or specific), and tertiary. these subtypes can be distinguished by buoyant density, when they are made during the neutrophil's maturation, their response to secretagogues, and their composition. about % to % of the protein content of the azurophil granules consists of defensins. relatively small amounts of defensins are released extracellularly after phagocytosis or secretagogue exposure, as the peptides are preferentially delivered phagosomes that contain opsonized bacteria (joiner et al. ). this targeted delivery of defensins not only places them where they are most needed, but it also helps minimize any potential cytotoxicity to host tissues. the high concentrations of defensins in human neutrophils are not unique: α-defensins are similarly plentiful in the granules of rabbit neutrophils (~ - μg/million cells) and rabbit alveolar macrophages (~ μg/million cells). although myeloid defensins (hnps) - are most abundant in neutrophils, hnp- is also highly expressed by natural killer (nk) cells (obata-onai et al. ; agerberth et al. ) and can be produced by other lymphocytes (agerberth et al. ) . although human blood neutrophils contain about μg of defensins per million cells, defensin mrna is not detectable in northern blots of mature neutrophils, indicating that defensin synthesis is restricted to the neutrophil's bone marrow precursors. myeloid α-defensins are synthesized as - -aa prepropeptides with a generally well-conserved signal sequence, followed by an anionic propiece and the cterminally placed defensin domain. nevertheless, nearly all of the defensin in neutrophil azurophil granules is found to be completely processed to - -aa mature peptides . posttranslational defensin processing has been studied in metabolically labeled hl- and cml cells (valore and ganz ) . their earliest defensin intermediate contained aa and arose by cotranslational removal of the -residue signal sequence. considerable quantities of this -aa form were secreted into the medium, and the remainder was proteolytically processed over hours via a -aa intermediate into the mature -aa and -aa defensins. mature human neutrophils also contained minor amounts ( . % of total defensins) of incompletely processed -aa, -aa, and -aa defensins (harwig and ganz ) . this pattern of synthesis and posttranslational processing was substantially reproduced when the hnp- defensin cdna was transduced into defensinless murine d and d cl cells (liu and ganz ) . these transgenic lines accumulated mature defensin in acidified vacuoles corresponding to lysosomes or immature granules. in contrast, two transgenic, defensin-producing nonmyeloid cell lines (an embryonic nih t cell line and the pituitary adenoma cell line att- ) failed to process the -aa prodefensin, indicating that the requisite processing pathway may be tissue specific. to study the role of the propiece in preprohnp- , a series of in-frame deletions between the signal peptidase site and the aminoterminus of the mature defensin region (aa - ) was constructed, packaged, and transduced into the d cl granulocytic cell line. deletions in the aminoterminal two-fifths of the propiece had only minor effects on defensin biosynthesis and did not interfere with accumulation of mature defensin in the granules of d cl cells. deletions in the carboxyterminal three-fifths of the propiece diminished net defensin synthesis, blocked constitutive secretion of prodefensin, and interfered with defensin accumulation in cytoplasmic granules. these effects were reproduced by the smaller deletion Δ - , which contains a highly conserved secondary structure. therefore, propiece residues - appear to be essential for subcellular trafficking and sorting of human neutrophil α-defensins (liu and ganz ) . instead of containing α-defensins like those found in human, rabbit, rat, hamster, and guinea pig neutrophils, bovine neutrophils contain an impressive array of β-defensin peptides . whereas myeloid α-defensins have free amino termini, about half of the β-defensins isolated from bovine neutrophils had a pyroglutamate residue at their amino terminus. this moiety characteristically results from enzymatic modification of an amino-terminal glutamine residue and could confer resistance to proteolytic cleavage by local proteases or alter other biologic properties. the β-defensin peptides from neutrophils manifest antibacterial activity against gram-positive and gram-negative bacteria, but because different in vitro assay conditions were used, direct comparison of the relative activity of bovine epithelial and neutrophil β-defensins is not yet possible. β-defensins ("gallinacins") were also isolated from the leukocytes of chickens (harwig et al. ) and turkeys (evans et al. ) . two of the three chicken gallinacins differed at only three amino-acid residues and had comparable antibacterial and antifungal activities. the third gallinacin differed at over half of the amino acids and exerted antibacterial activity, but lacked the antifungal activity seen with the other two (harwig et al. ) . the β-defensins of turkeys showed good activity against c. albicans, salmonella enteriditis, and campylobacter jejuni, but were relatively ineffective against pasteurella multocida at μg/ml, the highest peptide concentration tested. they did not neutralize infectious bronchitis virus, an enveloped coronavirus (evans et al. ) . in normal chickens, the expression of gallinacin- was especially prominent in the tongue, bursa of fabricius, and trachea. it also occurred in other organs, including skin, esophagus, air sacs, large intestine, and kidney. the tracheal expression of gallinacin- increased significantly in chickens experimentally infected with haemophilus paragallinarum ). despite continual entry of microorganisms from swallowed food and oral secretions, the normal small intestine contains only a sparse resident flora. why? although multiple factors contribute, the ability of intestinal enterocytes and paneth cells to secrete antimicrobial molecules is likely to play a significant role. through their differential antimicrobial activities, these molecules could contribute to selecting the normal intestinal flora, which may afford protection from pathogenic bacteria that enter the intestine. since the intestinal epithelium stem cells reside in the intestinal crypts, secretion of paneth cell defensins into the crypt lumen (figure . ) could provide a protective barrier for this vital proliferative compartment. paneth cells, first described over years ago, are located at the base of the crypts of lieberkühn. they are more abundant in the region of the ileum and jejunum and less so in the duodenum. the high density of paneth cells in the distal small intestine might signify a role in preventing adverse consequences from reflux of colonic flora into the ileum. paneth cells contain an extensive endoplasmic reticulum and golgi network and are filled with numerous, apically located eosinophilic secretory granules whose secretion is stimulated by cholinergic agonists or the entry of bacteria or lipopolysaccharide into the intestinal lumen (satoh ; qu et al. b ). ouellette and colleagues (ouellette et al. ) identified an α-defensin among several prominently expressed rna messages in the postnatal mouse small intestine. because this mrna was expressed in paneth cells at the base of small intestinal crypts, the corresponding peptides were named "cryptdins." cryptdin mrna levels were normal in germfree and nude mice, suggesting that expression was independent of t-cell signals or acquisition of the intestinal flora. independently, two α-defensin genes (hd- and hd- ) expressed in human paneth cells were detected bevins , ) . in contrast to the six characterized murine enteric defensin genes, which had very high ( %) nucleotide similarity, hd- and hd- were not as closely related, presumably having duplicated and diverged before their murine counterparts . the striking difference in murine and human enteric defensin gene numbers remains enigmatic. paneth cells also secrete larger antimicrobial polypeptides, including lysozyme and secretory phospholipase a (see sections on "identification of enteric α-defensins in paneth cells" and "developmental regulation of enteric α-defensin expression" in this chapter). antimicrobial activity of enteric α-defensins native mature defensins (cryptdins) have antibacterial and activity comparable with myeloid defensins (selsted et al. ; eisenhauer et al. ) and can kill the protozoan parasite giardia lamblia (aley et al. ) . recombinant human small intestinal defensin hd- was effective against listeria monocytogenes, s. typhimurium, and c. albicans (porter et al. b; ghosh et al. ) . it showed minimal cytotoxic activity against human intestinal cell lines (porter et al. a) . morphologically identifiable paneth cells appear in the mouse intestine immediately before birth. thereafter, murine α-defensin mrna levels and defensin immunoreactivity increase gradually and reach adult levels by the fourth postnatal week, paralleling expansion of the paneth cell population. mice reared in a germfree environment express comparable levels of enteric α-defensins (ouellette et al. ; putsep et al. ) . in humans, a sensitive rt-pcr assay can detect mrna encoding human enteric defensins hd- and hd- at . weeks' gestation, close to the time that morphologically distinguishable paneth cells can be identified by electron microscopy. at approximately weeks' gestation, hd- and hd- mrna was detected in paneth cells of the small intestinal crypt by in situ hybridization. by northern blot analysis, the levels were approximately -fold lower than in adults. the ability to detect human enteric defensin mrna prenatally indicates that its expression is at least partially constitutive and governed by a developmental program that operates without direct stimulation by microbes or their products. like their hematopoietic counterparts, intestinal αdefensins are initially synthesized as prepropeptides. however, unlike the myeloid defensins, paneth cell defensins are stored as propeptides, exclusively so in humans (porter et al. ; ouellette et al. ; ghosh et al. ) . the posttranslational processing of these epithelial α-defensins appears to be an important regulatory step in the generation of bioactive peptides in the small intestine. however, the pathways of processing appear to be quite different in mice and humans. in mice, the matrix metalloprotease matrilysin (matrix metalloproteinase ) has been identified as an essential enzyme in the processing of small intestinal α-defensins . in humans, the orthologous small intestine α-defensins are processed to mature peptides during or after secretion into the small intestinal lumen by the serine protease trypsin (ghosh et al. ) . in both species, the proteases mediating this α-defensin processing are expressed by paneth cells. the hypothesis that intestinal defensins contribute to the regulation of microbial flora in the small intestine received support from studies in matrilysin-deficient mice. matrilysin (matrix metalloproteinase ) is expressed specifically in paneth cells. in mice with a targeted disruption of the intestinal prodefensin-processing protease, matrilysin, paneth cell defensin precursors were not processed to active mature peptides. the mice had increased susceptibility to intestinal infections with gram-negative organisms . detailed analysis of secretions from the isolated crypts of normal and matrilysin-deficient mice indicated that the antimicrobial activity of crypt secretions was largely the result of defensins and that it was greatly impaired in matrilysin-deficient mice (ayabe et al. ) . in another model, the transgenic expression of human defensin- in the paneth cells of mice increased their resistance to orally administered s. typhimurium but, as might be expected, did not protect against infection by the peritoneal route (saltzman et al., ) . in the aggregate, these models point to a central role for paneth cell defensins as regulators of small intestinal flora and as enteroprotective molecules. bovine tracheal extracts contain an abundant peptide called "tap," or tracheal antimicrobial peptide, that exhibited antimicrobial activity in vitro against klebsiella pneumoniae, s. aureus, and c. albicans (diamond et al. ) . sequence analysis revealed similarity of this molecule to defensins expressed in leukocytes and to its designation as the first epithelial β-defensin. tap expression was found in the pseudostratified columnar epithelial cells of conducting airway and nasal mucosa (diamond et al. ) . a hallmark of tap expression, like several more recently described epithelial β-defensins of humans and mice, is inducible expression in response to bacterial lps and some inflammatory cytokines russell et al. ; diamond et al. ) . a canonical nf-kb recognition site in the ′-flanking region of the tap gene was important in the transcriptional regulation of this gene by bacterial products (diamond et al. ) . in vivo inoculation of pathogenic bacteria into the bovine lung caused a rapid and dramatic increase in β-defensin expression in the conducting airway epithelium, which was not seen in the adjacent control lobe . in bovine tracheal epithelial cells, β-defensin induction by lps appears to be dependent on epithelium-derived cd , a well-characterized lps-binding protein . this suggests that local expression of cd- might provide epithelial cells with the capacity to recognize bacterial products at mucosal surfaces and initiate local defense responses such as antimicrobial peptide production. several additional sites of β-defensin expression were identified in cattle, including squamous epithelial cells of the tongue (schonwetter et al. ) and epithelial cells of the small intestine and colon (tarver et al. ). mrna encoding a bovine lingual β-defensin ("lap") was markedly increased in epithelia surrounding naturally occurring tongue lesions, suggesting an integral role for antimicrobial peptides in local inflammatory responses. the antimicrobial activity of lap and tap was very similar, and lap expression was enhanced in bovine trachea after lps or tnf-α application . in mice, epithelial expression of β-defensins is also observed in the mucosal epithelium of the respiratory and digestive tracts (morrison et al. ; bals et al. ; jia et al. ) . similar to humans, mice express a noninducible βdefensin in kidney tubule cells and mucosal epithelia of several organ systems (huttner et al. ) . recent studies of mice rendered deficient in this epithelial β-defensin through targeted homologous recombination have shown a minimal phenotype (moser et al. ; morrison et al. ) . further studies of these mice may be necessary to elucidate the functional role of this β-defensin. a systematic characterization of the peptides present in human blood ultrafiltrates provided the first evidence for the existence of hbd- , the first characterized human β-defensin (bensch et al. ) . hbd- shared the nine invariantly conserved amino acids present in β-defensins from bovine and avian cells. although free hbd- was present in nanomolar concentrations in human plasma, an abundant hbd- message occurred in the kidney and vagina (bensch et al. ) . several hbd- peptides, generated by differential posttranslational proteolysis at the n-terminus, were the predominant cationic peptides in urine. when tested, using urine as a growth medium, they displayed antibacterial activity against e. coli. in situ hybridization and immunostaining localized their site of production to the distal tubules of the kidney as well as the various epithelia of the female genital tract (valore et al. ) . hbd- is also present in human conducting-airway epithelia (mccray and bentley ) as well as in epithelia of many other tissues and in certain glands (zhao et al. ) . expression of hbd- by lung epithelia is developmentally regulated, in a manner reminiscent of the α-defensins found in rabbit lung macrophages (mccray and bentley ) . circumstantial evidence implicates the impaired function of hbd- , consequent to high bronchial fluid salinity (smith et al. ) , as a contributing factor in respiratory tract colonization by p. aeruginosa in cystic fibrosis patients (goldman et al. ) . hbd- was first detected as an abundant cationic peptide and an antimicrobial component in the flaking epidermis of patients with psoriasis (harder et al. ) . it is produced by differentiated keratinocytes in the epidermis under the influence of inflammatory signals, principally il- (liu et al. (liu et al. , and secreted in lamellar bodies into the space between keratinocytes, along with the lipids that make the epidermis impermeable to water . there, hbd- reaches concentrations sufficient for antimicrobial activity (liu et al. ) . hbd- is active against a broad range of microbes, with the significant exception of the skin pathogen s. aureus (harder et al. ; liu et al. ) . hbd- was detected more or less simultaneously by genomic methodologies (jia et al. ; garcia et al. a) and by direct extraction of the peptide from psoriatic skin (harder et al. ) . hbd- is one of the most cationic defensins known, with a charge of + at neutral ph. unlike hbd- , hbd- is active against s. aureus and even b. cepacia, at least under low ionic strength conditions.the skin and the tonsils appear to have the highest levels of expression, but hbd- is detectable in other epithelial and nonepithelial tissues as well. the mechanisms of its regulation have not yet been reported. genomic analysis indicates that many additional human β-defensin genes and related genes exist, but they remain to be characterized at the peptide level . the α-defensin and β-defensin families almost certainly arose by divergence from an ancestral premammalian defensin and not by convergent evolution. this conclusion is supported by the proximity of human α-defensin and β-defensin genes within kb on chromosome p (liu et al. ) . defensinlike molecules not assignable to either defensin family have been identified in invertebrates. hemocytes of the horseshoe crab, tachypleus tridentatus, contain an antibacterial peptide ("big defensin") whose cterminal residues have a cysteine structure resembling that of mammalian β-defensins and a primary sequence similar to rat myeloid α-defensins (saito et al. ) . when the unusually long, -residue amino-terminal extension of big-defensin was cleaved by tryptic digestion, both the defensinlike domain and the amino-terminal extension had antimicrobial properties. an extremely defensinlike antiviral peptide was also identified in the sea anemone, anemonia sulcata (driscoll et al. ) . the primary structural resemblance between avian βdefensins (gallinacins) and certain cytostatic (marquardt et al. ) and myotoxic peptides in snake venoms (fig. . ) also suggests that the progenitors of β-defensins had potential to become weapons of offense, as well as of host defense. the same can be said about the antimicrobial insect defensins, which show striking structural similarity to certain toxic peptides found in scorpion venom (cociancich et al. ) . given such findings, it is reasonable to postulate the antiquity of defensins as host-defense molecules. an evolutionary relationship between vertebrate defensins and the structurally more distant defensins of insects and plants is possible, but lacks convincing supporting data. calprotectin, a member of the widespread calcium-binding s- protein family, is present in remarkably high concentration in the cytoplasm of human neutrophils (brandtzaeg et al. ) . the calprotectin molecule is composed of light (mrp ) and heavy (mrp ) subunits. although not secreted from intact neutrophils, calprotectin release from dead and dying neutrophils creates high concentrations of the protein in inflammatory or abscess fluids and in the intestinal tract lumen of patients with inflammatory bowel disease. calprotectin is also found in reactive tissue macrophages, in nonkeratinizing squamous epithelia, and in reactive epidermal cells. the c-terminal portion of calprotectin's heavy chain, mrp , is identical to a peptide called neutrophil immobilizing factor, "nif." calprotectin concentrations of - μg/ml inhibit growth by s. aureus, s. epidermidis, and e. coli. even lower concentrations ( - μg/ml) inhibit growth by c. albicans, possibly by depriving the candida cells of zinc. calprotectin purified from rat inflammatory peritoneal cells was markedly cytotoxic for mitogenstimulated lymphocytes and for various tumor cell lines, in fig. . . from serpent's tooth to chicken soup. myotoxin a is a myotoxic peptide from the venom of crotalus viridis viridis, the prairie rattlesnake (fox et al. ) . svgap stands for snake venom growth arresting peptide (marquardt ) , and is described in u.s. patent . gal- is the chicken β-defensin gallinacin- , originally isolated from chicken heterophils (harwig et al. ) and later found in chicken soup (lehrer unpublished) . the disulfide pairing patterns ( : , : , : ) of myotoxin a (fox et al. ) and avian β-defensins (harwig, unpublished) are identical. which it induced apoptosis. epithelial cell calprotectin might protect host cells from microbes that invade the cytoplasm directly or by lysing phagosomal membranes. the murine mrp protein (also called cp or s a ) is chemotactic for myeloid cells but this property is not shared by human calprotectin. homozygous disruption of mrp (s a ) gene in mice causes embryonic death by day . but mrp (s a )-deficient mice are viable and healthy (manitz et al. ) even though their neutrophils lack both mrp / partners. initial studies of these mice detected only very mild defects in neutrophil cytoskeletal organization and migration, but the activity of these neutrophils in microbicidal assays has not been reported. lysozyme was discovered over years ago by fleming, who called it a "remarkable bacteriolytic ferment" and did not patent it-or penicillin, which he discovered a few years later. this very cationic . -kda protein enzymatically attacks the β - glycosidic bonds between n-acetylglucosamine and n-acetylmuramic acid, which stabilize bacterial peptidoglycans. gram-positive bacteria susceptible to the lytic action of lysozyme include bacillus subtilis, bacillus megaterium, and micrococcus luteus (nee lysodeicticus). lysozyme also exerts bactericidal activity by nonenzymatic, nonbacteriolytic mechanisms. it is a remarkably abundant component of phagocytic leukocytes, and also of tears, saliva, and many other secretions. the detection of abundant lysozyme in paneth cell granules yielded the first clue that these intestinal epithelial cells were important in host defense (peeters and vantrappen ) . most gram-negative bacteria are resistant to lysozyme, except under very low ionic strength conditions. this is caused largely by the protective effects of their outer membrane, which covers and masks their peptidoglycan layer. lysozyme may function most effectively in conjunction with other antimicrobial factors, especially those capable of permeabilizing the outer membrane. recent experiments in mice deficient in lysozyme-m showed delayed killing of the lysozymesensitive m. luteus, but the major abnormality in these mice was the highly exaggerated inflammatory response to these bacteria and to peptidoglycan. peptidoglycan is a potent inflammatory stimulus, in part through its binding to tlr- and by peptidoglycan recognition proteins. whatever lysozyme may contribute to antibacterial activity, it appears to be critically important for eliminating bacterial peptidoglycan, thus extinguishing the strong signal to innate immunity and inflammation generated by this characteristic bacterial macromolecule. lactoferrin is a -kda single-chain glycoprotein that can bind one or two molecules of ferric iron. it is found in various secretions, including tears, milk, and semen, and also in the secretory granules of neutrophils. intestinal cells and mononuclear phagocytes have surface receptors for lactoferrin, but do not synthesize it. certain bacteria (e.g., n. gonorrhoeae, neisseria meningitidis, and moraxella catarrhalis) also possess lactoferrin receptors and use them to acquire iron. lactoferrin or lactoferricin-treated gram-negative bacteria release lps, develop electron-dense "membrane blisters," and become sensitized to lysozyme-all consistent with outer membrane damage. lactoferrin binds bacterial lipopolysaccharide with high affinity and also binds various other proteins. exposure of lactoferrin to pepsin (its normal fate when ingested in milk) releases an antimicrobial polypeptide, "lactoferricin" comprising the amino-terminal lactoferrin residues (tomita et al. ) . lactoferricin kills many bacteria and c. albicans, but is ineffective against proteus and serratia species and p. cepacia. lactoferrin also inhibits microbial adherence to and invasion of epithelial cells. lactoferrin has a higher affinity for iron than its plasma homolog transferrin, and its iron binding is less affected by acid ph. these characteristics may allow it to function as an iron-sequestering substance in mucosal secretions and inflammatory fluids. because iron is an essential metal for all living organisms, sequestration of iron is an effective means of inhibiting microbial growth. iron starvation also interferes with bacterial biofilm formation. even under conditions that otherwise promote biofilm formation, lactoferrin-exposed bacteria remain in the planktonic form, presumably allowing the more mobile bacterial population to disperse and reach iron sources (singh et al. ) . phospholipase a enzymes remove fatty acids from the middle (sn- ) carbon atom of phosphoglycerides. several phospholipase a enzymes occur in mammalian cells, but only the -kda secretory pla (spla ) has potent microbicidal properties, principally against gram-positive bacteria (harwig et al. ; weinrauch et al. ) . spla differs from pancreatic pla structurally and in preferring phospholipids prominent in bacterial membranes [e.g., phosphatidylglycerol (pg) and phosphatidylethanolamine (pe)] over phosphatidylcholine as substrates. pla is present in granules of human neutrophils and small intestinal paneth cells. spla is released from neutrophils exposed to secretagogues, such as phorbol esters or calcium ionophores, and by paneth cells exposed to cholinergic agonists, bacteria, or lps. serum spla levels are greatly elevated in patients with sepsis and rise sharply within hours after the experimental administration of lipopolysaccharide to uninfected patients. high concentrations of spla are also present in human tears. the antimicrobial specialization of this phospholipase is probably a result of its unusually high net positive charge and a cluster of positively charged residues near its n-terminus that facilitate its interaction with bacteria and access to its phospholipid target (weiss et al. ) . slpi is a -aa ( . -kda) nonglycosylated peptide with a bipartite structure. it is very abundant in many human secretions, including seminal plasma, cervical mucus, nasal secretions, and tears. slpi has received many alternative names, including human seminal plasma inhibitor-i (husi-i), cervix uteri secretion inhibitor (cusi), bronchial secretory inhibitor (bsi), and bronchial mucus inhibitor (mpi). mice deficient in slpi show impaired wound healing (ashcroft et al. ; zhu et al. ) , but their response to infection has not been reported yet. in vitro, slpi shows low-level activity against bacteria and fungi (hiemstra ) and may also have anti-hiv activity. if the high concentrations of slpi in many epithelial secretions compensate for its low intrinsic potency, its antimicrobial activity could be biologically important. slpi is synthesized as a -aa prepropeptide with a aa signal peptide. each of its two structurally similar domains contains four intradomain disulfide bonds in the "four-disulfide-core" pattern also present in wheat germ agglutinin and neurophysin.the c-terminal domain of slpi (residues - ) shows homology to the second domain of chelonianin, a basic protease inhibitor from red sea turtle, and is responsible for the molecule's prominent antiprotease activity (masuda et al. ) . slpi holoprotein and its nterminal domain (residues - ) have microbicidal properties (hiemstra et al. ) . other protease inhibitors with antimicrobial properties include aprotinin, a -aa protease inhibitor in bovine mast cells (pellegrini et al. ) , and enap- , a four-disulfide core protein found in equine granulocytes (couto et al. ) . multifunctionality may be a common attribute of other proteins involved in host defense. eosinophils, as well as other myeloid and epithelial cells, contain cationic antimicrobial proteins belonging to the ribonuclease family (rosenberg and domachowske ) . in humans, eosinophil cationic protein (ecp) and eosinophil-derived neurotoxin (edn) are among the principal components of eosinophil granules. ecp is broadly antimicrobial (lehrer, b) and toxic to helminths (hamann et al. (hamann et al. , ), but edn is nearly inactive in these assays. both ecp and edn show antiviral activity (rosenberg and domachowske ) against respiratory rna viruses: respiratory syncytial virus (rsv) and a related murine virus. new members of the antimicrobial ribonuclease family likely to be important in defending body surfaces include rnase , an abundant component of human epidermis (harder and schroder ) and angiogenin , an antimicrobial protein of murine paneth cell granules (hooper et al. ) . the specific role of these proteins in host defense remains to be defined. bactericidal permeability-increasing protein (bpi) (elsbach and weiss ) is found in the azurophil (primary) granules of human and rabbit neutrophils, but like defensins, may be absent from mouse neutrophils. it is a -kda cationic protein and a member of a family of lipid-binding proteins, two of which, bpi and lipopolysaccharide-binding protein (lbp) avidly bind to bacterial lipopolysaccharide. in vitro, bpi is specifically active against selected gram-negative bacteria at concentrations as low as nanomolar, and this activity is wholly contained in a -kda amino terminal fragment. the mechanism of activity of bpi against bacteria depends on the initial high-affinity interaction with lps in the outer membrane. the resulting rapid permeabilization of the outer membrane is followed by a slower process that culminates in the disruption of the inner membrane of gram-negative bacteria with attendant loss of viability (wiese et al. ). more recently, bpi was also identified as a component of human eosinophil granules and as a lipoxin-inducible protein in mucosal epithelia (canny et al. ) . in vitro studies suggest that bpi contributes to the killing of gram-negative bacteria in isolated epithelia and neutrophils. the neutrophils of humans, pigs, cattle, sheep, mice, and other mammals contain a variety of structurally diverse antimicrobial peptides collectively called "cathelicidins." these peptides were grouped together because they are synthesized at the carboxy-terminal portion of a precursor containing a highly conserved domain, approximately amino acids long, called "cathelin" (zanetti et al. ; ganz and weiss ) . some of these peptides have amphipathic α-helical structures, others form compact, defensin-like β-sheets stabilized by intramolecular cystine disulfide bonds, and still others are remarkably rich in proline, arginine, or tryptophan residues.the genes for cathelicidins contain four exons, the first three of which encode most of the conserved cathelin domain. exon specifies the last few cathelin residues, including the cleavage site and the mature peptide.the ′ flanking sequences of this gene family contain motifs for binding nf-κb, il- , gm-csf, and nf- , suggesting that synthesis responds to mediators generated early during infection. the sole known human cathelicidin is named hcap or fall /ll (agerberth et al. ; cowland et al. ; larrick et al. ) . unlike defensins, which are fully processed before they are stored in the azurophil granules of the neutrophil, the human cathelicidin peptide is stored in the specific (secretory) granules as hcap , a cathelin-containing, -residue, -kd propeptide. during or after secretion, hcap undergoes proteolytic processing by proteinase (sorensen et al. ) to liberate ll- , the -residue, α-helical antimicrobial peptide at its c-terminus (gudmundsson et al. ) . the analogous proteolytic processing of bovine cathelicidins (probac and probac ) and porcine cathelicidins (proprotegrins - ) is mediated by trace amounts of neutrophil elastase (panyutich et al. ; scocchi et al. ) . mrna for hcap is found in testis (malm et al. ) , inflamed keratinocytes (frohm et al. ; agerberth et al. ) , and airway epithelia (bals et al. ) . in vitro, ll- displayed both lps binding and microbicidal activities (agerberth et al. ) against e. coli. the abundance of ll- in neutrophil specific granules is about a third that of lactoferrin or lysozyme, the principal proteins of specific granules . in addition to microbicidal activity, certain cathelicidins also have chemoattractant activity. for example, the human cathelicidin ll- is chemotactic for neutrophils, monocytes, and t cells, but not for dendritic cells. this activity is likely mediated through binding to the formyl-peptide receptorlike receptor (de et al. ) . the proline-rich porcine cathelicidin pr- can induce upregulation of syndecans, heparan sulfate proteoglycans involved in the repair process at the site of skin wounds (gallo et al. ) . although the pig has a large number of cathelicidin genes, the most active antimicrobial peptides in porcine skin wounds are protegrins, secreted from neutrophils as proprotegrins and activated by proteolytic cleavage by neutrophil elastase (panyutich et al. ) . in vitro, inhibition of neutrophil elastase by specific inhibitors blocked the conversion of proprotegrins to protegrins and largely ablated the stable antimicrobial activity secreted by porcine neutrophils (shi and ganz ) . the application of neutrophil elastase inhibitor to porcine wounds decreased the concentration of mature protegrin in wound fluid and impaired the clearance of bacteria from wounds. the deficit could be restored by supplementing the wound fluid with synthetic protegrin in vitro or in vivo (cole et al. ). thus protegrins act as natural antibiotics that contribute to the clearance of microbes from wounds. strong evidence for the antibacterial function of cathelicidins in the skin comes from studies of dermal infections in mice with an ablated gene for cramp (cathelin-related antimicrobial peptide). cramp, normally the main murine cathelicidin (nizet et al. ) , is the murine homolog of human hcap . in cramp-knockout mice, the skin lesions caused by experimental group a streptococcus inoculation were larger lesions and had higher numbers of viable bacteria than those seen in control mice. as in pigs, infiltrating neutrophils appeared to be the chief source of cathelicidins in inflamed skin. epithelial cells also produced cathelicidins, but it is not clear if their production was quantitatively significant. how or if epithelial cathelicidins undergo proteolytic processing remains to be defined. many of our previous and ongoing studies have been supported by grants from the national institutes of health, including: ai , , and to c. b.; ai and to r. i. l.; and hl , ai , and ai to t. g. the continuing support of the will rogers institute is gratefully acknowledged. we thank ken miyasaki for his artistic rendering of the human defensin dimer, all our past and present collaborators for putting up with us, and antimicrobial peptides for being there. fall- , a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis the human antimicrobial and chemotactic peptides ll- and alpha-defensins are expressed by specific lymphocyte and monocyte populations killing of giardia lamblia by cryptdins and cationic neutrophil peptides secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing secretion of microbicidal α-defensins by intestinal paneth cells in response to bacteria the peptide antibiotic ll- /hcap- is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface mouse beta-defensin is an inducible antimicrobial peptide expressed in the epithelia of multiple organs human alpha-defensin (hnp- ) inhibits adenoviral infection in vitro the isolation and characterization of a novel corticostatin/defensin-like peptide from the kidney hbd- : a novel beta-defensin from human plasma human enteric defensin genes: chromosomal map position and a model for possible evolutionary relationships toll-like receptor -dependent activation of dendritic cells by beta-defensin contribution of rabbit leukocyte defensins to the host response in experimental syphilis biosynthesis of granule proteins in normal human bone marrow cells: gelatinase is a marker of terminal neutrophil differentiation the leucocyte protein l (calprotectin): a putative nonspecific defence factor at epithelial surfaces lipid mediator-induced expression of bactericidal/ permeability-increasing protein (bpi) in human mucosal epithelia mg + sensing by the mg + sensor phoq of salmonella enterica identification of defensin- , defensin- , and cap /azurocidin as t-cell chemoattractant proteins released from interleukin- -stimulated neutrophils purification and characterization of a scorpion defensin, a kda antibacterial peptide presenting structural similarities with insect defensins and scorpion toxins inhibition of neutrophil elastase prevents cathelicidin activation and impairs clearance of bacteria from wounds retrocyclin: a primate peptide that protects cells from infection by t-and m-tropic strains of hiv- staphylococcus aureus strains lacking d-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice enap- , a novel cysteine-rich bactericidal peptide from equine leukocytes hcap- , a cathelin/pro-bactenecin-like protein of human neutrophil specific granules direct inactivation of viruses by human granulocyte defensins ll- , the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like (fprl ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells tracheal antimicrobial peptide, a cysteinerich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cdna airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells determination of the three-dimensional solution structure of the antihypertensive and antiviral protein bds-i from the sea anemone anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing cryptdins: antimicrobial defensins of the murine small intestine role of the bactericidal/permeabilityincreasing protein in host defence salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity isolation of antimicrobial peptides from avian heterophils antimicrobial activity of chicken and turkey heterophil peptides chp , chp ,thp , and thp opsonic activity of mcp- and mcp- , cationic peptides from rabbit alveolar macrophages amino acid sequence and disulfide bond assignment of myotoxin a isolated from the venom of prairie rattlesnake (crotalus viridis viridis) the expression of the gene coding for the antibacterial peptide ll- is induced in human keratinocytes during inflammatory disorders syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds antimicrobial peptides of phagocytes and epithelia increased inflammation in lysozyme m-deficient mice in response to micrococcus luteus and its peptidoglycan identification of a novel, multifunctional beta-defensin (human beta-defensin ) with specific antimicrobial activity: its interaction with plasma membranes of xenopus oocytes and the induction of macrophage chemoattraction human beta-defensin : a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity paneth cell trypsin is the processing enzyme for human defensin- human beta-defensin- is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy the human gene fall and processing of the cathelin precursor to the antibacterial peptide ll- in granulocytes a phop-regulated outer membrane protease of salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides comparative toxicity of purified human eosinophil granule proteins for newborn larvae of trichinella spiralis in vitro killing of microfilariae of brugia pahangi and brugia malayi by eosinophil granule proteins a peptide antibiotic from human skin isolation and characterization of human beta-defensin- , a novel human inducible peptide antibiotic rnase , a novel innate immune defense antimicrobial protein of healthy human skin phagocytosis and killing of mycobacterium avium complex by human neutrophils characterization of defensin precursors in mature human neutrophils gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes bactericidal properties of murine intestinal phospholipase a antibacterial activity of antileukoprotease novel roles of protease inhibitors in infection and inflammation crystal structure of defensin hnp- , an amphiphilic dimer: mechanisms of membrane permeabilization structure and functions of endotoxin-binding peptides derived from cap angiogenins: a new class of microbicidal proteins involved in innate immunity the structure of human beta-defensin- shows evidence of higherorder oligomerization the structure of human beta-defensin- . new insights into structural properties of beta-defensins the mouse genome encodes a single homolog of the antimicrobial peptide human beta-defensin a novel murine beta-defensin expressed in tongue, esophagus, and trachea discovery of new human betadefensins using a genomics-based approach the opsonizing ligand on salmonella typhimurium influences incorporation of specific, but not azurophil, granule constituents into neutrophil phagosomes paneth cells of the human small intestine express an antimicrobial peptide gene defensin- mrna in human paneth cells: implications for antimicrobial peptides in host defense of the human bowel intracellular survival of burkholderia pseudomallei antimicrobial defensin peptides form voltage-dependent ionpermeable channels in planar lipid bilayer membranes tumor necrosis factor alpha stimulates killing of mycobacterium tuberculosis by human neutrophils mprf-mediated lysinylation of phospholipids in staphylococcus aureus leads to protection against oxygen-independent neutrophil killing effect of defensin on the process of healing of aseptic skin wound and on the permeability of blood vessels) human cap : a novel antimicrobial lipopolysaccharide-binding protein interaction of human defensins with escherichia coli: mechanism of bactericidal activity antibacterial properties of eosinophil major basic protein and eosinophil cationic protein defensins: antimicrobial and cytotoxic peptides of mammalian cells circular minidefensins and posttranslational generation of molecular diversity mechanisms for induction of acquired host immunity by neutrophil peptide defensins the structure of neutrophil defensin genes a -kb contig of defensin genes on human chromosome p the pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting the human βdefensin- and α-defensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry structure and mapping of the human β-defensin hbd- gene and its expression at sites of inflammation human beta-defensin- production in keratinocytes is regulated by il- , bacteria, and the state of differentiation by il- signaling, monocyte-derived cells dramatically enhance the epidermal antimicrobial response to lipopolysaccharide borrelia burgdorferi are susceptible to killing by a variety of human polymorphonuclear leukocyte components human enteric defensins: gene structure and developmental expression the human cationic antimicrobial protein (hcap- ) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa loss of s a (mrp ) results in reduced interleukin- -induced cd b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro snake venom growth arresting peptide the outer membranes of brucella spp. are resistant to bactericidal cationic peptides pharmacological activity of the c-terminal and n-terminal domains of secretory leukoprotease inhibitor in vitro human airway epithelia express a beta-defensin characterization of defensin resistance phenotypes associated with mutations in the phop virulence regulon of salmonella typhimurium in vitro activity of the antimicrobial peptides human and rabbit defensins and porcine leukocyte protegrin against mycobacterium tuberculosis a novel mouse beta defensin, defb , which is upregulated in the airways by lipopolysaccharide characterization of the mouse beta defensin , defb , mutant mouse model beta-defensin contributes to pulmonary innate immunity in mice defensins are mitogenic for epithelial cells and fibroblasts innate antimicrobial peptide protects the skin from invasive bacterial infection comprehensive gene expression analysis of human nk cells and cd (+) t lymphocytes activity of defensins from human neutrophilic granulocytes against mycobacterium avium-mycobacterium intracellulare in human epidermis, beta defensin- is packaged in lamellar bodies developmental regulation of cryptdin, a corticostatin/defensin precursor mrna in mouse small intestinal crypt epithelium paneth cell defensins: endogenous peptide components of intestinal host defense characterization of luminal paneth cell alphadefensins in mouse small intestine. attenuated antimicrobial activities of peptides with truncated amino termini porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins the paneth cell: a source of intestinal lysozyme identification and isolation of the bactericidal domains in the proteinase inhibitor aprotinin discovery of new human betadefensins using a genomics-based approach nmr solution structure of murine ccl /mip- alpha, a chemokine that specifically chemoattracts immature dendritic cells and lymphocytes through its highly specific interaction with the betachemokine receptor ccr localization of human intestinal defensin in paneth cell granules broadspectrum antimicrobial activity of human intestinal defensin isolation of human intestinal defensins from ileal neobladder urine germ-free and colonized mice generate the same products from enteric prodefensins susceptibility of neisseria gonorrhoeae to protegrins secretion of type ii phospholipase a and cryptdin by rat small intestinal paneth cells eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha a novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin effect of live and heat-killed bacteria on the secretory activity of paneth cells in germ-free mice interaction of macrophage cationic proteins with the outer membrane of pseudomonas aeruginosa genomics-based approaches to gene discovery in innate immunity the solution structures of the human beta-defensins lead to a better understanding of the potent bactericidal activity of hbd against staphylococcus aureus epithelial antibiotics induced at sites of inflammation discovery of five conserved beta-defensin gene clusters using a computational search strategy proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins enteric defensins: antibiotic peptide components of intestinal host defense purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils modulation of neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family dna as the intracellular secondary target for antibacterial action of human neutrophil peptide-i against mycobacterium tuberculosis h ra antibacterial activity of human neutrophil peptide- against mycobacterium tuberculosis h rv: in vitro and ex vivo study the role of protegrins and other elastaseactivated polypeptides in the bactericidal properties of porcine inflammatory fluids a component of innate immunity prevents bacterial biofilm development np- , a rabbit alpha-defensin, prevents the entry and intercellular spread of herpes simplex virus type cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid human cathelicidin, hcap- , is processed to the antimicrobial peptide ll- by extracellular cleavage with proteinase epithelial antibiotic induced in states of disease a cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins enteric beta-defensin: molecular cloning and characterization of a gene with inducible intestinal epithelial cell expression associated with cryptosporidium parvum infection monocyte-chemotactic activity of defensins from human neutrophils permeabilization of fungal membranes by plant defensins inhibits fungal growth antimicrobial peptides of lactoferrin posttranslational processing of defensins in immature human myeloid cells human beta-defensin- : an antimicrobial peptide of urogenital tissues effect of defensins on interleukin- synthesis in airway epithelial cells effect of small cationic leukocyte peptides (defensins) on the permeability barrier of the outer membrane the potent anti-staphylococcus aureus activity of a sterile rabbit inflammatory fluid is due to a -kd phospholipase a conversion of pig pancreas phospholipase a by protein engineering into enzyme active against escherichia coli treated with the bactericidal/permeability-increasing protein mechanisms of action of the bactericidal/permeability-increasing protein bpi on endotoxin and phospholipid monolayers and aggregates regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores rk- : a novel rabbit kidney defensin and its implications for renal host defense purification, primary structure, and biological activity of guinea pig neutrophil cationic peptides beta-defensins: linking innate and adaptive immunity through dendritic and t cell ccr human neutrophil defensins selectively chemoattract naive t and immature dendritic cells cathelicidins: a novel protein family with a common proregion and a variable c-terminal antimicrobial domain contribution of human alpha-defensin , , and to the anti-hiv- activity of cd antiviral factor widespread expression of beta-defensin hbd- in human secretory glands and epithelial cells gallinacin- , an inducible epithelial betadefensin in the chicken isolation and mode of action of rabbit corticostatic (antiadrenocorticotropin) peptides conversion of proepithelin to rpithelins. roles of slpi and elastase in host defense and wound repair key: cord- - gsmkmk authors: doll, tais a. p. f.; dey, raja; burkhard, peter title: design and optimization of peptide nanoparticles date: - - journal: j nanobiotechnology doi: . /s - - -z sha: doc_id: cord_uid: gsmkmk background: various supra-molecular structures form by self-assembly of proteins in a symmetric fashion. examples of such structures are viruses, some bacterial micro-compartments and eukaryotic vaults. peptide/protein-based nanoparticles are emerging in synthetic biology for a variety of biomedical applications, mainly as drug targeting and delivery systems or as vaccines. our self-assembling peptide nanoparticles (sapns) are formed by a single peptide chain that consists of two helical coiled-coil segments connected by a short linker region. one helix is forming a pentameric coiled coil while the other is forming a trimeric coiled coil. results: here, we were studying in vitro and in silico the effect of the chain length and of point mutations near the linker region between the pentamer and the trimer on the self-assembly of the sapns. identical peptide chains co-assemble to form a spherical nanoparticle displaying icosahedral symmetry. we have stepwise reduced the size of the protein chain to a minimal chain length of amino acids. we first used biochemical and biophysical methods on the longer constructs followed by molecular dynamics simulations to study eleven different smaller peptide constructs. we have identified one peptide that shows the most promising mini-nanoparticle model in silico. conclusions: an approach of in silico modeling combined with in vitro testing and verification yielded promising peptide designs: at a minimal chain length of only amino acids they were able to self-assemble into proper nanoparticles. this is important since the production cost increases more than linearly with chain length. also the size of the nanoparticles is significantly smaller than nm, thus reducing the immunogenicity of the particles, which in turn may allow to use the sapns as drug delivery systems without the risk of an anaphylactic shock. electronic supplementary material: the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. proteins of varied structures have evolved in nature to self-assemble into spherical nanoparticles. examples of such supra-molecular structures are viruses [ ] , some bacterial micro-compartments [ ] or eukaryotic vaults [ ] , which form by self-assembly of folded proteins in a symmetric fashion. they often perform sophisticated cellular functions. such nanoparticles have a central cavity that can be exploited as a simple encapsulation system for the transport and controlled release and delivery of drugs and genes to targeted cells [ ] [ ] [ ] . they can also be used in protein separation, enzyme immobilization, and in blood cell substitution [ , ] . virus-like particles have long been used as vaccine platforms for infectious diseases but they could potentially also be used as therapeutic vaccines to treat addiction and other diseases such as cancer [ , ] . in our group we are designing self-assembling peptide nanoparticles (sapns) as vaccines for infectious diseases like malaria [ ] , hiv [ ] or influenza [ ] , but they can be used for many other diseases [ ] . our sapns are formed by a single peptide chain that consists of two helical coiled-coil segments connected by a short linker region. one helix is forming a pentameric coiled coil while the other is forming a trimeric coiled coil (fig. a, b) . coiled coils are an ubiquitous protein folding and oligomerization motif that exhibits abundance in sequence and function [ ] . for instance, coiled coils like bzip transcription factors contain a dna-binding sequence whereas other coiled coils like intermediate filaments and spectrin can be structural components of the cell [ ] . furthermore, coiled coils can have dynamic functions such as myosin and dyneins, which act as "movement" proteins [ ] . the majority of α-helical coiled coils are based on a heptad sequence repeat, composed of seven amino acids usually denoted abcdefg [ ] . from two to seven strands of α-helices wrap around each other to create a superhelical twist that is normally left-handed [ ] . the driving force for the interaction between these α-helices are the hydrophobic amino acids located in positions a and d of the heptad repeat. ionic interactions between residues, for example in position e and g of the heptad repeat (i to i + ) in parallel dimers and trimers, also further stabilize coiled-coil interfaces. coiled coil stability can further be influenced by a number of factors including helical propensity of the residues, helix dipole, helix capping effects, translational and rotational entropy contributions and length of the coiled coil [ ] . litowski et al. investigated the effect of chain length in their heterodimeric coiled-coil system used in applications such as biosensors and affinity chromatography. their study showed that an increase in chain length led to an increase in stability of heterodimeric coiled coils, however, the effect was nonlinear [ ] . in a departure from studies of chain length effects, we have previously aimed to design the shortest possible coiled coil peptide using principles that play a part in monomeric α-helical stability or oligomeric packing. interestingly, a two heptad-repeat peptide was produced that was % dimeric under physiological buffer conditions [ , ] . here we have further investigated the influence of the length of the coiled coil for the self-assembly of our sapns, notably the length of the de novo designed trimer. there are many factors that govern the selfassembly of the peptide nanoparticles such as the nature of the linker region, constitution of pentamer and trimer. in a first step we decided to investigate the effect of the trimer length on the self-assembly of the sapns. apart from inherent scientific curiosity, useful applications of this study would be potential chemical syntheses of . a parent sequence t i is shown below that has been followed to build the initial model of the mini-nanoparticle peptide. computational model of the starting construct (peptide ) (b), neighboring pentamer-trimer complex (c), and a mini-nanoparticle (d) were built using the initial model of the peptide the monomeric polypeptide instead of production with recombinant protein technology. chemically synthesizing a long polypeptide is expensive, hence a shorter polypeptide displaying the same properties would be very advantageous and it would also allow for easier chemical functionalization. three constructs were designed, expressed, purified and refolded under different buffer compositions (figs. and ). it was found that even with only . heptad repeats in the trimer peptide nanoparticles were formed. furthermore, ph and salt concentration were shown to greatly affect the size and aggregation state of the sapns. apart from the length of the coiled coils designing and optimizing the correct linker has a pivotal role in the development of a nanoparticle that correctly refolds in vitro. we have done an exhaustive search in the protein data bank to find a template for a helix-turn-helix motif in which the angle between the two helices is about °, corresponding to the angle between the fivefold and threefold axes in an icosahedron. our recent study [ ] has shown that such a motif of the crystal structure of colicin e [pdb: i ] gives the optimum template for the linker (fig. a) , which can covalently connect two neighboring helices to produce correctly folded nanoparticles. in a second step we try to further optimize this linker region by the use of molecular dynamics simulations of the small peptide. all these different mutants along with the initial parent peptide (fig. ) were studied by molecular dynamics (md) simulations using the program charmm b in an attempt to further optimize the linker region and find the best peptide building block for the sapns. from the linker constitution study it was found that a malaria nanoparticle vaccine construct self-assembled into almost spherical nanoparticles [ ] . the de novo trimeric coiled coil in this construct had . heptad repeats ( residues). this led us to the question: if the trimeric coiled coil was shortened would nanoparticles still be formed? to answer this question the original construct was genetically engineered by pcr-mediated site-directed mutagenesis to create three new constructs. their sequences can be found in fig. and molecular models of the three designs are shown in fig. . the sds-page gel for hr showed that this construct expressed well. elution from the ni-affinity column took place at ph . and also in a buffer containing mm imidazole. the predicted molecular weight for hr is , . da. stepwise refolding at ph . and ph . was attempted but resulted in aggregation. direct refolding at ph . ( mm capso, mm nacl, % glycerol) resulted in nanoparticles as can be observed from tem and dls (fig. a , additional file : figure s ). however, it is evident from both, the dls and the tem data, that the preparation is significantly heterogeneous. the sds-page gel for . hr showed a similar pattern to hr except there also seemed to be elution at ph . . the predicted molecular weight for . hr is . da. the first refolding scheme attempted for . hr was direct refolding (dialysis against a buffer with no urea). direct refolding at ph . ( mm capso, mm nacl, % glycerol) resulted in nanoparticles as can be observed from tem and dls but also aggregation ( fig. b ; additional file : figure s ). to further characterize . hr a ph screening was performed. briefly, the quick refolding method, where the protein is concentrated to mg/ml then diluted times to a buffer with no urea, was used. for the buffers used for dilution the salt concentration ( mm nacl) and glycerol volumes ( %) were kept constant, only varying the buffering agent for each ph (ph . - mm hepes, ph . - mm tris, ph . - mm capso). at ph . precipitation in the dialysis bag was observed. at ph . there seemed to be no aggregation visible to the naked eye. however, investigation by tem revealed clusters of aggregation. only at ph . were there very nice nanoparticles visible (fig. c) . we decided to further screen different salt concentrations at this ph to confirm nanoparticle assembly. the quick refolding method described above was used. the different buffers had the same ph . ( mm capso) and same glycerol concentration ( %) only the salt concentration varied: no salt, mm nacl, mm nacl and mm nacl. it seems that as the salt concentration is increased the nanoparticle diameter also increases (additional file : figure s ). the construct with the shortest trimer was hr. elution from the ni-affinity column occurred largely at ph . but also at ph . and at ph . . however, direct refolding at ph . ( mm capso, mm nacl, % glycerol) resulted in aggregation. refolding of hr under phs . and . also resulted in aggregation. since this aggregation was visible to the naked eye it wasn't evaluated by dls or tem so results were not included. sapn was settled less than h for dls measurement and the suspension was not diluted. for accuracy purposes five scans were collected. the goal of this study was to characterize sapns immediately after refolding and long term stability was not evaluated. previous research efforts in our group shows that both long term settling of sapns and their concentration might or might not affect the morphology of sapns. it depends upon protein sequence, storage temperature and buffer [ ] . based on our construct design and the biophysical results of the . hr we ran a series of molecular dynamics simulations on the eleven short versions of . hr including only five helical turns (i.e. . heptad repeats) around the linker region of the nanoparticle peptide (figs. , a) in an attempt to further optimize the sequence that would yield the best refolding behavior. the angle between the pentameric and trimeric helices, the overall charge, the charge distribution along the peptide chain, and the different non-covalent interactions within the peptide nanoparticle, along with the solvent condition, are the key parameters that govern the quality of the nanoparticle formed in solution. figure represents different mutations of the parent sequence of the model peptide that we have studied by molecular dynamics simulation. mutations have been introduced close to the glycine residue of the linker region of all the constructs. a disulfide bond has been introduced by double mutations t c and s c in peptide , peptide , and peptide to see how this constraint affects the peptide conformation. a single mutation s t on the trimeric side has been introduced in peptide , peptide , and peptide to see how this bulkier residue (threonine versus serine) can affect the overall conformation of these peptides. another single mutation s n on the trimeric side has been tested in peptide . additional mutations have been introduced a little farther away from the linker region to modify the long range electrostatic interactions between the oligomerization domains along the fivefold and threefold axes of the icosahedron. figure a represents the d models of all the initial peptides to with the mutant residues in red color. the overall charge in both the pentameric and trimeric domains along with the overall charge of the construct are depicted in fig. . simulating a complete nanoparticle with spherical water around it is very time-consuming as the whole solvated system contains about half a million variables (fig. b) . in contrast, using only an asymmetric unit of the icosahedron during molecular dynamics runs and simulating the rest of the particle by the icosahedral symmetry operations applied to the asymmetric unit, the system becomes computationally much more efficient. an asymmetric unit can be built by cutting the whole system along the four planes passing through the rotational axes fivefold and twofold, twofold and threefold, threefold and twofold, and twofold and fivefold (fig. a) . the side and top views of such a wedge-shaped asymmetric unit containing only one nanoparticle peptide, water, and ions are shown in fig. c , d. during md simulations only the interactions within this wedge and its symmetry-related neighbors are calculated, which makes the md runs feasible. the resulting peptide structures after a ns md simulation have been superposed on their corresponding initial models (fig. b) . variations of rmsd with time of simulation have been calculated in mm sodium chloride solution (fig. a) for the eleven models chosen initially. another structural characteristics is the radius of gyration (rgyr), which is a combined measure of its overall size and shape. here, we have calculated the rgyr in mm salt environment, which are plotted in fig. b . panels a and b show two clusters of molecules in the salt environment, one with a diverging tendency (peptides , , and ) and the other eight constructs (peptides , , , , , , , and ) with a converging tendency. eight converging peptides show the variation of rmsd within Å with respect to the energy minimized structure, whereas the variation of rmsd for the three diverging structures is about Å. again, fig. b shows that the rgyr are pretty stable with simulation time, and the value ( . Å) for the converging cluster is lower than that of the diverging cluster ( Å). md simulations have also been run in water environment (data not shown) showing a tendency of divergence indicating unstable conformation even after ns. thus, adding salt has an important role in folding and converging to a stable nanoparticle. figure c shows the superposition of diverging constructs (peptides , , and ) after ns of md simulation. peptides and show deformation at the linker region, while peptide shows deformation at the n-terminus of the pentameric helix. in both models, where the linker region contains either a disulfide bond between two cysteine residues (peptide ) or a hydrogen bond between threonine and asparagine (peptide ), a deformation of the helices can be observed. similarly, an unbalanced situation in the start model (peptide ) results in a structural deformation as well (fig. c) . the upper clusters in fig. a , b that shows the divergence behavior of three model peptides (peptides , , and ) during ns molecular dynamics simulation in presence of mm nacl, were discarded for further analysis. figure d shows the superposition of the remaining eight constructs (peptides , , , , , , , and ), which are converging during ns md simulation in presence of mm nacl as shown in the lower clusters of the fig. a , b. additional file : figure s shows the effect of additional mutation on peptide , while additional file : figure s shows the effect of an additional mutation on peptide . in the model of peptide (with an overall charge of − ), where the serine residue in the trimeric domain was replaced by threonine (s t), no deformation of the helices was observed during a ns md simulation. rather, it converged into a stable helical conformation. although peptide converged to a reasonably good structure at the end of md simulation, when supplemented with an additional mutation (e a or r e) (peptides and ), it showed some degree of deformation in the d structure and also a deviation in rmsd and radii of gyration (additional file : figure s ). specifically, in peptide , where the mutation r e causes a loss of salt bridge interaction between e and r in peptide , a deformation developed at the n-terminus of the pentameric helix (additional file : figure s d ). although peptide and peptide did not show much structural deformation during ns md simulation, the angles between the pentamer and trimer helices are . ° and . ° respectively, which deviate by . ° and . ° from the angle ( . °) between the corresponding fivefold and threefold axes in an icosahedron (fig. a) . the angle between the two helices in peptide is . °, which is . ° smaller than the value in the icosahedron. final models of peptides and after ns md simulation are shown in fig. . alanine substitution in place of glu causes a deformation at the n-terminus of the pentameric helix in peptide . however, the reduced electrostatic interaction caused by the r e mutation in the trimeric helix of peptide results in maintaining the overall conformation with a slight bending at the c-terminus of this helix (fig. b) . the double mutation e r/e d introduced in peptide (fig. b) causes a deformation in the trimeric helix while maintaining the helical conformation of the pentameric domain. the mutation e r on the pentameric domain probably repels r on the trimeric side, thereby initiating both intra-and inter-domain salt bridge interactions r to d and r to e respectively that lead to a distinct conformational change at the c-terminus of the trimeric helix in peptide . by carefully analyzing the converging models in d, we were able to identify the two peptide models (peptide and ) showing lower rmsd and radii of gyrations with a reasonable angle between the trimer and pentamer while maintaining helical conformations as their secondary structure. finally, these two models, peptides and , were selected based on the nature of convergence (additional file : figure a , b) and in silico visualization on which we further performed md simulations for a longer period of ns (fig. ) . interestingly, the peptide model that shows deformation in md simulation because of the disulfide bond at the linker region, adopts an adequate helical conformation when supplemented with the same additional mutation e a or r e (peptides and ) beyond the linker region as that studied in peptides and respectively. the best model has been selected based on a ns md simulation on these two peptides and (fig. ) . in fig. a , peptide shows a slightly lower rmsd than that observed in peptide during the first . ns simulation. slightly lower radius of gyration for peptide (fig. b) also indicating more compactness than peptide . the angle between the pentameric helix and the trimeric helix of our spherical nanoparticle can be greater than the theoretical value . ° (calculated between the fivefold and the threefold axes in an icosahedron) depending on the flexibility of the linker region. here, in peptides and these inter-helical angles are . ° and . °. while, the minimum value . ° corresponds to the perfect alignment of pentameric and trimeric helices along the fivefold and threefold axes of the icosahedron respectively, the coiled coil add additional twisting to those helical axes. analyzing all these observations, we concluded that peptide is much closer to an asymmetric unit of an ideal t icosahedron and considered to be the best model out of all constructs. the purpose of this study was to investigate the effect of trimer length on the self-assembly of peptide nanoparticles. given that original malaria construct [ ] formed nice nanoparticles this construct was selected to have its trimer shortened and the epitope removed. the strategy used was to engineer a stop codon by pcrmediated site-directed mutagenesis at specific locations in the trimer resulting in three different lengths ( , . and ) of the heptad repeats in the trimer. just before the stop codon an arginine residue was introduced so as to further stabilize the short trimeric coiled coil. the hypothesis was that the arginine's positive charge would counteract the negative charge of the carboxy terminus and make the coiled-coil interaction as strong as possible. it is clear from the results with all three designs the crucial role ph plays in the self-assembly of the sapns. the monomeric peptide consists of a fusion protein where the pentameric coiled coil is connected by a short linker to the trimeric coiled coil. because of icosahedral symmetry the pentamer and trimer are at an angle where residues close to the linker region from the pentamer can interact with residues from the trimer. if the overall charge in the pentamer is the same as the overall charge in the trimer then they will repel each other. on the other hand, if the pentamer and trimer have opposite overall charges then they will attract each other (see fig. ). another interesting effect is salt concentration on nanoparticle size (additional file : figure s ). for . hr it seems that as the concentration of nacl is increased, larger nanoparticles are obtained. given that the trimer length for sapns to self-assemble are and . heptad repeats, the next logical step was to investigate if self-assembly occurs with a shorter pentamer. how short can you make the pentamer and trimer and still get nanoparticle assembly? to assess the feasibility of future lab work on such minimized peptides, we have performed molecular dynamics studies. our observation on peptide-based mininanoparticles based on molecular dynamics simulations elucidated the dependencies of point mutations close to and nearby the linker region on the assembly of simulated nanoparticles. flexibility in the linker region induced by a g can be controlled by the design and optimization of a single/double mutation near the linker region. we also studied the effect on the nanoparticle assembly of some point mutations further away from the linker region. a molecular dynamics simulation of designed constructs showed the variation of convergence into helical conformation of the two oligomerization domains as well as that of the inter-helical angle. peptide folded into the bestassembled nanoparticle after ns molecular dynamics simulation. in this construct we incorporated a disulfide bond by double mutations t c and s c together with a third point mutation a e in the trimeric domain. although the disulfide bond alone (peptide ) distorted the peptide conformation, when the additional mutation this computational study on peptide mini-sapns could potentially benefit the nanotechnology of therapeutic development, especially in vaccine design, drug delivery, and bio-imaging. if you could get a so-called "mini nanoparticle" chemical peptide syntheses might be useful to manufacture the peptide instead of relying on recombinant production with e. coli. for vaccine research chemical syntheses of peptide nanoparticles would be advantageous, as traditional contaminants inherent to protein purification such as lps and proteases would be eliminated. this in turn would allow the peptide nanoparticle to be on "fast-track" to phase i clinical trials and major hurdles with the fda could be avoided. for example audran et al. have led a synthetic peptide derived from a malaria antigen into a phase i malaria vaccine trial [ ] . our investigation demonstrated that the protein conformation is dependent on point mutations within and nearby the linker region to allow proper self-assembly of the sapns. flexibility in the linker region induced by glycine residues is needed to allow for the helix-turn-helix motif, but on the other hand the protein chain can be stabilized in this conformation by the choice of optimal neighboring residues. we studied the effect of point mutations within and somewhat more distant from the linker region on the nanoparticle assembly. a molecular dynamics simulation of designed peptide constructs showed the variation of convergence into a helical conformation of the two oligomerization domains and a stabilization of the angle between the two coiled-coil domains. peptide folded into the best-assembled nanoparticle after a ns molecular dynamics simulation. engineering a disulfide bond into the linker region to stabilize the relative orientation of the pentamer and the trimer distorted the peptide conformation. but when combining a third mutation with the disulfide bond it converged into a well-folded nanoparticle. in conclusion, the particular amino acid sequences of the linker region has a significant effect on the assembly properties of the sapns. combining mutations within the linker itself with mutations more distant from the linker can provide a powerful way to design and optimize its peptide sequence. this computational and biophysical study may allow the engineering of devices that can be used for drug targeting, drug delivery, and bio-imaging. in order to introduce an arginine followed by a stop codon in t i - -pf [ ] site-directed mutagenesis pcr was used. the resulting plasmids were transformed into escherichia coli strain dh α by heat shock. the resulting ampicilin-resistant colonies were screened for the recombinant gene by sequencing after miniprep (promega, madison, wi, usa). plasmids harboring the desired genes were transformed into the escherichia coli strain bl (de ) expression cells (novagen, gibbstown, nj, usa) and a % glycerol stock of each construct ( hr, . hr and hr) was stored at − °c. an overnight culture ( ml) of bl (de ) (novagen, gibbstown, nj, usa) cells containing the expression construct was added to lb ( l) supplemented with ampicillin (fisher, pittsburgh, pa, usa, ml, mg/ml). the culture was incubated ( rpm, °c) until the od reached ~ . - . , at which time protein expression was induced (iptg, fisher, pittsburgh, pa, usa, ml, m) and the culture incubated further ( rpm, °c, h). the cells were isolated by centrifugation ( g, min), and the pellet was stored at − °c. purification was done under denaturing conditions ( m urea). cell pellet was thawed on ice, resuspended in lysis buffer a which is composed of m urea, mm nah po , mm tris and mm β-mercaptoethanol, ph . and lysed by sonication (sonicator → ultrasonic liquid processor, cycle of s pulse at % amplitude followed by a s rest repeated for a min period). the insoluble cell debris was cleared by centrifugation ( min at , g). the supernatant was then incubated with nickel beads (qiagen, valencia, ca, usa) for one hour. possible dna contamination was removed by a wash with buffer b at ph . : m urea, mm nah po , mm tris, mm imidazole. protein contaminants were washed from the column using a ph gradient and mm imidazole in the buffers. the first wash was done with lysis buffer a and the second and third washes at phs . and . respectively with a buffer containing m urea, mm nah po , mm sodium citrate, mm imidazole and mm β-mercaptoethanol. elution was done at ph . and ph . again with m urea, mm nah po , mm sodium citrate, mm imidazole and mm β-mercaptoethanol. any protein that did not elute at the lower ph washes was eluted with buffers containing high concentration of imidazole (buffer c: m urea, mm nah po , mm tris, mm imidazole, ph . or buffer d: m urea, mm nah po , mm tris, mm imidazole, ph . ). protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( % gel). following purification, the denatured monomeric peptides were refolded using one of the following three methods: ( ) dialysis performed in a stepwise manner (stepwise refolding) ( ) one step dialysis against a buffer with no urea (direct refolding) or ( ) concentrated (amicon ultra centrifugal filter devices, millipore, billerica, ma, usa) and diluted to a buffer with no urea (quick refolding). the proteins were filtered with a . μm polyvinylidene fluoride membrane filter before and after dialysis (millipore, billerica, ma, usa, #slvv rs). for all three designs ( hr, . hr and hr) the protein concentration was calculated using the absorbance at nm with the extinction coefficient (m − cm − ) and molecular weight (daltons) of the protein. the extinction coefficient and molecular weight were obtained with expasy's protparam tool (http://ca.expasy.org/tools/ protparam.html). the hydrodynamic diameter was obtained with a malvern zetasizer nano s equipped with a nm laser using a mm path length quartz suprasil cell. the measurements were done at °c and for each protein five scans were collected. transmission electron microscopy samples were negatively stained with % uranyl acetate (spi) at a peptide concentration of μg/ml. electron micrographs were taken with a philips em transmission electron microscope at an accelerating voltage of kv. the micrographs were scanned at dpi. a starting model of the nanoparticle peptide was built using the crystal structures of the helix-turn-helix motif of the channel-forming domain of colicin e [ ] , the tryptophan zipper [ ] and a de novo designed trimeric coiledcoil peptide [ ] as templates for the linker, the n-terminal pentameric domain and the c-terminal trimeric domain, respectively (fig. a) . the model building (fig. b) was done in silico using the graphics program 'o' version . . [ ] . eleven different constructs were studied by molecular dynamics (md) simulations with charmm b [ ] installed on a linux cluster at the biotech center of the university of connecticut. the whole md simulation procedure is divided into five steps: vacuum minimization, solvation, energy minimization, heating and equilibration, and production dynamics. the peptide molecule was solvated and ions were added to achieve a particular salt concentration. in the energy minimization step, we used the steepest descent (sd) method for steps followed by the adopted basis newton-raphson (abnr) method for , steps, where each step is fs. after the energy minimization of the whole system, the temperature was raised slowly to k from an initial temperature of k at a rate of °/ steps to relax the molecule and then to run the equilibration for a total time of ps, including the time to raise the temperature. production dynamics for ns were run thereafter on the whole system for initial screening. long ns production dynamics were also performed on selected models to find the best one. to analyze the final models, we have calculated root mean square deviation (rmsd) and radius of gyration (rgyr) with respect to the energy minimized structure using charmm algorithm [ ] . natural supramolecular building blocks: from virus coat proteins to viral nanoparticles atomic-level models of the bacterial carboxysome shell development of the vault particle as a platform technology natural strategies for the spatial optimization of metabolism in synthetic biology towards an artificial cell biological containers: protein cages as multifunctional nanoplatforms drug delivery systems: entering the mainstream micellar nanocontainers distribute to defined cytoplasmic organelles immunodrugs: therapeutic vlp-based vaccines for chronic diseases developments in virus-like particle-based vaccines for infectious diseases and cancer a nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria conformation-specific display of e and f epitopes on self-assembling protein nanoparticles as a potential hiv vaccine a novel vaccine using nanoparticle platform to present immunogenic m e against avian influenza infection peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine coiled coils: a highly versatile protein folding motif self-assembly of coiled coils in synthetic biology: inspiration and progress peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems designing heterodimeric two-stranded alphahelical coiled-coils: the effect of chain length on protein folding, stability and specificity improving coiled-coil stability by optimizing ionic interactions design of a minimal protein oligomerization domain by a structural approach optimizing the design of protein nanoparticles as carriers for vaccine applications optimizing the refolding conditions of self-assembling polypeptide nanoparticles that serve as repetitive antigen display systems phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein antigen a mechanism for toxin insertion into membranes is suggested by the crystal structure of the channel-forming domain of colicin e atomic structure of a tryptophan-zipper pentamer design of a minimal protein oligomerization domain by a structural approach improved methods for building protein models in electron density maps and the location of errors in these models submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution support by the nih/nigms (award p gm ) and the nih/nida (award dp da ) for this work is gratefully acknowledged. authors' contributions pb designed the molecular structures and the experiments; td carried out the work related to the constructs hr, . hr and hr; rd carried out the work related to peptides - . all authors contributed to manuscript writing. all authors read and approved the final manuscript. pb has an interest in the company alpha-o peptides that has patents or patents pending on the technology. additional file . figure additional file . figure s . molecular dynamics simulation of three peptides , , and for ns. rms deviations (a) and radius of gyrations (b) of the three peptides , , and relative to their corresponding energy minimized structures after ns of md simulation. (c) superposition of the molecular structure of peptide on peptide after ns of md simulation. (d) superposition of the molecular structure of peptide on peptide after ns of md simulation. key: cord- -ix un c authors: teixeira, maria c.; carbone, claudia; sousa, maria c.; espina, marta; garcia, maria l.; sanchez-lopez, elena; souto, eliana b. title: nanomedicines for the delivery of antimicrobial peptides (amps) date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: ix un c microbial infections are still among the major public health concerns since several yeasts and fungi, and other pathogenic microorganisms, are responsible for continuous growth of infections and drug resistance against bacteria. antimicrobial resistance rate is fostering the need to develop new strategies against drug-resistant superbugs. antimicrobial peptides (amps) are small peptide-based molecules of – amino acids in length, with potent and broad-spectrum antimicrobial properties. they are part of the innate immune system, which can represent a minimal risk of resistance development. these characteristics contribute to the description of these molecules as promising new molecules in the development of new antimicrobial drugs. however, efforts in developing new medicines have not resulted in any decrease of drug resistance yet. thus, a technological approach on improving existing drugs is gaining special interest. nanomedicine provides easy access to innovative carriers, which ultimately enable the design and development of targeted delivery systems of the most efficient drugs with increased efficacy and reduced toxicity. based on performance, successful experiments, and considerable market prospects, nanotechnology will undoubtedly lead a breakthrough in biomedical field also for infectious diseases, as there are several nanotechnological approaches that exhibit important roles in restoring antibiotic activity against resistant bacteria. the discovery of new antimicrobial molecules in the early s was a landmark in the field of pharmacology allowing the reduction of morbidity and mortality from infectious diseases, which were among the main causes of death worldwide [ ] . however, the widespread and indiscriminate use of nanomaterials , , of burrer et al. have used several strains of murine coronavirus in cell culture and in vivo in mouse models for the assessment of the antiviral properties of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (p-pmos) [ ] . the authors have reported the targeting effect of p-pmos against various target sites in the viral genome in cell culture and protected mice against virus-induced tissue damage. ykeda et al. have used an antimicrobial peptide isolated from the skin of xenopus tropicalis (pxt- ), and its modified peptide (modify-pxt- ) to produce self-assembled discoidal nanoparticles composed of amphiphilic alpha-helical scaffold proteins or peptides organised in a lipid bilayer [ ] . both the peptides pxt- , having hydrophobic and hydrophilic faces, behaved like general surfactants and can be used as carriers. bacteriocins, while very active at low concentrations, usually have low in vivo stability, being susceptible to degradation by proteolytic enzymes [ ] . another major limitation on the use of these peptides is the difficulty encountered in their large-scale production, compromising altogether their clinical application. an approach to overcome their limited stability in vivo is their loading into nanoparticles. nanomedicine is currently a well-established approach intimately related to the design and development of nanomaterials with unique therapeutic and diagnostic properties [ ] . nanotechnologies have also shown great potential in almost every aspect of the management of microbial infection with more than ten nanoparticles (nps)-based products marketed for bacterial diagnosis, antibiotic delivery and medical devices in . with unique physicochemical characteristics, nanomaterials are sensitive and selective in the detection of bacterial signalling and may also exbibit intrinsic antimicrobial properties. furthermore, nanomaterials can be used for antimicrobial drug delivery, and the incorporation of antimicrobial nanomaterials in medical devices and implants can prevent microbial adhesion and infection [ , ] . all these facts are instrumental against antimicrobial resistance by compromising bacterial mechanisms of resistance [ ] . focusing on nanomaterials-based drug delivery systems, these offer an improved strategy to increase the therapeutic index, by decreasing the dosage and frequency of administration. besides, nanomaterials promote intracellular drug delivery, mitigating the development of drug-resistant bacteria and also allowing targeted organ accumulation by functionalized surface modifications, thus limiting systemic side effects and immunosuppression [ ] . despite these promising outcomes, the main challenge of establishing clinical use is related to the evaluation of interactions of nano-antibiotics with cells, tissues and organs achieving information about their possible toxic effects, together with the production on a large scale [ , , ] . several types of nanomaterials have shown potential in the pharmaceutical field. they have also been studied as potential drug carriers with applications in the delivery of amps, promising antimicrobial molecules which, due to their nature and physicochemical characteristics, have limited bioavailability. the aim of this work is to revise the state-of-the-art on the approach that combines the advantages of the design of new drug delivery systems for the improvement of antimicrobial bioavailability, taking into account the recent developments in nanomaterials for antimicrobial peptide delivery. amps are small natural oligopeptides that have recently showed a potential activity against antibiotics resistance mechanisms, due to their ability in lysing bacterial membranes, thus providing broad-spectrum effects, targeting microorganisms from viruses to parasites. the main interesting property of amps is their ability to selectively disrupt bacterial membranes without affecting mammalian cells, thus being safe. in addition, amps are referred to in the literature as host-defence peptides with anti-inflammatory and anticancer activities, because they are synthesized molecules that take action on the defence mechanisms against biological threats of the living organism of origin [ ] . the discovery of amps dates back to the first half of the th century when in , dubos extracted an antimicrobial agent from a soil bacillus strain, proven to be effective in mice pneumococci infection. this extract was then fractioned allowing the identification of gramicidin. despite some systemic toxicity, gramicidin has shown to be effective in the topical treatment of wounds and ulcers. the first animal-originated amp to be reported is defensin, isolated from rabbit leukocytes in [ ] . nowadays, more than amps have been described and current molecular developments can be consulted in a series of databases available on the web (table ) , including natural identified molecules, as well as peptidomimetic molecules and analogues that are pharmacologically designed thanks to the use of bioinformatics [ ] . current food and drug administration (fda)-approved amps with well-established use include bacitracin, colistin and polymyxin b (although only for topical administration) [ ] . despite the lack of consensus on the influence of peptide sequence in biologic activity of amps, some common characteristics seem to be important and fairly related to their antimicrobial property. the main one is primarily charge, with % of amps being cationic, and secondly, hydrophobicity or amphipathicity, influencing solubility profiles and consequently bioavailability [ ] . associated with structural characteristics, these are also the main physicochemical properties that should also be taken into account in the design of new synthetic amps [ ] . compared to conventional antibiotics, amps demonstrate significant advantages such as potency and board spectrum of activity, as well as an additional activity to modulate the immune system responses and low resistance rates. they also show some limitations that impair their safe therapeutic use, such as sensitivity to proteolysis, influencing stability and undefined toxicological data for systemic use [ ] . in addition, their cationic and amphiphilic nature lead to high binding to serum proteins after parenteral administration, with consequent rapid elimination from bloodstream circulation and accumulation in the reticuloendothelial system, thus resulting in toxic effects and reduced activity [ ] . to overcome these limitations, some strategies have been developed, maximizing the proven therapeutic potential of amps. there are numerous methods for obtaining amp delivery systems. amps could be immobilized onto a variety of materials or onto a variety of surfaces and still retain their antibacterial activity. also, amps can be targeted through loading them in nanoparticulate systems with selective delivery capacities, including polymers, liposomes, hydrogels, nanospheres, nano-capsules and carbon nanotubes [ ] . as practical examples on of these of strategies, the studies of mishra et al. were focused on the development of an amp-coated surface, specifically immobilizing lassioglossin-iii onto silicone-based catheters and its activity against urinary tract infections [ ] . at the same time, water et al. developed the possibility of encapsulating plectasin, a cationic amp, into polymeric nps and evaluated their efficacy on s. aureus strains [ ] . bacteria show resistance to antibiotics drugs through a variety of mechanisms. moreover, the development of even new mechanisms of resistance has resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (mdr) bacterial strains [ , ] . however, when bacteria are drug-resistant it does not mean that they stop responding to an antibiotic, but that occurs only at higher concentrations [ , ] . of greater concern are cases of acquired resistance, where initially susceptible populations of bacteria become resistant to an antibacterial agent, in particular antibiotics, and proliferate and spread under the selective pressure of use of that drug. one approach to address this challenge is to design analogues of drugs [ , ] that are already in clinical use and that have activity against resistant organisms. however, bacteria are constantly succeeding to develop a resistant mechanism to new antibiotic drugs as well as to their analogues [ , ] . nanotechnology offers opportunities to re-explore the biological properties of already known antimicrobial compounds such as antibiotics by manipulating their size to modify their effect. the combination of amps with nanomaterials is not new. out of the available , papers indexed in the web of science, a total of , combining amps with nanoparticles or nanomaterials have been published over the last years in a range of disciplines ( figure ). nanomaterials , , x for peer review of water et al. developed the possibility of encapsulating plectasin, a cationic amp, into polymeric nps and evaluated their efficacy on s. aureus strains [ ] . bacteria show resistance to antibiotics drugs through a variety of mechanisms. moreover, the development of even new mechanisms of resistance has resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (mdr) bacterial strains [ , ] . however, when bacteria are drug-resistant it does not mean that they stop responding to an antibiotic, but that occurs only at higher concentrations [ , ] . of greater concern are cases of acquired resistance, where initially susceptible populations of bacteria become resistant to an antibacterial agent, in particular antibiotics, and proliferate and spread under the selective pressure of use of that drug. one approach to address this challenge is to design analogues of drugs [ , ] that are already in clinical use and that have activity against resistant organisms. however, bacteria are constantly succeeding to develop a resistant mechanism to new antibiotic drugs as well as to their analogues [ , ] . nanotechnology offers opportunities to re-explore the biological properties of already known antimicrobial compounds such as antibiotics by manipulating their size to modify their effect. the combination of amps with nanomaterials is not new. out of the available , papers indexed in the web of science, a total of , combining amps with nanoparticles or nanomaterials have been published over the last years in a range of disciplines ( figure ). evidence collected in the review work of huh and kwon [ ] , pelgrift and friedman [ ] , brooks and brooks [ ] , diab et al. [ ] and shimanovich and gedanken [ ] clarifies microbial drug resistance mechanisms and how nanotechnology may be considered a tool against this issue. development of drug resistance occurs in (at least) three steps: (i) acquisition by microbes of resistance genes; (ii) expression of those resistance genes; (iii) selection for microbes expressing those resistance genes. first, bacteria acquire resistance to single and multiple drugs through horizontal gene transfer by transformation, conjugation and transduction [ ] . bacteria can also acquire resistance genes by spontaneous mutation of existing genes [ ] . mdr is acquired when a bacterial cell already containing one type of drug resistance gene acquires another type of drug resistance gene [ , ] . second, in response to exposure to an antimicrobial drug, microbes express the resistance gene [ ] . third, resistance becomes widespread when there is selection for microbes that express resistance genes against the antimicrobial drug. this selective pressure in favour of resistance occurs whenever evidence collected in the review work of huh and kwon [ ] , pelgrift and friedman [ ] , brooks and brooks [ ] , diab et al. [ ] and shimanovich and gedanken [ ] clarifies microbial drug resistance mechanisms and how nanotechnology may be considered a tool against this issue. development of drug resistance occurs in (at least) three steps: (i) acquisition by microbes of resistance genes; (ii) expression of those resistance genes; (iii) selection for microbes expressing those resistance genes. first, bacteria acquire resistance to single and multiple drugs through horizontal gene transfer by transformation, conjugation and transduction [ ] . bacteria can also acquire resistance genes by spontaneous mutation of existing genes [ ] . mdr is acquired when a bacterial cell already containing one type of drug resistance gene acquires another type of drug resistance gene [ , ] . second, in response to exposure to an antimicrobial drug, microbes express the resistance gene [ ] . third, resistance becomes widespread when there is selection for microbes that express resistance genes against the antimicrobial drug. this selective pressure in favour of resistance occurs whenever microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug nanomaterials , , of followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials , , x for peer review of microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: i) controllable and relatively uniform distribution in the target tissue; ii) improved solubility; iii) sustained and controlled release; iv) improved patientcompliance; v) minimized side effects; and vi) enhanced cellular internalization [ , ] . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: (i) controllable and relatively uniform distribution in the target tissue; (ii) improved solubility; (iii) sustained and controlled release; (iv) improved patient-compliance; (v) minimized side effects; and (vi) enhanced cellular internalization [ , ] . nanomaterials , , x for peer review of microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: i) controllable and relatively uniform distribution in the target tissue; ii) improved solubility; iii) sustained and controlled release; iv) improved patientcompliance; v) minimized side effects; and vi) enhanced cellular internalization [ , ] . current advances in the use of inorganic nanoparticles for amp delivery involve essentially the development of silver nanoparticles (agnps) and gold nanoparticles (aunps), as well as silicon derivates nano-systems [ ] . the studies revised in this work are summarized in table . the development of polymeric nanoparticles for amp delivery may offer an excellent technological strategy to improve drug bioavailability and safety, avoiding drug chemical and enzymatic degradation, preventing aggregation, enhancing controlled release. chitosan (cs) nps (csnps) are particularly interesting as the broad spectrum of antibacterial activity of cs is well known and documented, offering the possibility of synergistic effects with antimicrobial molecules. moreover, due to its biocompatibility properties, cs nanostructures have been extensively studied for drug delivery, and that is no different for amp delivery. in recent years, an increasing number of papers reporting on a new generation of antimicrobial metallic nps has been published [ ] . consequently, many of the information on the application of nanotechnology in the infectious disease field regards the use of silver (ag) and gold (au) nps [ , , ] . recently, derivatives of other metals have been studied for antimicrobial applications, and the antibacterial effects of zero-valent bismuth nps and uncoated au, nickel (ni) and silicon (si) nps were reported [ , ] . despite the demonstrated intrinsic antimicrobial properties, dispersed metallic nps tend to aggregate and separate in solution, resulting in a decrease in their antimicrobial efficiency. with the aim of improving antibacterial properties, functionalization of nps has been attempted with surfactants, polymers or antibiotics resulting in more stable, less aggregated nps suspension and innovative, synergistic antibacterial agents. for instance, silver nps stabilized by polymers(polyvinylpyrrolidone) and surfactants (sds and tween ) exhibit enhanced antibacterial activities [ ] . nps can also act as drug-carriers able to pass through cell membranes [ , ] . widely used antibiotics such as ciprofloxacin may benefit from the association with nps, and conjugation may result in an antibacterial effect also against micro-organisms resistant to the same molecule in the naturally occurring form [ ] . when the antimicrobial agents are covalently linked to or contained within, nps, a higher drug concentration is attained in the area of interest, resulting in better efficacy at comparable doses and in slower release over time that may be exploited for preventing bacterial colonization [ , ] . moreover, specific biological sites can be attacked after modification of nps with target molecules [ , ] . as the nps themselves may have antibacterial properties, the combination of nps and loaded drugs exert a synergistic action [ ] . current advances in the use of inorganic nanostructures for amp delivery involve essentially the development of ag and aunps, as well as silicon derivate nano-systems. the studies revised in this work are summarized in table . research on gold nps is increasing thanks to their many advantages, such as their ease of synthesis and conjugation to biomolecules, their capability to maintain their own structure when in circulation and their improved effectiveness against bacteria, thus demonstrating their high potential in the field of nanomedicine. even if at the beginning, the research in this field was focused on the possibility to exploit gold nps in combination to laser radiation, thus significantly reducing bacteria viability due to cell lysis and mechanical disruption [ ] . currently, two recent studies have been reported exploring the use of au nanostructures for amp delivery. photoluminescent au nanodots (aunds) were prepared by chen et al. [ ] . these aunds were functionalized with hybridized ligands, an antimicrobial peptide (surfactin; sft), and -dodecanethiol (dt), on aunps. ultrasmall sft/dt-au nds (size ≈ . nm) were achieved and exhibited highly efficient antimicrobial activity. the photoluminescence properties and stability as well as the antimicrobial activity of sft/dt-au nds were also studied, and it was shown that these characteristics are highly dependent on the density of sft on au nds. relative to sft, sft/dt-aunds exhibited greater antimicrobial activity, not only to non-multidrug-resistant bacteria but also to the multidrug-resistant bacteria. the minimal inhibitory concentration values of sft/dt-aunds were much lower (> -fold) than that of sft. the authors considered that the antimicrobial activity of sft/dt-aunds was mainly achieved by the synergistic effect of sft and dt-aunds on the disruption of the bacterial membrane. in vitro cytotoxicity and hemolysis, analyses were also performed and had revealed superior biocompatibility of sft/dt-aunds than that of sft. moreover, in vivo methicillin-resistant s. aureus infected wound healing studies in rats showed faster healing, better epithelialization. this study suggested that the sft/dt-aunds system may be a promising antimicrobial candidate for preclinical applications in treating wounds and skin infections [ ] . rai et al. also reported a one-step methodology to generate amp-conjugated (aunps) [ ] . the amp-conjugated aunps prepared showed controlled size ( nm) and low polydispersity and allowed the inclusion of high concentration of amps. further, these systems demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble amp, as well as low cytotoxicity against human cells [ ] . interestingly, akrami et al. showed the possibility to exploit gold nps as a novel anticancer platform [ ] . results of this research confirm the improvement of cell internalization of gold nps, with higher cytotoxicity and cellular uptake for smaller nps compared to larger nanospheres and nanorods, suggesting that anticancer effects of the selected peptides were modulated by the size and shape of the gold nanoparticles [ ] . geilich et al. developed novel delivery systems by combining silver nps and ampicillin so to achieve a synergistic dose-dependent effect on bacterial cells [ ] . the obtained polymersomes were safe on human fibroblasts and more effective in inhibiting bacterial cells with a silver-to-ampicillin ratio of one to . , respectively. recent advances in amp delivery by ag nanostructures, pazos et al. [ ] reported on supramolecular assemblies of novel peptide amphiphiles (pas) containing aldehyde functionality in order to reduce ag ions and subsequently nucleate ag metal nps in water. this proposed system spontaneously generates monodisperse ag particles at regular distances along the length of the filamentous organic assemblies. the metal−organic hybrid structures exhibited antimicrobial activity and significantly less toxicity toward eukaryotic cells. metallized organic nanofibers of the type described offer the possibility to create other structures. for instance, hydrogels, that can be potentially applied in wound dressing development [ ] . also addressing the wound infection problem, salouti et al. investigated the synergistic antibacterial effect of plant peptide mbp- and agnps on infected wounds caused by s. aureus [ ] . the mic and mbc of mbp- and agnps both on their own and in combination form were determined against s. aureus via macrodilution and microdilution methods. the mic and mbc of mbp- were found to be . and . mg/ml, respectively. mic and mbc of agnps were determined to be . and . mg/l, respectively. mic and mbc of the agnps and mbp- combination were found to be . mg/ml, . mg/l; and . mg/ml, . mg/l, respectively. the synergistic antibacterial effect of ag nps and mbp- was investigated on infected wounds caused by s. aureus in a mouse model, and the infected wound healed properly after the combined use of mbp- and agnps [ ] . it is worth to note recent findings by chaudhari et al. concerning the conjugation of silver-coated carbon nanotubes with the antimicrobial peptide tp . herein, authors underline the occurrence of an additive effect of silver-coated nanotubes and tp regarding antibacterial activity. the innovative nano-system was found to be safe to murine macrophages and hep cells at the mic concentrations [ ] . as delivery systems for amps, silicon and silicon derivates nanostructures have also been investigated recently. membrane interactions are critical for the successful use of mesoporous sinps. in order to elucidate these, braun et al. have studied the effects of np charge and porosity on amp loading and release, as well as consequences of this for membrane interactions and antimicrobial effects [ ] . anionic mesoporous sinps were found to incorporate considerable amounts of the camp ll- , whereas loading was found to be much lower for non-porous or positively charged sinps. the results also demonstrated that due to preferential pore localization, anionic mesoporous particles, but not the other particles, protects ll- from degradation by infection-related proteases. for anionic sinps, membrane disruption is mediated almost exclusively by peptide release. in contrast, non-porous sinps built up a resilient ll- surface coating due to their higher negative surface charge, and display largely particle-mediated membrane interactions and antimicrobial effects. for positively charged mesoporous sinps, ll- incorporation promoted the membrane binding and disruption displayed by the particles in the absence of peptide, but also caused toxicity against human erythrocytes. thus, the use of mesoporous sinps as amp delivery systems requires consideration of membrane interactions and selectivity of both free peptide and the peptide-loaded nps [ ] . the properties of amps adsorbed on inorganic or organic surfaces are of interest for their potential applications in intracellular drug delivery. in the work of syryamina et al., continuous-wave (cw) electron paramagnetic resonance (epr) and pulsed electron-electron double resonance (peldor) techniques were applied to study the adsorption of the amps trichogin ga iv and ampullosporin a on monodisperse colloidal silica nanospheres (sins) of nm diameter [ ] . the results obtained by cw epr supported the view that the adsorbed peptides form close-packed clusters. peldor data show that both trichogin and ampullosporin adsorbed on the silica surface possess a more disordered conformation as compared to that in solution. for ampullosporin, disordering is much more pronounced than for trichogin. after desorption, the peptides restored their conformations; upon adsorption, the peptides in some cases may lose partly their biradical character [ ] . these results may be of interest as the antimicrobial activity is often related to peptide conformation. nano-clays or layered silicates are an interesting nanostructure that has been used for remediation of environmental contaminants, delivery of drugs and various active molecules, and to enhance polymer mechanical and barrier properties in packaging films. they typically present a stacked arrangement of silicate layers with a nanometric thickness [ ] . meira et al. studied three different nano-clays (bentonite, octadecylamine-modified montmorillonite, and halloysite) as potential carriers for the amps nisin and pediocin, known bacteriocins, the first referred above as having application as a food preservative. higher adsorption at room temperature of nisin and pediocin was obtained on bentonite. the antimicrobial activity of the resultant bacteriocin-nano-clay systems was analysed using skimmed milk agar as food simulant, and the largest inhibition zones were observed against gram-positive bacteria for halloysite samples. bacteriocins were intercalated into the interlayer space of montmorillonites as deduced from the increase of the basal spacing measured by x-ray diffraction (xrd) assay. these results indicate that nano-clays, especially halloysite, are suitable nano-carriers for nisin and pediocin adsorption, and the results may be considered interesting for the food industry [ ] . different biodegradable polymeric nano-systems have been explored as carriers for antimicrobial agents that exhibit a high bactericidal activity. the efficacy of this strategy is well proven, highlighting polymeric nano-systems can effectively improve the cellular penetration, intracellular retention and specific subcellular distribution of antimicrobial agents, and even evade intracellular inactivation of antimicrobial agents [ ] . controlled drug release using biocompatible and biodegradable polymers further emerged in the s [ ] . after the first polymer-based delivery of macromolecules using poly[ethylene vinyl acetate] polymer was demonstrated in [ , ] . antimicrobial drug delivery using polymeric nps offers several advantages: (i) structural stability in biological fluids and under harsh and various conditions for preparation; (ii) precisely tuneable properties, such as size, zeta-potentials, and drug release profiles, by manipulating polymer lengths, surfactants, and organic solvents used for np preparation [ ] , and (iii) facile and versatile surface functionalization for conjugating drugs and targeting ligands [ ] . concerning antimicrobial nanoparticulate delivery systems, two major types of polymers have been explored: linear polymers (e.g., polyalkyl acrylates and polymethyl methacrylate) and amphiphilic block copolymers. the majority of polymeric nps prepared with linear polymers are nano-capsules or solid nanospheres [ ] . in polymeric nano-capsules, a polymeric membrane that controls the release rate surrounds the drugs that are solubilized in aqueous or oily solvents. in solid nanospheres, drugs are homogeneously distributed in the polymeric matrices of variable porosities [ , ] . amphiphilic block copolymers form self-assemble micellar nps with the drug being encapsulated in the hydrophobic core and surrounded by a hydrophilic shield. this shied allows the core to be protected from degradation [ ] . several biodegradable polymers, including poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(lactide-co-glycolide) (plga), poly(caprolactone) (pcl), and poly(cyanoacrylate) (pca), have been used as the hydrophobic core of the amphiphilic copolymers, whereas peg has been the most commonly used hydrophilic segment [ , [ ] [ ] [ ] [ ] . targeting ligands can also be conjugated on the termini of peg for targeted and selective delivery [ , ] . polymeric nps have been explored to deliver various antimicrobial agents and greatly enhanced therapeutic efficacy in treating many types of infectious diseases has been reported. for instance, encapsulated ampicillin in polymeric nps was effective for s. typhimurium infection treatment [ ] and intracellular l. monocytogenes infection in mouse peritoneal macrophages [ ] . the use of polymeric nps can overcome the limited oral administration of unstable or inadequately absorbed drugs, [ ] and, in addition, pegylation of nps, can increase drug half-life in serum, and improve mucoadhesive capabilities by reducing phagocytosis [ ] thus, among nanoparticle platforms, polymer nps may be the most suitable system that can be used for antimicrobial drug delivery. biodegradable polymers and bioorganic polymers are also promising materials in the delivery of peptide-based drugs due to their compatibility, degradation behaviour, and nontoxic nature of administration [ ] . the development of polymeric therapeutic nanostructures for amp delivery may offer an excellent technological strategy to improve drug bioavailability and safety. cs-based nps (csnps) are particularly interesting as the broad spectrum of antibacterial activity of cs is well known and documented, offering the possibility of synergistic effects with antimicrobial molecules. moreover, due to its biocompatibility properties, cs nanostructures have been extensively studied for drug delivery, and that is no different for amp delivery. in fact, the majority of the studies performed so far involves cs nanostructures and was already revised in previous work [ ] from which we reproduce table . research has been covering, the technological development of new carrier systems and their full characterization, and the evaluation of their efficacy as drug delivery improvers. in addition to cs-based nanostructures, recent studies on other polymeric nanostructures for amp delivery have been developed (table ). cs-based nanoparticles vancomycin cs particles were prepared by ionic gelation and freeze-drying or spray-drying as recovery methods. antibacterial activity against s. aureus. cerchiara et al. [ ] cs-based nanoparticles lysozyme as model development of a nanoparticle model with commercially available cs loaded with lysozyme as antimicrobial protein drug model. piras et al. [ ] cs and poly-gammaglutamic acid composites ll- the results indicated that both ll- and no were co-loaded successfully in micro particles, and the composite particles could sustain ll- and no release at physiological ph, in vitro. cryptdin- preparation of cs tripolyphosphate (cs-tpp) nps by ionotropic gelation. the formulation was then characterized on the basis of particle size, zeta potential and polydispersity, and antimicrobial in vivo assays against salmonella enterica were performed. cs-based nanoparticles temporin b cs-nps were prepared based on the ionotropic gelation between cs and sodium tripolyphosphate. the nano-carrier evidenced a sustained antibacterial action against various strains of s. epidermidis. nanogel composites -prepation of agnps embedded in a biocompatible nanogel comprising degradable, natural polymers. in this study, hybrid nanogels were prepared with varying polymer content and their potential by determining their antibacterial properties against e. coliand s. aureus strains. coll ferrer et al. [ ] glycol-cs nanogels -study of the biocompatibility of a glycol cs nanogel by evaluation of effects on metabolic activity, cell cycles blood compatibility. overall, the results demonstrated the safety of the use of the gc nanogel as drug delivery system. nanofibers and films cs thin films hlf - immobilization performed onto cs thin films as a model for an implant coating due to its reported osteogenic and antibacterial properties. cs thin films were produced by spin-coating on au surfaces. activity against methicillin-resistant s. aureus (mrsa). nanofibers defensin- , dermaseptin ll- magainin alternate deposition of polycation (cs) and polyanion over cotton gauzes. antimicrobial assays were performed with two strains: s. aureus and k. pneumonia. food packaging systems study of the efficiency as antimicrobial carriers of hydroxypropyl methylcellulose [ ] , chitosan (cs), sodium caseinate (sc) and polylactic acid [ ] films, in the release rates of fluorescently labeled nisin z, evaluating their potential as food packaging polymers. imran et al. [ ] nanomaterials , , of plla-l -pllain situ gel ap- potential application of amps in wound healing, by developing a biodegradable poly (l-lactic acid)-pluronic l -poly (l-lactic acid) (plla-l -plla) in situ gel-forming system li et al. [ ] food preservation systems nisin study of a safe suitable antimicrobial system to be used in food industry. influence of pectin degree of acetylation on np properties. krivorotova et al. [ ] chitosan/pga nanoparticles nisin influence of chitosan coating on colloidal stability, loading capacity and encapsulation efficiency wu et al. [ ] d'angelo et al. designed and developed a system of nano-embedded microparticles (nem) for sustained delivery of cationic amps (camps) [ ] in the lung, studying its effect on p. aeruginosa, a known lung infection pathogen. to this purpose, plga nps containing a model camp, colistin (col), were produced by the emulsion/solvent diffusion technique, and then spray-dried in different carriers (lactose or mannitol), thus producing nem. the most promising nem formulations were selected from bulk and flow properties, distribution of nps in the carrier and aerosolization performance upon delivery through a breath-actuated dry powder inhaler. col-loaded nem were found to kill p. aeruginosa biofilms and to display a prolonged efficacy compared to the free col. [ ] . another camp, plectasin, was encapsulated into plga nps using the double emulsion solvent evaporation method, in the work of water et al. [ ] the plectasin-loaded nps displayed a high encapsulation efficiency ( - %) and mediated release of the peptide over h. the antimicrobial efficacy was investigated using bronchial epithelial calu- cell monolayers infected with s. aureus, and encapsulated plectasin displayed improved efficacy as compared to non-encapsulated plectasin. the author also assessed the subcellular localization of the prepared nps in different relevant cell lines: calu- epithelial cells, thp- macrophages and a epithelial cells. here the results have shown good patterns of penetration on calu- epithelial cell lines, as well as in thp- macrophages [ ] . hydrogels and nanogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. the biological performance of these systems, particularly those used as wound dressing; can be complemented with antimicrobial activity capable of preventing colonization of the wound site by opportunistic bacterial pathogens [ , , ] . these types of structures have also been studied recently for amp delivery. continuing their study of the antimicrobial activity of multi-domain camps (md-camps) in solution, jiang et al. investigated the same effect of self-assembled -d hydrogels supramolecular nanostructures and its rheological properties [ ] . among the studied md-camps solutions, the bactericidal activity of peptide hydrogels was found to be improved. the improved antimicrobial activity of the self-assembled peptide hydrogels was found to be related to the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. thus, the structure-property-activity relationship developed through this study may provide important knowledge for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications [ ] . the water et al. group also designed novel nanogel-based novicidin delivery system. the peptide novicidin was self-assembled with a nano-ctenyl succinic anhydride-modified analogue of hyaluronic acid, and this formulation was optimized using a microfluidics-based quality-by-design approach. the encapsulation efficiency of novicidin ( - %) and the zeta potential (− to − mv) of the nanogels could be tailored by changing the preparation process parameters, with a maximum peptide loading of ± %. the nanogels exhibited good colloidal stability under different ionic strength conditions and allowed complete release of the peptide over days. furthermore, self-assembly of novicidin with hyaluronic acid into nanogels significantly improved the safety profile at least five-fold and six-fold when tested in huvecs and nih t cells, respectively, while showing no loss of antimicrobial activity against e. coli and s. aureus [ ] . li et al. explored the potential application of amps in wound healing, by developing a biodegradable poly(l-lactic acid)-pluronic l -poly(l-lactic acid) (plla-l -plla) in situ gel-forming system [ ] . an injectable formulation composed of human amps (ap- ) loaded nps, and thermosensitive hydrogel was prepared. ap- peptides were enclosed with biocompatible nps (ap- -nps) with high drug loading and encapsulation efficiency. ap- -nps were further encapsulated in a thermosensitive hydrogel (ap- -nps-h) to facilitate its application in cutaneous wound repair. as a result, ap- -nps-h released ap- in an extended period and exhibited quite low cytotoxicity and high anti-oxidant activity in vitro. the in vivo wound healing assay using a full-thickness dermal defect model of sd rats indicated that ap- -nps-h could significantly promote wound healing. at day after an operation, the treated group showed nearly complete wound closure of , ± , % [ ] . other studies of nisin nanoencapsulation were performed, with the purpose of protection to ensure the stability of this amp during food processing and storage period. nisin-loaded pectin nps (nlp-nps) were prepared and analysed by krivirotova et al. by a simple complexation method [ ] . three types of pectin biopolymer were tested and it was found that the methoxylation degree of pectin influenced nisin loading efficiency and particle size. for the complex formation, both electrostatic and hydrophobic interactions were important. nlp-nps exhibited antimicrobial activity that was dependent on the type of biopolymer. overall, the results indicated that nlp-nps might be a suitable antimicrobial system to be used in the food industry [ ] . nisin nanoencapsulation in self-assembly chitosan-and poly-glutamic acid, was recently reported by wu et al. [ ] . herein, the authors showed that the use of a coating layer of chitosan improved colloidal stability, loading capacity and encapsulation efficiency. furthermore, chitosan layer composite nanoparticles were more effective compared to uncoated nps and nisin in inhibiting the growth of escherichia coli and listeria monocytogenes. solid lipid nanoparticles (sln) and nanostructured lipid carriers (nlc) stand for nanoparticles composed of lipid materials that melt at temperatures above • c [ ] . their main purpose is to modify the release profile of the payload attributed to their solid core. while sln are composed of solid lipids only, nlc matrix is a blend of solid and liquid lipids (which also melts above the body temperature) to improve the solubility of lipophilic compounds. both sln and nlc are biodegradable and non-toxic [ ] [ ] [ ] , and their versatility of is also attributed to their capacity to load a range of chemically different bioactives, including peptides and proteins [ ] [ ] [ ] [ ] , and be modified on their surface [ , ] . fumakia et al. have developed sln for the simultaneous delivery of an endogenous host defence peptide (ll ) with antimicrobial activity and an elastase inhibitor serpin a (a ) for the treatment of wound infections [ ] . the authors reported that sln could modify the release profile of simultaneous delivery of both bioactives, accelerate wound healing in bj fibroblast cells and keratinocytes and promote antibacterial activity against staphylococcus aureus and escherichia coli in comparison to ll or a alone. ll loaded into nlc have also been proposed by garcia-orue for the topical treatment of chronic wounds [ ] . ll -loaded nlc did not affect cell viability tested against human foreskin fibroblasts while the in vitro bioactivity assay showed that the peptide remained active after the loading into lipid nanoparticles as the system reversed the macrophages activation as happened with the ll in solution. ll -loaded nlc were also active against escherichia coli. the in vivo testing in mice also demonstrated the systems' capacity to improve healing by promoting wound closure, reepithelization grade and restoration of the inflammatory process. lewies et al. demonstrated that biodegradable nlcs enhance the antibacterial activity of antimicrobial peptides [ ] . the authors have studied the effect of nisin z when loaded into nanostructured lipid carriers on staphylococcus aureus, staphylococcus epidermidis and escherichia coli. nisin z exhibited additive interactions with numerous conventional antibiotics, in particular, with novobiocin. the presence of edta improved the antimicrobial activity of free nisin z towards escherichia coli significantly. nisin z-loaded nlcs were effective against gram-positive species at physiological ph, also showing an increased efficacy when adding edta. nisin z-loaded nlcs were shown potential to enhance the antibacterial activity of nisin z towards gram-positive bacteria commonly found in skin infections. other types of nanomaterials, such as dendrimers and carbon nanodots, have also been successfully proposed for the delivery of amps. due to their ease of synthesis and low manufacturing costs, antimicrobial polymers including dendrimers have been exploited to mimic the antibacterial mechanism host defence peptides, by compromising bacterial cell membranes [ ] . gide et al. developed nanomaterials-lipidated amphiphilic dendrimers which displayed potent and selective antimicrobial activity against both gram-positive and gram-negative bacteria, including multidrug-resistant strains [ ] . these dendrimers are also shown to inhibit bacterial biofilms effectively. carbon nanodots or carbon quantum dots were originally discovered due to their bright fluorescence emissions as those found in conventional semiconductor quantum dots, and can also be used for drug delivery [ ] . carbon dots modified with vancomycin were proposed for the treatment of gram-positive bacterial infections [ ] . zhong et al. synthesized surface-modified carbon dots with vancomycin and successfully tested them against bacillus subtilis, listeria monocytogenes, salmonella, pseudomonas aeruginosa and escherichia coli. the modified carbon dots showed affinity to the tested gram-positive bacteria owing to the ligand-receptor interactions between vancomycin and the cell walls. pramanik et al. synthesis red/blue fluorescent carbon dot-attached magnetic nanoparticles for selective separation and identification of superbugs from infected blood samples [ ] . the nanoparticles were capable of isolating methicillin-resistant staphylococcus aureus (mrsa) and salmonella dt superbug from whole blood samples, followed by accurate identification via multicolor fluorescence imaging. the authors also described the design of antimicrobial peptide-conjugated multicolor fluorescent magneto-carbon dots for separation, identification and disinfection of multidrug resistance superbugs from infected blood. amps are antimicrobial compounds recognized as among the most promising drug candidates against infections. they exhibit distinct mechanisms of action and may show additional biological activities, e.g., as signalling molecules and biomarkers, and even as tumoricidal agents. amps, however, show low stability and bioavailability. the formulation of amps into nanomaterials seems to offer a very appealing and effective manner to improve these limitations, and several systems have been designed and studied for this purpose (e.g., inorganic, polymeric and lipid nanoparticles, carbon dots and dendrimers). however, concerns on how to regulate the distribution of nanomaterials in the body or specific organs are also raised. nano-drugs are foreign substances to the body and may produce inflammation. therefore, safety data for long-term therapy or repeated dosage are needed to circumvent the potential risk. biodegradable nanomaterials have been proposed to reduce the risk of toxicological events both in vitro and in vivo. to date, few studies have investigated the toxicological and environmental effects of direct and indirect exposure to nanomaterials, and no clear guidelines exist to quantify these effects. therefore, there is an urgent need for developing guidelines, which can assure the safe use of nanomaterials. moreover, more powerful ex vivo models or animal models are needed to assess the safety issues and to comply with government regulations. how to extend the shelf life of nanodrugs is also a problem due to their agglomeration also being a problem. the production methods for nanostructures should also be improved, and scalable studies for industrial production are also of great importance in order to promote cost effectiveness of these new formulations. the cost and production of nanomaterials on a large scale is one of the hurdles in effective implementation of these products. hence, the scientific community should also pay attention to 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antimicrobial activity nano-sized lipidated dendrimers as potent and broad-spectrum antibacterial agents employing carbon dots modified with vancomycin for assaying gram-positive bacteria like staphylococcus aureus magnetic multifunctional carbon dots for selective separation, identification, and eradication of drug-resistant superbugs this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -tid a authors: basso, luis g. m.; vicente, eduardo f.; crusca jr., edson; cilli, eduardo m.; costa-filho, antonio j. title: sars-cov fusion peptides induce membrane surface ordering and curvature date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: tid a viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. the s subunit of the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus (cov) contains internal domains called fusion peptides (fp) that play essential roles in virus entry. although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. here we employed differential scanning calorimetry (dsc) and electron spin resonance (esr) to gather information on the membrane fusion mechanism promoted by two putative sars fps. dsc data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. esr showed that both fps increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. therefore, bending moment in the bilayer could be generated, promoting negative curvature. the significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the sars-cov-mediated membrane fusion are discussed. corresponding to residues - (immediately positioned n-terminally to hr ) and to residues - (immediately positioned c-terminally to a second, internal cleavage site s ' at r ) , and another less conserved region corresponding to a hydrophobic stretch located between residues and (near the s /s boundary region at site r ) . these putative fps are thought to destabilize host cell membranes, driving the refolding of the s subunit into the post-fusion -hb configuration, one of the late steps in the viral membrane fusion process . although membrane fusion promoted by class i viral glycoproteins, such as sars-cov spike, human immunodeficiency virus (hiv) gp or influenza virus hemagglutinin (ha), has been broadly studied in recent years [ ] [ ] [ ] [ ] , many aspects of the molecular mechanism behind the virus-host cell membrane fusion remain unknown, including conformational changes of the lipid bilayers during peptide-membrane interactions. elucidating the nature of protein-lipid interactions as well as the conformational properties of both the membranotropic segments of the viral fusion proteins and the lipids in cell membranes can help to dissect the major steps of the orchestrated membrane fusion mechanism promoted by those biological machines. however, structural and dynamics information at the molecular level of peptide-induced membrane fusion in the context of the whole spike protein is difficult to obtain. thus, synthetic peptides corresponding to the putative fusion peptides might be very useful in providing detailed information on the interaction of those segments with lipid model membranes not only because the peptides themselves support membrane fusion, but also because there is a direct correlation between the effects of mutations in the intact protein and in the peptide analogues for membrane fusion [ ] [ ] [ ] . in the present study, we investigated the effects of two putative fusion peptides from sars-cov s glycoprotein, corresponding to residues - (sars fp ) and - (sars ifp ) , , , , on the structural dynamics, physicochemical properties, and thermotropic phase behavior of lipid model membranes by differential scanning calorimetry (dsc), continuous wave (cw) and pulsed electron spin resonance (esr) along with nonlinear least-squares (nlls) spectral fitting . we found that both peptides increase the lipid packing and decrease the water content inside the lipid bilayer only for membranes containing negatively charged lipids, as well as generating opposing curvature stresses on highly curved membranes containing non-bilayer-forming phospholipids. the significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids for the fusion mechanism mediated by the sars-cov s glycoprotein are discussed. we are interested in investigating the extent of perturbation of model membranes caused by a functional concentration of the peptides, i.e, peptide concentration that is already known to promote fusion of model membranes. at this concentration, what are the changes in the structural dynamics, curvature and hydration of the lipids and in the thermodynamic parameters of the membranes that lead to membrane fusion? it has been shown that sars fp and sars ifp are able to induce membrane fusion only at high peptide-to-lipid molar ratio , . thus, we chose a : lipid/peptide molar ratio for most of the experiments and compared the effects of the peptides on model membranes that present very different physicochemical properties. to examine the effects of the putative sars fusion peptides on the structural integrity of lipid model membranes, the thermotropic phase behavior of different multilamellar vesicles (mlv) in the absence and in the presence of mol% of peptides were determined (fig. ) . the thermodynamic parameters obtained from the analysis of the dsc curves are shown in table . it is worth mentioning that the membrane-associated peptides remain folded in the liquid crystalline phase of the phospholipids , . multilamellar vesicles of dppc and dppg exhibited two endothermic events in the temperature range studied. the low-enthalpic, broad pretransition arises from the conversion of the lamellar gel phase, l β' , to the ripple gel phase, p β' , and is observed at about . °c for dppc and at . °c for dppg (table ) . on the other hand, the more energetic and more cooperative (narrow) main phase transition arises from the conversion of p β' to the liquid-crystalline phase, l α , and is centered at . °c for dppc and at . °c for dppg. membranes composed of unsaturated lipids presented only a very asymmetric, low-enthalpic, and broad main phase transition at . °c for popa, at . °c for pope, and at ~ . °c for pops. these results reasonably agree with the literature [ ] [ ] [ ] [ ] . the slight discrepancies observed in comparison with literature data are likely due to differences in lipid preparations, buffers, and ionic strength [ ] [ ] [ ] . by contrast, the main phase transition of dpps vesicles is split into two endothermic events, one centered at . °c and the other centered at . °c (table ). this two-peak feature has been attributed to different protonation states of the head group polar moiety . binding of drugs, peptides, and proteins to membranes can promote structural and dynamic changes of lipid bilayers that significantly affect their thermotropic phase behavior , . shifts of the melting transition temperature of the lipids, for instance, can be related to alterations of the entropy change between the gel and fluid states, whereas a broadening of the dsc thermogram may be the result of a decreased transition cooperativity (~ /Δ t / ). as can be observed in fig. , incorporation of mol% of fusion peptides into the lipid bilayers did not strongly affect the main phase transition temperature of the liposomes. however, the enthalpy and entropy changes of the main phase transition were significantly altered, especially for membranes containing negatively charged lipids ( fig. and table ) . sars fp and sars ifp only slightly perturbed zwitterionic dppc liposomes. it was observed a small increase of both the melting temperature t m (less than %) and the calorimetric enthalpy change of the transition Δ h (less than . %), indicating a slight stabilization of the peptide-bound dppc gel phase. the increased pretransition temperature of the peptide-containing dppc vesicles also indicates structural changes in the dppc polar head group , . on the other hand, more prominent effects on the enthalpy change and on the cooperativity of the transition of zwitterionic pope vesicles were observed. sars fp decreased the transition Δ h by about %, whereas sars ifp decreased it by % (table ) . pope-sars fp interaction also markedly broadened the dsc endotherm (increase of Δ t / by ~ %). this result indicates that sars fp intercalates within pope bilayer and thus decreases the intermolecular cooperativity of the transition. overall, more significant effects on the thermodynamic (or dsc) parameters of the bilayer phase transitions were observed for membranes containing negatively charged lipids ( table ) : reduction of the calorimetric Δ h by about % ( %) for dppg and by about % ( %) for pops, for instance, after incorporation of sars fp (sars ifp ). unlike the previous cases, endotherms containing multiple peaks appeared when the peptides were mixed with popa vesicles (fig. ) . in this case, the lower-temperature endothermic peak certainly arises from peptide-bound membranes, whereas the higher-temperature peak can be due to peptide-free popa vesicles in the popa/sars fp samples and due to a mixture of peptide-free and peptide-bound popa vesicles in the popa/sars ifp samples. the two-component feature of dpps main phase transition remained in the peptide-containing dpps samples. although the calorimetric enthalpy change of the whole transition decreased in the presence of the peptides, the area under the higher-temperature endotherm increased. the peptides also promoted opposing effects on the cooperativity of the two endotherms: while sars fp narrowed both transitions (thus increasing lipid cooperativity), sars ifp broadened the endotherms. this may be related to different locations and topology of the peptides in the membranes. another interesting feature is the appearance of a third, low-enthalpic peak at around °c in dpps/peptide vesicles. this result indicates a different mechanism of ps-peptide interaction compared to the other negatively charged lipids. the low-enthalpy, higher-temperature peak ( °c) was also observed in dmso-treated peptide-containing dpps liposomes, but was not observed in the acetonitrile-treated or dmso-treated peptide-free dpps vesicles. this result indicates that the new peak is not due to the binding of acetonitrile or dmso from the peptide stock solutions to the dpps mlvs (see fig. s and table s in supplementary information). interestingly, a low-enthalpy peak also appeared in the peptide-bound pops vesicles at a temperature slightly higher than the t m of the peptide-free pops vesicles (fig. ) . thus, this more stable and much less energetic endothermic peak may be due to specific ps-peptide interaction. taken together, these results suggest different mechanisms for the interaction of the peptides with zwitterionic and negatively charged phospholipids. electrostatic interactions seem to play an important role for the sars-cov fusion peptide-membrane interactions, since both peptides are positively charged at ph . : sars fp , + e sars ifp , + . moreover, since both peptides also significantly changed the thermotropic phase behavior of dppg mlvs at high ionic strength ( mm nacl; reduction of Δ h by % for sars fp and by % for sars ifp - table ), hydrophobic interactions may also play an important role in peptide binding and penetration into membranes. interestingly, the peptides promoted different effects on the thermograms of dppg, dpps, pops, and popa, suggesting that not only the charge but also the lipid packing (dpps vs. pops) and the lipid polar head group (pg, ps, or pa) may contribute to the energetics of peptide-membrane interactions. membrane curvature-promoting properties. since changes in membrane curvature have been associated with the potential mechanistic role of fusion peptides in inducing membrane fusion , , we tested the ability of the sars-cov fusion peptides to promote curvature strain on dipope vesicles. below the lα -h ii phase transition temperature, t h , phosphatidylethanolamines, such as dipope, spontaneously form lipid bilayers in the liquid-crystalline state, whereas above t h , they usually pack together in a highly curved hexagonally inverted structure with negative membrane curvature , . stabilization of this concave curvature due to peptide binding, for instance, will favor the nonbilayer h ii phase, which will translate into a t h reduction . conversely, peptides that induce positive membrane curvature will increase t h of the dipope/peptide samples and thus will stabilize the liquid-crystalline bilayer phase . figure shows dsc traces of dipope without and with . or . mol% of sars fp and sars ifp at both low ( fig. a) and high (fig. b) effects of the peptides on the structural dynamics of lipid vesicles. the local ordering and rotational dynamics of the lipid head group and acyl chains were investigated by cw esr using the nitroxide-labeled lipids dpptc, -pcsl, and -pcsl (see structure of the spin probes in supplementary fig. s ), which monitor different regions of the lipid bilayers. dpptc reports on the lipid/water interface, whereas -pcsl and -pcsl monitor the hydrophobic core of the membranes at different depths of penetration , [ ] [ ] [ ] . despite the perturbation of the dppc thermotropic parameters caused by the peptides, esr spectra of the spin labels embedded into dppc and dppc/cholesterol / (mol/mol) mlvs at different temperatures did not show noticeable changes in the lipid structural dynamics of the peptide-containing vesicles as compared to the peptide-free lipid bilayers (see supplementary fig. s ). this means that the membrane-bound peptides do not significantly perturb the local structural dynamics of the head group or the hydrophobic core of the zwitterionic mlvs as investigated by cw esr. on the other hand, remarkable changes in the esr spectra of the spin labels in liposomes containing negatively charged lipids, such as dppg, dpps, and popa, were observed. the experimental and best-fit nlls simulations are presented in supplementary figs s and s and the best-fit magnetic tensor components, rotational diffusion rates, and order parameters are summarized in supplementary tables s -s . before analyzing the changes in the lipid ordering and mobility induced by the peptides, it is worth mentioning a general feature observed for the order parameter s of dpptc in the gel and the fluid phases of all lipid model membranes. we generally and consistently found a positive value for s in the gel phase of the lipids and a negative value in their fluid phase. for instance, we found s = − . for dpptc in dppg at °c (table s ) , s = − . in dpps at °c (table s ) , and s ~ − . in popa at °c and °c (table s ). the meaning of this negative value for s of dpptc was extensively discussed by ge and freed for this same spin label in the fluid phase of dppc dispersions (s = − . at °c) in terms of the analysis of the cartesian components of the restoring potential u(Ω) . in the gel phase, there is a molecular force field in the head group that tends to align the trimethyl ammonium (tma) group of the dpptc (supplementary fig. s ) perpendicular to the bilayer surface, i.e. parallel to the local director of the bilayer, giving rise to s > . however, in the fluid phase, that surface-orienting potential tends to align the tma group parallel to the surface, giving rise to a negative order parameter . by definition, s ≡ < ( cos θ − )/ > , hence s tends to − . when θ tends to ° and it is negative for ° < θ < ~ . °. thus, if we compare the s values of dpptc from our studies with that obtained by ge and freed, we might infer that the orientation of the tma group in dppc (s = − . ; ge and freed work) and in dpps (s = − . ; our work) lies in between the one in dppg (s = − . ) and the other in popa (s = − . ). those differences in the tma orientation are likely due to distinct orienting forces that arise from the particular hydrogen-bonding network provided by the pg, ps, and pa bilayers and the dipolar interactions between the zwitterionic phosphoryl-tempo-choline group of dpptc and the surrounding negatively charged head groups. additionally, irrespective of its sign, an increase of s (either by becoming more positive for s > or less negative for s < ) would lead to the same interpretation: a greater tendency of the preferential orienting axis of tma to orient along the local director of the membrane and an increased restriction of the amplitude of its rotational motion. figure shows the plots of the rotational diffusion rates r ⊥ and the changes in the order parameter s fig. ). thus, the dpptc esr spectra at °c may be regarded as a single averaged spectrum from which both gel-like and fluid-like populations are not resolved. therefore, the effect of the peptides on r ⊥ and s should be analyzed with caution in this case. the ordering effect of the peptides on the dppg head group region in the gel and fluid phases was also observed in the hydrophobic core of the bilayer at all temperatures, but with the most prominent changes in the ripple gel phase (fig. b ,c). the more fusogenic sars fp consistently increased the ordering of -pcsl and -pcsl more than sars ifp . particularly, since the -pcsl probe is more sensitive to molecular motions than dpptc and -pcsl , , the structural dynamics of the gel-like and fluid-like lipid states could be resolved and characterized (table s ) . although the peptides shifted the equilibrium between the two states towards the fluid-like one (the population of the fluid-like component increased from to ~ %), the packing (s ) of the center of the bilayer was also increased for both lipid states. as for the mobility, the peptides reduced the rotational diffusion of -and -pcsl mainly in the fluid phase. only sars fp was able to decrease r ⊥ below t m , with the most significant change observed for -pcsl at °c (r ⊥ decreased from . × s − to . × s − ; table s ). figure shows the r ⊥ and Δ s values for dpptc and -pcsl embedded in dpps at temperatures corresponding to the gel ( and °c) and fluid ( °c) phases of the membrane. the rotational mobility and order parameter of dpptc were both affected by the peptides at all temperatures, but the most striking changes were observed in the very ordered dpps gel phase (fig. a ). in the fluid phase, r ⊥ decreased by % and % for sars ifp and sars fp , respectively. at °c, r ⊥ decreased by - %: from . × s − to . × s − for sars ifp , and to . × s − for sars fp (table s ). the most prominent change in the dpps head group region induced by the peptides, based on our nlls simulations, took place on the molecular alignment of the dpptc tma group. firstly, the s at °c found in our simulations was . , much lower than that found in pure dppg at the same temperature (s = . ; table s ). ge and freed reported a value of . for dpptc in dppc dispersions in the gel phase ( °c) and a similar value was found by barroso et al. for a similar pc spin probe in an equimolar mixture of dppc/dpps (s = . ). this result indicates that the phosphoryl-tempo-choline group of dpptc is more loosely packed in the gel phase of the pure dpps bilayer than in the other model membranes reported. interestingly, peptide addition to pure dpps remarkably increased the ordering of the head group in the gel phase: s jumped from . to about . for both peptides at °c and similar changes were observed at °c. in the fluid phase, though, just a slight variation was obtained (Δ s = . - . ) (fig. a ). peptide binding to dpps head group caused a large ordering effect, thus restricting the mobility of the lipids. the spectra of -pcsl in the peptide-free dpps obtained at temperatures below t m presented very broad resonance lines (supplementary fig. s b ; °c). even in high ionic strength condition, a very broad lineshape in the ordered dpps gel phase persisted (not shown). this line broadening effect is due to strong dipolar interactions that arise from possible cluster formation of the pc spin labels in the dpps milieu. to test this hypothesis, we prepared dpps/ -pcsl samples with either . mol% or . mol% of the spin label. at the higher probe concentration, even broader lines were obtained as compared with the esr lineshape of . mol%, whereas narrower, but still broad, lines were found with . mol%. this result shows that -pcsl does not partition very well in the dpps gel phase but does so in the fluid phase, as illustrated in supplementary fig. s b (spectrum acquired at °c). after unsuccessful attempts to fit the -pcsl spectra with the addition of a heisenberg exchange coupling, we presented only the best-fit parameters from nlls simulations of the spectra acquired at °c (supplementary table s ). both r ⊥ and s were only slightly affected by the membrane-bound peptides at that temperature: r ⊥ slightly decreased from . × s − to . × s − for sars fp and s increased from ~ . to ~ . or . for sars ifp and sars fp , respectively (table s ) . interestingly, binding of the peptides to dpps promoted a better partition of -pcsl in the membrane. the structural organization of the dpps/peptide membranes altered in such a way that the pc spin probe became miscible in the binary system. as for the -pcsl in dpps, no change in the esr lineshape at both °c and °c upon peptide addition was observed (not shown), indicating no long range effect of the peptides on the center of the bilayer, despite the fact that they do bind and perturb the head group and the region around c of the lipid acyl chain. however, at °c, sars fp and sars ifp promote the appearance of a second, disordered component in the esr spectra table s ). in the fluid phase, the only parameter affected by peptide binding was the rotational mobility, which was diminished from . × s − to . or to . × s − for sars fp or sars fp , respectively. our results indicate the peptides bind to both gel and fluid phases of dpps, but peptide insertion into the membrane seems to be activated either by temperature or ultimately by loosening of the hydrophobic packing of the highly ordered dpps gel phase. as the temperature is increased, long range effects on r ⊥ of the acyl chain were observed in the bilayer center. we also studied the effect of the peptide binding on the structural dynamics of popa mlvs in the fluid phase ( °c and °c). as shown in table s , sars fp and sars ifp only changed the ordering and r ⊥ of the spin labels at °c (fig. ) . at °c, the esr spectra from peptide-free and peptide-bound vesicles are almost the same. differently from the other model membranes, peptide binding to popa head group causes an increase of the mobility: r ⊥ changed from . to . × s − for sars ifp and to . × s − for sars fp (fig. a ). this result is probably related to the lack of a bulky head group of popa (the surface area per lipid of pa head group is smaller than that of pg or ps), which may facilitate the rotational diffusion of the phosphoryl-tempo-choline group of dpptc, despite the small decrease in the amplitude of the tma molecular motion (s increased by about . to . ; fig. b ). on the other hand, the best-fit nlls simulations of -pcsl spectra showed a prominent effect on r ⊥ rather than s : while the rotational mobility diminished % for sars ifp , it decreased % for sars fp , whereas Δ s was only about . (supplementary table s ). as for the -pcsl, we found a very high value for s (− . ) in the peptide-free popa liposomes, indicating a large deviation from cylindrical symmetry of the molecular alignment of the end-chain label to the local director of the membrane. to gain further insights on the molecular orientation of the end-chain label we derived the orienting potentials along the cartesian directions from the potential coefficients obtained by the best fits of the nlls simulations (section si ): u x ≡ u( °, °)/kt = . , u y ≡ u( °, °)/kt = − . , and u z ≡ u( °, °)/kt = − . . this result indicates a strong preference for the rotational diffusion axes z r (parallel to the pz orbital of the nitrogen) and y r of the nitroxide moiety to align along the normal to the bilayer, while x r , which is parallel to the n-o bond, is prevented to align along the membrane local director. therefore, the unsaturation of the oleyl acyl chain of popa would allow for a dynamic bending of the end chain of the spin label that favors much more the alignment of both z r and y r axes to table s ). since the fluid-state lipids ( °c) present lower order (s = . ) and high degree of molecular misalignment (s = − . ) with r ⊥ ranging from . to . × s − (supplementary table s ), these results allow us to infer that this second component does not present the same properties of fluid-state lipids but rather peptide-bound lipids. that is, peptide binding to popa bilayers gives rise to peptide-enriched and peptide-free domains, where the latter is barely affected by the peptides. affected by the peptides. these results are in good agreement with our dsc experiments, in which, as discussed earlier, thermograms with multiple peaks arise from peptide-associated and peptide-free popa vesicles. additionally, it is important to investigate the effects of membrane fusion promoters and inhibitors on the bilayer properties in an attempt to identify the changes in the physicochemical parameters of the membrane that might be relevant for membrane fusion. generally speaking, insertion of inverted cone-shaped molecules such as the lysophosphatidylcholine -palmitoyl- -hydroxyl-pc (lpc) into the outer monolayer would prevent membrane fusion supposedly by promoting positive membrane curvature , . conversely, insertion of cone-shaped molecules such as phosphatidylethanolamine, arachidonic acid (aa), and linoleic acid (la) into the outer monolayer would facilitate membrane fusion presumably by inducing negative curvature . using esr, ge and freed have found opposing effects of lpc and aa on the order parameter of a headgroup spin label embedded in dmpc model membranes . in their work, an ordering (disordering) effect was observed for the fusion promoter (inhibitor) aa (lpc), which may help to induce (prevent) membrane fusion. since the putative fusion peptides from sars-cov s protein promote substantial membrane fusion only in the presence of negatively charged lipids, it is important to verify whether that correlation is also valid for negatively charged lipid-containing vesicles. to do so, we prepared equimolar mixtures of dppc/dppg and dppc/popa membranes and studied the effects of sars fp , sars ifp , la, and lpc on the order parameter s of dpptc at °c. the best-fit parameters of the esr spectra shown in fig. s are summarized in table s in supplementary information. figure shows that both fusion peptides and la caused an ordering effect on the head group of both model membranes. in contrast, lpc promoted a disordering effect of dpptc. these results are thus in accordance with those found by ge and freed for the zwitterionic dmpc and indicate that sars fp and sars ifp might facilitate membrane fusion similarly to how the fusion peptide from influenza hemagglutinin does. since the ordering effect of the lipid head group has been attributed to bilayer dehydration , we used eseem spectroscopy to find out whether the peptide-induced ordering of anionic vesicles is possibly related to membrane dehydration, as suggested as a general fusion mechanism of other class i fusion peptides , . eseem has been successfully applied to examine the penetration depth profile of deuterium-substituted molecules, such as water, glycerol, and sugar inside lipid bilayers [ ] [ ] [ ] [ ] as well as to investigate the water permeation at protein/peptide-lipid interface of membrane-interacting peptides and membrane proteins [ ] [ ] [ ] . here we used a d o-containing buffer to probe the deuterium environment surrounding the spin labels doptc, -pcsl, and -pcsl ( supplementary fig. s ) embedded in popc/popg / (mol/mol) mlvs. the different spin labels allowed investigating the changes promoted by the peptides in the water content from the lipid/water interface down to the hydrophobic core of the bilayers. changes in the modulation depth of the time-domain eseem spectra or in the deuterium spectral density yields information about d o molecules situated near the nitroxide within a distance range of up to . nm, which is the known spatial reach of the method . figure a shows the spectral density of the spin-labeled lipids in the peptide-free and peptide-containing lipid vesicles (the normalized time-domain stimulated echo signals are given in the supplementary fig. s a ). all spectra are dominated by two signals, one centered at . mhz, arising from the hyperfine interaction of the nitroxide with surrounding deuterium nuclei ( h-larmor frequency at mt), and the other centered at . mhz, which arises from matrix protons near the spin label. since lipids, peptides and possibly residual h o contribute to the spectral density at . mhz, the h signal was not further analyzed. two additional weak peaks were observed in the ft-spectra of doptc and -pcsl: one at ~ . mhz, which is assigned to n , and the other at ~ . mhz, which corresponds to the larmor frequency of p (fig. a) . those nuclei belong to the choline group of popc ( n) and to the phosphate group ( p) of the lipids. both peaks are absent in the -pcsl eseem spectra because the distance between the spin probe moiety in the bilayer midplane and the head group is beyond the limit of the technique. the deuterium peak is composed of two spectral components: a low-intensity broad signal that arises from a direct deuterium bond between d o and the nitroxide radical; and a narrow doublet of intensity i( h) and amplitude Δ (fig. a) , which arises from the quadrupole interaction between the electron spin and non- h-bonded water molecules inside the bilayer . both Δ and i( h) have been found to depend linearly and nonlinearly, respectively, on the concentration of free water molecules at a distance of . to . nm . the parameters i( h), measured at . mhz, and Δ for all lipid spin labels are shown in fig. b . as expected, the deuterium spectral density decreases from the bilayer/water interface toward the membrane center. particularly, i( h) is reduced from ( ± ) ns in the water/lipid interface to ( ± ) ns around th carbon position and to ( . ± . ) ns near the bilayer midplane in the peptide-free vesicles, indicating reduced water content in the hydrophobic core as compared to the head group region. those i( h) values are somewhat higher than previously reported spectral densities for popc/popg / (mol/mol) bilayers but are in the same order of magnitude of those for dppc , . the discrepancies are most likely due to different freezing protocols, sample preparation, and experimental conditions such as the choice of τ , the first interpulse delay, and the frequency chosen to measure the intensity of the spectral density . sars fp and sars ifp decreased both i( h) and Δ for the spin labels that monitor the hydrophobic region of the bilayer (fig. b) . this finding implies that both peptides interact with popc/popg membranes and displace free water molecules from their hydrophobic core. theoretical calculations from milov et al. indicated that nitroxide-deuterons interactions spread to around nm . therefore, the spin label at c position is able to monitor water density around c up to the level of the carbonyl-glycerol and phosphocholine regions (distant about . nm from c ) , . thus, the contribution from bulk water molecules to the spectral density of -pcsl is negligible , . the significant % decrease of Δ for -pcsl in the peptide-containing membranes, relative to the peptide-free vesicles (supplementary table s , fig. b ), allows us to infer that the intramembrane water in the outer membrane region is dramatically reduced upon peptide incorporation. the same reasoning holds true for the -pcsl. as for the head group spin label doptc, we found that the peptides surprisingly promoted opposing effects on i( h) and Δ : while they slightly raised the magnitude of the d o signal, Δ values were reduced (fig. b) . the source of this opposing effect remains unclear to us, but it may be of some interest to speculate on that. considering the electron density profile of pc lipids , , we may infer that the nitroxide radical of the doptc phosphoryl-tempo-choline group is in direct contact with both membrane surface and bulk water molecules. changes in the orientation of the phosphoryl-tempo-choline must therefore locally disturb the water density around the spin label. incorporation of cholesterol into pc membranes, for instance, decreases the ordering of the lipid head group, i.e., makes it less aligned along the bilayer director . this effect allows for water molecules to move from the bulk into the membrane (down to ~c ), thus increasing both i( h) and Δ (fig. s b and table s in supplementary information) . on the other hand, if the phosphoryl-tempo-choline group becomes more aligned along the bilayer normal, i.e. the s of doptc increases, membrane-surface dehydration takes place . in this situation, the concentration of membrane-surface water molecules is reduced in the 'membrane side' . in contrast, realignment of the head group dipole makes the nitroxide radical become more exposed to interact with bulk water molecules in the 'solvent side' . milov et al. have theoretically shown that the nonlinear dependence of the i( h) on water concentration is due to the formation of nitroxide-water complexes . therefore, if the water distribution around the nitroxide is locally perturbed in such a way that it allows for formation of nitroxide-water complexes (i.e., disturbances take place closer than . nm), it must affect mainly the i( h), but not Δ . that is, reorientation of phosphoryl-tempo-choline due to peptide-lipid interactions may be such that it allows formation of nitroxide-water complexes, affecting mainly i( h). this may correspond to the source of the increased i( h). the contribution to Δ stems mainly from water molecules belonging to the second hydration shell (> . nm) up to ~ nm. from the 'solvent side' , the second water coordination sphere for the water-exposed nitroxide radical should not change, thus the major contribution to Δ of doptc may arise from free water molecules located in the 'membrane side' . the reduced level of the head group hydration accounts therefore for the decrease of Δ values. however, further detailed studies are needed to investigate this hypothesis. viral membrane fusion is a concerted mechanism that involves remarkable protein and lipid conformational changes. the molecular mechanism by which viral fusion proteins catalyze the membrane fusion reaction and the molecular details of lipid rearrangements in the lipid bilayer are not fully understood yet, although the current model has been constantly revisited and refined [ ] [ ] [ ] [ ] [ ] [ ] . crystal structures of pre-and post-fusion states of class i viral fusion proteins along with nmr structures of fusion peptides in membrane mimetics have provided a mechanistic view of the membrane fusion process. in this process, different protein segments act in an orchestrated way to achieve the complex and kinetically unfavorable task of bringing together and fusing two lipid bilayers , . however, there are still major gaps in the molecular details involved in the fusion process. they include: the sequential order and kinetics of the events, changes in the structure, dynamics, and physicochemical properties of different protein domains and lipid bilayers during the whole membrane fusion mechanism, free energies of the corresponding pre-, post-(fusion pore) and intermediate (hemifusion) fusion states as well as the modulation of all those parameters by lipid composition. sars-cov s subunit possesses various membranotropic segments, i.e., relatively short hydrophobic domains that are able to bind to and perturb membranes. these segments can act independently from each other and may help to stabilize the contact between viral and cell membranes , [ ] [ ] [ ] [ ] . however, monitoring the structural rearrangements of different segments of the intact s subunit as well as the dynamics of their interaction with viral and cell membranes on a molecular level constitute a challenging task. the use of synthetic peptides and phospholipids has provided important thermodynamic, structural, biochemical, and functional details of peptide-membrane interactions from both peptide and lipid perspectives, which makes them a good platform to study membrane fusion , , . our results indicate that both sars fp and sars ifp significantly perturb the thermotropic phase behavior as well as the molecular ordering and phospholipid rotational mobility of model membranes containing negatively charged lipids, but only cause moderate effects on zwitterionic membranes. these findings are in agreement with previously reported studies that indicated greater disturbance and higher affinity of both peptides for model membranes containing anionic rather than zwitterionic phospholipids , . even though the peptides do not partition well into zwitterionic membranes, they are able to perturb the thermodynamic parameters of the zwitterionic dppc and pope phase transitions as well as to change the membrane curvature properties of dipope. on the other hand, our cw esr experiments showed that the peptides do not affect the structural dynamics of zwitterionic lipid bilayers, but they do promote an ordering effect on the lipid head group and on the acyl chains of negatively charged membranes. this latter effect seems to be correlated to membrane dehydration, as shown by our eseem experiments. ordering and dehydration effects have also been observed for other class i viral fusion peptides such as those from hiv-i gp and influenza ha glycoproteins , . the relevance of membrane-curvature induction and lipid dehydration for the viral membrane fusion of the sars-cov fusion peptides will be discussed below. peptides induce bending moment and membrane dehydration of anionic membranes. lipid molecules in bilayers experience a restoring torque from neighboring lipids that tends to reorient the lipid chain and/or the head group along the normal to the bilayer. the order parameter s , which is associated with the restoring torque through the orienting potential u(Ω), is a measure of the extent of that molecular alignment. generally, the higher the s the lower is the angular amplitude of the wobbling motion of the spin probe, i.e. the more aligned along the local director is the lipid segment to which the probe is attached. a better alignment can enhance the molecular interactions between lipid molecules in the bilayer. therefore, s directly reports on the lipid packing density. in particular, slight changes in s of the lipid head group would have a greater impact on the structural organization of the membrane than changes in the ordering of the acyl chain . this is primarily due to the strong hydrogen-bonding network in the head group region (~ . to . kcal/mol) . thus, although the molecular structure of dpptc head group is different from those of the pg, ps, and pa head groups, reorientation of dpptc due to conformational changes in the polar region might be associated with changes in the hydrogen-bonding network . that is, the more ordered the dpptc, the more condensed is the bilayer, which leads to changes in the ionic interactions between the head groups and between the head groups and surface-bound water molecules. by contrast, the van der waals interactions between the lipid acyl chains in the hydrophobic core of the membrane are much weaker, i.e. the strength of the interaction is about . kcal/mol, which is even smaller than the thermal energy at k (k b t ~ . kcal/mol) . nevertheless, enhancement of lipid-lipid interactions due to condensation of the hydrophobic core leads to an increased chain-packing energy. in our studies, the peptides were added into preformed vesicle solutions. therefore, the changes observed here are primarily due to the interaction of the peptides with the outer leaflet of the bilayers. sars fp and sars ifp increase the s of -pcsl and -pcsl in dppg and popa membranes, making the nonpolar core of the outer leaflet more solid-like. furthermore, both peptides increase the ordering of head group spin label dpptc in pure dppg, dpps, and popa as well as in dppg-and popa-containing membranes, but not in the zwitterionic dppc and dppc/chol. therefore, an increase of both the head group and acyl chain packing densities of the outer leaflet is the major effect of the peptides on the negatively charged phospholipid membranes investigated in this work, which is in agreement with previous studies , . interestingly, sars fp shows higher membrane fusion activity in pure pg or in ps-or phosphatidylinositol (pi)-containing membranes, but not in zwitterionic ones . thus, ordering of the lipid head group seems to be an important structural change in the bilayer necessary to induce membrane fusion , . indeed, we found the same ordering effect on the head group of membranes containing negatively charged lipids for the membrane fusion promoter la and an opposite effect for lpc, a known fusion inhibitor. this ordering or condensation effect on the outer monolayer leads to a shrinkage of its surface area that compresses the inner leaflet. because of the mechanical coupling between the two leaflets in a lipid bilayer vesicle, the inner layer counteracts the compressive force exerted by the outer monolayer and therefore creates a nonuniform tension in the two leaflets, which redistributes the stress profile across the bilayer. as a result, a uniform membrane bending moment toward the condensed leaflet is induced, which ultimately generates negative (positive) membrane curvature if the outer (inner) monolayer is condensed . due to the properties of the stress profile across the bilayer and to the nature of the molecular interactions in the head group and acyl chain regions, the largest contribution to the bending moment arises from the ordering of the head group region . thus, the increase of s of dpptc in the outer leaflet of negatively charged lipid membranes promoted by the sars-cov fusion peptides could potentially induce negative membrane curvature (please see section si of supplementary information for further details). this negative curvature effect induced by bending moment has also been proposed as the putative membrane fusion mechanism of other class i fusion peptides such as those from influenza ha or hiv- gp , . another important aspect to consider is the inverse correlation between head group ordering and hydration of lipid bilayers . if an increased head group packing density leads to membrane dehydration, this effect may help to overcome the high hydration repulsive energy that arises from membrane surface-bound water molecules . prior to fusion, two apposed lipid bilayers must approach. when the distance between the approaching bilayers is within to nm, a high hydration repulsion arises and dominates the interactions between them , thus preventing the membranes to make contact and to proceed to fusion. therefore, molecules that have the ability to overcome this hydration barrier could succeed in promoting membrane fusion. bilayer dehydration can be accomplished by different mechanisms depending on the molecular nature of the fusogenic molecule [ ] [ ] [ ] . particularly, hiv- gp and influenza ha fusion peptides promote membrane dehydration by increasing the ordering of the lipid bilayers , . that conclusion was inferred, though, from the aforementioned s /dehydration correlation and not by actually measuring the water content inside the bilayer. our eseem experiments, on the other hand, have undoubtedly shown that partial membrane dehydration actually takes place upon binding of the sars-cov fusion peptides to popc/popg membranes. this finding also provided a direct evidence of the correlation between head group ordering and membrane dehydration. however, in contrast to our eseem data, guillén et al have suggested that sars fp increases the water penetration depth into zwitterionic and anionic large unilamellar vesicles . this conclusion was based on the analysis of the fluorescence decay of diphenylhexatriene (dph) embedded in membranes, whose multi-component lifetimes were shortened in the presence of the peptide. although the increase of water penetration depth is a possible interpretation for their results , since the quantum yield of dph decreases in water, probe location and orientation might also influence the fluorescence lifetime of dph in lipid bilayers. conflicting results in the literature have indicated there is no consensus yet regarding the exact location of dph in pure model membranes [ ] [ ] [ ] [ ] . particularly, konopásek and coworkers , have shown that the short-lived component of dph embedded in lipid bilayers originates from a probe population located at the membrane-water interface. therefore, the subnanosecond short-lived component of dph in the very ordered gel phase of dimyristoyl phosphatidylglycerol (dmpg) found by guillen et al. in the absence of sars fp could thus potentially be due to a shallower interfacial location of dph in the dmpg bilayer. thus, the decrease of the dph lifetime in the presence of sars fp found by guillen and coworkers could potentially stem from the contact of the fluorophore with water molecules at the membrane-water interface due to a reorientational distribution of dph in the bilayer upon the condensing effect induced by the peptide. lastly, it is important to emphasize that the alterations of the bilayer structure observed for sars fp and sars ifp take place at high peptide concentrations and thus are most likely due to a cooperative behavior of membrane-bound self-associated β -sheet peptides. in fact, both peptides present membrane fusion activity only at high peptide-to-lipid molar ratio and regular extended β -sheet aggregates are the most populated membrane-bound peptide conformation , , . peptide oligomerization in membranes as extended β -like aggregates seems to play an important role in peptide-induced membrane fusion . peptide self-association in small areas of the membrane surface might actually be important in the very first stages of the membrane fusion process . indeed, since a high amount of work is required to merge large surface areas of two lipid bilayers, lipid merging most likely proceeds through local points of contact , , . thus, bending moments promoting negative curvature and membrane dehydration may not only help to decrease the hydration barrier between the proximal leaflets of the approaching bilayers but also minimize the work necessary for merging the monolayers. the latter is a consequence of the possible formation of point-like membrane protrusions, also referred to as local 'nipples' , a critical step that theoretically precedes the formation of the intermediate hemifusion stalk , , . peptides change membrane curvature of non-bilayer lipids in opposing ways. accumulating evidence suggests that membrane fusion involves the formation of strongly curved lipid bilayers in the pre-fusion, intermediate, and post-fusion states , . it is therefore tempting to investigate the potential ability of fusion peptides to generate membrane curvature, since that information can help to elucidate the role played by those molecules in the viral membrane fusion process. dsc experiments with non-bilayer-forming lipids such as phosphatidylethanolamines (pe) have been successfully used to indirectly probe changes in the intrinsic spontaneous curvature properties of pe vesicles by a wide variety of peptides , , . shift of the l α -to-h ii transition temperature is an excellent indicator of lipid phase changes and curvature alterations . we found that both sars fp and sars ifp peptides have the ability to bend dipope lipid bilayers. while sars fp induces positive curvature, sars ifp causes opposing stresses on the membrane depending on the ionic strength: it promotes positive (negative) curvature strain at low (high) ionic strength. the capacity of the sars fusion peptides to generate different curvature stresses on pe vesicles might actually be important in the context of the whole sars-cov s -mediated membrane fusion. the small stressed protrusions formed in the pre-fusion state are characterized by lipid domains possessing positive curvature flanked by negatively-curved lipid patches that stabilize the local 'nipple' and allow for the establishment of close intermembrane contact , , . therefore, both peptides can hypothetically act in this early stage by stabilizing the two curved domains depending on the ionic strength of the environment and on the exposure to pe or to negatively charged lipids . membrane fusion proceeds with the formation of the so-called hemifusion intermediate or lipidic stalk, which is characterized by lipid mixing between the outer leaflets of the two apposed bilayers with the distal monolayers remaining unfused. the resultant mixed outer leaflet presents a high degree of negative curvature , . stabilization of this intrinsic negative curvature has been traditionally interpreted as the common role played by various fusion peptides in the viral membrane fusion process , . the hemifusion intermediate would then subsequently progress to the formation of the fusion pore state , , which is characterized by lipid mixing of both the outer and inner leaflets of the merged bilayers, thus establishing the opening of a pore that allows content mixing between the two apposed membranes. in this pore state, both monolayers present strong and opposite curvature strains , . based on our findings, the outer, negatively-curved monolayer of both the lipidic stalk and the fusion pore states may be stabilized by the sars fusion peptides depending on the lipid composition: sars fp and sars ifp could act in membranes containing anionic lipids, and sars ifp could play a major role in pe-rich membranes at high ionic strength. on the other hand, the intrinsic positive curvature of the inner leaflet of the pore state can be stabilized by sars fp in the presence of pe lipids. that is, the more fusogenic sars fp peptide can also support membrane fusion by stabilization of porous structures. experimental and computational studies have also indicated stabilization of the pore state by fusion peptides from influenza ha , and parainfluenza virus (piv ) f protein . thus, our findings imply that both sars fusion peptides can act at different stages of the fusion process to facilitate membrane fusion. putative sars-cov membrane fusion model. taken together, our results reveal a functional plasticity of sars fp and sars ifp in helping to promote membrane fusion. both peptides can act in the early and late stages of the membrane fusion reaction by changing three properties of lipid bilayers, namely spontaneous curvature, hydration, and lipid packing density. incorporation of our findings into the currently proposed membrane fusion model induced by class i viral fusion proteins yields the following. upon receptor binding, s domain of sars-cov spike glycoprotein undergoes a large conformational change that releases and exposes sars fp and sars ifp to interact with target membranes . both peptides insert into but not significantly disturb (our data and data in references , and ) the highly-ordered, zwitterionic outer leaflet of the plasma membrane bilayer . this binding process bridges viral and cell membranes and thus facilitates trimerization of other s subunits. trimers are, in general, the fusion-active oligomeric state of class i fusion proteins . as a result, a trimeric extended prehairpin conformation is formed. at this point, it is important to emphasize that the actual conformational state of the sars-cov fusion peptides remains elusive. sars fp and sars ifp adopt, respectively, a v-shaped and a linear helical conformation in dodecylphosphatidylcholine micelles , but have a high tendency to aggregate and to form intramolecular β -sheets and extended β -strands stabilized by intermolecular interactions in models of lipid bilayers as well as to adopt, in small fractions, α -helical and unordered structures , . in the context of the intact protein, however, the structure and oligomerization state of those peptide segments still need to be addressed, although it has been proposed that membrane-bound self-associated peptides may provide the major driving force for trimerization of the whole protein , . peptide binding, conformational change and possibly aggregation into the membrane may trigger s refolding into a trimeric hairpin conformation. as a result, a six-helix bundle would form, bringing not only viral and target membranes into close proximity, but also the internal fusion peptide (sars ifp ) and the pretransmembrane (sars ptm ) domain of the s subunit . due to the high hydration repulsion of the closely apposed lipid bilayers and the requirement for bending membranes to minimize areas of strong interbilayer repulsion , , displacement of water molecules from the membrane surface and changes in membrane curvature seem to be the prerequisites for allowing close intermembrane contact and subsequent formation of the high-energy hemifusion intermediate state. interaction of sars fp and sars ifp with pe or with negatively charged lipids contained either in the plasma (via nonendocytic pathway) or in the endosome (via endocytic pathway) membranes may be important for the formation of point-like protrusions or for stabilization of the hemifusion stalk . since anionic phospholipids are mostly located in the inner leaflet of the membrane, it would be possible that the action of lipid flippases and scramblases could be endorsed by the peptide perturbation on the outer leaflet of the plasma membrane . the major effects of the peptides at the pre-fusion state could be the following: induction of positive curvature on pe-rich membranes, as indicated by our dsc data; and membrane dehydration and induction of bending moment on the outer leaflet of bilayers comprised of anionic lipids, as suggested by our esr data. the latter effects may also be responsible for triggering stalk formation, which is further stabilized by exposure of pe on the outer leaflet to sar ifp . hemifusion could be further facilitated by membrane interaction of a loop peptide segment located in between hr and hr domains and by a possible heteroligomerization of sars ifp with sars ptm . juxtaposition of sars ifp and sars ptm leads to a synergistic and cooperative action of both peptides that causes membrane destabilization and further peptide insertion . exposure of sars fp to the inner leaflet of the merged viral and cell membranes could have a great impact in the post-fusion state. indeed, sars fp could act by promoting positive curvature and stabilizing the high positively-curved inner leaflet that characterizes the porous state, thus facilitating pore formation (our dsc data). however, the molecular details of the above processes still need to be investigated. overall, the two putative fusion peptides from sars-cov s protein may help to regulate membrane fusion by acting in the early and late stages of the membrane fusion process. our main findings were: ( ) sars fusion peptides increase the ordering of the headgroup and acyl chain regions of mlvs containing negatively-charged, but not zwitterionic phospholipids; ( ) membrane fusion promoters induce similar effects on the head group ordering than do the fusion peptides, whereas membrane fusion inhibitors cause opposing effects; ( ) changes in the order parameters of the lipids are generally greater for the more fusogenic sars fp peptide than for sars ifp ; ( ) both peptides promote dehydration of pg-containing membranes and this effect is well correlated with the increased head group ordering; and ( ) dsc data support a hypothesis that sars fp induces positive curvature on dipope vesicles, whereas sars ifp promotes opposing stresses on the intrinsic negative curvature of dipope depending on the ionic strength. peptide-induced chain-packing energy and membrane surface ordering of the outer leaflet of negatively charged lipid bilayers promote partial membrane dehydration and could generate bending moment, as suggested by our esr studies. both effects may induce negative curvature and decrease the hydration repulsion of apposed bilayers. possible peptide involvement on the formation of the pre-fusion point-like protrusions and intermediate hemifusion stalk as well as on the stabilization of the fusion pore state suggest that the sars fusion peptides might play important roles in the whole membrane fusion process. taken together, our findings suggest that the sars fusion peptides have the ability to change the physicochemical properties of model membranes depending on the lipid composition and on the ionic strength. therefore, they can act in the early and late stages of the membrane fusion process, conferring them a functional plasticity that might be important to help overcome the high kinetic barrier involved in the sars-cov-induced membrane fusion. materials. n-terminally acetylated and c-terminally amidated sars fp ( mwktptlkyfggfnfsqil ) and sars ifp ( gaalqipfamqmayrf ) peptides were either purchased from genscript (piscataway township, nj) or manually synthesized according to the standard fmoc solid-phase peptide synthesis method on a rink-amide resin . the details of peptide synthesis are described in vicente et al. . purification was performed as described in supplementary section si . the phospholipids -palmitoyl- -hydroxy-sn-glycero- -phosphocholine (lpc), , -dipalmitoyl-snglycero-phosphatidylcholine (dppc), , -dipalmitoyl-sn-glycero- -phospho-( '-rac-glycerol) (dppg), , -dipalmitoyl-sn-glycero- -phospho-l-serine (dpps), , -dipalmitoleoyl-sn-glycero- -phosphoethanolamine (dipope), -palmitoyl- -oleoyl-sn-glycero- -phosphocholine (popc), -palmitoyl- -oleoyl-sn-glycero- -phospho-( '-rac-glycerol) (popg), -palmitoyl- -oleoyl-sn-glycero- -phospho-l-serine (pops), -palmitoyl- -oleoyl-sn-glycero- -phosphate (popa), and the spin labels -palmitoyl- -stearoyl(n-doxyl)-snglycero- -phosphocholine (n-pcsl, where n = and ), , -dioleoyl-sn-glycero- -phospho(tempo)choline (doptc), and , -dipalmitoyl-sn-glycero- -phospho(tempo)choline (dpptc) were purchased from avanti polar lipids, inc. (alabaster, al). cholesterol (chol) and linoleic acid (la) were obtained from sigma-aldrich (st. louis, mo). all reagents were used without further purification. sample preparation. phospholipids ( . mg for dsc and . mg for esr) and spin labels ( . mol% for cw esr and mol% for pulsed esr) either in chloroform or chloroform/methanol : (v/v) stock solutions were mixed in a glass tube. after dried under a n flow, the lipid film was ultracentrifuged under vacuum overnight to remove traces of solvent. for cw esr experiments, the sample was hydrated in mm potassium phosphate buffer, ph . , sonicated in a bath type sonicator for a few seconds and maintained at a temperature above the main phase transition of the lipid for at least two hours for complete hydration. samples were then subjected to at least six freeze-thaw cycles. a measured volume of sars fp or sars ifp stock solutions in dimethyl sulfoxide (dmso) was added to the preformed multilamellar lipid dispersions. for dsc experiments, peptides dissolved in either acetonitrile/water : (v/v) or in dmso solutions were diluted into buffer and added to the lipid film for hydration. samples were vortexed for few seconds, maintained at a temperature above the phase transition for each lipid during at least min, and subjected to six freeze-thaw cycles. the amount of phospholipid (final lipid concentration of mg/ml for esr and mg/ml for dsc) and peptides used provided a : lipid/peptide molar ratio for most of the experiments. it is worth mentioning that the same amount of dmso or acetonitrile/water : added in the peptide-containing samples was also used in the peptide-free samples as controls for the esr and dsc experiments. the control samples were prepared using the same protocol as those of the peptide-containing samples. for dipope/peptide samples, peptides and lipids dissolved in chloroform/methanol : (v/v) stock solutions were mixed in a glass tube, dried to a lipid film under n gas and lyophilized overnight. samples were hydrated in mm sodium phosphate buffer, ph . , with or without mm sodium chloride, and freeze-thaw cycled six times below the liquid crystalline-to-inverted hexagonal (lα -h ii ) phase transition temperature (t h ) of the lipid. dipope concentration was mg/ml and peptide concentration varied from . to . mol% ( : and : lipid/peptide molar ratio, respectively). for eseem experiments, popc/popg : mol/mol and peptides ( : lipid/peptide molar ratio) were prepared as above, but hydrated in mm sodium phosphate, mm nacl d o buffer, pd = . (actual ph measurement). peptide concentration was confirmed spectrophotometrically by using the theoretical molar extinction coefficients of , m − cm − for sars fp esr experiments. cw-esr experiments were carried out on a varian e- spectrometer operating at . ghz. temperature was controlled by a homemade temperature control unit coupled to the spectrometer, whose accuracy is about . °c. samples were transferred to glass capillaries ( . mm i.d.), which were set into a quartz tube containing a mineral oil bath to help stabilize the sample temperature. the following acquisition parameters were used: center field, , g; scan width, to g; modulation amplitude, . or . g; modulation frequency, khz; microwave power, or mw; time constant, ms, and acquisition time, s. nonlinear least-squares simulations (nlls) of the cw-esr spectra were performed using the multicomponent labview (national instruments) software developed by dr. christian altenbach (university of california, los angeles, california) , . the rotational diffusion rates (r ⊥ , r ∥ ) and order parameters (s , s ) were obtained as described in ref. with further details in the section si of supplementary information. seed values for the magnetic parameters of both -pcsl and -pcsl were obtained from earle et al. and those of dpptc were taken from ge and freed . the strategy of the nlls simulation was performed as described elsewhere . pulsed esr experiments were performed on a bruker elexsys x-band pulsed esr spectrometer equipped with the bruker flexline er x-ms split-ring resonator and the itc oxford cryogenic system for temperature control. samples were immersed into liquid nitrogen prior to the measurements at k. three pulse electron spin echo envelope modulation (eseem) experiments were carried out with the π / -τπ / -t -π / -τecho pulse sequence and using a four-step phase cycling to suppress unwanted echoes . the microwave power was adjusted to give ns π / pulses and an interpulse delay τ of ns, kept constant in all experiments, was chosen to maximize deuterium modulations at the magnetic field where the echo intensity is maximum. starting at time delay t = ns, points were recorded with Δ t = ns steps to obtain the three-pulse stimulated echo decays. the integration gate length was ns and the shot repetition time was , μ s. the number of accumulations varied from to depending on the signal-to-noise ratio and on the modulation depth. data analysis was performed as described in bartucci et al. . briefly, the contribution of the spin relaxation to the eseem signal was eliminated by dividing the time-dependent echo amplitudes, v(τ , t), by a bi-exponential decay, 〈 v(τ , t)〉 , followed by subtraction of unity, as v norm (τ , t) = v(τ , t)/〈 v(τ , t)〉 − . the remained oscillations about zero were apodized with a hamming window and zero-filled to increase the total number of points to about k. numerical fourier transformation was performed and the resultant magnitude spectrum was multiplied by the dwell time Δ t = ns to provide a spectral density in ns units. sars -beginning to understand a new virus characterization of a novel coronavirus associated with severe acute respiratory syndrome angiotensin-converting enzyme is a functional receptor for the sars coronavirus cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus the sars-cov s glycoprotein: expression and functional characterization sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway the life cycle of sars coronavirus in vero e cells the spike protein of severe acute respiratory syndrome (sars) is cleaved in virus infected vero-e cells furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry mechanisms of coronavirus cell entry mediated by the viral spike protein structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s fusion protein roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein s domain with characteristics of a viral fusion peptide identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein viral membrane fusion the structural biology of type i viral membrane fusion mechanisms of virus membrane fusion proteins virus membrane-fusion proteins: more than one way to make a hairpin structure and function of membrane fusion peptides are fusion peptides a good model to study viral cell fusion? structural and dynamic characterization of the interaction of the putative fusion peptide of the s sars-cov virus protein with lipid membranes a second sars-cov s glycoprotein internal membrane-active peptide. biophysical characterization and membrane interaction nonlinear-least-squares analysis of slow-motion epr spectra in one and two dimensions using a modified levenberg-marquardt algorithm a differential scanning calorimetric study of the thermotropic phase behavior of model membranes composed of phosphatidylcholines containing linear saturated fatty acyl chains calorimetric and spectroscopic studies of the thermotropic phase behavior of lipid bilayer model membranes composed of a homologous series of linear saturated phosphatidylserines interaction of poly(l-arginine) with negatively charged dppg membranes: calorimetric and monolayer studies interaction of the c domain from protein kinase c epsilon with model membranes network formation of lipid membranes: triggering structural transitions by chain melting some aspects of the phase behavior of charged lipids lipid bilayer pre-transition as the beginning of the melting process membrane fusion between liposomes composed of acidic phospholipids and neutral phospholipids induced by melittin: a differential scanning calorimetric study effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylethanolamine bilayers: differential scanning calorimetric and fourier transform infrared spectroscopic studies nature of the thermal pretransition of synthetic phospholipids: dimyristolyl-and dipalmitoyllecithin thermal analysis of lipids, proteins and biological membranes. a review and summary of some recent studies molecular view of the role of fusion peptides in promoting positive membrane curvature fusion peptides and the mechanism of viral fusion stability of lyotropic phases with curved interfaces the mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms reciprocal effects of apolipoprotein and lytic peptide analogs on membranes. cross-sectional molecular shapes of amphipathic alpha helixes control membrane stability interactions of the antimalarial amodiaquine with lipid model membranes the two sides of a lipid-protein story -ghz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes polarity profiles in oriented and dispersed phosphatidylcholine bilayers are different: an electron spin resonance study fusion peptide from influenza hemagglutinin increases membrane surface order: an electron-spin resonance study hiv gp fusion peptide increases membrane ordering in a cholesterol-dependent fashion chain configuration and flexibility gradient in phospholipid membranes -comparison between spin-label electron spin resonance and deuteron nuclear magnetic resonance, and identification of new conformations an electron-spin-resonance study of interactions between phosphatidylcholine and phosphatidylserine in oriented membranes inhibition of membrane fusion by lysophosphatidylcholine lysophosphatidylcholine inhibits vesicles fusion induced by the nh -terminal extremity of siv/hiv fusogenic proteins control of baculovirus gp -induced syncytium formation by membrane lipid composition hydration, structure, and molecular interactions in the headgroup region of dioleoylphosphatidylcholine bilayers: an electron spin resonance study water concentration profiles in membranes measured by eseem of spin-labeled lipids glycerol penetration profile in phospholipid bilayers measured by eseem of spin-labelled lipids eseem measurements of local water concentration in d( ) o-containing spin-labeled systems membrane-sugar interactions probed by pulsed electron paramagnetic resonance of spin labels investigation of model membrane disruption mechanism by melittin using pulse electron paramagnetic resonance spectroscopy and cryogenic transmission electron microscopy electron spin-echo envelope modulation (eseem) reveals water and phosphate interactions with the kcsa potassium channel water penetration profile at the protein-lipid interface in na, k-atpase membranes structure of lipid bilayers intramembrane water associated with toac spin-labeled alamethicin: electron spin-echo envelope modulation by d o structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains a d-eldor study of the liquid ordered phase in multilamellar vesicle membranes the influenza hemagglutinin fusion domain is an amphipathic helical hairpin that functions by inducing membrane curvature the atomic structure of the hiv- gp transmembrane domain and its connection to the immunogenic membrane-proximal external region order and disorder control the functional rearrangement of influenza hemagglutinin viral fusion protein transmembrane domain adopts beta-strand structure to facilitate membrane topological changes for virus-cell fusion real-time intermembrane force measurements and imaging of lipid domain morphology during hemifusion hiv gp -mediated membrane fusion occurs at edges of cholesterol-rich lipid domains identification of the membrane-active regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using a / -mer peptide scan: implications for the viral fusion mechanism interaction of a peptide from the pre-transmembrane domain of the severe acute respiratory syndrome coronavirus spike protein with phospholipid membranes membrane insertion of the three main membranotropic sequences from sars-cov s glycoprotein interaction of a peptide corresponding to the loop domain of the s sars-cov virus protein with model membranes viral fusion peptides: a tool set to disrupt and connect biological membranes intermolecular and surface forces mechanics and thermodynamics of biomembranes hydration forces between phospholipid bilayers membrane fusion: overcoming of the hydration barrier and local restructuring modulation of poly(ethylene glycol)-induced fusion by membrane hydration: importance of interbilayer separation thermodynamic and structural properties of phosphatidylserine bilayer membranes in the presence of lithium ions and protons a mechanism of divalent ion-induced phosphatidylserine membrane fusion fluorescence techniques for probing water penetration into lipid bilayers location of diphenylhexatriene (dph) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth recent developments in molecular dynamics simulations of fluorescent membrane probes the origin of the diphenylhexatriene short lifetime component in membranes and solvents short-lived fluorescence component of dph reports on lipid-water interface of biological membranes mechanics of membrane fusion induction of negative curvature as a mechanism of cell toxicity by amyloidogenic peptides: the case of islet amyloid polypeptide induction of non-lamellar lipid phases by antimicrobial peptides: a potential link to mode of action membrane lipids: where they are and how they behave wild-type and mutant hemagglutinin fusion peptides alter bilayer structure as well as kinetics and activation thermodynamics of stalk and pore formation differently: mechanistic implications conformation and lipid interaction of the fusion peptide of the paramyxovirus piv in anionic and negative-curvature membranes from solid-state nmr nmr structures and localization of the potential fusion peptides and the pre-transmembrane region of sars-cov: implications in membrane fusion tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of sars-cov spike protein protein-lipid interplay in fusion and fission of biological membranes biophysical properties of lipids and dynamic membranes synthesis of the antibacterial peptide cecropin-a( - ) n-terminal microdomain peptide from human dihydroorotate dehydrogenase: structure and model membrane interactions labview programs for the analysis of epr data polarity profiles in oriented and dispersed phosphatidylcholine bilayers are different -an electron spin resonance study elimination of unwanted echoes and reduction of dead time in three-pulse electron spin-echo spectroscopy key: cord- -ldkuwcc authors: he, hui-qiong; ye, richard d. title: the formyl peptide receptors: diversity of ligands and mechanism for recognition date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: ldkuwcc the formyl peptide receptors (fprs) are g protein-coupled receptors that transduce chemotactic signals in phagocytes and mediate host-defense as well as inflammatory responses including cell adhesion, directed migration, granule release and superoxide production. in recent years, the cellular distribution and biological functions of fprs have expanded to include additional roles in homeostasis of organ functions and modulation of inflammation. in a prototype, fprs recognize peptides containing n-formylated methionine such as those produced in bacteria and mitochondria, thereby serving as pattern recognition receptors. the repertoire of fpr ligands, however, has expanded rapidly to include not only n-formyl peptides from microbes but also non-formyl peptides of microbial and host origins, synthetic small molecules and an eicosanoid. how these chemically diverse ligands are recognized by the three human fprs (fpr , fpr and fpr ) and their murine equivalents is largely unclear. in the absence of crystal structures for the fprs, site-directed mutagenesis, computer-aided ligand docking and structural simulation have led to the identification of amino acids within fpr and fpr that interact with several formyl peptides. this review article summarizes the progress made in the understanding of fpr ligand diversity as well as ligand recognition mechanisms used by these receptors. the formyl peptide receptors (fprs) are a group of g protein-coupled chemoattractant receptors that play important roles in host defense and inflammation [ , ] . they are so named because their cognate agonists are peptides bearing formylated methionine (fmet), such as those derived from bacterial and mitochondrial proteins [ , ] . the present review focuses on the fpr family members in humans and mice and their interaction with a wide variety of ligands. the reader is referred to recently published reviews on fpr signaling [ ] , synthetic non-peptide ligands for fprs [ ] and characterization of fpr knockout mice [ ] . the reader is also referred to a general overview of fprs and their nomenclature [ ] . three genes coding for human g protein-coupled formyl peptide receptors have been cloned, including fpr , fpr and fpr [ ] [ ] [ ] [ ] [ ] [ ] . among them, fpr and fpr share an overall high sequence identity ( figure ) and certain overlapping functions. [ ] in three fpr isoforms are shown in color; (b) schematic diagram for the location of the positive selection sites in the formyl peptide receptors, based on the sequence of human fpr . the amino acids at these positions in each of the three receptors are marked on the right. fpr and fpr are expressed in both monocytes and neutrophils, while fpr is found in monocytes but not neutrophils. besides myeloid cells, fpr is expressed in astrocytes, microglial cells, hepatocytes and immature dendritic cells. fpr shows an even wider distribution pattern than fpr and is expressed in a variety of non-myeloid cells including astrocytoma cells, epithelial cells, hepatocytes, microvascular endothelial cells, neuroblastoma cells, in addition to phagocytic leukocytes. in recent studies, fpr has been associated with anti-bacterial inflammation and metastasis of malignant glioma cells, while fpr is implicated in the pathogenesis of chronic inflammatory diseases such as systemic amyloidosis, alzheimer's disease, atherosclerosis, systemic lupus erythematosus and ovarian cancer metastasis et al [ ] [ ] [ ] [ ] [ ] . more examples and details of fpr involvement in multiple diseases are provided in recent reviews [ , ] . since the human fpr gene (fpr ) was first cloned, orthologs have been identified in other mammalian species, including rabbits, guinea pigs, horses, rats and mice (reviewed in [ ] ). the genes coding for fpr members comprise a large family that implicates a complex evolutionary path with evidence of positive selection [ ] . despite overall sequence homology, these genes vary considerably in their numbers and sequences between humans and other species. for example, the mouse fpr gene family has eight known members (mfpr , mfpr , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs and mfpr-rs ) clustered on mouse chromosome a . . among these genes, mfpr-rs (ψmfpr-rs ) is found as a pseudogene that does not code for a functional receptor. although several mouse fpr genes are present in neutrophils, work on mfprs has been focused mostly on the gene products of mfpr and mfpr , which are widely expressed in mouse phagocytic leukocytes and show high similarity to their human counterparts. targeted deletion of mfpr or mfpr renders mice more susceptible to bacterial infection without changing their viability and fertility [ , ] . under unstimulated conditions, mice lacking mfpr or mfpr behave normally. however, in models of human diseases, both mfpr −/− and mfpr −/− mice respond differently from their wildtype littermates, [ ] in three fpr isoforms are shown in color; (b) schematic diagram for the location of the positive selection sites in the formyl peptide receptors, based on the sequence of human fpr . the amino acids at these positions in each of the three receptors are marked on the right. fpr and fpr are expressed in both monocytes and neutrophils, while fpr is found in monocytes but not neutrophils. besides myeloid cells, fpr is expressed in astrocytes, microglial cells, hepatocytes and immature dendritic cells. fpr shows an even wider distribution pattern than fpr and is expressed in a variety of non-myeloid cells including astrocytoma cells, epithelial cells, hepatocytes, microvascular endothelial cells, neuroblastoma cells, in addition to phagocytic leukocytes. in recent studies, fpr has been associated with anti-bacterial inflammation and metastasis of malignant glioma cells, while fpr is implicated in the pathogenesis of chronic inflammatory diseases such as systemic amyloidosis, alzheimer's disease, atherosclerosis, systemic lupus erythematosus and ovarian cancer metastasis et al. [ ] [ ] [ ] [ ] [ ] . more examples and details of fpr involvement in multiple diseases are provided in recent reviews [ , ] . since the human fpr gene (fpr ) was first cloned, orthologs have been identified in other mammalian species, including rabbits, guinea pigs, horses, rats and mice (reviewed in [ ] ). the genes coding for fpr members comprise a large family that implicates a complex evolutionary path with evidence of positive selection [ ] . despite overall sequence homology, these genes vary considerably in their numbers and sequences between humans and other species. for example, the mouse fpr gene family has eight known members (mfpr , mfpr , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs and mfpr-rs ) clustered on mouse chromosome a . . among these genes, mfpr-rs (ψmfpr-rs ) is found as a pseudogene that does not code for a functional receptor. although several mouse fpr genes are present in neutrophils, work on mfprs has been focused mostly on the gene products of mfpr and mfpr , which are widely expressed in mouse phagocytic leukocytes and show high similarity to their human counterparts. targeted deletion of mfpr or mfpr renders mice more susceptible to bacterial infection without changing their viability and fertility [ , ] . under unstimulated conditions, mice lacking mfpr or mfpr behave normally. however, in models of human diseases, both mfpr −/− and mfpr −/− mice respond differently from their wildtype littermates, indicating regulatory roles of these receptors in host defense and inflammation. targeted gene disruption of mfpr also suggested a homeostatic role for the mfpr in the production of glucocorticoids and anxiety-like behavior [ ] . as the suggested ortholog of human fpr , mfpr was identified as a low-affinity receptor for the classical formyl tri-peptide fmet-leu-phe (fmlf) [ ] , but this receptor responds well to several n-formyl peptides derived from other bacteria (e.g., fmifl, fmivil, fmivtlf) and mitochondria (e.g., fmmyalf) [ , ] . in general, mfpr is not a high-affinity receptor for either fmlf or other native formyl peptides tested so far [ ] . however, it responds to endogenous peptide agonists for fpr , including the amyloidogenic proteins saa (serum amyloid a) [ ] and aβ [ ] . likewise, this receptor can be inhibited by fpr -specific antagonists when expressed in neutrophil or by stably transfected cells. besides this, mouse mfpr has also been reported as a receptor for f l [ ] that is a potent agonist for human fpr [ ] . published reports indicate that other members of the murine mfpr family are not coding for stereotypic formyl peptide receptors. in recent studies, mfpr-rs (ψmfpr-rs ) has been characterized as a constitutively expressed gene associated with mouse longevity [ ] . the gene products of five other mfprs, including mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs , and mfpr-rs , are recently described as mouse vomeronasal sensory receptors [ , ] . phylogenetic analysis suggested that they belong to a chemosensory gpcr family, which is characterized with an olfactory function and has the ability to identify pathogenic states. among them, the property of mfpr-rs is still uncertain as it is also expressed in neutrophils. it was believed that mfpr-rs might have some functional overlap with human fpr because a variant of mfpr-rs was reported to encode a mouse receptor for lipoxin a (lxa ) [ ] , an eicosanoid ligand for fpr . however, functional and pharmacology assays conducted in stably transfected cells suggested that mfpr-rs responds poorly to most agonists that activate human and murine fpr receptors [ ] . these observations add to studies using knockout animal models and together indicate that murine fprs (especially mfpr and mfpr ) share numerous structural and pharmacological properties with human fprs, but the evolutionary correlation between them is by no means linear. the fpr family is well known for the structural diversity of their ligands, including a variety of ligands with different chemical properties and origins ranging from natural peptides to synthetic non-peptide compounds [ , ] (table ) . fprs are therefore classified as a group of pattern recognition receptors (prrs) that recognize pathogen-associated molecular patterns (pamps) and damage-associated molecular pattern (damps) [ ] [ ] [ ] . among all the ligands for the fprs, n-formylated peptides, particularly fmlf (an e. coli-derived chemotactic peptide), are most often studied. fmlf is among the first characterized and also the shortest formyl peptides with full agonistic activities [ ] [ ] [ ] . these peptides are initiated with n-formyl methionine and are generally cleavage products of bacterial and mitochondrial proteins. n-formylated peptides constitute the most commonly studied class of fpr agonists that trigger a variety of biological activities in myeloid cells, such as chemokinesis, chemotaxis, calcium flux, cytokine production and superoxide anion generation. through binding with the high affinity receptor fpr , fmlf and other n-formylated peptides serve as potent chemoattractants, which also include activated complements (c a, c a) and chemokines, in recruiting and guiding leukocytes to the site of bacterial infection and to damaged tissues. a wealth of literatures have described the proinflammatory properties of n-formyl peptides. as early as years ago, right after the discovery of n-formyl peptides, studies have shown that fmlf was involved in the pathogenesis of multiple inflammatory diseases such as colitis [ ] [ ] [ ] , pouchitis, ulcerative colitis and crohn's disease [ , ] and juvenile peridotitis [ ] . studies also reported that inhalation or injection of fmlf can cause rapid neutropenia and bronchial inflammation in human and other mammals [ ] [ ] [ ] [ ] . [ ] f. n-phenylureas ag- [ ] fpr > fpr [ , ] e. pyridazin- ( h)-one derivative [ ] molecules , , of [ ] [ ] human fpr is a low affinity receptor for fmlf and many potent formyl peptide agonists for fpr . however, fpr displays relatively high affinity for n-formylated peptides of specific composition and longer length. for instance, fpr responds better to peptides carrying positive charges at the c-terminus (e.g., fmlfk, fmlfik) than peptides with negative charges (e.g., fmlfe and fmlf) [ ] . studies also showed that fpr responds well to formyl peptides of microbial origin other than e. coli, such as fmifl (s. aureus) and fmivil (l. monocytogenes) in calcium mobilization and camp release assays [ ] . in general, most bacteria-derived formyl peptides are more potent at fpr than fpr . a prominent exception is the recently described cytolytic peptides psm (phenol-soluble modulins) that are α-helical peptides composed of - amino acids [ ] . although they are predominantly secreted from community-associated methicillin resistant staphylococcus aureus (ca-mrsa) in formylated form, psmα peptides are more selective for fpr than for fpr [ ] [ ] [ ] . another exception is mitocryptide- , a neutrophil-activating formyl peptide produced from mitochondrial cytochromes. mitocryptide- can directly bind to fpr and activate it with an ec of . nm in calcium flux assay, whereas it manifests no interaction with fpr [ ] . in addition, another group of formyl peptides derived from mitochondria (fmmyalf, fmyfiniltl, and fmlkliv) were found equally potent on fpr and fpr with ec values ranging from nm to nm [ ] . despite the different potency and selectivity of these formyl peptides, the n-formyl group is important for optimal interaction with fpr and fpr . it is suggested that fpr is a highly effective receptor recognizing formyl peptides and mediating phagocyte functions, while fpr has an overall lower affinity but it can discriminate between formyl peptides with different sizes and features. in contrast, no bacterial or mitochondrial formyl peptide has been described as agonist for fpr by far. noting that the prototypic fpr agonist fmlf is not a potent activator for murine fprs, including mfpr and mfpr , when compared to human fpr . in agreement with the research that murine neutrophils are more susceptible to bacteria other than e. coli [ ] , mfpr was found more responsive to formyl peptides derived from listeria monocytogenes (fmivtlf), staphylococcus aureus (fmifl), and mitochondria (fmmyalf) [ , ] . except for formyl peptides, a number of different peptides/proteins, including microbial and host-derived non-formyl peptides/proteins and peptides from synthetic libraries, have been identified to be agonists for fprs. it appears that the absence of n-formyl group and lack of overall sequence similarity with formyl peptides mentioned above do not impair their agonistic activity at fprs. in general, the ligand diversity is more prominent for fpr than fpr . the non-formylated peptides/proteins derived from microbe are represented by virus envelope proteins (gp and gp ) of the human immunodeficiency virus type (hiv- ), which contains at least five fpr-recognizing peptide sequences composed of - amino acids. most of these sequences were found more potent at fpr than at fpr and fpr [ ] [ ] [ ] [ ] [ ] , except only one is selective for fpr and mfpr [ , , ] . a cecropin-like peptide (hp - ) from helicobacter pylori was also identified as an agonist for fprs [ , ] . the studies demonstrated that in h. pylori infection, hp - was able to induce chemotaxis of monocytes and basophils, and evoke superoxide release via signaling through fpr and fpr . recently, two antimicrobial peptides (amps) isolated from scolopendra subspinipes mutilans were reported to elicit neutrophil chemotactic migration through fpr [ ] . the host-derived non-formyl peptide agonists for fprs are generally peptides and proteins associated with human diseases and inflammation. this group of agonists activates fprs independently of the n-formyl group and they also show a preference for fpr . a prominent example of them is serum amyloid a (saa), an acute-phase protein that is implicated in chronic inflammation and amyloidosis. as the first identified endogenous peptide agonist for fpr [ ] , documented studies have shown that it exerts pro-inflammatory activities through fpr in phagocytes, epithelial cells and t lymphocytes, including stimulating production of inflammatory mediators and enhancing the expression of cytokine receptors [ ] [ ] [ ] [ ] [ ] . recently, questions have been raised regarding the proinflammatory cytokine-like properties of native saa, since most studies of saa and fpr are performed using a commercially available recombinant protein that contains amino acid substitutions of saa in saa [ , , ] . however, research conducted with saa isoforms purified from an e. coli expression system suggested that major saa isoforms have cytokine-inducing activity and minor substitutions of amino acids affected their potency at fpr [ ] . in vivo studies have shown th -induction activity of saa in gut epithelium, although whether these activities are attributed to fpr or other saa receptors remain unclear [ , ] . another amyloidogenic disease-associated fpr agonist is the prion protein fragment prp , which was reported to interact with fpr in glial cells and induce calcium mobilization, chemotaxis as well as production of pro-inflammatory cytokines [ ] . in addition to saa and prp , other two amyloidogenic disease-associated peptides were found to be agonists for fpr : the -amino acid form of aβ amyloid peptide (aβ ) and humanin. although both peptides use fpr to induce migration and activation of monocytic phagocytes in the brain, aβ and humanin display divergent roles in the development of alzheimer's disease: aβ is a major cause of fibrillary formation and deposition in brain of ad patients [ , ] , whereas humanin is neuroprotective on the contrary [ ] . studies suggested that humanin reduces aggregation and fibrillary formation by inhibiting aβ /fpr interaction in mononuclear phagocytes [ ] . fpr and mfpr are also functional receptors for humanin in humans and mice, respectively [ , ] . annexin a , a glucocorticoid-regulated protein, and its n-terminal peptides (ac - and ac - ) are fpr agonists that have dual roles in inflammation. at high concentrations, the annexin a peptides fully activate fpr like the conventional agonists that induce pro-inflammatory responses. in contrast, at low concentrations they only show partial activity at fpr [ ] , leading to neutrophil desensitization and inhibiting neutrophil migration induced by other chemoattractants. all three fpr members are implicated in the various functions of annexin a peptides. some researchers thought that these peptides use fpr for anti-inflammatory actions [ ] , while others suggested the presence of receptors other than fpr and fpr as being responsible for the resolving effects [ ] . the exact mechanism for the pro-/anti-inflammatory activity of annexin a peptides is not entirely clear. the antimicrobial peptide ll- and its murine homolog cramp (cathelicidin-related anti-microbial peptide) were identified as fpr agonists [ , ] . ll- is a cleavage product of the neutrophil granule protein cathelicidin found in leukocytes and epithelial cells. as a pro-inflammatory mediator, ll- activates fpr and evokes superoxide generation in human fibroblasts [ ] . it also induces directional migration of human monocytes, neutrophils, and t lymphocytes [ ] . however, ll- also inhibits saa signaling in human neutrophils, including il- production and chemotaxis through fpr [ ] . ll- seems to be a pleiotropic peptide and its interaction with fpr is implicated in wound healing, cell proliferation [ ] , angiogenesis [ ] and anti-or pro-tumorigenesis [ ] [ ] [ ] . in a recent study, the proinflammatory circuits of ll- and leukotriene b was reported. in human neutrophils, ll- /fpr promotes leukotriene b production, which in turn elicits further ll- release through blt (leukotriene b receptor ) [ ] . upar is the cell surface receptor for urokinase-type plasminogen activator (upa) that is a serine protease involved in regulation of fibrinolysis, cell adhesion, migration and tissue repair. upar can be cleaved by different proteases, including upa, generating soluble upar fragments that can be recognized by different fpr members. for instance, upar d d - , a cleaved peptide corresponding to residues from to of upar, binds to and activates fpr in monocytes, inducing cell migration [ ] . moreover, upar - ( srsry ) interacts with fpr [ ] , while upar [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] activates both fpr and fpr in basophils [ ] . in a more recent study, a new host-derived chemotaxis agonist fam d, for the fpr and fpr was identified [ ] . fam d, a cytokine-like protein, is constitutively expressed in the gastrointestinal tract with significant induction in dextran sulfate sodium (dss)-induced colitis [ ] . fam d was found to strongly attract the chemotaxis of human peripheral blood neutrophils and monocytes. through functional screen of a group of chemoattractant receptors, fpr and fpr were determined to be the receptors for fam d. in hek cells transfected to express fpr or fpr , fam d induced significant cell chemotaxis and calcium mobilization that was desensitized by other fpr agonists (fmlf and wkymvm), and vice versa. the activation of erk / and p mapk signaling caused by fam d in neutrophils was blocked by an antagonist of fpr (wrw ). this study suggested that fam d/fpr interaction might play a role in intestinal homeostasis and gastrointestinal inflammation. the fpr receptors also have other endogenous peptide ligands, such as cathepsin g for fpr , chemokine ccl (amino acids and its n-terminal fragment shaagtide for fpr , a vasoactive intestinal neuropeptide vip (vasoactive intestinal polypeptide) for fpr , and a heme-binding protein fragment f l for fpr (table ). in addition, exogenous allergens including house dust mite and birch pollen extracts were recently shown as being agonistic for fpr and fpr . the detailed properties of these fpr agonists are discussed in previous reviews [ , , ] . among all the synthetic peptide agonists for fprs, a hexapeptide, trp-lys-tyr-met-val-d-met-nh (wkymvm) isolated from a random peptide library [ ] , was identified to be a strong activator for fpr , fpr and fpr [ , ] . wkymvm conjugated with fitc in the second residue lys was shown slightly more efficacious in binding to fpr than to fpr [ ] . wkymvm is by far the most potent peptide agonist for fpr , being able to activate fpr at picomolar concentrations in chemotaxis assays. wkymvm, a derivative of wkymvm with the substitution of l-methionine at the carboxyl terminus, becomes highly selective for fpr and is weakly agonistic for fpr [ ] . another peptide identified from library screen, mmk- (lesifrsllfrvm), is a highly selective chemotactic agonist for fpr [ , ] . cgen- a, a amino acids anti-inflammatory peptide obtained from a computational platform, was identified to be agonist for fpr and fpr [ ] . in another study, a tethered library was screened and a peptide with the sequence mmwll was identified as an fpr agonist [ ] . this peptide becomes -fold more potent when the first met is n-formylated, consistent with the preferential recognition of fmet-containing peptides by fpr . in a recent study [ ] , a series of aapeptides, a class of peptidomimetics based on n-acylated-n-aminoethyl amino acid residues, were designed and synthesized to mimic the structure and function of the prototypic tripeptide fmlf, the reference agonist of fpr . three of these fmlf-mimicking aapeptides were found effective in activating fpr-expressing cells in calcium mobilization assay, albeit at concentrations above µm. certain aapeptides were shown to be more efficacious than fmlf at a higher concentration ( µm). similar to fmlf, these aapeptides have receptor preference for fpr over fpr . in comparison with peptide/protein fpr agonists, the small molecule agonists screened from chemical library are more stable and highly selective for certain fpr members. these properties make them valuable tools for detailed characterization of fprs, which is important for a better understanding of the complex role of fprs in vivo. fprs are involved in a variety of signaling systems and their distribution and functions are not limited to the immune system. as a result, these small molecules screened by targeting fprs as agonists or antagonists are expected to serve as potentially useful agents in therapeutic treatment. the first reported non-peptide agonist for fpr from library screening is a quinazolinone derivative named quin-c [ ] . quin-c ( -butoxy-n-[ -( -methoxy-phenyl)- -oxo- , -dihydro- h-quinazolin- yl]-benzamide) was found highly selective for fpr as opposed to fpr . it was identified as a biased agonist, as it stimulated calcium mobilization through fpr but did not induce substantial neutrophil superoxide generation, even at concentrations up to µm. a later study revealed an anti-inflammatory role for quin-c in bleomycin-induced lung injury in a mouse model [ ] . the murine receptors for quin-c were determined to be mfpr and mfpr [ ] . competitive binding analysis showed that quin-c does not share the same recognition sites with formyl peptides in mfprs [ ] . given that quin-c is non-competitive with formyl peptides, it is interesting to surmise that quin-c may act at fpr as an ago-allosteric modulator. however, the precise mechanism for the agonistic activity of quin-c requires further examination. since the first synthetic non-peptide fpr agonist was reported, an increasing number of small-molecule agonists have been discovered in recent years [ ] . so far, a series of structurally divergent small-molecules with high-affinity have been identified as fpr ligands via cell-based assays for high throughput screening (hts), as well as structure-activity relationship (sar)-directed design and synthesis [ ] [ ] [ ] , ] . in general, the identification of new molecules relies on known structures, sar analysis and computer-aided design. using this strategy, around one hundred fpr agonists with high potency and clear selectivity have been identified and optimized in the reported research. based on the structures with varying scaffolds and sar-directed evaluation, these small compounds fall into at least groups: benzimidazoles, pyrazolones (e.g., compound ) , n-substituted benzimidazoles, pyridazin- ( h)-ones, chiral pyridazines, n-phenylureas, chiral -( h-indol- -yl)- -[ -( -nitrophenyl) ureido] propanamides, -(n-piperazinyl)acetamide derivatives, quinazolinones (exemplified by quin-c ), and other derivatives (reviewed by [ ] ). among these molecules, the most potent fpr -selective agonist was identified to be a polyphenylure derivative with an ec of nm in calcium flux assays and a ki of nm in binding assays. this compound, [ -phenyl- -((r)- -( -phenyl- -((s)- -( -phenylureido)propan- -yl)ureido)hexan- -yl)- -( -phenylbutyl)urea], coded as - is also the most potent synthetic non-peptide agonist for fpr known to date [ ] . in addition to - , other chemical agonists with high fpr specificity, such as the benzimidazole derivates [ ] and the pyridazinone-based compounds [ ] , have been described, although they act with ec above micromolar (> . µm). compared with fpr , fpr have more specific and potent agonists identified from screening of compound library and through rational design. currently, more than fpr -slective agonists have been reported to have an ec in the nanomolar range in functional assays, including three pyrazolone derivatives [ ] , an n-substituted benzimidazole derivative [ ] , an n-phenylurea derivative [ ] , a chiral n-phenylurea derivative [ ] , two benzodioxole derivatives of n-phenylureas [ ] , six chiral ureidopropanamido derivatives [ , ] , a phenylsubstituted urea compound [ ] , a compound with a unique -( -phenyl- -((phenylamino)methyl)- , , -triazol- -ylthio) acetamide scaffold [ ] , and a pyrrolidine bis-diketopiperazine derivative [ ] . as expected, there are numerous agonists with dual specificity (fpr /fpr ) (reviewed by [ ] ). noting that a pyrazolone derivative, designated in most publications as "compound ", was first identified as a fpr -specific agonist [ ] , but it was redefined to be a mixed fpr /fpr agonist in an independent screen recently [ ] . it is not clear for most of the reported small molecule agonists whether they interact with murine formyl peptide receptors since they are designed and screened to target human fprs and murine fprs have distinct properties compared to their human counterparts. lipoxin a (lxa ), derived from arachidonic acid, has highly potent anti-inflammatory and pro-resolving activities based on in vivo studies [ ] [ ] [ ] [ ] . this eicosanoid was reported to directly bind to fpr and a variant of mouse mfpr-rs [ , , , ] . lxa was shown to have a high affinity (k d of~ . nm) in binding to fpr -expressing chinese hamster ovary cells [ ] . despite the high-affinity binding, lxa is an atypical ligand for fpr as it cannot activate proinflammatory activities such as chemotaxis, enzyme release and ros production. lxa also fails to activate fpr -dependent signaling, such as calcium mobilization, in transfected cell lines [ ] [ ] [ ] . these findings do not support the original report that lxa could stimulate gtpase activity in fpr -expressing cho cells [ ] . it appears difficult to determine the role of lxa as an fpr agonist without establishing a standard in compound preparation. moreover, fpr specific antagonists, stereoselective analogs, antibodies, and transgenic approaches are required in further studies, and the interaction of lxa with other receptors such as the cannabinoid receptor cb [ ] , aryl hydrocarbon receptor (ahr) [ ] and estrogen receptor [ ] will have to be considered. resolvins, protectins and maresins are novel pro-resolving mediators that are biosynthesized from omega- fatty acids, including docosahexaenoic acid (dha) and eicosapentaenoic acid (epa). these lipid mediators block neutrophil recruitment, promote monocyte activation, and enhance macrophage phagocytosis. resolvin d , a biosynthetic product from dha, was reported to activate fpr and gpr , an orphan gpcr. similar with lxa , resolvin d cannot evoke calcium mobilization through fpr or gpr . the interaction of resolvin d with these gpcrs was examined by a gpcr-β-arrestin-coupled system [ ] . selective knock down or inhibition of fpr reduces the resolvin d response, such as macrophage phagocytosis [ ] and salivary epithelium migration [ ] . resolvin d and fpr are also implicated in pulmonary inflammation [ ] and obesity [ ] . oxidized low-density lipoprotein (oxldl), an atherogenesis associated molecule, was recently found to stimulate macrophages via fpr signaling [ ] . the oxldl-induced foam cell formation and tnf-α production could be inhibited by an fpr antagonist wrw , but not affected by an fpr antagonist. in fpr -expressing rbl- h cells, oxldl also stimulated significant calcium mobilization and chemotaxis [ ] . in another study, the mimetic peptide l- pa (table ) of apoa-i, a major component of circulating high-density lipoprotein (hdl), was reported to induce calcium flux and chemotaxis through fpr . l- pa is probably a biased agonist of fpr as it fails to induce superoxide generation in human neutrophils at a concentration up to µm [ ] . d- pa, the d-stereoisomer of l- pa, blocks l- pa signaling and induces chemotaxis but not calcium flux. it is unclear whether d- pa is an anti-inflammatory antagonist of fpr . it has been noted that apoa-i itself and apoe, another important component of hdl, are described as being anti-inflammatory, as they suppress chemotaxis of leukocytes in the airway. currently, there has been an increasing interest in a group of newly emerging molecules that modulate gpcr activity by entirely novel mechanisms. these molecules, known as pepducins, comprise a lipid moiety conjugated with a peptide that is derived from a sequence of the cytoplasmic loops or the c-terminal tail of the target gpcr [ , ] . with the facilitation of the lipid moiety, pepducins are thought to pass across plasma membrane and anchor in the cytosolic interface to activate or inhibit signaling of receptors [ ] . these lipidated peptides have been proposed as allosteric modulators, because the binding sites and action modes are different from those of conventional ligands that generally interact with extracellular domains of receptors [ , ] . despite that the 'allosteric modulation' mechanism is not yet fully clarified, several receptor-specific pepducins have been identified, including an fpr agonistic pepducin f pal . f pal is composed of palmitic acid linked to a -mer peptide with sequence identical to the third intracellular loop of fpr . f pal was found to activate fpr , like conventional fpr agonists, in phagocytes and stably transfected hl- cells [ , ] . a shorter variant f pal , with higher potency compared to f pal , was shown to act as a partial agonist for direct activation of fpr but as a full agonist for cross-talk triggered hpfr reactivation generated by paf receptor and atp receptor (p y r) [ , ] . it is noteworthy that pepducins are not always specific for their designated target receptor. for example, the pepducins p y palic and p y palic that contain sequences of the second and third intracellular loops of p y r, were identified to be fpr agonists and could activate neutrophils with responses inhibited by fpr antagonists but not p y r antagonist [ ] . in a recent study, a pepducin (ati- ) designed for cxcr , a chemokine receptor, was found to activate neutrophil functions through fpr -dependent signaling [ ] . the selectivity of pepducins for the fpr appears not entirely relying on the segment of the cognate receptor. specific sequence and amino acid are required for the action of fpr pepducins. it is also notable that pepducin derived from another intracellular loop has no effect [ ] and pepducin with an amino acid substitution lost all activity [ ] . more efforts are needed to delineate the mechanisms of action by which pepducins recognize and modulate fpr . of interest, although fpr and fpr share very similar sequence in the intracellular loops, there are no fpr specific pepducins so far. this suggests not only the divergence in the g protein signaling triggered from cytosolic domains of fpr and fpr , but also the presence of different forms (such as homo-or heterodimers) involved in the activation of these two major human fprs. however, this speculation needs verification. it also remains a question whether the pepducins mentioned above can interact with murine fprs. as fprs are potentially attractive drug target, molecules that inhibit cell responses induced by fpr agonists are thought to have therapeutic value for fpr-related diseases. these molecules, generally originating from natural peptides and secondary metabolites, can interact with fprs directly or interfere with their downstream signaling pathways. in addition to drug development, radio-labeled fpr antagonists can be used as tracking probes for in vivo imaging of infiltrating neutrophils [ , ] . the first fpr antagonists were obtained by modifying the amino termini of the classical tripeptide fmlf. early studies have shown that the n-formyl group is important for the formyl peptides to be recognized by fpr . freer et al. substituted this group with tert-butyloxycarbonyl group (t-boc) and found the resulting peptide (t-boc-mlf, boc- ) exhibiting fpr antagonistic properties [ ] . the switch from an agonist to an antagonist was also observed when other carbamates (such as iso-butyloxycarbonyl and benzyloxycarbonyl group) were used to replace the formyl group at the amino terminus [ ] . further c-terminal modification of iso-butyloxycarbonyl-mlf (i-boc-mlf) did not alter the antagonistic potency, implying the different roles of amino and carboxyl terminus in conferring the activities to these peptides. the t-butyloxycarbonyl analog of formylated peptide f-flflf (t-boc-flflf, boc- ) is another antagonistic peptide that potently inhibits neutrophil nadph-oxidase activity induced by fmlf [ , , ] . there is evidence that boc- and boc- begin to lose their receptor preference to fpr at high concentrations (> µm) and display inhibitory effects on fpr and even the complement component a (c a)-induced responses [ ] . thus, despite their fairly specific activity at low concentrations, these boc peptides may be classified as pan-antagonists for fprs. however, other disagree because receptor overlap was seen at high concentrations of all antagonists [ ] . in a recent study, optimization at the phe residues of boc- following ala-scanning led to a more potent fpr -selective antagonist [ ] . nevertheless, among the available formyl peptide-derived antagonists, boc- and boc- are still most potent and commonly used in experiments. chemotaxis inhibitory protein of s. aureus (chips) is another native protein ( . kda) with fpr antagonistic activity. the full length chips ( amino acids) was first reported to be an antagonist for both fpr and the c a receptor [ ] . however, the n-terminal peptides ftfepfptneeiesn and ftfepf (the first six residues) display only anti-fpr properties [ ] , suggesting a mechanism of s. aureus invasion through inhibiting fmlf-induced neutrophil chemotaxis. accordingly, neutralizing antibodies against chips may be useful for treating s. aureus infection [ ] given the fact that chips can bind to fpr with high affinity (k d~ nm) [ ] . in addition, another s. aureus-derived -amino acid protein flipr (fpr-like inhibitory protein), was reported to selectively inhibit the binding of and activation by fpr agonists [ ] . several peptides derived from viruses were also found inhibitory to fpr activation. these peptides include a retroviral p e-derived hexapeptide (ldllfl) that can inhibit fmlf-induced response in monocytes and granulocytes [ ] , and a coronavirus e-derived -mer peptide (etyikpwwvwl) that was identified as a potent antagonist of fpr with a k i of nm [ ] . additional peptides isolated from hiv- , hiv- , severe acute respiratory syndrome coronavirus and ebola virus were reported as fpr antagonists with lower affinities [ ] . it is interesting that n-formylation of etyikpwwvwl transforms this -mer peptide into an fpr agonist, which is consistent with the notion that modification of size and shape at the amino terminus can change the fpr agonistic/antagonistic property of some peptides. among the antagonistic cyclic peptides from natural sources, cyclosporin h (csh) is one of the most potent and specific fpr antagonists. csh and another cyclic undecapeptide csa are derived from peptide metabolites of the fungus tolypocladium inflatum [ , , ] . several cyclosporins were synthesized based on csh and sar analysis [ ] . competition assays conducted with radionuclide binding or n-acetyl-β-d-glucosaminidase release assay demonstrated that cyclosporin h and its derivatives are potent fpr antagonists with nanomolar ic values [ , ] . these compounds were shown to be specific to fpr only at low concentrations. the blocking effect on fpr signaling is also observed with csh at high concentrations (> . µm) [ ] . it was reported that csh could attenuate mouse acute inflammation evoked by cigarette smoking [ ] . another study revealed that administration of csh reduced opioid peptide secretion and analgesia associated with neutrophil activation during mycobacteria butyricum infection [ ] . csh and derivatives are anticipated to be useful in the treatment of airway, gastrointestinal tract and skin inflammatory disorders associated with neutrophil and eosinophil infiltration. an endogenous allergen uteroglobin (ug) was previously identified as an fpr antagonist. ug, known as clara cell kda protein (cc ) in humans, is an anti-inflammatory and anti-chemotactic protein that is constitutively expressed in mammalian airway epithelium. before molecular cloning of fpr, ug was found to inhibit fmlf-induced chemotaxis of monocytes and neutrophils [ ] . after molecular cloning of the fpr genes, the high affinity binding of ug to fpr was determined with a k d of nm in hek cells stably expressing fpr [ ] . the interaction of ug with fpr inhibits saa expression and saa-driven inflammation [ , ] . the activity of ug at fpr or other receptors has not been examined yet, thus the selectivity of this allergen is not fully understood. an additional natural peptide with antagonistic activity is a urokinase receptor-derived cyclic peptide. note that the linear form of this peptide ser-arg-ser-arg-tyr is actually a dual agonist for fpr and fpr [ ] . the cyclized srsry was reported to inhibit the binding of fluorescence labeled fmlf to fpr -expressing rbl- h cells and it inhibits fmlf-directed monocyte migration with a picomolar ic [ ] . however, the activity of cyclized srsry on fpr was not verified in the study. an extract of secondary metabolites from a marine bacillus sp. was also found to have fpr antagonistic properties [ ] . the precise molecule responsible for the inhibitory activity has not yet been identified. other endogenous non-cyclized peptides with antagonistic activity include spinorphin (lvvypwt) [ ] and sarpopeptate, an aurantiamide isolated from plants and fungi [ ] . spinorphin is an fpr antagonist [ , ] . sarpopeptate and its dipeptide derivatives were shown to have potent inhibitory effects on fmlf-stimulated neutrophil responses [ , ] and exhibit a receptor preference for fpr over fpr [ ] . however, the receptor specificity needs further identification because of the lack of data for binding analysis. in a ligand screen of hexapeptide libraries, several peptides were found to inhibit the binding of fpr agonist wkymvm. the peptide wrwwww (wrw ) is the most potent of these peptides with antagonistic activity against fpr agonists [ ] . in addition, wrw is the first fpr antagonist that can completely inhibit the activation of fpr by f l in fpr -transfected hek cells [ ] . it inhibits f l-induced response in mature monocyte-derived dendritic cells, which express fpr but not other fprs. recently, a cell permeable peptide of amino acids conjugated with a rhodamine group (rhob-qrlfqvkgrr, pbp ) was shown to have potent inhibitory activity on fpr . the peptide sequence of pbp is derived from pip -binding domains of the cytoskeletal protein gelsolin, and the rhodamine group is essential for the peptide to pass through plasma membrane and exert antagonistic activity. this rhodamine-linked decapeptide can completely inhibit ros production mediated by fpr but not by fpr [ ] . in a shorter form, rhob-qrlfqvg maintains potent antagonistic activity for fpr and displays the ability to partially inhibit fpr . since pbp peptides bind to the intracellular domain of receptors, the difference in length may reflect divergent structural features of the cytosolic side of fpr and fpr . it is notable that pbp is not an fpr -specific antagonist as it also affects non-fpr mediated signaling [ ] . the pepducin f pal , derived from a segment of the third intracellular loop of fpr , was found to inhibit fpr -mediated cellular response, but had no effect on fpr signaling [ ] . the fpr antagonistic property of f pal was confirmed by selective inhibition in the binding of a conventional fpr agonist (cy -wkymvm). this is not the first pepducin designed to target another gpcr but instead was found to hijack fpr ; however it is the first fpr pepducin with antagonistic activity. pam-(lys-βnspe) -nh , a lapidated α-peptide/β-peptoid oligomers was recently found to be a cross-species antagonist with selectivity for fpr and mfpr [ , ] . pam-(lys-βnspe) -nh belongs to a group of immunomodulatory host defense peptides (hdps), however, unlike most hdps, this synthetic hdp mimic is resistant to proteolysis. it was revealed that both the lipid and peptidomimetic portions are important for its immunomodulatory function. pam-(lys-βnspe) -nh can directly bind to fpr and prevent the binding of fluorescently labeled fpr -specific agonist (cy -wkymwm) but not the fpr -specific agonist (fitc-fnlfnyk) [ ] . the inhibitory potency of pam-(lys-βnspe) -nh is close to that of pbp . both fpr antagonists are membrane permeable and display a reversible inhibition pattern [ ] . despite the similarity, their action mechanisms appear divergent, because the rhodamine group in pbp cannot be exchanged for hexadecanoic acid in the hdp mimic peptide [ ] . it was suggested that pam-(lys-βnspe) -nh and its structural analogs are allosteric modulators of fpr , because they can inhibit neutrophil responses induced by the fpr pepducin f pal , which is assumed to act in a different way from conventional fpr agonists by anchoring to the intracellular loop of the receptor [ ] . however, like other fpr antagonists such as wrw , pbp and flipr were also found to inhibit f pal at fpr , an assumption requiring further verification. as fprs are potential drug targets, the search for novel fpr antagonists/inhibitors with high affinity and selectivity remains one of the fundamental aims of biomedical research in this field. the first reported fpr competitive antagonist is a non-steroidal anti-inflammatory drug (nsaid), sulfinpyrazone and its derivative , -diphenyl- -( -( -naphthyl)-propyl)- , -pyrazolidinedione (dnp) [ , ] . however, sulfinpyrazone is a low affinity (k i = µm) and non-specific ligand at fpr [ , ] . in recent years, numerous small molecule compounds have been discovered to have antagonistic/inhibitory effects using hts of either mixture-based combinatorial libraries or natural product-based drug designs combined with sar analysis and computational modeling. although there are hundreds of antagonistic/inhibitory small molecules in published reports, the fully characterized chemical antagonists with high potency (k i < µm) are no more than compounds after excluding those without binding analysis and/or agonistic activity assays. the h-chromone compound ( -hexyl- -methyl- -( -methyl- h-benzimidazol- -yl)- -oxo- hchromen- -yl acetate) was identified as the most potent fpr antagonist (k i~ nm) among the related synthetic and natural isoflavones. this isoflavone and analogs were found to suppress agonist-stimulated calcium flux and chemotaxis of neutrophils with ic values of . ~ µm [ ] . the competitive analysis showed that they are specific for fpr and did not inhibit fpr -, fpr -, cxcr -or murine mfpr -dependent responses. they have been also shown to be inactive as direct agonists of fpr /fpr and mfpr [ ] . a hederagenin smg- [ -o-( , -odi-acetylα-l-arabinopyranoside)-( → )-α-l-rhamnopyranosyl-( → )-α-l-arabinopyranoside], a saponin isolated from sapindus mukorossi, was reported to block binding of fluorescently labeled fpr ligand fnlfnyk (k i < µm) and inhibit fpr -mediated response in neutrophils and fpr -expressing hl- cells [ , ] . it has been verified that smg- has no direct agonist effects. in another study, a lignin pp- [( r, r)- -( , -dihydroxybenzyl)- -( , "-dimethoxybenzyl) butyrolactone], isolated from piper philippinum, was found to inhibit fmlf-induced neutrophil response and block fitc-fmlf binding to neutrophil with a k i of~ . µm. in the studies of a pharmacophore model of fpr antagonists and molecular docking, pp- displays impressive properties of fpr antagonism [ ] . however, it is difficult to consider pp- a competitive antagonist for fpr based on the experimental data because the antagonistic effect of pp- appears non-competitive and reversible [ ] . further studies are also necessary to address the specificity of this lignin. by screening combinatorial compound libraries, a pyrolidine bis-diketopiperazine-based compound - was recently identified as the most potent non-peptide fpr antagonist with a k i of nm and an ic of nm in calcium mobilization assays. the compound - [(r)- -(cyclohexylmethyl)- -( hydroxybenzyl)- -((r)- -((s)- -(((s)- -isopropyl- , -dioxopiperazin- -yl)methyl)pyrrolidin- -yl)- -(naphthalen- -yl)propan- -yl)piperazine- , -dione]) showed no agonistic activity at concentrations up to µm [ ] . a quinazolinone derivative was previously reported to suppress fpr agonist-stimulated inflammatory responses in vitro and in an in vivo model [ ] . this quin-c related quinazolinone, namely quin-c , was proven to have no agonist activity. it could displace [ i]wkymvm binding to fpr with an estimated k i of . µm. it did not inhibit the binding of [ h]fmlf to fpr at concentrations up to µm, indicating a high preference for fpr over fpr [ ] . quin-c was described as an fpr -specific antagonist, albeit its activity at other inflammation-related receptors (such as c ar and cxcr) has not been fully examined. additional fpr -specific antagonists with an -phenylimidazo[ , -a]pyrimidine scaffold were discovered through the use of a high-throughput hypercyt flow cytometric platform [ ] [ ] [ ] [ ] . they were reported to competitively bind to fpr with k i values of . , . and . µm. the subsequent functional analysis revealed no direct agonistic effects in one of the most potent compounds [ ] . more information and analysis are provided in recent reviews [ , ] . in summary, future studies of binding properties and off-target effects are needed to determine the bona fide antagonists among compounds that have been identified to suppress fpr agonist-stimulated cell responses in functional activity assays. moreover, most of the antagonists mentioned above have been characterized as human ligands, and their potency and specificity for the murine fprs remain to be determined. despite the lack of crystal structures, efforts in understanding fpr structure-function relationship has been propelled with approaches of molecular modification (including construction of receptor chimera and site-directed mutagenesis) and computational docking studies. several clusters of key residues have been identified to be crucial for the interaction between fprs and various modulators with highly divergent profiles and properties. mutational studies have confirmed the individual functions of some of these residues in fpr-ligand interaction [ ] . using receptor chimeras constructed by domain swapping between fpr /fpr [ ] and fpr /c ar [ ] , early studies demonstrated that the first, second and third extracellular loops and adjacent membrane regions seem to be important for recognition of fmlf. this approach was also taken to study chemotaxis mediated by fprs [ ] . following these studies, point mutations were introduced into both fprs, resulting in the identification of multiple charged amino acids that are important for the interaction with fmlf [ ] [ ] [ ] [ ] . these non-contiguous residues include arg , lys , arg , arg , and asp in fpr (figure ). among them, lys in the second transmembrane segment (tm ) and asp in tm were proven essential for the high-affinity binding of fmlf [ , ] . it is suggested that binding of fmlf to fpr disrupts the electrostatic interaction between lys and asp , which might contribute to the receptor activation [ , , ] (figure ) . the lack of a 'salt bridge' formed by lys/asp in fpr (becoming met and asn in corresponding positions) is responsible for the low affinity binding of fmlf and most formyl peptide, although it cannot explain the relatively effective recognition by formyl peptides of other compositions and lengths. arg , arg , and asp in fpr (figure ). among them, lys in the second transmembrane segment (tm ) and asp in tm were proven essential for the high-affinity binding of fmlf [ , ] . it is suggested that binding of fmlf to fpr disrupts the electrostatic interaction between lys and asp , which might contribute to the receptor activation [ , , ] (figure ) . the lack of a 'salt bridge' formed by lys/asp in fpr (becoming met and asn in corresponding positions) is responsible for the low affinity binding of fmlf and most formyl peptide, although it cannot explain the relatively effective recognition by formyl peptides of other compositions and lengths. figure ). reproduced from [ ] with permission. using a similar approach of site-directed mutagenesis aided by computer modeling, another charged residue asp in fpr (gly in fpr ) was found to be crucial for the interaction of fpr with certain formyl peptides that contain a positively charged c-terminal residue (figure ), such as residues referenced in the manuscript are boxed. conserved side-chains between both receptors are shown in black, and major differences in amino acids are highlighted in green for n-formyl peptide binding to fpr ) or gray (for n-formyl peptide binding to fpr ). b and c, molecular electrostatic potential on the inner surface of the binding cavity of a computational model of fpr (b) and fpr (c), viewed from top of the receptors. fpr and fpr were modeled using the structure of the cxcr chemokine receptor as template. arrows show key positive area in fpr due to arg and lys , and negative area in fpr due to asp sequence alignment and location of positive selection sites in fpr , fpr and mfpr (see also figure ). reproduced from [ ] with permission. using a similar approach of site-directed mutagenesis aided by computer modeling, another charged residue asp in fpr (gly in fpr ) was found to be crucial for the interaction of fpr with certain formyl peptides that contain a positively charged c-terminal residue (figure ), such as lys in fmlfk [ ] . in this case, the formation of 'salt bridge' seems still important because the d g substitution of fpr alone failed to restore the potency of fmlf to the level in fpr [ ] . studies have also shown that longer formyl peptides with α-helical and amphipathic properties favor fpr over fpr [ , , ] . a simple explanation is that the binding pocket of fpr is larger and deeper than the relatively shallow and narrow one in fpr , thus providing an added advantage in accommodating longer peptides. to understand the structural basis for the selective activation of fpr by pro-resolving modulators, chimeric receptors were constructed between fpr and other receptors, including fpr and blt . the study using these chimeras demonstrated that tm and adjacent regions of fpr are essential for the recognition of lxa . in contrast, the extracellular loops were required for high affinity binding for pro-inflammatory peptide agonists such as mmk and the mhc peptides. in addition, conserved n-glycosylation sites in fpr (asn and asn ) were also implicated in the interaction with these peptides but not with lxa [ , ] . in another study, a computational model of fpr -annexin peptide interaction was built and it suggested that the transmembrane portions (tm , tm , tm , tm ) of the binding pocket of fpr is important to optimal binding of the n-terminal residue in ac- qawf , the core structure of active annexin peptides [ ] . using the same set of chimeric fpr /fpr receptor [ ] stably expressed in hek cells [ ] , the n-terminal region and the second extracellular loop of fpr were identified as required for annexin a -mediated signaling [ ] . chimeric receptors were also constructed to dissect the mechanisms of action by which cell-penetrating lipopeptides (e.g., pepducins and pbp ) use unconventional access to modulate fpr . it was suggested that the pepducins f pal and f pal act primarily through the third intracellular loop of fpr . the third intracellular loops of fpr and fpr differ in only two amino acids. however, a substitution of these amino acids from fpr to fpr did not affect the selectivity of these pepducins [ ] . another study focused on pbp , an antagonist against fpr agonists, suggested that neither the third intracellular loop nor the presumed signaling cytoplasmic tail of fpr alone is responsible for the specific inhibition of pbp [ ] . clearly, the current knowledge on the action mode and binding sites of these lipopeptides is still very limited. perhaps new strategies should be exploited to understand the structure-function relationship of fpr in association with these allosteric modulators before crystal structures become available. the relatively shallow and narrow one in fpr , thus providing an added advantage in accommodating longer peptides. to understand the structural basis for the selective activation of fpr by pro-resolving modulators, chimeric receptors were constructed between fpr and other receptors, including fpr and blt . the study using these chimeras demonstrated that tm and adjacent regions of fpr are essential for the recognition of lxa . in contrast, the extracellular loops were required for high affinity binding for pro-inflammatory peptide agonists such as mmk and the mhc peptides. in addition, conserved n-glycosylation sites in fpr (asn and asn ) were also implicated in the interaction with these peptides but not with lxa [ , ] . in another study, a computational model of fpr -annexin peptide interaction was built and it suggested that the transmembrane portions (tm , tm , tm , tm ) of the binding pocket of fpr is important to optimal binding of the n-terminal residue in ac- qawf , the core structure of active annexin peptides [ ] . using the same set of chimeric fpr /fpr receptor [ ] stably expressed in hek cells [ ] , the n-terminal region and the second extracellular loop of fpr were identified as required for annexin a -mediated signaling [ ] . due to the lack of crystal structures for fprs, homology models and molecular docking analysis based on the mutagenesis/chimera studies represent an alternative approach for explaining ligand binding and receptor activation. the current homology models of fpr receptors (including fpr and fpr ) are generally created by using the crystal structures of two classes of receptors: ( ) the bovine rhodopsin receptor, that has a relatively low sequence identity to fprs (~ %) and was selected as a template because of its higher resolution ( . Å) [ ] ; ( ) the cxcr -based templates, usually cxcr alone, which has a higher sequence identity with fprs ( %) but has a lower resolution ( . Å) compared to that of rhodopsin [ ] . a dual template strategy (cxcr and µor opioid receptor) was developed in a recent docking study focused on fpr and non-peptide ligands [ ] . despite questions about the low similarity of rhodopsin receptor with fprs, several interesting points have been made by using the homology models based on this receptor. the homology model derived from the rhodopsin template shows that the fpr ligand binding site comprises two channels, two cavities, and the bottom [ ] . several amino acids are found to be involved in the interaction of fpr with agonists, including asn , thr , arg , tyr , and thr [ , , , ] . docking studies with fpr non-peptide antagonists (including the bile acids dca and cdca) suggested that thr , tyr , and thr are involved in the formation of three stable h-bond interactions with dca and cdca in the docked pose [ , ] . a pharmacophore model based on best docking poses of four fpr antagonists including csh described three anchor points: two acceptors for h-bonding and one hydrophobic point [ , , ] . in comparison with the agonists, fpr antagonists are characterized as containing more oh groups, which can serve as h-bond donors and/or acceptors upon binding to fpr [ ] . however, the three-point model was further proved to be not only fitted by fpr antagonists but also by fpr agonists [ , ] . thus, this model may be helpful in prediction for structures of fpr ligands without discriminating agonists and antagonists. in another study, a cxcr -based homology model of fpr suggested an important role of water molecule in discrimination between fpr agonists and antagonists [ ] . in a comparative model, the potent antagonist boc- was found to interact with fpr by forming h-bonds with arg and lys , similarly to the interaction between fmlf and fpr . however, boc- directly forms an h-bond with asp while water molecule mediates h-bonding between the fmlf carbonyl group and asp [ ] . the rhodopsin-based model with five fpr agonists described the binding pocket of fpr as dumb-bell shaped, with a large cavity and a small cavity linked by a narrow channel. the proposed model comprises three sub-pockets, the pharmacophore sub-pocket i (within the small cavity) surrounded by positive residues, and the hydrophobic sub-pockets ii and iii (within the large cavity) surrounded by polar residues [ ] . the fpr -specific peptide agonist wkymvm fits well with this model by occupying all three sub-pockets, with the n-terminal indole moiety located in sub-pocket i in the best docked pose [ ] . in another docking simulation, residues his , val , asp , leu , and trp were identified as being critical for agonist binding [ ] . in a more recent study, comparative docking analysis was performed on a homology model of fpr that used cxcr and µor as a dual template [ ] . the model proposed that three hydrophobic clusters are important in binding non-peptide ligands, including agonists and antagonists. the first cluster is formed by tm and tm while his is crucial in the interaction with docked ligands. the second cluster is located between tm and tm with phe as the key residue in binding to aromatic groups of docked compounds. the third cluster is buried in the protein consisting of residues phe , val , phe and phe [ ] . the docking model suggested that the proper orientation is required for penetration into the binding site: the peptide ligands are placed upright while non-peptide ligands prefer a more horizontal position. these findings have yet to be substantiated by mutational studies. still, the differences in binding modes between antagonists and agonists to fpr are not fully understood. to date, there are no reports describing modeling of fpr and mouse mfprs. the fprs have evolved to be a class of receptors that not only recognize bacteria-derived formyl peptides but also ligands with drastically different structures, including non-formyl peptides of microbial origins, endogenous peptides and synthetic small molecules. because of these features, the fprs are highly 'promiscuous' in terms of ligand recognition. how fprs recognize these different ligands remains unclear at present without available crystal structures. molecular docking and computer simulation approaches combined with site-directed mutagenesis, however, have provided clues to our understanding of how formyl peptides interact with fpr and to a lesser extent, fpr . over a very long period of time, adaptive evolution has occurred in fprs, especially fpr , with positive selection for receptor interaction with the bona fide or orthosteric ligands. indeed, several residues involved in h-bonding with amino acids in formyl peptides are located within the predicted binding sites undergoing positive selection, suggesting that human fpr is under selection pressure for its binding of formyl peptides. as for other fpr agonists as well as for fpr , the picture remains unclear. there is no doubt that some agonists for the fprs have been in existence only recently and they are likely candidates for allosteric or ago-allosteric modulators of the fprs. with respect to fpr , it apparently has diverged from the evolutionary path taken by fpr , resulting in a different recognition profile including binding of longer formyl peptides and a wide range of endogenous and microbial peptides. a better understanding of the mechanisms by which fprs interact with their ligands will help to sort out agonists and antagonists that are crucial for the biological functions of these receptors, and to speed up identification of therapeutically important molecules. author contributions: h.q.h. and r.d.y. initiated and designed the study, collected the literature and wrote the manuscript. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: formyl peptide receptors: a promiscuous subfamily of g protein-coupled receptors controlling immune responses international union of basic and clinical pharmacology. lxxiii. nomenclature for the formyl peptide receptor (fpr) family n-formylmethionyl peptides as chemoattractants for leucocytes mitochondrial n-formylmethionyl proteins as chemoattractants for neutrophils basic characteristics of the neutrophil receptors that recognize formylated peptides, a danger-associated molecular pattern generated by bacteria and mitochondria development of small molecule non-peptide formyl peptide receptor (fpr) ligands and molecular modeling of their recognition new development in studies of formyl-peptide receptors: critical roles in host defense synthesis and use of a novel n-formyl peptide derivative to isolate a human n-formyl peptide receptor cdna the human n-formylpeptide receptor. characterization of two cdna isolates and evidence for a new subfamily of g-protein-coupled receptors isolation of a cdna that encodes a novel granulocyte n-formyl peptide receptor a structural homologue of the n-formyl peptide receptor. characterization and chromosome mapping of a peptide chemoattractant receptor family mapping of genes for the human c a receptor (c ar), human fmlp receptor (fpr), and two fmlp receptor homologue orphan receptors (fprh , fprh ) to chromosome cloning of a cdna encoding a receptor related to the formyl peptide receptor of human neutrophils adaptive evolution of formyl peptide receptors in mammals potential role of the formyl peptide receptor-like (fprl ) in inflammatory aspects of alzheimer's disease bacterial lipopolysaccharide selectively up-regulates the function of the chemotactic peptide receptor formyl peptide receptor in murine microglial cells interferon and granulopoiesis signatures in systemic lupus erythematosus blood role of formyl peptide receptor-like (fprl /fpr ) in mononuclear phagocyte responses in alzheimer disease leukocyte chemoattractant receptor fpr may accelerate atherogenesis formyl peptide receptors at the interface of inflammation, angiogenesis and tumor growth impaired antibacterial host defense in mice lacking the n-formylpeptide receptor formylpeptide receptors are critical for rapid neutrophil mobilization in host defense against listeria monocytogenes reduced fear memory and anxiety-like behavior in mice lacking formylpeptide receptor n-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two n-formylpeptide receptor (fpr) subtypes. molecular characterization of fpr , a second mouse neutrophil fpr identification of formyl peptides from listeria monocytogenes and staphylococcus aureus as potent chemoattractants for mouse neutrophils functional characterization of three mouse formyl peptide receptors serum amyloid a is a chemotactic agonist at fpr , a low-affinity n-formylpeptide receptor on mouse neutrophils amyloid-beta induces chemotaxis and oxidant stress by acting at formylpeptide receptor , a g protein-coupled receptor expressed in phagocytes and brain f l, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating fpr , the low-affinity n-formylpeptide receptor identification and characterization of an endogenous chemotactic ligand specific for fprl characterization of fpr-rs , an atypical member of the mouse formyl peptide receptor gene family formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ aspirin-triggered -epi-lipoxin a (lxa ) and lxa stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors characterization of two new members of the formyl peptide receptor gene family from s mice intravascular danger signals guide neutrophils to sites of sterile inflammation circulating mitochondrial damps cause inflammatory responses to injury intravital imaging of neutrophil recruitment reveals the efficacy of fpr blockade in hepatic ischemia-reperfusion injury the isolation and partial characterization of neutrophil chemotactic factors from escherichia coli further studies on the structural requirements for synthetic peptide chemoattractants purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by escherichia coli acute colitis produced by chemotactic peptides in rats and mice chemotactic peptide-induced acute colitis in rabbits guinea pig ileum motility stimulation elicited by n-formyl-met-leu-phe (fmlf) involves neurotransmitters and prostanoids chemotactic peptides. mechanisms, functions, and possible role in inflammatory bowel disease increased neutrophil receptors for and response to the proinflammatory bacterial peptide formyl-methionyl-leucyl-phenylalanine in crohn's disease defective polymorphonuclear leukocyte formyl peptide receptor(s) in juvenile periodontitis formyl-methionyl-leucyl-phenylalanine causes bronchoconstriction in rabbits haematological effects of inhalation of n-formyl-methionyl-leucyl-phenylalanine in man hemodynamic and metabolic effects of intravenous formyl-methionyl-leucyl-phenylalanine (fmlp) in rabbits endotoxin augments hemodynamic and metabolic effects of formyl-methionyl-leucyl-phenylalanine (fmlp) in rabbits structural changes of the ligand and of the receptor alters the receptor preference for neutrophil activating peptides starting with a formylmethionyl group human mitochondria-derived n-formylated peptides are novel agonists equally active on fpr and fprl , while listeria monocytogenes-derived peptides preferentially activate fpr mitocryptide- , a neutrophil-activating cryptide, is a specific endogenous agonist for formyl-peptide receptor-like a proinflammatory peptide from helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis a seven-transmembrane, g protein-coupled receptor, fprl , mediates the chemotactic activity of serum amyloid a for human phagocytic cells structural mechanism of serum amyloid a-mediated inflammatory amyloidosis amyloid (beta) activates a g-protein-coupled chemoattractant receptor, fpr-like- endogenous lipid-and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin a receptor annexin and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement ll- , the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like (fprl ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells cross-talk between fmlp and vitronectin receptors triggered by urokinase receptor-derived srsry peptide urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like and -like the neurotoxic prion peptide fragment prp( - ) is a chemotactic agonist for the g protein-coupled receptor formyl peptide receptor-like proinflammatory proteases liberate a discrete high-affinity functional fprl (ccr ) ligand from ccl vip differentially activates beta integrins, cr , and matrix metalloproteinase- in human monocytes through camp/pka, epac, and pi- k signaling pathways via vip receptor type and fprl a peptide with unique receptor specificity: stimulation of phosphoinositide hydrolysis and induction of superoxide generation in human neutrophils utilization of two seven-transmembrane, g protein-coupled receptors, formyl peptide receptor-like and formyl peptide receptor, by the synthetic hexapeptide wkymvm for human phagocyte activation the synthetic peptide trp-lys-tyr-met-val-met-nh specifically activates neutrophils through fprl /lipoxin a receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor fprl identification of surrogate agonists for the human fprl- receptor by autocrine selection in yeast apolipoproteins and apolipoprotein mimetic peptides modulate phagocyte trafficking through chemotactic activity activation of human monocytes by a formyl peptide receptor -derived pepducin the leukocyte chemotactic receptor fpr , but not the closely related fpr , is sensitive to cell-penetrating pepducins with amino acid sequences descending from the third intracellular receptor loop identification of a human cdna encoding a functional high affinity lipoxin a receptor a novel nonpeptide ligand for formyl peptide receptor-like identification of novel small-molecule agonists for human formyl peptide receptors and pharmacophore models of their recognition new 'chemical probes' to examine the role of the hfprl (or alxr) receptor in inflammation potent hfprl (alxr) agonists as potential anti-inflammatory agents formyl peptide receptor and dual agonist inhibits human neutrophil chemotaxis by the induction of chemoattractant receptor cross-desensitization synthesis and pharmacological evaluation of new pyridazin-based thioderivatives as formyl peptide receptor (fpr) agonists synthesis, enantioresolution, and activity profile of chiral -methyl- , -disubstituted pyridazin- ( h)-ones as potent n-formyl peptide receptor agonists -( h-indol- -yl)- -[ -( -nitrophenyl)ureido]propanamide enantiomers with human formyl-peptide receptor agonist activity: molecular modeling of chiral recognition by fpr novel -( h-indol- -yl)- -[ -( -methoxyphenyl)ureido]propanamides as selective agonists of human formyl-peptide receptor selective agonists and antagonists of formylpeptide receptors: duplex flow cytometry and mixture-based positional scanning libraries n-terminal residues of the chemotaxis inhibitory protein of staphylococcus aureus are essential for blocking formylated peptide receptor but not c a receptor uteroglobin suppresses allergen-induced th differentiation by down-regulating the expression of serum amyloid a and socs- genes cyclosporin h is a potent and selective formyl peptide receptor antagonist. comparison with n-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine and cyclosporins a, b, c, d, and e cyclization of the urokinase receptor-derived ser-arg-ser-arg-tyr peptide generates a potent inhibitor of trans-endothelial migration of monocytes identification of peptides that antagonize formyl peptide receptor-like -mediated signaling structural characterization and inhibitory profile of formyl peptide receptor selective peptides descending from a pip -binding domain of gelsolin a neutrophil inhibitory pepducin derived from fpr expected to target fpr signaling hijacks the closely related fpr instead the proteolytically stable peptidomimetic pam-(lys-betanspe) -nh selectively inhibits human neutrophil activation via formyl peptide receptor the peptidomimetic lau-(lys-betanspe) -nh antagonizes formyl peptide receptor expressed in mouse neutrophils antagonism of human formyl peptide receptor (fpr ) by chromones and related isoflavones pharmacological characterization of a novel nonpeptide antagonist for formyl peptide receptor-like structural determinants for the interaction of formyl peptide receptor with peptide ligands community-associated mrsa: what makes them special? human formyl peptide receptor senses highly pathogenic staphylococcus aureus formyl peptide receptor-mediated proinflammatory consequences of peptide deformylase inhibition in staphylococcus aureus receptor-dependent and -independent immunomodulatory effects of phenol-soluble modulin peptides from staphylococcus aureus on human neutrophils are abrogated through peptide inactivation by reactive oxygen species t /dp , a synthetic leucine zipper-like domain of the hiv- envelope gp , attracts and activates human phagocytes by using g-protein-coupled formyl peptide receptors t /dp , an ectodomain peptide of human immunodeficiency virus type gp , is an activator of human phagocyte n-formyl peptide receptor a synthetic peptide derived from human immunodeficiency virus type gp downregulates the expression and function of chemokine receptors ccr and cxcr in monocytes by activating the -transmembrane g-protein-coupled receptor fprl /lxa r n , a synthetic n-terminal heptad repeat domain of the hiv- envelope protein gp , is an activator of human phagocytes activation of the chemotactic peptide receptor fprl in monocytes phosphorylates the chemokine receptor ccr and attenuates cell responses to selected chemokines the hiv- cell entry inhibitor t- potently chemoattracts neutrophils by specifically activating the n-formylpeptide receptor basophils infiltrate human gastric mucosa at sites of helicobacter pylori infection, and exhibit chemotaxis in response to h. pylori-derived peptide hp( - ) promotion of formyl peptide receptor -mediated neutrophil chemotactic migration by antimicrobial peptides isolated from the centipede scolopendra subspinipes mutilans serum amyloid a induces il- secretion through a g protein-coupled receptor, fprl /lxa r local expression of the serum amyloid a and formyl peptide receptor-like genes in synovial tissue is associated with matrix metalloproteinase production in patients with inflammatory arthritis opposing regulation of interleukin- and nf-kappab responses by lipoxin a and serum amyloid a via the common lipoxin a receptor differential production of leukotriene b or prostaglandin e by wkymvm or serum amyloid a via formyl peptide receptor-like serum amyloid a opposes lipoxin a( ) to mediate glucocorticoid refractory lung inflammation in chronic obstructive pulmonary disease serum amyloid a mediates human neutrophil production of reactive oxygen species through a receptor independent of formyl peptide receptor like- endogenous acute phase serum amyloid a lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors serum amyloid a isoforms display different efficacy at toll-like receptor and formyl peptide receptor an il- r/il- circuit regulates epithelial serum amyloid a to promote local effector th responses th cell induction by adhesion of microbes to intestinal epithelial cells pleiotropic roles of formyl peptide receptors beta amyloid peptide (abeta ) is internalized via the g-protein-coupled receptor fprl and forms fibrillar aggregates in macrophages humanin, a newly identified neuroprotective factor, uses the g protein-coupled formylpeptide receptor-like- as a functional receptor n-formylated humanin activates both formyl peptide receptor-like and an annexin n-terminal peptide activates leukocytes by triggering different members of the formyl peptide receptor family neutrophil nadph-oxidase activation by an annexin ai peptide is transduced by the formyl peptide receptor (fpr), whereas an inhibitory signal is generated independently of the fpr family receptors mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like /mouse formyl peptide receptor-like as the receptor and acts as an immune adjuvant fprl -mediated induction of superoxide in ll- -stimulated imr human fibroblast ll- inhibits serum amyloid a-induced il- production in human neutrophils human endogenous antibiotic ll- stimulates airway epithelial cell proliferation and wound closure the pro-inflammatory peptide ll- promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells leucine leucine- uses formyl peptide receptor-like to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells the expression of beta-defensin- , and ll- induced by candida albicans phospholipomannan in human keratinocytes leukotriene b /antimicrobial peptide ll- proinflammatory circuits are mediated by blt and fpr /alx and are counterregulated by lipoxin a and resolvin e the fibrinolytic receptor for urokinase activates the g protein-coupled chemotactic receptor fprl /lxa r identification of fam d as a new endogenous chemotaxis agonist for the formyl peptide receptors distinct signaling cascades elicited by different formyl peptide receptor (fpr ) agonists the synthetic peptide trp-lys-tyr-met-val-d-met inhibits human monocyte-derived dendritic cell maturation via formyl peptide receptor and formyl peptide receptor-like synthetic peptide mmk- is a highly specific chemotactic agonist for leukocyte fprl a novel peptide agonist of formyl-peptide receptor-like (alx) displays anti-inflammatory and cardioprotective effects tethered ligand library for discovery of peptide agonists design, synthesis and characterization of fmlf-mimicking aapeptides characterization of quin-c for its anti-inflammatory property in a mouse model of bleomycin-induced lung injury stable formyl peptide receptor agonists that activate the neutrophil nadph-oxidase identified through screening of a compound library a non-peptide receptor inhibitor with selectivity for one of the neutrophil formyl peptide receptors, fpr molecular docking of -(benzimidazol- -ylthio)-n-phenylacetamide-derived small-molecule agonists of human formyl peptide receptor further studies on -arylacetamide pyridazin- ( h)-ones: design, synthesis and evaluation of , -disubstituted analogs as formyl peptide receptors (fprs) agonists high-throughput screening for small-molecule activators of neutrophils: identification of novel n-formyl peptide receptor agonists gastrin-releasing peptide/neuromedin b receptor antagonists pd , pd , and related analogs are potent agonists of human formyl-peptide receptors lipoxins and aspirin-triggered -epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution the lipoxin receptor alx: potent ligand-specific and stereoselective actions in vivo lipoxins: update and impact of endogenous pro-resolution lipid mediators resolution of inflammation: state of the art, definitions and terms identification, cloning, and functional characterization of a murine lipoxin a receptor homologue gene lipoxin a( ) metabolites/analogues from two commercial sources have no effects on tnf-alpha-mediated priming or activation through the neutrophil formyl peptide receptors what formyl peptide receptors, if any, are triggered by compound and lipoxin a ? heterologously expressed formyl peptide receptor (fpr /alx) does not respond to lipoxin a( ) anti-inflammatory lipoxin a is an endogenous allosteric enhancer of cb cannabinoid receptor lipoxin a : a new class of ligand for the ah receptor lipoxin a is a novel estrogen receptor modulator resolvin d binds human phagocytes with evidence for proresolving receptors resolvin d prevents tnf-alphamediated disruption of salivary epithelial formation aspirin-triggered resolvin d reduces mucosal inflammation and promotes resolution in a murine model of acute lung injury resolvin d and resolvin d govern local inflammatory tone in obese fat oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor insider access: pepducin symposium explores a new approach to gpcr modulation turning receptors on and off with intracellular pepducins: new insights into g-protein-coupled receptor drug development a pepducin derived from the third intracellular loop of fpr is a partial agonist for direct activation of this receptor in neutrophils but a full agonist for cross-talk triggered reactivation of fpr a pepducin designed to modulate p y r function interacts with fpr in human neutrophils and transfers atp to an nadph-oxidase-activating ligand through a receptor cross-talk mechanism data on human neutrophil activation induced by pepducins with amino acid sequences derived from β ar and cxcr . data brief localization of radiolabeled chemotactic peptide at focal sites of escherichia coli infection in rabbits: evidence for a receptor-specific mechanism a novel neutrophil-specific pet imaging agent: cflflfk-peg- cu selective inhibition of n-formylpeptide-induced neutrophil activation by carbamate-modified peptide analogues antibiotics and peptides with agonist and antagonist chemotactic activity cyclosporin h, boc-mlf and boc-flflf are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor development of potent antagonists for formyl peptide receptor based on boc-phe-d-leu-phe-d-leu-phe-oh chemotaxis inhibitory protein of staphylococcus aureus binds specifically to the c a and formylated peptide receptor characterisation of receptor binding by the chemotaxis inhibitory protein of staphylococcus aureus and the effects of the host immune response a new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like an immunosuppressive retrovirus-derived hexapeptide interferes with intracellular signaling in monocytes and granulocytes through n-formylpeptide receptors peptides derived from hiv- , hiv- , ebola virus, sars coronavirus and coronavirus e exhibit high affinity binding to the formyl peptide receptor cyclosporins: structure-activity relationships for the inhibition of the human fpr formylpeptide receptor the immunosuppressant cyclosporin a antagonizes human formyl peptide receptor through inhibition of cognate ligand binding genetic ablation of the fpr gene confers protection from smoking-induced lung emphysema in mice mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils inhibition of phagocyte chemotaxis by uteroglobin, an inhibitor of blastocyst rejection sodin-semrl, s. uteroglobin, a possible ligand of the lipoxin receptor inhibits serum amyloid a-driven inflammation bioactive secondary metabolites of a marine bacillus sp. inhibit superoxide generation and elastase release in human neutrophils by blocking formyl peptide receptor . molecules the endogenous opioid spinorphin blocks fmet-leu-phe-induced neutrophil chemotaxis by acting as a specific antagonist at the n-formylpeptide receptor subtype fpr two trypanocidal dipeptides from the roots of zapoteca portoricensis (fabaceae) inhibitory effects of spinorphin, a novel endogenous regulator, on chemotaxis, o -generation, and exocytosis by n-formylmethionylleucyl-phenylalanine (fmlp)-stimulated neutrophils constituents and bioactive principles of polygonum chinensis design and synthesis of new n-(fluorenyl- -methoxycarbonyl) (fmoc)-dipeptides as anti-inflammatory agents design and synthesis of tryptophan containing dipeptide derivatives as formyl peptide receptor antagonist trp-arg-trp-trp-trp-trp antagonizes formyl peptide receptor like -mediated signaling receptor-directed inhibition of chemotactic factor-induced neutrophil hyperactivity by pyrazolon derivatives. definition of a chemotactic peptide antagonist the interaction of , -pyrazolidinedione drugs with receptors for f-met-leu-phe on human neutrophil leukocytes: a study of the structure-activity relationship. can inhibition of human platelet cyclo-oxygenase activity by sulfinpyrazone and three of its metabolites high-throughput screening with hypercyt flow cytometry to detect small molecule formylpeptide receptor ligands the hederagenin saponin smg- is a natural fmlp receptor inhibitor that suppresses human neutrophil activation antagonism of human formyl peptide receptor with natural compounds and their synthetic derivatives r, r)- -( , -dihydroxybenzyl)- -( , -dimethoxybenzyl)butyrolactone suppresses fmlp-induced superoxide production by inhibiting fmlp-receptor binding in human neutrophils high-content screening: flow cytometry analysis biomolecular screening of formylpeptide receptor ligands with a sensitive, quantitative, high-throughput flow cytometry platform a novel fluorescent cross-reactive formylpeptide receptor/formylpeptide receptor-like hexapeptide ligand duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes multiple domains of the n-formyl peptide receptor are required for high-affinity ligand binding. construction and analysis of chimeric n-formyl peptide receptors species and subtype variants of the n-formyl peptide chemotactic receptor reveal multiple important functional domains identification of functional domains in the formyl peptide receptor-like for agonist-induced cell chemotaxis the ligand binding site of the formyl peptide receptor maps in the transmembrane region identification of an n-formyl peptide receptor ligand binding domain by a gain-of-function approach human formyl peptide receptor function role of conserved and nonconserved charged residues characterization of the binding site on the formyl peptide receptor using three receptor mutants and analogs of met-leu-phe and met-met-trp-leu-leu identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry ligand-specific regulation of the extracellular surface of a g-protein-coupled receptor linking agonist binding to histamine h receptor activation insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (psm) alpha peptide peptide length and folding state govern the capacity of staphylococcal beta-type phenol-soluble modulins to activate human formyl-peptide receptors or activation of lipoxin a( ) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation the annexin i sequence gln( )-ala( )-trp( )-phe( ) is a core structure for interaction with the formyl peptide receptor annexin a interaction with the fpr /alx receptor: identification of distinct domains and downstream associated signaling crystal structure of rhodopsin: a g protein-coupled receptor structures of the cxcr chemokine gpcr with small-molecule and cyclic peptide antagonists non-peptide ligand binding to the formyl peptide receptor fpr -a comparison to peptide ligand binding modes integration of virtual screening with high-throughput flow cytometry to identify novel small molecule formylpeptide receptor antagonists pharmacophore model for bile acids recognition by the fpr receptor c-and n-terminal residue effect on peptide derivatives' antagonism toward the formyl-peptide receptor identification of novel formyl peptide receptor-like agonists that induce macrophage tumor necrosis factor alpha production the role of water in activation mechanism of human n-formyl peptide receptor (fpr ) based on molecular dynamics simulations stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -xys g b authors: shichijo, s.; keicho, n.; long, h.t.; quy, t.; phi, n.c.; ha, l.d.; ban, v.v.; itoyama, s.; hu, c.‐j.; komatsu, n.; kirikae, t.; kirikae, f.; shirasawa, s.; kaji, m.; fukuda, t.; sata, m.; kuratsuji, t.; itoh, k.; sasazuki, t. title: assessment of synthetic peptides of severe acute respiratory syndrome coronavirus recognized by long‐lasting immunity date: - - journal: tissue antigens doi: . /j. - . . .x sha: doc_id: cord_uid: xys g b abstract: in order to determine highly immunogenic severe acute respiratory syndrome coronavirus (sars‐cov) epitope peptides capable of inducing long‐lasting immunity, we first screened immunoglobulin‐g (igg) antibodies reactive to different overlapping ‐mers from the sars‐cov proteins in the sera of three infected patients. forty‐two peptides among them were reactive to the sera from all three patients. consequently, we tested for the reactivity of these peptides to patients' sera (n = ) at ‐month post‐infection. the significantly higher levels of igg antibodies specific to three (s , m and n ) of peptides were detectable in the post‐infection sera from ( %), ( %) and ( %) of patients, respectively. these three peptides, recognized by their long‐lasting immunity, may provide a better understanding of the immunogenicity of sars‐cov. . each peptide was dissolved in dimethylsulfoxide (dmso) and was then stored at À c until use. these peptides were tested for their reactivity to the sera of early stages of three taiwanese sars-cov-infected patients by using flowmetry analysis with luminex tm (luminex corp., austin, tx) ( ) . the sera were collected from jen-ai municipal hospital, saan district, taipei, taiwan. the patients' sera showed significantly higher levels of immunoglobulin-g (igg) (p < . ) activities reactive to of peptides tested, including spike (s)-, seven membrane (m)-and nucleocapsid (n)-derived peptides, when the means of the scores of fluorescence intensity (fi) from the sera ( -fold dilution) of the three patients (closed bar) were compared to those of the three healthy donors (hd) (open bar). the peptides were coupled to colour-coded beads, according to the modified manufacturer's instructions (luminex corp.). in brief, ml of colour-coded beads were mixed with ml of peptide ( mg/ml in . m morpholinoehanesulfonic acid (mes) buffer, ph . ). the peptide-loaded beads were then incubated with -ethyl- -[ -dimethylaminopropyl]carbodiimide -(n-morpholino)ethanesulfonic acid (edc) ( mg ml À ) at room temperature for min in darkness, and the beads were washed with tween- phosphatebuffered saline (pbs). the beads were treated with -aminoethanol for min at room temperature in darkness, washed twice and then re-suspended with ml of . % block ace (snow brand milk products co., ltd, hokkaido, japan) in tween- pbs. two microlitres of serum at dilutions of - , times was incubated with ml of the peptide-coupled colour-coded beads for h at room temperature on a plate shaker in a -well filter plate (multiscreen tm -bv, millipore co., bedford, ma). after incubation, the plate was washed by using a vacuum manifold apparatus and was incubated with ml of biotinylated goat anti-human igg (gamma-chain-specific: vector laboratory inc., burlingame, ca) for h at room temperature on a plate shaker. the plate was then washed, and ml of streptavidin-pe (molecular probes, eugene, or) was added into wells, followed by incubation for min at room temperature on a plate shaker. the bound beads were washed three times followed by the addition of ml of tween- pbs into each well, and the plate was placed for min on a plate shaker. fifty microlitres of sample was analysed by using the luminex tm system with the help of the method reported previously ( , , ) . (table ). in contrast, there were no significant differences in the reactivity against any of peptides between the hd and the contact persons. anti-n anti-s (table ) . however, the positive cases showing fi scores of greater than the mean plus sd were only six of patients ( %). in contrast to these four peptides, significant levels of igg reactive to the remaining peptides were either scarcely or not detected in the patients (table ) fig. . absorption test. the immunoglobulin-g (igg) activity to each of the s , m and n peptides was absorbed by using a triplicate assay with an immobilized corresponding peptide and each of the five different irrelevant peptides. the method for the preparation of immobilized peptides was the same as the method used for elisa plate preparation, as described in the legend of fig. . the results of the absorption test were analysed by means of a two-tailed student's t-test. all tests of significance were two-sided. in order to test the specificity of anti-peptide igg in the serum samples, ml/well of serum samples ( : dilution with . % pbst) was absorbed with the immobilized peptide ( mg/well: closed bar or mg/well: open bar, as final concentrations) in wells kept for h at room temperature. the absorption was repeated three times, and then the level of peptide-specific igg in the resultant supernatant was measured. pbst, tween- pbs. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus effects of a sars-associated coronavirus vaccine in monkeys angiotensin-converting enzyme is a functional receptor for the sars coronavirus expression of the monoclonal antibody against nucleocapsid antigen of sars-associated coronavirus in autopsy tissues from sars patients new multiplexed flow cytometric assay to measure anti-peptide antibody: a novel tool for monitoring immune responses to peptides used for immunization analysis of false-positive associated with antibody tests for sars-cov in sle patients antibodies to sars-like virus hint at repeated infections dynamic observation igg and igm antibodies in patients with severe acute respiratory syndrome microbiologic characteristics, serologic responses, and clinical manifestations in severe acute respiratory syndrome detection of the anti-sars coronavirus-specific antibody levels in sars patients multiplexed quantification of human igg, iga, and igm with the flowmetrix tm system severe acute respiratory syndrome (sars) igg reactive to ctl-directed epitopes of selfantigens is either lacking or unbalanced in atopic dermatitis patients assessment of sars peptides recognized by long-lasting immunity the authors thank dr nguyen le hang, ms. nguyen thu ha and pham phuong thuy for supporting the co-ordination and implementation of this research project in vietnam, and drs masamichi koujiro and akira yamada of kurume university school of medicine, asahi-machi, kurume, fukuoka, japan, for co-ordinating this research. key: cord- - p x lwx authors: melnik, lilia i; garry, robert f; morris, cindy a title: peptide inhibition of human cytomegalovirus infection date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: p x lwx background: human cytomegalovirus (hcmv) is the most prevalent congenital viral infection in the united states and europe causing significant morbidity and mortality to both mother and child. hcmv is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (hiv)- infected patients with aids, and solid organ and allogeneic stem cell transplantation recipients. current treatments for hcmv-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit hcmv infection. the aim of this study was to develop therapeutic peptides targeting glycoprotein b (gb), a major glycoprotein of hcmv that is highly conserved across the herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing hcmv entry and infection. results: using the wimley-white interfacial hydrophobicity scale (wwihs), several regions within gb were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. the ability of synthetic peptides analogous to wwihs-positive sequences of hcmv gb to inhibit viral infectivity was evaluated. human foreskin fibroblasts (hff) were infected with the towne-gfp strain of hcmv ( . moi), preincubated with peptides at a range of concentrations ( nm to μm), and gfp-positive cells were visualized hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. peptides that inhibited hcmv infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. peptide - showed % inhibition of viral infection at a concentration of μm, and % and % inhibition at concentrations of μm and . μm, respectively. peptide - inhibited infection by % and % at concentrations of μm and μm, respectively, and % at a concentration of . μm. while peptides - and - , individually failed to inhibit viral infection, when combined, they showed % inhibition of hcmv infection at a concentration of . μm each. conclusions: peptides designed to target putative fusogenic domains of gb provide a basis for the development of novel therapeutics that prevent hcmv infection. human cytomegalovirus (hcmv) is a ubiquitous opportunistic pathogen that belongs to the betaherpesviridae. the virulence of this pathogen is directly linked to the immune status of its host. primary hcmv infection is generally asymptomatic in immunocompetent individuals, although it causes a mononucleosis-like syndrome in some. after primary hcmv infection, the virus establishes lifelong latency and periodically reactivates with notable pathological consequences. in contrast, hcmv infection in immunocompromised patients such as aids patients and solid organ and allogeneic stem cell transplantation recipients causes serious disease [ ] . primary infection of women during or right before pregnancy with hcmv is the most common cause of congenital viral infection leading to significant morbidity and mortality. congenital hcmv infection is also associated with spontaneous abortion, premature delivery, intrauterine growth restriction (iugr), and pre-eclampsia. the risk of primary infection in a seronegative mother is to %, which carries a to % risk of congenital infection [ , ] . the majority of congenitally infected babies are asymptomatic at birth; however, to % subsequently develop hearing defects or neurodevelopmental sequelae [ ] . although the most serious clinical sequelae are seen in cases where a mother acquires a primary infection during pregnancy, downstream side effects are also seen in cases where latent hcmv is reactivated [ ] and where a mother is reinfected with a different strain of the virus [ ] . hcmv has a double-stranded dna genome of kb encoding approximately genes [ ] . it has a very broad cellular tropism resulting in potential infection of nearly every organ system. the ability of hcmv to enter a wide range of cell types involves a complex interaction between several viral envelope glycoproteins and host cell surface receptors, although the entry of herpesviruses into host cells is still poorly understood. the hcmv virion envelope contains at least virusencoded glycoproteins that are involved in cell attachment and penetration [ ] . of these, glycoprotein b (gb) is the most abundant glycoprotein [ ] and is highly conserved among the herpesviridae [ ] . glycoprotein b plays a critical role in the hcmv entry process. initially, gb along with gm/gn, is involved in tethering of virions to heparan sulfate proteoglycans (hspg) on the surface of host cells. the short interaction of hcmv with hspg is followed by more stable interactions with one or more viral cellular receptors, namely epidermal growth factor receptor (egfr) [ ] , platelet-derived growth factor receptor (pdgfr) [ ] , and toll-like receptor tlr- [ ] . glycoprotein b also interacts with integrin αvβ , a coreceptor that enhances hcmv entry [ ] . integrins are known to synergise with egfr as well as with other receptors to activate signal transduction pathways [ ] [ ] [ ] . to complete the entry process, both viral and cellular membranes fuse, allowing the release of virion-associated tegument and capsid proteins into the cytoplasm. this final step of viral entry into host cells requires gb and the gh/gl complex [ ] [ ] [ ] [ ] . antibodies to hcmv gb have been shown not only to block penetration of virions into cells, but also to limit cell-to-cell infection, implying that gb plays a role in virion penetration into cells, cell-to-cell transmission, as well as fusion of infected cells [ , ] . recently, isaacson and coworkers used genetic complementation to confirm that gb is required for the fusion of viral and cellular membranes, virus entry, and cell-to-cell spread of hcmv [ ] . the importance of gb for viral infection suggests that this viral envelope protein may be a rational target for novel drug design. hcmv infection is highly prevalent in the population due to the ability of the virus to efficiently transmit between hosts that harbour and periodically shed the virus. hcmv is transmitted through direct exposure to infected bodily secretions, including saliva, urine and breast milk. following infection, hcmv enters the bloodstream and spreads to various organs including kidney, liver, spleen, heart, brain, retina, esophagus, inner ear, lungs, colon, and salivary glands [ ] . the ability of hcmv to infect a wide variety of cell types is not due to the presence of high plasma levels of extracellular virus, but is primarily due to cell-to-cell transmission between mononuclear phagocytes (possibly macrophages or dendritic cell precursors) and uninfected tissues [ ] . the lack of a successful hcmv vaccine as well as the toxicity and drug-induced resistance associated with current therapeutics for hcmv indicate that this virus continues to pose a significant public health problem. current treatments for hcmv disease target viral replication and can fail due to the emergence of drug-resistant virus variants and induction of adverse effects. hence, a new approach in drug design against hcmv is required [ , ] . since hcmv and other herpesviruses establish a lifelong latency in humans, antiviral therapy that inhibits viral entry may serve as an alternative to the already existing and inadequate therapeutic agents. here, we report the design, development and characterization of peptides that specifically inhibit viral infection and/or entry as a novel approach to prevent hcmv infection. structural studies place herpes simplex virus type gb- [ ] and epstein-barr virus gb into class iii viral fusion proteins (vfp) [ ] , which also includes vsv g [ ] , members of the gp superfamily (baculovirus and thogotovirus) [ ] and tentatively bornavirus g [ ] . because gb is the most highly conserved envelope protein amongst the mammalian and avian herpesvirues [ , ] , gb of hcmv is likely to be a class iii vfp and shares structural features with gb of other members of the herpesviridae. class iii viral fusion proteins share certain characteristics found in class i or class ii viral fusion proteins. the class iii viral fusion proteins contain an extended α-helix that trimerizes in the postfusion forms of the proteins [ , , ] , as has been well-documented for the post-fusion forms of the class i viral fusion proteins of orthomyxoviruses, retroviruses, paramyxoviruses, arenaviruses, and coronaviruses [ ] . similarly, the class ii viral fusion proteins of flaviviruses and alphaviruses contain a fusion domain comprised principally of β-sheets and "fusion loops." class iii viral fusion proteins also possess a fusion domain, as well as several other features of class ii viral fusion proteins, suggesting that these two classes of proteins may share a common progenitor [ ] . the class iii domain nomenclature used here can apply to both class ii and class iii viral fusion proteins: domain i (green), domain ii (yellow), domain iii (blue), domain iv (stem domain, indigo) ( figure ). this unified nomenclature assigns domain ii appellation to the following: vsv g domain iv in the nomenclature of roche et al. [ ] , hsv- gb- and baculovirus gp domain i in the nomenclature of heldwein et al. [ ] and kadlec et al. [ ] as the class iii fusion domain, which is structurally similar to class ii viral fusion proteins. in addition to minor adjustments in the ends of domains, the current class iii viral fusion protein numbering also combines two interacting domains into domain iii (i + ii in roche's vsv g nomenclature, iii + iv in heldwein's hsv- gb- nomenclature and kadlec's baculovirus nomenclature). the wimley-white interfacial hydrophobicity scale (wwihs) is an experimentally determined hydrophobicity scale that provides a quantitative description of a protein partitioning and folding into membrane interfaces. wwihs score-positive sequences may also interact with hydrophobic surfaces within proteins, and are often sequestered within pre-fusion forms of viral fusion proteins. in addition to similarities in the overall structure of the post-fusion forms of class iii vfp, there are additional similarities in the distribution of wwihspositive sequences (figure , red). the similarities include at least one extended "fusion loop" in the fusion domain (domain ii), and one or more wwihs scorepositive sequences in domain iii. with the exception of the acnpv gp , each of these proteins contains another wwihs positive domain ii sequence near the "hinge" region adjacent to the domain. herpesvirus gb proteins have an additional wwihs scale score-positive sequence in domain i. in the case of class ii and iii viral fusion proteins, the fusion loops in the fusion domain often contain sequences with positive wwihs scores. previous studies have suggested that synthetic peptides corresponding to or overlapping with sequences in viral fusion proteins that have positive wwihs scores can sometimes serve as viral entry inhibitors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, enfurvitide (fuzeon) is a -amino acid peptide that overlaps with a wwihs score-positive sequence in the transmembrane protein (tm) of hiv- , and prevents viral fusion and entry of the virus. to identify regions of hcmv gb that have a high propensity to interact with the lipid bilayer of cell membranes and which potentially may serve as hcmv entry inhibitors, we employed membrane protein explorer version . (http://blanco.biomol.uci.edu/mpex), a computer program based on the wwihs. nine sequences with significant positive wwihs scores were identified ( figure ). as expected, several of these wwihs sequences corresponded to the predicted fusion domain of hcmv gb, including the predicted fusion loops. one of the wwihs score-positive sequences spanned amino acids to (peptide - ) that had a Δg score of . kcal/mol. this sequence was split into two smaller peptides: - and - . a second large figure wimley-white interfacial hydrophobicity scale score-positive sequences in class ii and iii viral fusion proteins. sequences of a representative class ii viral fusion protein (dengue virus e) and of class iii viral fusion proteins with high potential to interface with lipid membranes (red) were identified using membrane protein explorer software (mpex version . ). as discussed in the text, a class iii domain nomenclature is used here that can apply to both class ii and iii viral fusions proteins. the alternative domain number schemes used by roche et al. and heldwein et al. are noted in parentheses. the dengue virus (denv e) stem domain sequence that was not included in the protein used to determine the crystal structure has been added. the denv stem has a positive wwihs scale score, and corresponds to a previously determined inhibitor of denv and west nile virus [ ] . segment within the fusion domain of hcmb gb had a Δg score of . , and was split, consequently, into smaller peptides: - and - . an additional peptide, - corresponding to another fusion domain sequence, was also synthesized, along with an additional peptides corresponding to other wwihs-positive domains of hcmv gb. to prevent dimer formation, cysteines were replaced with alanines. nine synthetic peptides corresponding to sequences with significant wwihs scores were synthesized and examined for their ability to inhibit hcmv infection of hff cells. peptides that were most effective are presented here ( table ) . all synthetic peptides were tested at the following concentrations: μm, μm, μm, the wwihs is a computational approach, based upon an experimentally determined algorithm to estimate the propensity of an amino acid sequence to interact with lipid membrane interfaces [ ] . using this method, we identified several regions of hcmv gb with high interfacial hydrophobicity. peptides that are analogous to several of these regions inhibited hcmv infectivity at low μm concentrations (figure , , , , and ). when tested in combination certain combinations of peptides (peptides - and - ) displayed increased inhibition of infectivity at concentrations of nm (figure ) . these results suggest that the hcmv inhibitory peptides identified here may serve as figure determination of regions within gb that display a high propensity to interact with the lipid surface of cell membranes by using wimley-white interfacial hydrophobicity scale (wwihs). wwihs identifies segments of proteins that prefer a transbilayer helix conformation to an unfolded interfacial location. we used the interface scale of the membrane protein explorer (mpex version . ) computer program to identify these particular segments of hcmv gb. the interface scale measures a residue's free energy of transfer within an unfolded polypeptide chain from water to a phosphocholine bilayer. we identified nine segments of hcmv gb that display high propensity to interact with the lipid surface of cell membrane, and designed peptides, ranging from to amino acids in length, that are analogous to the identified regions of gb. the basis for potential antiviral therapies. the success of the inhibition of fusion greatly depends, not only on biophysical properties of synthesized peptides and their concentrations, but also on the size and shape of the binding pocket of hcmv gb. it is possible that the potency of the peptides, either alone or in combination, can be increased by modifying the sequence of the peptides or by conjugating the peptide(s) to other molecules. akkarawongsa and coworkers prepared a library of overlapping peptides homologous to the ectodomain of herpes simplex virus type (hsv- ) gb- and screened for the ability of these peptides to block infection [ ] . seven out of -mer peptides inhibited infection by more than % at a concentration of μm. three peptides (gb , gb and gb ) with % effective concentrations below μm were studied further. peptide gb (residues to in hsv- gb- ) was identified as a specific entry inhibitor (ec , μm). the gb peptide (residues to in gb- ) blocked viral entry (ec ,~ μm), protected cells from infection (ec ,~ μm), and inactivated virions in solution (ec ,~ μm). of the seven inhibitory peptides identified in the akkarawongsa et al. study, three of them, corresponding to residues to (gb ), to (gb ), and to (gb ), were in regions of hsv- gb- with positive wwihs scores. the success of their study affirms our strategy of targeting the wwihs score-positive sequences as inhibitors of hcmv. two overlapping peptides spanning the residues to (gb ) and to (gb ) were not in wwihs score-positive sequence, but the analogous sequence in hcmv is wwihs score-positive. additionally, comparisons across viral families could potentiate identification of entry inhibitors. for instance, hsv- inhibitory peptide gb corresponding to domain iv, also known as a stem, is analogous to the hrobowski dengue inhibitory peptide for the class ii viral fusion proteins [ ] (figure ) . the most potential inhibitors of hcmv infection were all in domain ii. hsv- inhibitory peptide gb (residues to ) identified by akkarawongsa et al. corresponds to this region, with hcmv inhibitory peptide - being the analogous peptide ( figure ). it is possible that some of the inhibitory peptides identified for hsv- could be optimized to work for hcmv and vice versa. the fusion domain of hcmv gb may be the initial site of interaction directly with the lipids of the cell bilayer [ ] . the hcmv inhibitory peptides could competitively block these interactions, or trigger downstream fusion events, and conformational changes in gb, prematurely. inhibitory peptides corresponding to other domains likely do not block lipid interactions. inhibitory peptides may employ several mechanisms of action, and therefore those that are analogous to domain iii could block receptor interactions. as the result of this study we found that the effects of several of the hcmv inhibitory peptides did not follow a linear dose-dependent inhibition curve. this trend was also reported previously describing peptide inhibitors of dengue and west nile viruses [ ] . possibly, when the concentration of one or more peptides is too high, the peptides may self-associate, preventing them from interfering with virus infectivity. in addition to screening synthetic peptides for their ability to inhibit hcmv infection alone, we tested some peptides in combination. it is possible that peptides - and - block hcmv entry at distinct steps in the fusion process. any two peptides that work additively to block the fusion of the virion with the host cell membrane must have unique amino acid sequences, biophysical properties and be present at certain concentrations that will allow them to interact with each other, with gb and, possibly, with other glycoproteins that are instrumental in the fusion event. peptides - and - tested together showed only % inhibition at the concentration of μm each (figure ) , and did not work in an additive fashion. this result may be due to peptide-peptide interactions that do not allow interference with gb trimer formation and with necessary conformational changes of the virion required for successful fusion to occur. several drugs, including ganciclovir, its oral prodrug valganciclovir, foscarnet, cidofovir, and fomivirsen have been approved for the treatment of hcmv-associated disease. all of these drugs, with the exception of fomivirsen, have a common target, the viral dna polymerase [ ] . the above listed anti-hcmv drugs provoke not only drug-specific side effects, which include leukopenia, thrombocytopenia, anemia, bone marrow hypoplasia, diarrhea, and renal toxicity, but also the emergence of clinically relevant drug-resistant hcmv [ ] . new drugs that are more efficacious and are not toxic in treatment of hcmv infection are urgently needed. the inhibitory peptides identified here can serve as the basis for the development of a novel therapeutic against hcmv. these antiviral agents could be used as an antiviral treatment to reduce the viral load in pregnant women and neonates. it is not clear how hcmv infects the fetus during pregnancy, but some studies demonstrate that placental infection with hcmv occurs before the transmission of the virus to the fetus and suggest that the placenta plays a role in vertical transmission of hcmv from mother to fetus. also, placental viral infection has been implicated in spontaneous abortion during early pregnancy that occurs in fifteen percent of women with primary hcmv infection. placental pathology as a result of hcmv infection during pregnancy may also cause premature delivery, intrauterine growth restriction (iugr), or pre-eclampsia [ ] [ ] [ ] [ ] . enfurvitide, a -amino acid peptide also known as fuzeon works by inhibiting the structural rearrangement of hiv- gp to block the fusion of hiv- virions with their target cell membrane. brennan-benson et al. showed that fuzeon prevents vertical transmission of hiv- in pregnancy, but does not cross the placenta [ ] . we have shown that some peptides are effective at preventing hcmv infection. those lead peptides will be studied further and modified to increase their efficacy, solubility and delivery. currently available drugs to treat hcmv infection are not approved to treat pregnant women due to their potential high toxicity. hcmv infected neonates are treated with these toxic compounds only in cases of high morbidity. the antiviral peptide-based agents that may be developed as a result of this study would not require activation by virally encoded proteins, further phosphorylation by cellular enzymes or incorporation into the growing viral dna by viral dna polymerase as the current therapeutics do. such peptide therapeutics would be predicted not to provoke drug-induced resistance that is a significant problem with existing fdaapproved therapeutics to treat hcmv infection, since they employ a different mechanism of action. our cell viability assays demonstrate that the effective peptides have no statistically significant toxicity at the highest concentrations tested in our studies (data not shown). consequently, we do not expect adverse effects or toxicity due to treatments developed from these synthetic peptides. wwihs is an experimentally determined algorithm that can be used to estimate the propensity of amino acid sequences to interact with lipid membrane interfaces [ ] . sequences of hcmv gb with positive wwihs score were identified using membrane protein explorer (mpex version . ) (http://blanco.biomol.uci.edu/mpex), a computer program based on wwihs. hcmv gb synthetic peptides were synthesized by solid phase conventional n-α- -fluorenylmethyloxycarbonyl chemistry by genemed synthesis inc. (san francisco, ca). peptides were purified by reverse-phase high performance liquid chromatography and confirmed by amino acid analysis and electrospray mass spectrometry. peptide stock solutions were prepared in % dimethyl sulfoxide (dmso, spectroscopy grade): % (v/v) h o. peptide concentrations were determined by absorbance of aromatic side chains at nm (smart-spec ™ , biorad, hercules, ca). the towne strain of hcmv containing the green fluorescent protein (gfp) expression cassette was obtained from dr. daniel streblow at the oregon health science university and was propagated in human foreskin fibroblasts (hff). viral supernatants were collected days after % cpe was observed, centrifuged to clear cell debris, and filtered through a . μm filter. hff were grown in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), penicillin g ( u/ml), streptomycin ( mg/ml), and glutamax ( mm). hff were seeded at a density of . × cells in each well of a -well plate 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preeclampsia and normotensive intrauterine growth restriction? surveillance for congenital cytomegalovirus disease: a report from the national congenital cytomegalovirus disease registry enfurvitide prevents vertical transmission of multidrugresistant hiv- in pregnancy but does not cross placenta the pymol molecular graphics system peptide inhibition of human cytomegalovirus infection the authors would like to thank dr. daniel streblow at the oregon health science university for kindly supplying the virus strain used in this study, and dr. william wimley for helpful discussions. authors' contributions lm participated in the experimental design, performed viral propagation and all experiments, and drafted the manuscript. cm and rg conceived of the study, and participated in its design and assisted lm in the interpretation of results. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -c cjxq authors: gwyer findlay, emily; currie, silke m.; davidson, donald j. title: cationic host defence peptides: potential as antiviral therapeutics date: - - journal: biodrugs doi: . /s - - - sha: doc_id: cord_uid: c cjxq there is a pressing need to develop new antiviral treatments; of the drugs currently available, half are aimed at hiv- and the remainder target only a further six viruses. this demand has led to the emergence of possible peptide therapies, with currently in clinical trials. advancements in understanding the antiviral potential of naturally occurring host defence peptides highlights the potential of a whole new class of molecules to be considered as antiviral therapeutics. cationic host defence peptides, such as defensins and cathelicidins, are important components of innate immunity with antimicrobial and immunomodulatory capabilities. in recent years they have also been shown to be natural, broad-spectrum antivirals against both enveloped and non-enveloped viruses, including hiv- , influenza virus, respiratory syncytial virus and herpes simplex virus. here we review the antiviral properties of several families of these host peptides and their potential to inform the design of novel therapeutics. abstract there is a pressing need to develop new antiviral treatments; of the drugs currently available, half are aimed at hiv- and the remainder target only a further six viruses. this demand has led to the emergence of possible peptide therapies, with currently in clinical trials. advancements in understanding the antiviral potential of naturally occurring host defence peptides highlights the potential of a whole new class of molecules to be considered as antiviral therapeutics. cationic host defence peptides, such as defensins and cathelicidins, are important components of innate immunity with antimicrobial and immunomodulatory capabilities. in recent years they have also been shown to be natural, broad-spectrum antivirals against both enveloped and non-enveloped viruses, including hiv- , influenza virus, respiratory syncytial virus and herpes simplex virus. here we review the antiviral properties of several families of these host peptides and their potential to inform the design of novel therapeutics. viral diseases are a leading cause of morbidity and mortality worldwide, particularly of children [ ] , and yet development of effective therapies is slow. in particular, progress is hampered by the fact that the majority of antiviral drugs are specific for only one virus. current approaches are expensive, require rapid identification of the virus before therapy and, at the initial stages of development, involve enormous redundancy of research effort. this also results in efforts being concentrated on a few viruses; of the antiviral drugs that have so far been approved by the us food and drug administration (fda), almost half target hiv- ; the remaining half are used for the treatment of hepatitis b virus (hbv), herpes simplex virus (hsv), varicella-zoster virus (vzv), cytomegalovirus (cmv), influenza (iav) and hepatitis c virus (hcv) infections [ ] . these factors, in combination with the rate of development of drug resistance, mean that there is an urgent need for new broader-spectrum intervention strategies. it is therefore exciting that in recent years a new class of antiviral therapeutic peptides are emerging, with peptidebased intervention strategies against viruses currently in various stages of clinical trials [ ] . peptide-based strategies are proposed to be cost-effective, with peptides having low molecular weights, rapid elimination following treatment, and low levels of side effects [ ] . an exciting current area of advancement is in understanding the antiviral properties of naturally occurring cationic host defence peptides (chdps) and the capacity of this to inform the design of novel synthetic antiviral analogues. in this review we will give an overview of the antiviral activities of chdps and consider their potential in the development of broad-spectrum antiviral therapeutics. defence against infection is emphasised by their conservation across plants, insects, reptiles, birds and fungi [ ] . in mammals the two major families of chdps are defensins and cathelicidins. defensins are cationic, amphipathic peptides generated from prepropeptides via proteolysis and are categorised within three subfamilies: a, b and h. defensins were first characterised as ''natural peptide antibiotics'' with the discovery of a-defensins from the granules of neutrophils [ ] . the a-defensin family has been identified in a range of higher eukaryotes (including primates, mice, rats, guinea pigs and rabbits) and comprises six distinct peptides in humans (human neutrophil peptides (hnp) - and human defensins (hd) - ), expressed from five defa genes [ ] . all have a triple-stranded b-sheet core stabilised by three intramolecular disulphide bonds, and are made first as a prepropeptide which is proteolytically cleaved to the active form [ ] . in the case of hd and hd the key protease is trypsin. hnp - are produced mainly by neutrophils, where they comprise - % of total neutrophil protein [ ] , and neutrophil precursors in the bone marrow [ ] . hnp - are also described in nk cells, b cells, cd t cells, macrophages and immature dendritic cells [ ] , but can be acquired from neutrophils [ ] . the release of active hnps from neutrophil azurophilic granules can be induced by a range of stimuli, including chemokines, fc gamma receptor cross linking, pma and tlr stimulation [ ] . in contrast, hd and are expressed by paneth cells of the small intestine [ , ] and epithelial cells of the female genital tract [ , , ] . interestingly, although mice express a large number of intestinal a-defensins (cryptdins) [ ] , in contrast to humans they do not express a-defensins in their neutrophils [ , ] . a-defensins have well-described broad spectrum antimicrobial activity against both gram-positive and -negative organisms in vitro [ ] , with cationicity and hydrophobicity being shown to be key determinants of these properties [ , ] . their cationic charge is proposed to enable interaction with the net negative charge on the surface of the gram-negative bacteria and the teichoic acids of the gram-positive organisms, while their amphipathic structure enables insertion into and disruption of the bacterial membranes, leading to lysis of the cells. in addition, various a-defensins are described as having additional, non-microbicidal properties, including chemotaxis for effector cells of the innate and adaptive immune systems [ , ] , inhibition of macrophage pro-inflammatory cytokines [ ] , modulation of the intestinal microbiome [ ] and the formation of protective peptide nanonets [ ] . the b-defensin family contains more than members in humans and more than in mice, and are widely expressed across many species, in particular being the only defensins found in birds and with close homologues present in snakes, platypus and sea anemones [ ] . they are also triple-stranded b-sheet proteins, but differ from the a-family members in the organisation and arrangement of the three disulphide bonds [ ] . the most well characterised human b-defensins are human b-defensin (hbd) - , expressed by epithelial cells [ ] , monocytes, macrophages and macrophage-derived dc [ ] . hbd is encoded by defb and is expressed constitutively [ ] , whereas expression of hbd (defb a) and hbd (defb a) are up-regulated in response to various inflammatory stimuli, including microbes [ ] , tlr and nod proteins [ ] and pro-inflammatory cytokines [ ] . b-defensins are also described as having broad-spectrum antimicrobial activity in vitro [ ] , with potency varying in different family members. interestingly, the weak microbicidal properties of hbd were recently shown to be greatly enhanced upon reduction of its disulphide bonds [ ] . although relatively mild phenotypes were found in mouse models deficient in defb [ , ] , redundancy in this multi-gene family may be responsible for this observation. in addition b-defensins are described as having a range of immunomodulatory properties, including chemotaxis of cd ? memory t cells, macrophages and immature dendritic cells [ ] , enhancement of wound healing [ ] and the modulation of inflammatory cytokine responses (with the capacity both to promote and to suppress inflammation in different settings) [ ] . h-defensins are circular octadecapeptides, the only circular peptides of mammalian origin [ ] , formed by the splicing of two nonapeptides, each of which contributes three cysteines to a series of disulphide bonds in the mature peptide [ ] . three h-defensins have been found in the leukocytes of rhesus macaques and named rtd - , but although six rna transcripts homologous to rtd are found in the human bone marrow they contain a premature stop codon preventing their expression [ , ] . however, an artificially constructed peptide based on these pseudogenes, called retrocyclin, has been studied with respect to its function and therapeutic potential and shown to kill escherichia coli in the same way as a-defensins, by permeabilising its membrane [ ] . the cathelicidin family is quite distinct from defensins; cathelicidins are defined by a conserved cathelin domain and with a variable c-terminal region, which is proteolytically cleaved to produce a mature functional peptide, with a range of structural forms in different family members [ ] . in contrast to the extensive defensin family, humans (and mice, rats and rabbits) express a single cathelicidin, whereas multiple cathelicidins are found in other species (e.g. protegrins in pigs). the sole human cathelicidin, human cationic antimicrobial peptide of kda (hcap- ; encoded by the camp gene), is cleaved by proteinase into its active form, ll- , which is a cationic, amphipathic peptide of . kda with an a-helical structure [ , ] . hcap- is stored in neutrophil-specific granules, inducible in epithelial cells, macrophages and other leukocytes to a lesser extent, and detectable in a range of body fluids, including airway surface liquid, plasma, urine, breast milk and sweat [ ] . it is up-regulated in response to infectious and inflammatory signals [ ] and wounding [ ] and its expression can be increased by vitamin d metabolites [ ] and compounds such as butyrate [ ] . ll- has well-documented antibacterial potential; however, when studied in the presence of physiological concentrations of cations or serum, ll- has high minimum inhibitory concentrations compared to levels described in vivo [ ] . mice deficient in mcramp (encoded by the camp gene, the orthologue of camp) have increased susceptibility to infections in multiple systems, including the skin, intestinal tract and lung [ ] [ ] [ ] . although these models clearly demonstrate the critical role for cathelicidin in host defence against infection, it remains unclear to what extent this is due to microbicidal or modulatory properties of the peptide. ll- has been shown to have a broad range of immunomodulatory and inflammomodulatory properties [ ] . these include chemotactic activity for neutrophils, monocytes and t cells [ ] , modulation of cytokine production [ ] , effects on dendritic cell differentiation and function [ , ] , promotion of wound healing [ ] and angiogenesis [ ] , and modulation of cell death [ , ] . although the field of antimicrobial peptide research has been dominated by evaluation of the antibacterial activities of these peptides, early studies evaluating the antiviral potential of human a-defensins showed promise and have been followed by an increasing level of research interest (fig. ). the first paper detailing an antiviral role was published years ago, describing inhibition of a number of viruses including hsv types and , cytomegalovirus and vesicular stomatitis virus by hnp in vitro [ ] . in particular, it demonstrated direct antiviral activity of hnp against hsv- in a temperature-and ph-dependent manner, inhibited by serum, but interestingly less sensitive to the inhibitory effects of cations than the more broadly studied antibacterial properties. hnp - , hd and hd have subsequently been found to be active against hsv- (up to approximately log decrease at lg/ml) by preventing viral binding to either glycoprotein b (gb ) or heparan sulphate (the primary receptor for hsv) [ ] . gb binding capacity was found to be primarily determined by peptide sequence rather than net cationic charge [ ] , with lectin-like properties likely to be key to the glycoprotein binding functions [ ] . interestingly, those defensins (hnp - and hbd ) which bound viral envelope gb were also found to be effective at preventing infection if added after viral entry, even up to h post-infection (with hnp or hd ), suggesting additional later stage effects on viral replication [ ] . furthermore, cumulative effects were observed with repeated application of peptide, reducing infection with hsv by greater than logs at lg/ml. these features make a-defensins of considerable interest as potential exogenous vaginal microbicides, with a proof of concept study in mice demonstrating protection against hsv following gel-based application of hd [ ] . a-defensins have been demonstrated to directly inactivate hiv- , with the observation of reduced cytopathogenicity in a cd ? t cell line [ ] . although confusion existed following the retraction of a paper proposing that adefensins were the key component of the cd antiviral factor caf [ , ] , it is worth noting that the retraction related to the source of the a-defensins (probably ''imported'' into cd cells from co-cultured cells), and the anti-hiv- properties of the peptides were not called into question. indeed, the potential of a-defensins to treat hiv was reinforced when it was found that breast milk a-defensin concentration is significantly associated with a decreased risk of intrapartum and postnatal hiv transmission [ ] . recent studies demonstrate that multiple steps in the entry of hiv- virus into cells are disrupted by hnp [ ] . this peptide has been shown to bind to cd and to the env glycoprotein on the virus, thus inducing the down-regulation of cd and cxcr and blocking interaction of env with the co-receptors. by targeting particular conformations of env it also inhibited late fusion steps [ ] . hnp - can bind to cd and hiv- gp with high affinity; however, hnp , which is a much weaker binder, is a more potent inhibitor, meaning this aspect of direct inhibition is not, currently, entirely clear [ ] . mechanistic studies have shown that hnp - can also inhibit steps following reverse transcription and integration by inhibiting pkc activity; pkc is important for hiv replication as it upregulates transcription through nf-jb activation and tat phosphorylation, as well as regulating fusion and assembly of the virions [ ] . it is worth noting that in these studies the direct effects on the virus particles occurred only in the absence of serum; in its presence, these mechanisms were inhibited and effects are instead on the host cells, resulting in inhibited replication of the virus [ , [ ] [ ] [ ] . defensins can also stimulate an antiviral state in cells by promoting secretion of chemokines [ ] . in macrophages this upregulation of chemokines also contributes to inhibition of hiv through competition for receptors [ ] . a potential issue with using defensins as a topical antiviral treatment is that, despite chdps (including defensins) being required for in vitro anti-hiv activity of vaginal fluid from healthy women, defensins may also cause immune activation and subsequent loss of cd ? t cells by apoptosis, and may indirectly enhance hiv transmission [ ] . in particular, it is known that hnp and hbd are both chemotactic for dc and induce infiltration of dc into cultures of hpv-transformed keratinocytes and subsequent immune activation [ ] . in addition, hd and , which are produced by cervico-vaginal epithelial cells [ ] and present at up to lg/ml in the vaginal fluid of healthy women [ ] , enhance hiv infectivity in vitro by promoting the virus attachment to target cells [ , ] . thus, the balance of these effects remains to be determined. a-defensins are also found in high concentrations in the inflamed lung, generating interest in their potential activities against respiratory viruses. hnp - are the most abundant antimicrobial peptides in airway fluid [ ] and are up-regulated further following infection or inflammation [ ] as they are produced by both immigrating neutrophils and airway epithelial cells [ ] . daher et al. [ ] first described antiviral effects of hnp against the wsn strain of iav in vitro, showing a fairly modest approximately . log reduction at lg/ml. this property of hnp and was later confirmed in other strains of iav (with decreased infectivity of approximately log at lg/ml in a fluorescent focus assay of infectivity). the peptides were shown to have no activity in a haemagglutinin inhibition assay [ ] , but to induce aggregation of iav [ ] . interestingly hnp aggregation of the pr viral strain was much higher than that of the phil strain, which has greater surface glycosylation [ ] [ ] [ ] , and neutralising activity of hnps was greater against pr than against phil [ ] , suggesting the reduced carbohydrate attachments on the envelope proteins allow greater interaction with the defensins. however, direct effects of hnp on iav viral particles were found to have no impact on viral growth in infected cultures [ ] . maximal antiviral effects in this study ( - logs at - lg/ml hnp in a range of cell lines) required interaction of hnp with the eukaryotic cells before infection and the continued presence of peptide in the culture system. nevertheless, in contrast to pretreatment of virus, pretreatment of the cells did induce some protection. this suggests the predominant mechanism of action is cell-mediated, and has been proposed to be a consequence of hnp-mediated inhibition of pkc activity (essential for endosomal trafficking of the iav) [ , ] . the capacity of a-defensin to protect against iav was lost in its linearised analogue [ ] . in addition to effects on iav infection of epithelial cells, hnp and at approximately lg/ml (but not hnp , hbd or ) can enhance the uptake of iav by neutrophils, following pre-incubation of either the cells or the virus with the defensins [ ] . however, this modulation of neutrophil phagocytic capacity was also observed using bacteria, and was not virus specific. these properties of a-defensins suggest potential as templates for novel therapeutics. however, their ability to bind surfactant protein d (sp-d) may be of concern, having mixed competitive or co-operative, and iav straindependent effects on the antiviral activities of sp-d [ ] . hnps (but not b-defensins) can bind and precipitate bronchoalveolar lavage (bal) fluid sp-d [ , ] and may account for sp-d depletion from the lung in diseases with chronic neutrophilic inflammation. it was originally suggested that a-defensins had no activity against non-enveloped viruses, on the basis of the absence of effects against echovirus type ii and reovirus type [ ] . however, more recent work has demonstrated inhibition of non-enveloped viruses such as adenovirus, human papillomavirus (hpv) [ ] [ ] [ ] and bk virus [ ] . the mechanisms underpinning these antiviral effects against non-enveloped viruses appear to be distinct from those targeting enveloped viruses. a study of hpv (utilising pseudovirus particles) found normal binding, uncoating and internalisation in the presence of a-defensins; however, the peptides prevented the virion from escaping the endocytic vesicles [ ] . the antiviral activity was observed using hnp - (and the cathelicidin ll- ), was maximal using hd , but was not observed using hd . the sensitivity of adenoviruses to a-defensin-mediated neutralisation is serotype-dependent [ , ] . hnp and hd have been shown to inhibit human adenovirus infection in both lung and conjunctival epithelial cells by inhibiting an early step in viral entry [ , , , ] . two arginine residues on one face of hd were found to be critical for antiviral activity, with arg- necessary for killing of both adv and hpv and arg- for adv only [ ] . viral aggregation is not sufficient for neutralisation and binding of a-defensin to the adenoviral virus capsid appears to be critical, preventing uncoating in the cell and hence entry of the viral genome into the nucleus [ , ] . in contrast, the antiviral effects of hnp and hd on the bk polyomavirus appear to primarily relate to viral aggregation preventing receptor binding on the host cells [ ] . [ ] . similarly, human rhinovirus (hrv) replication in human bronchial epithelial cells induces nf-jb-dependent hbd and expression (but not hbd ) [ , ] , whereas respiratory syncytial virus (rsv) can induce hbd in an nf-jb-dependent, but ifn type -independent manner in human lung epithelial cells [ ] . the extent to which these innate responses are functionally effective against the viral pathogens is starting to be elucidated (fig. ). in contrast to the effects of a-defensins, only one of the bdefensins tested (hbd ) had appreciable effects against hsv [ ] . hbd was found to inhibit hsv- infection of human cervical epithelial cells (by approximately log at lg/ml) by interfering with the viral binding and penetration processes by binding both gb and heparan sulphate. in contrast, hbd and hbd had low affinity to gb and cellular glycosaminoglycans, and were not able to reduce hsv- infectivity. however, only hd was applied in vivo in this study [ ] . hiv- infection of epithelial cells induces hbd and expression in vitro [ ] and in buccal mucosal cells in vivo [ ] , and both inhibit hiv transmission through multiple mechanisms [ , ] . both down-regulate the co-receptor cxcr expression (but not ccr ) on the surface of cd ? t cells via an increase in internalisation [ ] . in addition, there are both direct effects on the virions in a concentration-dependent manner [ ] and on intracellular, post-viral entry inhibition [ ] . however, in large-scale studies, copy number variation of total b-defensin gene number positively correlates with hiv load in ethiopian and tanzanian patients [ ] . the authors suggest that the chemoattractant nature of bdefensins may bring the target th cells into mucosal sites where they can be readily infected by virus. other work, however, has recently shown that exposed but seronegative individuals have much higher hbd and copy numbers in oral mucosa (but not vaginal mucosa) than healthy controls, indicating a role for these defensins in combating infection [ ] . although not normally regarded as inducible, expression of hbd (but not hbd , or ) has been observed in primary human blood-derived plasmacytoid dc and monocytes following infection with iav [ ] . in contrast, an early decrease in expression of this peptide was found in iav-infected epithelial cell lines [ ] . recombinant hbd was found to have antiviral effects in vitro (approximately . log decrease at lg/ml). however, despite evidence of a significant defect in host defence against iav in defb -deficient mice, this did not extend to differences in viral load, suggesting a primary role of immunomodulatory properties in vivo [ ] . iav has been found to up-regulate expression of murine defensins mbd and mbd in upper and lower airways, as well as transcription of the genes encoding mbd and mbd in the lung [ ] , also suggesting protective roles for these peptides. recombinant mbd [ ] and mbd [ ] protected mdck cells against infection with iav pr strain (up to approximately log decrease at lg/ml); this protection was effective during binding or internalisation of the virus, but not after viral entry. furthermore, repeated intranasal administration of recombinant mbd ( mg/kg; optimal when premixed with virus before infection) or intravenous delivery of recombinant mbd ( mg/kg) was found to be protective in murine lethal infection models [ , ] . the latter study also suggested that the effects may relate to immunomodulatory properties, with systemic mbd treatment up-regulating ifn-c and il- and reducing levels of tnf. thus, although b-defensins can inhibit influenza virus infectivity (albeit less potently than the a-defensins or ll- ) [ ] , immunomodulatory properties, perhaps also including up-regulation of iav uptake by neutrophils [ ] , may prove to be key to their protective function against this virus in vivo and future therapeutic developments. in addition to effects on iav, evidence has been found for b-defensin activity against rsv, another important respiratory virus, for which no effective vaccine or antiviral treatments exist. in studies using the a human lung cell line, hbd was identified as a component of an nf-jbdependent, ifn-a/b-independent antiviral response [ ] . epithelial cell hbd production was induced in response to rsv replication and also in response to the tnf secreted by infected epithelial cells. hbd , but not hbd , was found to protect against rsv infection (approximately log decrease at lg/ml), by blocking viral cellular entry [ ] . destabilisation of the viral envelope was proposed to occur upon contact with soluble hbd in solution, or following exposure to plasma membrane-associated hbd during cell entry. in addition to lung epithelial cells, myeloid cells can produce high levels of tnf in response to rsv infection [ ] . this has the potential to up-regulate hbd expression in the airway lumen and might consequently modulate protection against rsv infection and limit virus spread. interestingly mbd (but not mbd ; both considered homologues to hbd ) was found to be upregulated in vivo in the murine lung in response to rsv infection [ ] . hbd has been proposed to have antiviral activity against vaccinia virus [ ] . however, hnp , hbd and hbd are unable to neutralise the virus [ ] . pre-exposure of virus to synthetic hbd for h decreased infection of the bsc- monkey kidney cell line, shown both by reduced levels of dna-dependent rna polymerase expression and plaque formation (approximately log decrease at lm) [ ] , although the mechanism remains to be elucidated. up-regulation of hbd expression was observed in response to vaccinia virus infection in primary human keratinocytes, but this could be inhibited by il- and il- . interestingly, these cytokines are associated with pathology in atopic dermatitis, a condition in which b-defensin expression is reduced [ ] and patients are at risk of developing eczema vaccinatum caused by vaccinia virus. circular h-defensins were identified in the leukocytes and bone marrow of macaques (rhesus h-defensins; rtd) [ ] and discovered to have effective antibacterial activity. humans were found to have at least six h-defensin genes (deft genes) [ ] , but none produce a translated protein, owing to insertion of a premature stop codon. putative ancestral human h-defensins, named retrocyclins (rc), were developed and their antimicrobial properties were tested [ ] . in addition to activity against pseudomonas aeruginosa, e. coli, listeria monocytogenes and staphylococcus aureus, antiviral potential has also been described (fig. ) . both rhesus h-defensins and retrocyclins (including rtd , rc and rc ) have been found to inhibit hsv- and hsv- infection of human cervical epithelial cell lines following pre-incubation of virus and peptide [ ] . however, rc was found to have no direct virucidal properties [ ] , but to be active irrespective of pre-incubation by blocking attachment and cell penetration of hsv [ ] . this activity resulted from peptide binding to gb , in a manner dependent on the presence of sialic acid and carbohydrate moieties in the glycoprotein's o-and n-linked glycans. prophylactic application of rc in a murine hsv-mediated ocular keratitis model demonstrated the capacity of rc to modestly reduce viral titres in vivo and reduce blepharitis, corneal vascularization and stromal disease. however, rc had no effect upon disease pathology when applied post-infection [ ] . initial studies on retrocyclins found no direct inactivation of hiv- , but demonstrated strong inhibition of proviral dna formation and protection of primary human cd ? t cells from t-and m-tropic hiv- strains in vitro [ , ] . these observations led to a significant body of work evaluating retrocyclins as hiv treatments. rc has been characterised as a lectin, which protects peripheral blood [ ] . this peptide prevents viral entry into cells [ , ] , blocking formation of the -helix bundle required for fusion, by binding to hiv gp and cellular cd through interactions with their o-and nlinked sugars [ ] . activity of many of the initial retrocyclins produced was inhibited by serum, minimising usefulness in serum-containing anatomical compartments [ ] . however, analogues, each differing from rc by a single amino acid substitution, show greater potential as topical microbicides [ ] . of these, rc was found to inhibit primary cd ? t cell infection by hiv- similarly to rc , but was active in the presence of vaginal fluid, and rc is significantly more potent against hiv than rc ; both have potential as topical microbicides [ ] . interestingly, the rate of escape mutations of rc -treated hiv was low; treatment altered sites in hr and hr of gp but these only reduced susceptibility by -fold, compared to , - , -fold for ccr blockade [ ] . using an organ culture model, rc was found to block transmission of two strains of hiv- across cervical mucosa. antiviral activity was retained in the presence of semen and vaginal fluid, there was no cytotoxicity to cervical tissue and, importantly, rc did not induce a pro-inflammatory response, with no chemotactic activity for immune cells [ ] . in addition, topical intra-vaginal rc application in pigtailed macaques was found to be safe and well tolerated, with peptide retained in the cervical and vaginal tissue for up to days post-application, no changes in vaginal ph observed and minimal effects on commensal microbiota [ ] . the application of retrocyclins to pathogenic respiratory viruses has also been evaluated. expression of recombinant rc in both mdck cells and chicken embryos has been shown to inhibit replication of an h n influenza virus [ ] . in addition, rc , rc and rc all had antiviral activities in studies using mdck and a cell lines (approximately log decrease at lg/ml against iav phil , but less effective against pr strain) [ ] . the retrocyclins were more effective than hbd and hbd , with potency equivalent to hnp - . this study noted the capacity of the rc peptides to induce aggregation of the virus and increase viral uptake by neutrophils and macrophages [ ] . rc was also found to inhibit influenza a/x infection of cell lines in vitro by blocking the haemagglutinin-mediated fusion of viral and endosomal membranes [ ] . the inhibition of hemifusion formation was shown to be a lectin property which involved cross linking and immobilising surface glycoproteins and blocking the protein displacement required to bridge the bilayers for fusion. rc was only effective when given before viral internalisation, but had antiviral properties even when only applied to the host cell membrane. interestingly, despite the capacity to bind sp-d, retrocyclins increased, rather than inhibited, the antiviral activity of sp-d [ ] . this is in contrast to the a-defensins [ ] , and promising for therapeutic use at mucosal sites. to further improve the antiviral activity of synthetic retrocyclins against influenza and simplify their structure, a series of analogues containing - amino acids, termed hapivirins and diprovirins, were designed [ ] . some of these analogues, including hpv , , - , , and dpv , , and proved to be more effective than rc and rc against iav phil (h n ) and pr- (h n ). mechanistically analogous to the retrocyclins, these peptides were found to be active before viral internalisation, induce viral aggregation, opsonise virus, and have an additive effect with sp-d. in addition, hapivirins and diprovirins were shown to inhibit iav-induced macrophage tnf production, adding immunomodulatory mechanism to their therapeutic potential. antiviral activity of retrocyclins has also been reported against sars virus, a coronavirus which infects the alveolar epithelial cells and induces rapid severe lung pathology [ , ] . in a mouse model of sars infection, rtd- treatment increased survival from to %. interestingly, this was in the absence of any impact on viral titres. instead the cytokine profile of the infected animals was modified, with increased early (day ) production of il- and gm-csf, but decreased il- a, il- b, il- , mip a, and il- p by day . these data highlight the possibility that novel peptide therapeutics may be productively targeted at modulation of the inflammatory response to infection, rather than developed for virucidal properties per se. such an approach may prove to have broad-spectrum applicability and lends further support to the potential for these peptides as novel immunomodulatory antiviral agents. cathelicidins have been widely studied with regard to their antibacterial properties and broad array of immunomodulatory activities [ ] . although known to be up-regulated in inflammation and released by neutrophils, their roles in defence against viral infection and properties as antiviral agents are less well understood (fig. ). hcap- is expressed in human epididymal epithelium and is present at high concentrations in seminal plasma [ ] . it is also expressed in cervico-vaginal secretions and upregulated in participants with bacterial sexually transmitted infections [ ] . cervico-vaginal ll- levels in hivnegative individuals who were in hiv sero-discordant relationships were also found to be greatest in those whose hiv-positive partners had the highest viral load [ ] . ll- has been shown to inhibit the replication of a range of hiv- isolates in primary cd ? t cells [ ] . this occurred in a manner that was independent of change in expression of any hiv- receptors in these cells. a recent study demonstrated that ll- was capable of a dosedependent suppression of hiv reverse transcriptase activity [ ] . this study also demonstrated that this function was retained by a amino acid fragment of ll- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , with implications for production of smaller synthetic analogues. thus, epithelial expression of ll- has been proposed to contribute to local protection against hiv- infection. however, although the cationic peptide fraction of cervico-vaginal secretions was found to have hiv- neutralising activity, which could be enhanced by addition of recombinant ll- , this property did not correlate with levels of endogenous ll- detected [ ] . in addition, in one study ll- levels were independently associated with increased hiv acquisition, although both observations might be the result of a high prevalence of sexually transmitted infections in these individuals [ ] . therefore, the in vivo significance of ll- in hiv remains unclear. we recently demonstrated that ll- has antiviral effects against iav, both in vitro and in vivo [ ] . both human and murine cathelicidins had antiviral activity when preincubated with influenza viruses in vitro [approximately log decrease at lg/ml against a/pr/ / (h n ), but somewhat less effective against a/udorn/ / (h n )]. in addition, ll- has recently been shown to bind iav, without aggregating or affecting haemagglutination activity, and to have maximal effects in vitro when pre-incubated with virus (although delayed addition of peptide or treatment of the cells, with washing before infection also has some antiviral effects) [ ] . surprisingly, ll- was found not to alter the binding or initial uptake of virus by cells, but peptide-mediated disruption of viral membranes (shown by electron microscopy) was proposed to affect viral propagation or survival downstream of this [ ] . we demonstrated that aerosolised therapeutic administration of either ll- or mcramp (given every day for week, with an additional pre-infection dose) provided protection against infection in a mouse model [ ] . peptide-treated mice showed increased survival and decreased weight loss compared to control infected animals, and were similarly protected to those treated with zanamavir (a neuraminidase inhibitor currently used therapeutically in humans). the cathelicidin-treated mice showed some decrease in viral loads, but more striking reductions in lung cytokines (in particular gm-csf, il- b, kc and ccl ), again suggesting the possibility of a key immunomodulatory roles in the antiviral efficacy of such peptides. scrambled control peptides did not have these effects but interestingly the d-enantiomers did, indicating the actions of ll- are not solely related to charge and are likely not reliant on specific receptor interactions. the mechanisms by which cathelicidins modulate inflammation in iav infection remain unclear, but may relate to the observation that ll- can modulate tolllike receptor signalling [ ] . induction of rapid antiviral responses depends, at least in part, on tlr recognition of the viral genome, with ssrna and dsrna viruses recognised by tlr / and tlr [ ] . ll- complexed with self dna and rna has been shown to induce tlr -, tlr -and tlr -dependent inflammatory responses to otherwise non-immunostimulatory nucleic acids [ , ] . in addition, ll- has been proposed to enhance [ , ] or inhibit [ ] tlr -dependent responses to viral rna or synthetic mimics. these modulatory properties may well prove to underpin the in vivo effects. the expression of ll- /hcap by airway epithelial cells can be induced in vitro by infection with rsv [ ] , in a manner that is significantly enhanced by the , oh metabolite of vitamin d. in addition, a recent study found that median serum hcap- levels are significantly lower in children with rsv bronchiolitis than in children with bronchiolitis caused by human rhinovirus [ ] . furthermore, rsv-infected children with hcap- levels lower than the median are more likely to be hospitalised for prolonged periods than those with hcap- levels above the median. these findings suggest an important antiviral role for ll- in host innate immune response against rsv. in keeping with this hypothesis, we have recently demonstrated that ll- exhibits effective, dose-dependent and timing-specific anti-rsv activity in vitro in a number of cell lines [ ] . these data indicate that therapeutic use of cathelicidin or strategies to up-regulate cathelicidin expression in vivo (particularly during the winter when low vitamin d levels may lead to diminished cathelicidin expression) may be protective against rsv infections. individuals with atopic dermatitis (ad) have low levels of ll- expression and increased susceptibility to skin infections, in contrast to those with psoriasis, who have high levels of ll- expression and are less prone to skin infections, despite similar disruption to skin barrier function [ ] . the low levels of ll- expression in ad are proposed to be important in the susceptibility to eczema vaccinatum, a disseminated viral skin infection that follows inoculation with vaccinia virus (vv). ll- expression was induced in response to vv in normal and psoriatic skin biopsies, but not those from ad skin [ ] . both ll- and mcramp have been shown to have antiviral activity against vaccinia virus in vitro (approximately log decrease at lm) [ ] . these peptides were shown to damage the integrity of the double-layered viral envelope [ ] , by removing the outer membrane [ ] . in addition, camp-/-mice were found to develop significantly more pox skin lesions following infection with vv, demonstrating the antiviral effects of cathelicidins in vivo [ ] . interestingly these effects against vv were found to be specific to cathelicidins, with hnp , hbd and hbd unable to neutralise the virus [ ] . this implies that the different chdps may have different (if sometimes overlapping) targets in order to successfully deal with the enormously wide range of human pathogens. the antiviral activities of host defence peptides have been somewhat neglected until recently, in contrast to the study of their antibacterial effects. yet, as discussed in this review, a strong basis exists for future evaluation of the physiological roles of chdps as key components of innate antiviral host defences and to develop synthetic analogues as novel antiviral therapeutics. the precise mechanisms involved clearly vary in a peptide-and virus-specific manner, from direct effects on the viruses through to primarily immunomodulatory properties. these need to be examined in detail and tested in vivo against specific viral infections. such research is expected to reveal therapeutic strategies that range from simple administration of more potent antiviral and/or immunomodulatory analogues, to therapeutic measures to enhance natural levels of expression, replace down-regulated peptides or provide peptide at an earlier, critical ''tipping point'' stage in infection. although some peptides may prove to be effective when administered after infection is established, the optimal use of other peptide therapeutics may be prophylactic administration in high-risk populations (e.g. infants and elderly in an outbreak of a novel influenza strain for which a vaccine is not available). challenges remain in optimising peptide therapeutics to develop the shortest, cheapest analogues that are stable and function at lower concentrations, in physiological environments, without cytotoxic effects or the generation of resistance. in this regard, the h-defensins appear particularly exciting as potential topical anti-hiv agents. they are well tolerated, certain members of the family appear to function in the presence of serum, they rapidly and directly kill hiv- without inducing large-scale immune activation and escape variants are infrequent. engineering of peptides is a simple process, which enables development of more suitable compounds. the difference in the antiviral capabilities and rc values of hnp and , despite them differing by only a single amino acid, hints at what is possible. the engineering of rc from rc (again a single amino acid change) leading to significantly higher anti-hiv- activity also suggests that further enhancements should be possible. likewise, linear, disulphide bond-free defensins have been generated [ ] , which retain potent antimicrobial activity. the development of linear peptides would be significantly easier than recreating their tertiary structure. however, the stability of such peptides against protease degradation remains uncertain and loss of antiviral properties following linearisation has been described in some instances [ , ] , highlighting the need for further research. thus, in an era of increasing concern about the resurgent threats of infectious diseases, a very limited repertoire of antiviral drugs and fears about the rapidity with which new viruses can spread globally, peptide therapeutics have potential that is clearly worth pursuing. acknowledgments djd is supported by a mrc senior non-clinical research fellowship (g ). smc is supported by a university of edinburgh college of medicine and veterinary medicine studentship. the authors have no conflicts of interest that are directly relevant to the content of this article. open access this article is distributed under the terms of the creative commons 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hnp- analogs without cysteines key: cord- -hi z nob authors: huang, jian; ru, beibei; li, shiyong; lin, hao; guo, feng-biao title: sarotup: scanner and reporter of target-unrelated peptides date: - - journal: j biomed biotechnol doi: . / / sha: doc_id: cord_uid: hi z nob as epitope mimics, mimotopes have been widely utilized in the study of epitope prediction and the development of new diagnostics, therapeutics, and vaccines. screening the random peptide libraries constructed with phage display or any other surface display technologies provides an efficient and convenient approach to acquire mimotopes. however, target-unrelated peptides creep into mimotopes from time to time through binding to contaminants or other components of the screening system. in this study, we present sarotup, a free web tool for scanning, reporting and excluding possible target-unrelated peptides from real mimotopes. preliminary tests show that sarotup is efficient and capable of improving the accuracy of mimotope-based epitope mapping. it is also helpful for the development of mimotope-based diagnostics, therapeutics, and vaccines. in , smith pioneered phage display technology, an in vitro methodology and system for presenting, selecting and evolving proteins and peptides displayed on the surface of phage virion [ ] . since then, phage display has developed rapidly and become an increasingly popular tool for both basic research such as the exploration of protein-protein interaction networks and sites [ ] [ ] [ ] , and applied research such as the development of new diagnostics, therapeutics, and vaccines [ ] [ ] [ ] [ ] [ ] [ ] . usually, the protein used to screen the phage display library is termed as target and the genuine partner binding to the target is called template. peptide mimicking the binding site on the template and binding to the target is defined as mimotope, which was first introduced by geysen et al. [ ] . one type of the most frequently used targets is monoclonal antibody. in this situation, the template is the corresponding antigen inducing the antibody, and the mimotope is a mimic of the genuine epitope. in fact, the original definition of mimotope given by geysen et al. goes "a mimotope is defined as a molecule able to bind to the antigen combining site of an antibody molecule, not necessarily identical with the epitope inducing the antibody, but an acceptable mimic of the essential features of the epitope [ ] ." mimotopes and the corresponding epitope are considered to have similar physicochemical properties and spatial organization. the mimicry between mimotopes and genuine epitope makes mimotopes reasonable solutions to epitope mapping, network inferring, and new diagnostics, therapeutics, and vaccines developing. powered by phage display technology, mimotopes can be acquired in a relatively cheap, efficient and convenient way, that is, screening phage-displayed random peptides libraries with a given target. however, not all phages selected out are target-specific, because the target itself is only one component of the screening system [ ] . from time to time, phages reacting with contaminants in the target sample or other components of the screening system such as the solid phase (e.g., plastic plates) and the capturing molecule (e.g., streptavidin, secondary antibody) rather than binding to the actual target are recovered with those target-specific binders (displaying mimotopes) during the rounds of panning. peptides displayed on these phages are called target-unrelated peptides (tup), a term coined recently by menendez and scott in a review [ ] . the results from phage display technology might be a mixture of target-unrelated peptides and mimotopes, and it can be difficult to discriminate tup from mimotopes since the binding assays used to confirm the affinity of peptides for the target often employ the same components as the journal of biomedicine and biotechnology initial panning experiment [ ] . therefore, target-unrelated peptides might be taken into study as mimotopes if the researchers are not careful enough. undoubtedly, this will make the conclusion of the study dubious. several such examples have been discussed in references [ , ] . obviously, target-unrelated peptides are not appropriate candidates for the development of new diagnostics, therapeutics, and vaccines. for mimotope-based epitope mapping, targetunrelated peptides are main noise. if tup is included in the mapping, the input data is improper and the result might be misleading [ ] . there are now quite a few programs for mimotope based epitope mapping, none of them, however, has a procedure to scan, report and exclude target-unrelated peptides [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we describe a web server named sarotup, which is an acronym for "scanner and reporter of target-unrelated peptides". sarotup was coded with perl as a cgi program and can be freely accessed and used to scan peptides acquired from phage display technology. it is capable of finding, reporting, and precluding possible target-unrelated peptides, which is very helpful for the development of mimotope-based diagnostics, therapeutics, and vaccines. the power and efficiency of sarotup was also demonstrated by preliminary tests in the present study. scott reviewed a collection of target-unrelated peptides recovered in the screening of phage-displayed random peptide libraries with antibodies [ ] . they divided their collection into several categories according to the component of the screening system to which target-unrelated peptides bind. they also derived one or more tup motifs for each category. very recently, brammer et al. reported a completely new type of target-unrelated peptides [ ] . in the review of menendez and scott, target-unrelated selection is due to the binding to contaminants or components other than target; however, in the report of brammer et al., target-unrelated selection is due to a coincident point mutation in the phage library [ , ] . we compiled a set of tup motifs from the above two references [ , ] , including motifs specific for the capturing agents, motifs specific for the constant region of antibody, motifs specific for the screening solid phase, motifs specific for the contaminants in the target sample, and motif for a mutation in phage library (table ) . all motifs are presented in patterns according to prosite format [ ] . the sarotup was implemented as a free online service, powered by apache and perl. three pages are designed and integrated into a tabbed web interface with cascading style sheets codes. the core program of sarotup was sar.pl, a cgi script coded with perl. in this script, the tup motifs were converted to regular expressions, which were then used to match each input peptide sequence. we constructed two-test data sets from [ , , - , , ] . the first data set contains cases; of them are sourced from test cases used in extant programs for mimotope-based epitope mapping [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; the left are cases studies published recently [ , ] . as shown in table , the target of each case in the first data set is monoclonal antibody and the structure of corresponding antigen-antibody complex has been resolved, which is used to derive its structural epitope as the golden standard for evaluation. for each case, there is one or more sets of peptides recovered from phage display technology. these peptides have been used in mimotope-based epitope mapping by other researchers. we scanned each set of peptides with sarotup. if target-unrelated peptides were found, a new panel of peptides excluding tup was produced. the old and the new panel of peptides were then used to predict epitope using mapitope or pepsurf [ , , ] . finally, the results were compared to show if sarotup could improve the performance of mimotope-based epitope mapping. the second data set is composed of peptides in raw sequence format. it has two groups. the first group has sequences compiled from the first data set without any known tup motifs; the second group has sequences sourced from [ , ] with various tup motifs. the mixture of the two groups of sequences made the second data set, which was then used as the sample input and can be used to evaluate the efficiency of sarotup. . . web interface of sarotup. as a free online service, the web interface of sarotup has successfully been implemented as a tabbed web page. the left tab is the default page, providing a brief introduction to this web service. the right tab is a more detailed help page. click the middle tab will display a web form. the upper section of the form is for basic input (figure ). the users can either paste a set of peptide sequences in the text box or upload a sequence file to the sarotup server for scanning. as shown in figure , a panel of peptides in raw sequence format taken from the b test case was pasted in the text box. besides the raw sequences, sarotup also supports peptides in fasta format. however, only the standard iupac one-letter amino acid codes are accepted at present. the lower section of the form has a series of options ( figure ). it includes three drop lists for the screening target, screened library, and screening solid phase, respectively. it also has two groups of check boxes for the capturing reagents and contaminants in the target sample or screening system. by default, sarotup will scan each peptide against all the known tup motifs. however, the users can customize their scan according to their experiment at this section. after the users submit their request, the scanning results of sarotup will be displayed on the middle tabbed page. if any target-unrelated peptides are found, they will be reported in a table. at the same time, a new panel of peptides excluding target-unrelated peptides is produced and can be downloaded from the hyperlink created by the sarotup server ( figure ). the file of the new panel of peptides will be stored on the server for a month and then automatically deleted. we have tested sarotup on the internet explorer (version . ), mozilla firefox (version . . ), and google chrome (version . ). although sarotup looks a little bit table , the first test data set has panels of peptides acquired from phage display libraries screened with targets. in the panels of peptides sarotup scanned, there were target-unrelated peptides in panels from cetuximab, r, and b test case, respectively (table ). this result suggested it was not rare that target-unrelated peptides sneaked into biopanning results and then were taken as mimotopes in study. in all, target-unrelated peptides were found; of them were due to binding to plastic; the left were due to binding to the fc fragment (table ) . for the above cases, the genuine epitopes recognized by cetuximab, r, and b monoclonal antibodies are compiled according to the ced records [ ] and pdbsum entries [ ] . mapitope or pepsurf [ , , ] were used to perform mimotope-based epitope prediction with or without sarotup procedure. for mapitope and pepsurf algorithm, the library type was set to "random"; the stop codon modification was set to "none"; and all other options were in default. the cluster with best score was taken as the predicted epitope. in the cetuximab case, pepsurf was used because there are only four or three peptides in the panel, statistically too few for mapitope. in the case of r and b , mapitope was used because many peptides in the two cases exceeding the length limit of pepsurf, that is, amino acids. if a predicted residue is identical with a residue in the true epitope, it is underlined ( table ) . as shown in table , the number of true positives improved from zero to four in the cetuximab case with sarotup procedure. when it came to the b case, the number of true positives increased from one to eight. sarotup did not improve the number of true positives in the r case when the parameters are same to the cetuximab and b cases. however, when the distance parameter was adjusted from default (i.e., Å) to Å, sarotup did increase the number of true positive residues from eight to eleven. these results indicate: ( ) epitope prediction based on mimotope will be interfered if target-unrelated peptides are taken as mimotopes; ( ) sarotup can improve the performance of mimotope based epitope mapping through cleaning the input data. we also scanned the second data set to evaluate the efficiency of sarotup. the second data set has peptides, varying from to residues long. suppose that matching each pattern to each peptide manually costs seconds, then it would take a researcher more than hours ( , seconds) to look through the second data set for targetunrelated peptides, even if he is as prompt during the whole period. however, it took only one second for sarotup to complete this work. besides, a table of target-unrelated peptides and a new panel of peptides excluding tup was produced at the same time by sarotup. it is true that some target-unrelated peptides can be identified through control and binding competition experiments. however, using sarotup first will certainly save a lot of labor, money, and time for researchers in this area. although the target of all tests described previously were monoclonal antibodies, sarotup can be customized and used in scanning the results from phage display technology using other targets such as enzymes and receptors. this is because their screening systems are similar. for the same reason, we can also expect that sarotup will extend its use to other similar in vitro evolution techniques, such as ribosome display [ ] [ ] [ ] , yeast display [ ] , and bacterial display [ ] [ ] [ ] . furthermore, sarotup will not only benefit the mimotope-based epitope mapping, but also the development of new diagnostics, therapeutics, and vaccines. targetunrelated peptides are not appropriate candidates for mimotope based diagnostics, therapeutics, and vaccines, since they are mimics to components or contaminants of the screening system rather than target. therefore, it is reasonable to find and exclude possible target-unrelated peptides from the candidate list of new diagnostics, therapeutics, and vaccines. take the cetuximab as an example. riemer et al. screened a phage-displayed random peptides library with the cetuximab and got four different peptides, that is, qfdl-strrlk, qynlssralk, vwqrwqksyv, and mwdrfs-rwyk [ ] . as described previously, we scanned the four "mimotopes" with sarotup and the result suggested that the peptide vwqrwqksyv might be a tup. indeed, the dot blot analysis of riemer et al. showed that qynlssralk bound the cetuximab with high affinity but vwqrwqksyv was less reactive with the cetuximab [ ] . trying to develop a mimotope vaccine, riemer et al. synthesized two-vaccine constructs with the peptide qynlssralk and vwqr-wqksyv, respectively. after immunization mice with these constructs, they found that either the cetuximab or the antibodies induced by the qynlssralk vaccine construct inhibited the growth of a cancer cells significantly. the inhibition of the antibodies induced by the vwqrwqksyv vaccine construct however, was not statistically significant when compared with the inhibition caused by the isotype control antibody [ ] . h , v , p , f , s , p , d , g , k , p , c , t , p , p , a , l , n , c , y r , s , y , k , y , y , p , p , a , l , c , y , w , l , n , d , g , y , t , t , t , g ,y , q l , r , h , i , s , n , v , p , f , s , p , d , g , k , p , c , t , p , p , a , l , n , c , y b i , c , s , l , d , q , s , l , k , p , c , v , p , k , v , s , f , e , p , i , p , i , r , p , i , n , m , w , c , k , v n , a , s , g , g , d , p , i , v , t , y , n , p , r , v , g , k , t , r , g , g , d , m w , t , f , n , t , s , l , n , g , s , l , a , e , e , e , v , v , t , s , s , s , g , g , d , p , e , i , v , t , t , s . . cautions in using sarotup. sarotup must be used with caution since it is a tool only based on pattern matching at present. there are a lot of target-unrelated peptides bearing no known motifs [ ] . as these tups are not embedded in sarotup at present, it is possible that a true tup cannot be detected by sarotup. to reduce this kind of false negatives, we are constructing a database for targetunrelated peptides and mimotopes. besides the motif-based search, the database-based search can find out the known tup without known motifs. it is also possible that a sarotup predicted target unrelated peptide is actually target-specific. to decrease this kind of false positives, the users should customize the scan according to their experiment at the section of advance options. for example, the user should select "antibody without fc fragment" as the target if fab was used in biopanning; this will prevent sarotup from reporting peptides bearing the fc-binding motifs as tup. as described above, sarotup in future will also provide an exact match tool based on database search. in this way, a match might mean that different research groups have isolated the same peptide with a variety of targets. it is obvious that this peptide can hardly be a true target binder. thus, the false positive rate of sarotup can be decreased further when its new feature become available. at last, we must point out that the controlled experiment is still the gold standard to distinguish tups from the specific mimotopes. the report of sarotup should be verified with experiment. sarotup, a web application for scanning, reporting and excluding target-unrelated peptides has been coded with perl. it helps researchers to predict epitope more accurately based on mimotopes. it is also useful in the development of diagnostics, therapeutics, and vaccines. to our knowledge, sarotup is the first web tool for tup detecting and data cleaning. it is very convenient for the community to access sarotup through http://immunet.cn/ sarotup/. filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface searching for peptide ligands with an epitope library a combined experimental and computational strategy to define protein interaction networks for peptide recognition modules probing a protein-protein interaction by in vitro evolution epitope identification and discovery using phage display libraries: applications in vaccine development and diagnostics mimotope vaccines: epitope mimics induce anti-cancer antibodies evolution of phage display: from bioactive peptides to bioselective nanomaterials overview of mimotopes and related strategies in tumor vaccine development phage display derived therapeutic antibodies mimotope vaccination-from allergy to cancer a priori delineation of a peptide which mimics a discontinuous antigenic determinant the nature of target-unrelated peptides recovered in the screening of phage-displayed random peptide libraries with antibodies a target-unrelated peptide in an m phage display library traced to an advantageous mutation in the gene ii ribosome-binding site information loss and noise inclusion risk in mimotope based epitope mapping the mapping and reconstitution of a conformational discontinuous bcell epitope of hiv- a new method for mapping discontinuous antibody epitopes to reveal structural features of proteins sitelight: bindingsite prediction using phage display libraries d-epitope-explorer ( dex): localization of conformational epitopes within three-dimensional structures of proteins discontinuous epitope prediction based on mimotope analysis mimox: a web tool for phage display based epitope mapping epitope mapping using combinatorial phage-display libraries: a graphbased algorithm pepitope: epitope mapping from affinity-selected peptides pep- d-search: a method for b-cell epitope prediction based on mimotope analysis the years of prosite computational prediction of the cross-reactive neutralizing epitope corresponding to the monoclonal antibody b specific for hiv- gp mapping a neutralizing epitope on the sars coronavirus spike protein: computational prediction based on affinity-selected peptides ced: a conformational epitope database pdbsum new things rna-peptide fusions for the in vitro selection of peptides and proteins mrna display: diversity matters during in vitro selection in-vitro protein evolution by ribosome display and mrna display yeast surface display for screening combinatorial polypeptide libraries bacterial surface display: trends and progress display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines display of proteins on bacteria vaccination with cetuximab mimotopes and biological properties of induced anti-epidermal growth factor receptor antibodies the authors are grateful to the anonymous reviewers for their valuable suggestions and comments, which have led to the improvement of this paper. this work was supported in part by the national natural science foundation of china under the grant and the scientific research foundation of uestc for youth under the grant jx . key: cord- - bthqj authors: huang, jian; ru, beibei; dai, ping title: bioinformatics resources and tools for phage display date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: bthqj databases and computational tools for mimotopes have been an important part of phage display study. five special databases and eighteen algorithms, programs and web servers and their applications are reviewed in this paper. although these bioinformatics resources have been widely used to exclude target-unrelated peptides, characterize small molecules-protein interactions and map protein-protein interactions, a lot of problems are still waiting to be solved. with the improvement of these tools, they are expected to serve the phage display community better. phages, also known as bacteriophages, are viruses that infect bacterial cells. many phages such as m and fd are good expression vectors. in , george p. smith displayed foreign peptides on the virion surface by inserting the foreign dna fragments into the filamentous phage gene iii [ ] . it was demonstrated that foreign peptides in fusion proteins on the virion surface were in immunologically accessible form and specific fusion phage could be enriched by affinity selection [ ] . also in that paper, smith, considered the father of phage display technology, inferred that desired clones could be isolated from a phage library of random inserts in a fusion-phage vector by one or more rounds of selection [ ] . since the pioneering work described above, phage display technology has further been developed and improved by scientists from various fields, and its applications has extended from epitope mapping to antibody engineering and organ targeting [ ] [ ] [ ] [ ] . so far, phage display technology has been widely open access used in basic research such as studying the sites and the networks of protein-protein interactions [ ] [ ] [ ] [ ] , and in applied research such as developing new diagnostics, therapeutics and vaccines [ ] [ ] [ ] [ ] [ ] . the most common protocol of phage display technology can be summarized as: ( ) preparing phage library e.g. random library and cdna library; ( ) immobilizing the target; ( ) incubating the phage library with the immobilized target; ( ) washing away the unbound phage with buffer; ( ) eluting the bound phage with template or stronger buffer; ( ) amplifying the eluted phage by infecting bacteria; ( ) repeating steps ( )-( ) for more rounds; ( ) amplifying the eluted phage and randomly picking up some clones for binding test and dna sequencing. the foreign peptides displayed on the surface of virion can then be derived from the dna sequences. in the foregoing description, the target refers to the substance used to screen the phage library, which is also known as bait or selector; the template refers the natural partner binding to the target. the whole process of affinity selection is usually called biopanning or just panning. the acquired peptides mimicking the binding site on the template and binding to the target are defined as mimotopes. the term mimotope was first introduced by geysen et al. to describe peptides able to bind to the antigen combining site of an antibody but different from the epitope inducing the antibody [ ] . in fact, it was hard to get mimotopes using geysen's method because all their peptides were synthesized chemically. this situation has changed since the coming of the phage display technology. mimotopes can now be obtained in a relatively cheap, efficient and convenient way, i.e. screening phage-displayed random peptide libraries with a given target. however, not all eluted phage clones are target-specific, because the target itself is only one component of the screening system [ ] . from time to time, clones binding to contaminants in the target sample or other components of the screening system such as the solid phase (e.g. plastic plates) and the capturing molecule (e.g. streptavidin and secondary antibody) rather than binding to the actual target, are recovered with those target-specific binders during panning. peptides displayed on these phage clones are called target-unrelated peptides (tups), a term coined recently by menendez and scott [ ] . since all the tups mentioned above are favored during the affinity selection process itself, they are categorized as the selection-related tups. there is also another category of target-unrelated peptides, called the propagation-related tups, which are favored because of increased infectivity or productivity during phage propagations [ ] [ ] [ ] . mimotopes obtained from phage display experiments are very valuable. first, these peptides are candidates for new diagnostics, therapeutics and vaccines. second, they can be used to predict the networks or sites of protein-protein interactions. therefore, bioinformatics studies on integrating all available mimotope data and developing more powerful tools for mimotope analysis are of great importance. in this review, we will summarize the special databases, algorithms, programs, web servers and their applications in the phage display area, focusing on the tools for mimotope-based epitope mapping. as a high-throughput technology, the amount of peptide sequence data derived from phage display has been accumulating rapidly. however, the data related to phage display experiments has been scattered in separate resources for a long period of time. for example, the structure data of mimotopes were store in the pdb database; the sequence data of phage vectors might be stored in the genbank; the mimotope data were stored in various reference databases with the published papers. special databases for mimotopes and other associate information are urgently needed since data integration plays a fundamental role in bioinformatics. available databases for mimotope data are listed in table . [ ] aspd is short for artificially selected proteins/peptides database. it is the first special database for mimotopes [ ] . this database became available at the beginning of the st century, collecting panning results from random libraries, mutant libraries and cdna libraries. at present, the aspd database contains data on selection experiments, which were described in original papers. for each experiment, the following information is given: target, template, links to the external databases such as swiss-prot and pdb, aligned sequences of peptides retrieved through in vitro evolution and relevant native or constructed sequences, rounds of selection, occurrences of clones with each sequence. for each paper, a full reference, a link to the medline database and the name of the corresponding author with his email address are recorded. all curated data are stored in two flat files. one is for panning experiment; another is for literature. aspd has a user-friendly interface and can be searched by means of the srs system. there is a blast search tool against the aspd database for looking directly for homologous sequences. regretfully, the aspd database has not been updated for years. the relic peptides is a relational database created with oracle i and can be accessed through a web interface coded with asp. it currently houses over , peptide sequences that have been selected with small molecule metabolites such as atp, gtp and glucose and drugs such as taxol and taxotere, as well as random clones from parent libraries. as part of the relic suite, this database is indispensible because many programs in relic are dependent on the data of relic peptides [ ] . the motif database contains , peptides obtained from the public domain and by competitive screening of phage display libraries using antisera raised against allergens and industrial enzymes [ ] . it also contains , binding motifs derived from the sequence alignments of these mimotopes. this database was integrated into an epitope mapping tool (emt), which can search motifs in the database against a given antigen structure. matches indicate all possible epitopic regions on the antigen. the pepbank database also includes some peptide data from phage display technology [ ] . at the time of writing, it contains a total of , individual peptide entries. the major source of peptide sequence data comes from text mining of medline abstracts with a program coded with perl. another component of the pepbank database is the peptide sequence data from aspd and uniprot. an additional, smaller part of the database is manually curated from sets of full text articles and text mining results. the mimodb database is the newest database for mimotopes, which is developed by our team very recently [ ] . this database is scheduled to be revised and updated quarterly, collecting mimotopes from random libraries. peptides with sequences longer than residues or selected from phage display cdna libraries (e.g. antibody phage display libraries) are not included. in the current release, it has , peptides manually curated from publications, which were grouped into , sets. these peptides are selected with different targets. the type of targets is quite diverse, varying from small compounds to nucleic acids, proteins, cells, tissues, organs and even entire organisms. nonetheless, proteins including antibodies and receptors from human and mouse are the most used targets. at present, there are known templates in mimodb. for most of the peptides, their templates are not determined. the database also stores solved structures for target-template complex, which are related to mimotope sets. there are five solved structures for target-mimotope complex, which are related to four mimotope sets. for structures of target-template or target-mimotope complex, contact residues making the interface can be viewed interactively with jmolapplet in mimodb. the mysql relational database management system is used to store and manage all the data described above. the mimodb database can be browsed and searched through a user-friendly web interface coded with php. all the databases described here are important resources for the phage display community. with the large amount of sequences in these databases, it is feasible to find out new target-unrelated peptides. for example, in our preliminary analysis, among the , peptides in mimodb, , peptides appear only once; peptides appear times [ ] . svsvgmkpsprp, haiyprh and lpltplp are most frequent, which are seen in , and sets of mimotopes selected with different targets. svsvgmkpsprp and haiyprh have been proved to be propagation-related tups [ , ] . experimental biologists can search the mimodb database to verify if their results have appeared in the database. if so, the match might mean that different research groups have isolated the same peptide with different targets. in this situation, the peptide may not be a true target binder. it is also convenient for computational biologists to derive benchmarks and customized data sets from these databases, which are useful for new algorithm development and tool evaluation. mimotopes are considered to be similar with the corresponding template in some way since they bind the target competitively. it is only natural that the first thought would be their sequence similarity. it is true in some cases. mimotope sequence might be very similar or even identical to one part of its template sequence, indicating the location of the protein-protein interaction site [ , ] . eyes and tools are often used to align mimotope sequence to its template. even if the template is not determined (e.g. panning against a small molecule drug with no priori knowledge about the corresponding drug target), a local alignment search against the non-redundant protein database with blast might help to find it out. however, most existing sequence alignment tools are developed and optimized for long protein sequences. they are not good at the analysis of short peptides deduced from phage display. moreover, it is more often that the mimotope has little sequence similarity with its template, though they do have similar physicochemical properties and spatial organization locally. therefore, many computational tools have been developed to interpret mimotope data reasonably onto the structure of its template. to improve the performance of these tools, algorithms and programs for excluding tups have also been developed. all these algorithms, programs and web servers are listed in table . with these tools, phage display technology has shown its power in exploring the interactions between proteins, peptides and small molecule ligands. generally, all methods described in this section can be divided into two categories, i.e. the sequence-sequence alignment category and the sequence-structure alignment category. findmap, epimap and the mimalign method of the mimop belong to the first category. all methods left can be included in the sequence-structure alignment category, which requires mimotopes and the structure of template as input. in this category, there are motif-based methods (such as dex, peptide, mimox, mimop and etc.), pairs-based methods (such as mapitope and denisova method), patch-based methods (such as sitelight and episearch), graph-based methods (such pep- d-search and pepsurf) and all kinds of hybrid methods (such as mimopro). though different from each other, most mentioned methods mainly address on deciphering the protein-protein interactions, especially epitope mapping. although specific peptides can be selected from the phage-displayed random peptide libraries with various targets ranging from small compounds to whole organisms, the most frequently used targets are proteins, especially antibodies. actually, phage display technology was used for epitope mapping from its infancy [ ] . powered by special computational tools developed for phage display technology, not only linear epitope but also conformational epitope formed by discontinuous residues brought into spatial proximity by protein folding can be mapped reasonably. the tramontano lab from italy might be the first team that tried to map discontinuous epitope computationally based on mimotopes and the antigen structure [ ] . their method includes four steps. first, the solvent accessible area of each residue of the antigen is calculated with the program what if and then surface residues were determined. second, a program called peptide, which they developed, is used to create a file in pir format containing all the peptide sequences of a given length that can mimic a preselected number of side chains exposed on the surface of antigen structure. third, the commercial package such as gcg is used to search the pir file with the consensus sequence obtained from a set of mimotopes. last, energy minimizations and molecular dynamic simulations are performed with the commercial software packages such as insight and discover. as the commercial programs or the rd party software were expensive and not integrated well enough, this method were not widely used by the phage display community. in , the jensen-jarolim group from austria proposed a -dimensional epitope search method and applied it to localize the major ige epitope on bet v , the major birch pollen allergen [ ] . in brief, the -dimensional coarse-grained epitope search was based on the x-ray structure of bet v . each amino acid in the structure was localized using the coordinates of its cβ atom (for glycine, the cα atom). based on this cβ grid, neighboring amino acids were found in a distance smaller than . Å. fitting of only two amino acids consecutively induced a broader attempt, which allowed gaps. the model was further simplified by classifying all amino acids into four groups: polar, lipophilic, acidic and basic amino acids. all hits were statistically evaluated. using this method internally, they found two regions on bet v surface significantly similar with mimotope, though aligning the mimotope with bet v sequence using the gcg package failed to find any similarities. in , mumey et al. described the program findmap [ ] . this program aligns the mimotope sequence to its template sequence, allowing any permutations and local rearrangements (e.g. inversion) of the mimotope sequence. it is therefore different from traditional sequence alignment and has proved to be np-complete. a branch-and-bound algorithm was used to solve this alignment problem in practice and implemented with c++ originally. findmap is unique because it is only based on sequences. the sequences of mimotope and its template are enough; the structure of template is not needed. with this advantage, it has been widely used to explore the epitope and topology of membrane proteins, which lack solved structures due to difficulties in crystallization [ ] [ ] [ ] [ ] [ ] . recently, an improved version with the name epimap has been proposed and coded with java [ ] . while findmap can deal with only one mimotope or a consensus sequence, epimap is capable of: ( ) aligning each mimotope to the template and producing a set of the top-scoring alignments; ( ) selecting the most mutually compatible alignments and filtering out spurious alignments with the program epifilter. the program sitelight developed by halperin et al. might be the first patch-based method [ ] . briefly, the template surface is divided into overlapping patches based on geodesic distances between two cα atoms of amino acids; each mimotope is compared with each patch and a bipartite graph is created for each potential match scored by a similarity matrix; the best alignment of a mimotope and a patch represented in a bipartite graph is found by the maximal bipartite matching algorithm. the mapitope algorithm was originally proposed by enshell-seijffers et al. [ ] . the core idea of mapitope is that: ( ) the simplest meaningful fragment of an epitope is an amino acid pair; ( ) amino acid pairs on the template surface can be simulated by amino acid pairs in the mimotope sequence. two amino acids on the template surface can be considered as a pair when the distance between their cα atoms is less than a threshold, for example Å. to predict the epitope with a set of mimotopes, at first, each peptide is converted to overlapping sequence pairs. all amino acid pairs derived from the set of mimotopes are then pooled, and the frequency of each type is calculated and it is determined whether its representation in the pool is higher than the random expectation. once the most significant amino acid pairs of the pool are identified, the algorithm seeks the match pairs on the template surface and attempts to link them into clusters. the mapitope program was implemented with c++ and has been applied in predicting epitope recognized by monoclonal antibodies against viruses [ , , ] . as the above method typically predicts only a fraction of the epitope, denisova et al. added a filling step to improve the performance of mapitope [ ] . in the filling step, an additional set of amino acid pairs (separated by distance no longer than Å) for every predicted cluster based on the mimotope sequences, is created. this new dataset is then compared to the total amino acid pair composition of the mimotopes. new pairs that were present in the original dataset but were not selected initially because they did not meet the statistical threshold are indentified. the cluster analysis is then repeated using the new pairs and the largest clusters are predicted to be the epitope. the improved method was applied to the prediction of epitopes for five monoclonal antibodies against the west nile virus e protein. in the case of e monoclonal antibody, only three contact residues were uncovered by the original algorithm, while additional nine contact residues were found by the improved algorithm. in our opinion, there is quite a lot information loss while converting mimotopes to overlapping sequence pairs linked with a peptidyl bond [ ] . other pairs, perhaps all pairs, should be considered. according to our observation, other conformations such as helix, turn, hairpin and even globular shape can be seen in the structures of free and bound mimotopes besides the extended conformation. this means that two residues far away in a mimotope sequence may fold together to form a space pair. furthermore, based on our computations on representative structures in the pdb database, the cα distances between two residues separated by one amino acid in all types of conformations are below Å. this indicates that at least pairs separated by one residue in the mimotope sequence are space pairs and should not be ignored [ ] . the same fact might have also been noticed by other groups. in a recent method based on pattern recognition theory, all possible space pairs (for example, pairs separated by one residue, two residues, three residues, and so on) in mimotope sequences are taken into account [ , ] . this new method can be regarded as a derivative of mapitope. however, it is specially designed for elucidating epitope specificity within antiserum. the method consists of two phases: learning and identification. during the learning phase, a large set of mimotopes is collected through panning against specific monoclonal antibodies. the mimotopes are analyzed to identify epitope specific pairs. during the identification phase, mimotopes selected using patient antiserum are interrogated for the presence of the epitope specific pairs. in , the tool dex (short for d-epitope-explorer) was developed by schreiber et al. [ ] . to localize a mimotope on the structure of its template, dex maps each amino acid in the mimotope into a table, containing all the same amino acids on the template surface. the residues in each table are then connected one by one if their cα or cβ distances below the predefined threshold. gaps are allowed in the connecting process. in , a php program named mimop was developed by moreau et al. [ ] . it includes two methods. mimalign, the first method, combines results from four multiple sequence alignments of the template and its mimotopes. for each position, a frequency and a score are calculated. convergent positions are then selected and clustered based on their topology. the clusters obtained are considered as potential epitopic regions, then scored and ranked. the second method named mimcons, evaluates the similarity of the mimotopes and clusters them accordingly. consensus patterns are identified from mimotope sequences of each cluster. the template surface is scanned to look for all possible exposed consensus patterns. the two methods can be run independently or their results combined. mimox might be the first freely accessible web tool for mimotope-based epitope mapping [ ] . it was coded with perl using modules from the bioperl project. mimox has two sections. in the first section, it provides a simple interface for clustalw to align a set of mimotopes and a consensus sequence is derived from the alignments. in the second section, mimox can map a single mimotope or a consensus sequence, or part of them, onto the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. naccess is used to evaluate the surface accessibility of the candidate clusters; and jmol is embedded to view them interactively in their d context. mimox is an interactive rather than an automatic tool at present. the default parameters of the program are optimized to decrease the load of the server. thus, the users often need to adjust the parameters many times to get a reasonable result. mimox has been applied by immunologists to characterizing both monoclonal antibody and antiserum [ , ] . in , perschinka et al. proposed a structural alignment method to identify conformational epitopes on heat shock protein associated with atherosclerosis [ ] . in their method, each mimotope is divided into a set of overlapping -mer peptides. these peptides are then superimposed onto the template surface according to cβ atoms. an alignment score is calculated for every superimposition based on similarity of the superimposed amino acids and the distance between the superimposed cβ atoms. for each -mer peptide, the calculated structural alignment score of each surface exposed amino acid is plotted. a control plot can be done with a random peptide. peaks in the plot for mimotope with high scores can easily be observed and taken as the positive hits. the program was written in matlab . and the source code is available on request from the authors. mayrose et al. described a graph-based tool pepsurf for mapping a set of mimotopes onto the solved structure of the template [ ] . in pepsurf, the problem is converted into the task of aligning a set of query peptides to a graph representing the template surface. the best match of each mimotope is found by aligning it against virtually all possible paths in the graph. a clustering step then combines the most significant matches and a predicted epitope is inferred. the program was written in c++ and can be used directly through the pepitope web server [ ] . the mapitope and a combination of pepsurf and mapitope algorithm are also implemented in the pepitope. the pepsurf algorithm and the pepitope web server have been widely used in predicting epitopes on toxins, allergens and receptors recognized by monoclonal or polyclonal antibodies [ ] [ ] [ ] [ ] . in , huang et al. proposed another graph-based tool pep- d-search [ ] . in this method, a surface graph of all exposed residues on the template is created at first. then, the algorithm can be employed in two modes. the first mode is the mimotope mode, which searched for matching paths on the template surface with each query mimotope by the ant colony optimization (aco) algorithm. all paths were scored to the corresponding mimotope according to an amino-acid substitution matrix. putative candidate epitopes were then picked out by the p-value calculation algorithm and the depth-first search algorithm. the second mode is the motif mode, which directly mapped the motif onto the template surface using the aco algorithm and took the top-scoring paths as epitope candidates. all source code is in visual basic and can be downloaded freely. in , negi et al proposed another patch-based method called episearch [ ] . with a set of mimotopes (up to peptide sequences) and corresponding template structure as input, the algorithm first divides the surface of template into overlapping surface patches around each solvent accessible amino acid residue with a radius of Å. then it ranks all surface exposed patches according to the frequency distribution of similar residues in each mimotope and in each patch. episearch is fully automated and has shown an impressive performance in the reported test cases. the web server mimopro has been available on line very recently. it is a mixture of the patch-based method and the graph-based method coded with java. firstly, the template surface is divided into overlapping surface patches centered at the cα atom of each surface residue with a Å radius. then, the surface patches are converted into graphs by specifying two amino acids as neighbor amino acids using a fluctuating distance threshold guided by the compactness factor. for each patch, a complete search method is conducted to find the best alignment for each mimotope sequence. dynamic programming and branch-bound methods are adopted to avoid repeating search and narrow the search space. the floating distance threshold used in mimopro is quite new as all tools mentioned previously use a fixed distance threshold. different from the tools described above, the relic server was particularly designed for the study of the interaction of small molecules with proteins [ ] . by analyzing the sequence of a protein and the sequences of small molecule affinity-selected, phage-displayed peptides, relic can predict proteins or some residues on the protein that bind to drugs, drug candidates and small metabolites. relic is not a single program but rather a suite of computational tools. it currently includes programs: dna pro, aafreq, popdiv, aadiv, info, divaa, motif , motif , closecon, heteroalign, distsim, match, fastacon and fastaskan (see table ). the dna pro is designed to get mimotope sequence from the ph.d.- and ph.d.-c c phage libraries of new england biolabs. any other library of interest is supported if provided with the start and end dna sequences of the vector. aafreq, popdiv, aadiv, info and divaa are designed to analyze the statistical properties of a peptide population. these data are particularly valuable when calculated in conjunction with randomly chosen members of the unselected library, as some propagation-related tups can be identified and subtracted. motif and motif are designed to identify weak sequence motifs within short peptide sequence populations. closecon, heteroalign and distsim use pdb file of the template as the basis for analysis of protein-ligand interactions. if the structure of the template is not available, match, fastacon and fastaskan can be used to do optimal sequence alignments between mimotopes and its template sequence. all programs in the relic suite were developed in fortran with a dos based, command-line user interface. the dos based applications were then converted into web based applications by creating com+ wrappers around the legacy code. as complicated software package online, relic, especially its tools for population analysis, motif identification and sequence alignment are extensively used by the phage display community [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as discussed previously, the results from phage display technology are noisy. besides mimotopes, target-unrelated peptides often creep into and even dominate the biopanning results [ ] [ ] [ ] [ ] . although strict control and subtractive experiment might help to decrease tups, either selection-related tups or propagation-related tups can not be eradicated. undoubtedly, taking tups as mimotopes would make the experimental and computational conclusions misleading. to improve the accuracy of programs for mimotope-based analysis, procedures or special tools for excluding tups have been developed. when peptide population of both affinity-selected library and unselected library are analyzed by the program info in the relic suite, the propagation-related tups can be identified and theoretically subtracted from the affinity-selected library. the method is based on the theory of information by shannon. this virtual subtraction process is used not only by info, but is also an option in the relic programs heteroalign, match, and fastaskan to reduce the noise in affinity-selected peptide sequences and amplify the signal, i.e. mimotope [ ] . noise filtering procedure has also been implemented at the level of amino acid pairs. in the algorithm proposed by denisova et al. recently, three groups of peptides are used [ , ] . group a is a collection of mimotopes isolated by affinity selection using a series of specific monoclonal antibody. group b is the set of peptides obtained by antiserum, which needs characterizing. group c consists of irrelevant peptides to be used as a negative control. at the learning phase, the entire collection of group a peptides is used to filter amino acid pairs that are common to more than one antibody. we have developed a free web tool called sarotup, which can be used to scan, exclude and report possible target-unrelated peptides from mimotopes [ ] . at present, a set of tup motifs collected from literature are compiled in the program. among them, one motif indicates propagationrelated tups; motifs indicate selection-related tups, including motifs specific for the capturing agents, five motifs specific for the constant region of antibody, three motifs specific for the screening solid phase and two motifs specific for the contaminants in the target sample. these motifs are converted to regular expressions and then used to check input peptide sequence one by one. however, there are a lot of target-unrelated peptides bearing no known motifs [ ] . as these tups are not embedded in sarotup at present, it is possible that a true tup cannot be detected by sarotup. one way to reduce such false negatives is to search the mimodb database. as stated previously, analysis on peptides in the mimodb database can also revealed new tups. the existing bioinformatics resources and tools reviewed in this paper have benefited the phage display community. however, there are still a lot of problems need to be solved. for example, known tups, especially propagation-related tups are very limited. is there any pattern exists in propagationrelated tups? most tools ignore tups and only a few tools have integrated a filtering procedure. no tools can map epitope formed by two or more chains. the performances of available tools are far from satisfying, and etc. we believe all the mentioned problems will be solved in the coming years. with the advances of bioinformatics tools, the powerful phage display technology will become even more powerful. we can expect that some tools can also be used to other similar surface display technology such as ribosome display, yeast display and bacterial display, producing broader influence. filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface searching for peptide ligands with an epitope library random peptide libraries: a source of specific protein binding molecules phage antibodies: filamentous phage displaying antibody variable domains organ targeting in vivo using phage display peptide libraries phage display affinity selection from biological libraries a combined experimental and computational strategy to define protein interaction networks for peptide recognition modules probing a protein-protein interaction by in vitro evolution small peptides as potent mimetics of the protein hormone erythropoietin peptide agonist of the thrombopoietin receptor as potent as the natural cytokine display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines towards a solution for hepatitis c virus hypervariability: mimotopes of the hypervariable region can induce antibodies cross-reacting with a large number of viral variants a peptide-based erythropoietin-receptor agonist for pure red-cell aplasia a priori delineation of a peptide which mimics a discontinuous antigenic determinant the nature of target-unrelated peptides recovered in the screening of phage-displayed random peptide libraries with antibodies selection by phage display of peptides targeting the hiv- tar element a target-unrelated peptide in an m phage display library traced to an advantageous mutation in the gene ii ribosome-binding site corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures aspd (artificially selected proteins/peptides database): a database of proteins and peptides evolved in vitro relic--a bioinformatics server for combinatorial peptide analysis and identification of protein-ligand interaction sites an in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context pepbank--a database of peptides based on sequence text mining and public peptide data sources mimodb: a new repository for mimotope data derived from phage display technology identification of biologically active peptides using random libraries displayed on phage mapping epitopes on protein surfaces allergen mimotopes for -dimensional epitope search and induction of antibodies inhibiting human ige a new method for mapping discontinuous antibody epitopes to reveal structural features of proteins sitelight: binding-site prediction using phage display libraries the mapping and reconstitution of a conformational discontinuous b-cell epitope of hiv- stepwise prediction of conformational discontinuous b-cell epitopes using the mapitope algorithm d-epitope-explorer ( dex): localization of conformational epitopes within three-dimensional structures of proteins mimox: a web tool for phage display based epitope mapping discontinuous epitope prediction based on mimotope analysis filtering epitope alignments to improve protein surface prediction epitope mapping using combinatorial phage-display libraries: a graph-based algorithm pepitope: epitope mapping from affinityselected peptides identification of atherosclerosis-associated conformational heat shock protein epitopes by phage display and structural alignment pep- d-search: a method for b-cell epitope prediction based on mimotope analysis a novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: a case study using monoclonal antibodies against the west nile virus e protein deciphering epitope specificities within polyserum using affinity selection of random peptides and a novel algorithm based on pattern recognition theory applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes -relevance for vaccine design automated detection of conformational epitopes using phage display peptide sequences sarotup: scanner and reporter of target-unrelated peptides constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints equilibrium between metarhodopsin-i and metarhodopsin-ii is dependent on the conformation of the third cytoplasmic loop new insights into the membrane topology of the phagocyte nadph oxidase: characterization of an anti-gp -phox conformational monoclonal antibody c-terminal tail phosphorylation of n-formyl peptide receptor: differential recognition of two neutrophil chemoattractant receptors by monoclonal antibodies nfpr and nfpr new p -phox monoclonal antibodies: identification of a conformational probe for cytochrome b computational prediction of the cross-reactive neutralizing epitope corresponding to the monclonal antibody b specific for hiv- gp mapping a neutralizing epitope on the sars coronavirus spike protein: computational prediction based on affinityselected peptides information loss and noise inclusion risk in mimotope based epitope mapping towards the definition of a chimpanzee and human conserved cd domain epitope recognized by t monoclonal antibody induction of immunity in sheep to fasciola hepatica with mimotopes of cathepsin l selected from a phage display library characterization of a key neutralizing epitope on pertussis toxin recognized by monoclonal antibody b peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-egfr immune response characterization of ige epitopes of cuc m , the major melon allergen, and their role in crossreactivity with pollen profilins mapping protective epitopes in the tick and mosquito subolesin ortholog proteins phage display reveals multiple contact sites between fhua, an outer membrane receptor of escherichia coli, and tonb interactions between tonb from escherichia coli and the periplasmic protein fhud in vivo phage display selection yields atherosclerotic plaque targeted peptides for imaging t lytic phage-displayed peptide libraries exhibit less sequence bias than m filamentous phage-displayed peptide libraries trinucleotide cassettes increase diversity of t phage-displayed peptide library ifats collection: combinatorial peptides identify alpha beta integrin as a receptor for the matricellular protein sparc on adipose stromal cells efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a t phage display pool identification of microtubule-binding domains on microtubule-associated proteins by major coat phage display technique identification of peptides with targeted adhesion to bone-like mineral via phage display and computational modeling the adsorption of preferential binding peptides to apatitebased materials a d monoclonal antibody recognizes a new linear epitope from sag a toxoplasma gondii tachyzoites, identified by phage display bioselection phage-displayed combinatorial peptide libraries in fusion to betalactamase as reporter for an accelerated clone screening: potential uses of selected enzyme-linked affinity reagents in downstream applications novel beta-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy from combinatorial peptide selection to drug prototype (i): targeting the vascular endothelial growth factor receptor pathway from combinatorial peptide selection to drug prototype (ii): targeting the epidermal growth factor receptor pathway sample availability: contact the authors this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors are grateful to the anonymous reviewers for their valuable comments, which have led to the improvement of this review. this work was supported by the national natural science foundation of china under the grant and the scientific research foundation of uestc for youth under the grant jx . key: cord- -d shmb o authors: harrison, patrick l.; abdel-rahman, mohamed a.; miller, keith; strong, peter n. title: antimicrobial peptides from scorpion venoms date: - - journal: toxicon doi: . /j.toxicon. . . sha: doc_id: cord_uid: d shmb o the need for new antimicrobial agents is becoming one of the most urgent requirements in modern medicine. the venoms of many different species are rich sources of biologically active components and various therapeutic agents have been characterized including antimicrobial peptides (amps). due to their potent activity, low resistance rates and unique mode of action, amps have recently received much attention. this review focuses on amps from the venoms of scorpions and examines all classes of amps found to date. it gives details of their biological activities with reference to peptide structure. the review examines the mechanism of action of amps and with this information, suggests possible mechanisms of action of less well characterised peptides. finally, the review examines current and future trends of scorpion amp research, by discussing recent successes obtained through proteomic and transcriptomic approaches. over the last few decades an increasing number of pathogenic microorganisms have developed resistance to conventional antibiotics. this poses problems in the clinical management of infections, especially in immunocompromised individuals but also increasingly, at the community level. during the same period, more worryingly, the development of new antibiotics has decreased. selective pressures caused by the overuse of conventional antibiotics in the healthcare setting, as well as unprecedented increases both in global transport systems and migration has led to a situation of previously isolated forms of resistance being widespread. the ability of mobile genetic elements of bacterial plasmids to spread through a population is increasing the rate of resistance (kumarasamy et al., ) . while the media portrayal of an imminent crisis of untreatable infections is misguided, the position in reality is rather more complex. although treatment options for some pathogens have undoubtedly decreased, for others the position has actually reversed (livermore et al., ) . a major problem in modern antibiotic drug development is designing agents that are not easily susceptible to resistance and therefore a new approach to drug development is required (jenssen et al., ) . the need for new antimicrobials agents has been highlighted by the release of a consensus statement following discussion of leading academic, clinical and industrial experts (chopra et al., ) who discussed the growing problem of resistance within gram negative bacteria and the lack of new compounds readily available to treat such pathogens. the report highlighted that no novel classes of antimicrobial agents has been developed since the 's and with no compounds in clinical trials, the threat of untreatable infection in the near future is feasible. antimicrobial peptides (amps) represent an ancient defence mechanism that transverses the evolutionary spectrum and remains an effective strategy against invading pathogens. because of their selectivity for prokaryotic membranes and their membrane-disruptive mechanisms for which microbes have little natural resistance, the spotlight in recent years has turned towards the development of novel antibiotics from these peptides (zasloff, ) . previous attempts to develop amps into the clinical setting have proven frustrating, with a number of peptides being rejected during the latter stages of clinical trials (gordon et al., ) . however a recent commentary (fox, ) examined these failures and provided optimism for future amp development; in this light, a number of these previous attempts are discussed below. for example, pexiganan (lamb and wiseman, ), a residue peptide developed for the treatment of foot ulcers was stopped because of manufacturing difficulties and changes in the clinical trial design, leading to a non-approval letter been issued ( ) due to deficiencies with us chemistry manufacturing & controls. the united states federal drug administration (fda) also indicated that pexiganan was no more effective in treating foot ulcers than conventional antibiotics. similarly plectasin (nz ) (mygind et al., ) developed by novozymes for the treatment of gram positive infections and licenced ( ) to sanofi-aventis was abandoned because of commercial rather than scientific reasons. in another example, omiganan (sader et al., ) , which had good efficacy in inhibiting catheter associated infections, was rejected on the grounds of cost when it was demonstrated to show no clinical statistical difference when compared to the widely used povidone iodine. stringent regulations placed on new antimicrobials was also highlighted by chopra et al., who argued that, in contrast, other drugs, for example those used to treat cancer, are approved with a high degree of adverse side effects and that this has contributed to the lack of development of new antimicrobials. however recent acceptance of the lipopeptide, daptomycin ( ) and the vancomycin derived telavancin ( ) , which have relatively low therapeutic indices compared with conventional b-lactams, would suggest a possible change in regulation. in this changing climate, pexiganan is re-entering clinical trials, as locilex (fox, ) . scorpion venom has proved a rich source of bioactive molecules, especially ion channels blockers; in recent years it has been increasingly recognized that scorpion venoms also have an abundant supply of amps and it has been suggested (see inter alia, hern andez- aponte et al., ) that the presence of amps might protect the venom gland from infection and facilitate the action of other neurotoxins. scorpion venom amps are positively charged amphipathic peptides and can be conveniently divided into three structural categories: ( ) cysteine containing peptides with disulfide bridges; ( ) peptides with an amphipathic a-helix but lacking cysteine residues and ( ) peptides rich in certain amino acids such as proline and glycine. this review aims to provide an examination of amps from scorpion venom (table ; fig. ), with discussions on structure, biological activity and proposed mechanisms of action, together with a final discussion of recent and future strategies for mining new bioactive molecules. cysteine containing peptides are ubiquitous in scorpion venoms and usually contain or disulphide bridges. these peptides have been characterised as interacting with ion channels, namely na þ , k þ , ca þ and cl À channels, and make up the largest family of peptides in the venom with non-disulphide bridged peptides being a smaller family. whilst amps from scorpions have increasingly been found to belong to the non-disulphide bridged family, disulphide bridged peptides having interesting biologically activities that more than warrants mention. the noted similarity between insect defensins and scorpion toxins (bontems et al., ) led to the isolation of the first scorpion defensin from the haemolymph of the north african scorpion leiurus quinquestriatus. this . kda peptide contained residues, with the characteristic cysteines of many ion channel scorpion toxins and showed a high degree of homology to insect defensins within the order odonata. this peptide was active against grampositive m. luteus but inactive against gram-negative e. coli (cociancich et al., ) (details of mic values for all bacteria and fungi tested, against all peptides mentioned in this review are provided in table ). the first cysteineconstrained amp from scorpion venom (scorpine) was isolated by possani and colleagues (conde et al., ) from the venom of the african scorpion pandinus imperator. scorpine ( . kda, residues, disulphide bridges) had a unique structure, with n-terminal similarity to some insect cecropins and c-terminal similarity to some scorpion defensins. scorpine was active (mic e mm) against both grampositive (b. subtilis) and gram-negative (k. pneumonia) bacteria in agar diffusion assays. the anti-malarial properties of scorpine were also investigated against the causative parasite p. berghei (ed . mm and mm against ookinete and gamete stages, respectively). a second putative peptide (bmtxks ) was identified during this same period, as a full length cdna clone isolated from the venom gland of the chinese scorpion, buthus martensii karsch . bmtxks was predicted to have residues including cysteines and have similarity to haemolymph defensins. however there have been no subsequent reports of the translated peptide (either native or recombinant) being isolated and no biological data is available. in subsequent years, two more members of the scorpine family members have been identified e opiscorpine from opistophtalmus carinatus (zhu and tytgat, ) and heteroscorpine- from heterometrus laoticus (uawonggul et al., ) . six peptides have been isolated from the venom of tityus discrepans termed the bactridines (bacts) (diaz et al., ) , which unlike the scorpine family contain disulphide bridges. bacts and have been characterised more extensively with edman degradation and in silico analysis revealing charged peptides containing and residues respectively. bacts showed an mic range of e mm whilst bacts had an mic range of e mm (appendix table ). in hemolytic assays, bacts showed only . % at mm and . % at mm with bacts showing . and % at the zhao et al., same concentrations, respectively. whilst these peptides do not have potent antimicrobial activity, their mechanism of action is of most interest. using the pathogen y. enterocolitica loaded with mm of the na þ fluorescent indicator corona™ red the efflux of sodium ions was observed upon bacts and interactions. furthermore this could be blocked in the presence of the sodium channel blockers amiloride ( mm) and mibefradil ( mm) although no inhibition was observed in the presence of mm tetrodotoxin; furthermore these interactions were shown to be na þ channel specific with no interactions observed with k þ or ca þ channels. bacts showed no toxicity towards mice ( . mm/g) however was toxic to cockroaches ( . mm) induced sialorrhoea in crabs at . mm/g and death in out of crabs at . mm/g. bacts however was toxic to mice at the same concentration suggesting specificity towards different classes of sodium channels. peigneur et al. ( ) showed no modulation of the mammalian sodium channels na v . -na v . with bacts , however no modulation of the insect dmna v or the bacterial nachbac was seen either suggesting further research needs to be carried out. bacts showed modulation of na v . , na v . and na v . . bacts shares % homology to ardiscretin (d'suze et al., ) which is an insect sodium channel blocker from the same venom whilst bacts shares % homology to the first residues of tz and td from tityus zulianus and tityus discrepans respectively (borges et al., ) . structural modelling of the two peptides suggested a classical a-bmotif in which the peptides have an alpha helical region and conserved cysteine constrained beta sheet structure. the lesser characterised bacts peptides ( e ) have molecular masses of , , , and respectively and their antimicrobial activities are shown in supplementary table . in hemolytic assays at mm bacts showed only . % hemolysis whilst bacts showed . %; bacts and showed . and . %. sodium efflux studies were positive and again inhibited with amiloride whilst tetrodotoxin had no effect, interestingly bacts and showed only partial inhibition with mibefradil and no inhibition observed with bacts (appendix table ). toxicity testing revealed no effect on crabs with bacts , and and transient effects with whilst toxicity was observed with , and with mice and transient toxicity observed with (appendix table ). bacts represents a novel basis for an amp although further work is required before a sodium channel specific mechanism of action can be truly revealed however it would be of interest to the field of venom antimicrobials if these questions were answered and more bacteridine like amps were isolated (kuhn-nentwig, ; zeng et al., ) . whereas the original antimicrobial peptides isolated from scorpion venoms contained cysteine residues, various non-disulphide bridged peptides have subsequently been isolated (e.g. kuhn-nentwig, ; zeng et al., zeng et al., , gao et al., ) and these are now in the majority. in this review, peptides have been divided into three groups e long-chain peptides (> amino acids), intermediate-chain guo et al., in press peptides ( e amino acids) and short chain peptides (< amino acids). parabutoporin, isolated from the venom of the south african scorpion parabuidethus schlechteri is a highly basic peptide ( amino acids, overall charge of þ ). a structurally similar, basic molecule (charge þ ), existing as two isoforms, opistoporin and was isolated from another south african scorpion opistophtalmus carinatus (moerman et al., ) . these two isoforms ( amino acids) differ at position where a phenylalanine replaces a leucine. parabutoporin was predicted to have an a-helical structure between amino acids e in comparison to opistoporin and with two a-helical regions between residues e and e which are connected by a short random coil wnsep (moerman et al., ) . circular dichroism (cd) spectra of parabutoporin in % , , -trifluoroethanol (tfe), dimyristoylglycerophosphocholine (myr gro-pcho) showed the peptide adopts an a-helical structure in membrane mimicking conditions. under the same solvent conditions, opistoporin re-organises from an unordered state into a continuous a-helical structure (moerman et al., ) . the amphipathic natures of both parabutoporin and opistoporin were also predicted with a-helical wheel projections, which clearly show distinct hydrophobic and hydrophilic regions within each peptide, although comparison of these projections clearly shows that parabutoporin has a larger polar surface. antimicrobial assays for parabutoporin showed growth inhibition of all gram-negative organisms assayed with mic's generally varying from to mm. the peptide showed less activity towards gram-positive organisms with mic's generally greater than mm. interestingly, a decrease in growth inhibition was observed in the presence of mg þ ions (moerman et al., ) . opistoporin showed less inhibitory activity against gram-negative organisms (mic values generally ranging from to mm) than parabutoporin. on the other hand, differences between the effects of opistoporin and parabutoporin on gram-positive organisms were less marked, although gram-positive bacteria did appear slightly more sensitive to the former. in antifungal studies, both parabutoporin and opistoporin inhibited growth of a range of fungi (b. cinerea, f. culmorum and s. cerevisiae) at similar concentrations ( % growth inhibition between . and . mm). parabutoporin was more than twice as effective as opistoporin in disrupting eukaryotic cell membranes. using human erythrocytes, parabutoporin induced % haemolysis at mm; in comparison, opistoporin induced only % haemolysis at mm. hadrurin is the prototype of another cysteine-free amp family purified from the venom of the mexican scorpion hadrurus aztectus. hadurin ( da, amino acids), carries an overall positive charge at physiological ph and has the same structural profile as opistoporin, namely two a-helical regions connected by an undefined region between amino acid residues e (torres-larios et al., ) . the plot of an a-helical wheel shows opposite hydrophobic and hydrophilic regions and these two distinct regions allow the peptide to adopt an amphipathic conformation, as the carboxyl-terminal helix can rotate with respect to the amino-terminal helix. in antimicrobial activity studies with gram-negative organisms, several e. coli strains were very sensitive with mics less than mm; in comparison, pseudomona strains required mm for full inhibition (torres-larios et al., ) . haemolytic activity was observed at mm. studies with synthetic analogues have demonstrated that the (unnatural) d-isoform of hadrurin has a different activity profile from the native l-isomer, suggesting activity is a consequence of membrane disruption (torres-larios et al., ) . studies on two antimicrobial peptides isolated from pandinus imperator have shed further light on the contribution of the two a-helical regions discussed above, to biological function. pandinin (pin ) has two a-helical regions between residues e and e , separated by a random coil region containing a pro- kink. in contrast, nmr analysis of pandinin (pin ) in a % tfe solvent containing dpc micelles, showed that this second peptide had a singular helical structure between residues e with no significant kink at the equivalent proline residue (pro- ) (nomura et al., ) . the structural differences found between these two peptides are significant. while the antimicrobial activity of pin and pin against a range of gram-positive and gram-negative organisms was broadly similar, it is the haemolytic studies on eukaryotic membranes that are of most interest. pin lysed % of sheep erythrocytes at mm whilst pin only lysed . % at the same concentration . the authors suggest that the ability of pin to lyse both eukaryotic and prokaryotic membranes may be related more to the venom toxicity, in a similar manner to the bee venom peptide melittin, than a specific antimicrobial action. in contrast, the activity of pin is more in keeping with magainin , the prototype amp isolated from the skin of the south american toad, xenopus. a flexible hinge region is also important in successful amp design. replacing (structurally rigid) pro- in pin with a (flexible) gvg tripeptide reduces the haemolytic activity of pin without altering its antimicrobial activity . this substitution creates a more flexible region while retaining the essential amphipathic nature of pin which is more in tune with the dual helical structure of corzo, nakajima and colleagues have subsequently demonstrated that the haemolytic activity of pin , pin and other cationic amphipathic peptides (e.g. isct , see section . ) depends on the animal species studied (guinea pig > pig > sheep) and this is turn is related to the phosphatidylcholine:sphingomyelin ratio in the different erythrocyte membranes (belokoneva et al., ) . with such contrasting effects on prokaryotic and eukaryotic membranes, a comparison of the mechanisms of action of these two peptides is of particular interest and it is instructive to consider these mechanisms in greater detail. on examination of the effects of pin on phosphatidylcholine (pc) lipid vesicles using a calcian dye release assay, it was seen that the dose response curve for dye efflux was sigmoidal, suggesting peptide oligomerisation during the time course of the experiment. the effects of pin were seen at very low peptide: phospholipid concentrations (belokoneva et al., ) . when phosphatidylethanolamine (pe), known to promote negative membrane curvature (matsuzaki et al., ) , was incorporated into the pc vesicles, no change in pin activity was observed, supporting the hypothesis of a barrel-stave mechanism of pore formation (see fig. for a background explanation to current hypotheses for the mechanism of action of amps). dextranloaded liposome assays also showed great variability in the size ( . e nm) of the membrane pores generated by pin , dependent on peptide: phospholipid ratios (belokoneva et al., ) . based on nmr studies of pin , nomura and colleagues (nomura et al., ) proposed a pore forming mechanism of action, as a consequence of pin inducing clustering of hydrophobic residues which allows further incursion into the membrane hydrophobic core and interaction with the inner membrane. this process can be conveniently visualized in a step-wise fashion as shown in fig. . pin inserts into the membrane at a angle however is seen to be only at a angle around the leu residue. this causes a slight kink in the peptide when in the membrane, and is a phenomena seen in other pore forming amps, thus thought to be essential for activity. after insertion, oligomerisation occurs between the pin monomers and then pore oligomerisation is seen leading to membrane being trapped between these pores to be 'pinched off'. thus pin induces a fold attack on the target with loss of intracellular constituents through the pore lumen and loss of membrane through pore association. in contrast, nmr studies with pin (nomura et al., ) showed a radically different mode of action, with a detergent-like effect due to the formation of cubic phase structures. it was suggested (nomura et al., ) that pin sits within the interface of the membrane between the hydrophobic core and the polar phospholipid head groups. this can also be conveniently visualised (fig. ) . the pin nterminal helices are tilted at a angle with respect to the horizontal bilayer plane and the c-terminal helices. interactions between tryptophan residues at position , and on the tilted helices and the polar head groups cause the membrane disruption and the formation of cubic phase structures. the n-terminal helices rotates around the average helical axis (fig. ) . using primers originally designed to detect a bradykinin-potentiating peptide (k- ), zeng and colleagues have identified a full length cdna encoding a putative amp (bmkbpp) from the venom glands of the chinese scorpion, buthus martensii karsch. bmkbpp (ndbp- . family) is predicted to encode a amino acid mature peptide, the c-terminal region showing % homology to peptide k- . blast analysis of bmkbpp revealed high homology with a number of scorpion toxins such as ndb . ( %) from lychas murconatus, tx ( %) from buthus occintanus israelis and parabutoporin ( %), suggesting a novel family of amps . secondary structure predictions of bmkbpp show a similar structure as pin , with two a-helical regions (between residues e and e ) separated by a random coil region. also, a-helical wheel projections reveal a highly amphipathic molecule. as with pin low haemolytic activity is seen with only . % lyses of human red blood cells at a concentration of mm. antimicrobial assays of bmkbpp in liquid cultures showed preferential activity against gram-negative organisms (mic's typically in the range e mm) in comparison to gram-positive organisms (mic's typically > mm). using a bioassay-guided fractionation strategy, miyashita et al. ( ) isolated im- ( . kda, amino acids) from the venom of isometrus maculatus. im- belongs to the bpp family and showed % homology toward parabutoporin, especially at the n-terminus. secondary structure analysis indicated an a-helical structure between residues e which was confirmed by cd spectra in % tfe. synthetic im- had potent antimicrobial effects in liquid culture assays against both gramnegative and gram-positive bacteria (typical mic values . e . mm and . e . mm, respectively) (miyashita et al., ) . another long chain peptide, vejovine ( . kda, amino acids), has been isolated from the venom of vaejovis mexicanus (hernandez-aponte et al., ), with % homology to hadrurin. in antimicrobial assays performed in liquid broth cultures, both native and synthetic vejovine showed preferential activity towards gram-negative organisms (e. coli mic values . e mm) with no activity towards grampositive staphylococcus aureus. vejovine gradually breaks down when freshly milked venom is stored, producing a truncated derivative (vm ), lacking the first eight n-terminal amino acids of vejovine. in comparison to vejovine, vm showed no antimicrobial activity, demonstrating the importance of the n-terminal region of this and presumably other amps with similar structure. in cd studies, vejovine adopted an a-helical secondary structure in % tfe although it remained unordered in aqueous solution. in contrast, vm peptide adopted an a-helical structure in aqueous solution which may indicate that the increased flexibility of vejovine is a key factor in promoting antimicrobial activity. vejovine ( mm) induced % haemolysis of human erythrocytes in eukaryotic cytotoxicity studies. using a cdna cloning strategy, three peptides have been identified from the venom gland of heterometrus spinifer termed heterin- , heterin- and spiniferin (wu et al., in press). heterin- is residues in length and shares % homology with the opistoporins and % with pandinin with secondary structure prediction indicative of a mono-helical structure between residues e flanked by two random coil regions, however, unlike these other peptides heterin- has an amidated c-terminal. in antimicrobial assays it had good activity against the gram positive pathogens of b. megaterium and m. luteus ( . mm) whilst its potency against s. aureus and a range of gram-negative organisms was less so ( . e . mm) in cytotoxicity assays % hemolysis was seen at . mm. a completely new class of scorpion amps has been reported from the venom gland of the asian scorpion heterometrus spinifer . clones encoding four highly homologous peptides were characterised and one peptide (hsap) was synthesized by solid state methods. hsap ( amino acids) showed no significant homology to any other class of scorpion amps. it has broad spectrum antibacterial activity against both gram-positive (mics typically e mm) and gram-negative organisms (mics typically e mm), without clear target-cell specificity as well as anti-fungal activity (mic approx. mm). hsap was postulated to have an a-helical structure (residues e ) with coiled n and c terminals. it has classical amphipathic characteristics although its hydrophobic face is disrupted by two hydrophilic residues (ser and glu). nie and colleagues indicated that hsap is a bacterial infectionresponsive peptide because the genomic sequence of its gene is intronless. at present hsap has limited therapeutic potential because of its potent haemolytic activity against human erythrocytes ( % haemolysis at . mm). two antimalarial peptides (meucin- and meucin- ) have been identified by screening a venom gland cdna library from the asian scorpion mesobuthus eupeus (gao et al., ) . the two peptides have been synthesized by solid state methods. cd spectral analysis of synthetic meucin- ( . kda, amino acids) showed a disordered conformation in water; however a-helical formation increased in % tfe and nmr spectra revealed an a-helical structure between residues e and random coil regions at the n-and c-terminals (gao et al., ) . although fig. . the mechanism of action of amp's is postulated to occur in a number of stages (zasloff, ) : ( ) electrostatic interaction onto the membrane surface; ( ) a peptide threshold concentration is reached before membrane disruption can occur after which a number of models have been proposed. in the carpet model (pouny and shai, ) , peptides remain parallel to the bilayer causing a detergent like effect. in the barrel stave pore model peptides insert perpendicularly into the bilayer and self-association occurs forming a pore containing peptideepeptide interactions (baumann and mueller, ) . the more recent toroidal model (ludtke et al., ) indicates a pore forming mechanism in which the pore lumen is lined with both peptides and phospholipid in a less rigid association. fig. . interaction of pandinin interaction with phospholipid head groups. pin sits between the head groups (a) within the interface between the head groups and hc core (b). interactions between pin e (green) and the head groups causes negative membrane curvature (a) as pin e is at a tilt with respect to pin e (red) due to the presence of a proline at position . membrane disruption accrues when pin e rotates around the average helical axis, which is parallel to the lipid long axis (c) causing membrane disruption and dispersion of the lipid into the cubic phase (d). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) meucin- can be considered amphipathic, the hydrophobic face of the molecule contains only six residues; this reduction in hydrophobicity has been postulated to be responsible for the lack of antibacterial activity (gao et al., ) . the charged residues in this amphipathic molecule are unusually, predominantly anionic and therefore not thought to interact with negatively charged membrane phospholipids. the authors provide an excellent structural comparison to the classical amp, magainin , which has a larger hydrophobic face and a predominantly cationic hydrophilic domain (fig. ) . secondary structure analyses of meucin- ( . kda, amino acids) by cd and nmr revealed a radically different structure to that of meucin- (gao et al., ) . in water, meucin- revealed a b-sheet motif with % b-sheet and % a-helical content. however in % tfe, the proportion of a-helical structure increased to %; in contrast, the proportion of b-sheet was reduced to %. molecular modelling of meucin- was ambiguous, suggesting that meucin- may be able to fold in more than one way. both meucin- and meucin- (at mm) showed neither haemolytic activity nor antimicrobial activity against either fungi or gram-positive and gram-negative bacteria. only meucin À showed any cytotoxicity ( % reduction viability of gc- cells at mm). however both peptides ( mm) produced a e % reduction in the development of malarial parasite pandinus berghei ookinetes and at mm, completely eradicated pandinus falciparum ookinetes in h (gao et al., ) . the potent effects on intraerythrocytic p. falciparum yet little haemolytic or cytotoxic effects and no effect on bacterial and fungal membranes, is intriguing. one possible explanation that we suggest is an affinity-driven transfer from the erythrocyte membrane to the parasitic membrane. heterin- from the scorpion heterometrus spinifer is a residue c-terminally amidated peptide which shares % homology to pandinin secondary structure predictions revealed a single helical domain between residues e flanked by two random coil regions and is amphipathic in nature. moderate antimicrobial potency was observed against gram-positive pathogens ( . e . mm) and it was broadly less potent against gram-negative organisms ( . e> . mm), it also exhibited high cytotoxicity with . mm causing over % hemolysis. interestingly when the c-terminal random coil region is removed (kkd) the therapeutic index is improved with a halving of the haemolytic potential and an overall increase in the mic against grampositive organisms; however a reduction is seen against gram-negative pathogens (wu et al., in press). the first short chain amps were isolated from the venom of the african scorpion opisthacanthus madagascarienis. isct and isct (dai et al., (dai et al., , share % homology (both approx. . kda, amino acids), have the same net charge (þ ) and are both amidated at the c-terminus. the precursor of each peptide consists of a signal peptide and a c-terminal pro-sequence which contains the typical c-terminal processing signals gly-arg-arg or gly-lys-arg. both peptides showed a broad spectrum profile against most gram-positive and gram-negative bacteria on solid agar plates (mics typically . e mm) although gram-negative pseudomonas strains were unusually resistant (mics typically > mm). both peptides were weakly haemolytic ( % lysis of sheep erythrocytes at mm) (dai et al., ) although isct (approx. mm) was extremely effective, in comparison to mastoparan, at degranulating rat peritoneal mast cells, as measured by a histamine release assay (dai et al., ) . a pore-forming mechanism of action was postulated (dai et al., ) due to the threshold concentration kinetics during antimicrobial assays and calcian release assays with synthetic membranes. the latter revealed a membrane disruptive mechanism for both peptides. at low peptide concentrations a preferential affinity towards phosphatidic acid (pa) compared with phosphatidylcholine (pc) was noticed; however this selectivity was not seen with high concentrations of peptides. extensive structure-function analysis has been carried out on isct and isct (dai et al., (dai et al., , lee et al., ) . cd spectra and helical wheel predictions of both peptides revealed an a-helical organisation in membrane-mimicking environments ( and % tfe) which adopted a classical amphipathic structure. a sequence comparison (fig. ) between the two peptides shows differences at phe- (leu), ala- (lys) and asn- (gln) (isct residues in brackets). helical wheel analysis demonstrates that these differences cause changes within the hydrophilic face of isct , although there is no overall change in charge; this suggests that both overall ionic charge and amphipathic nature, as distinct from specific amino acid residues, are critical for amp function. mutation studies on isct (lee et al., ) showed the importance of the hydrophobic residue at position . substitution of trp for ala resulted in a remarkable decrease in both antimicrobial and haemolytic activities. dai and colleagues (dai et al., ) also identified two other peptides in the crude venom, analogous to isct and isct (isctf and isct f, respectively). these latter two peptides are thought to be proteolytic products of the parent molecules devoid of the two penultimate amino acids (leu and phe-nh ). isctf and isct f have no antimicrobial activity and have no a-helical structure as evidenced by cd spectral analysis, implicating these two c-terminal residues as critical for function. two amps, bmkb and bmkn , have been identified from the venom gland cdna library of buthus martensii (zeng et al., ) . these peptides are both basic (bmkb , charge þ ; bmkn , charge þ ) and like the isct peptides discussed earlier, also have amidated c-terminals. bmkb has a amino acid pre-pro sequence containing a amino acid signal sequence, which after post translational modification gives an amino acid mature peptide. in comparison, bmkn has a amino acid pre-pro sequence containing a amino acid signal sequence, finally resulting in a amino acid mature peptide. secondary structure predictions suggest that both bmkb and bmkn have ahelical regions (amino acids e and e respectively) flanked by two random coils; helical wheel projections demonstrate their amphipathic natures. the shorter and more highly charged bmkn is considerably more active than bmkb (mics for gram-positive organisms were typically . e mm for bmkn as compared with e mm for bmkb ; mics for gram-negative organisms were typically e mm for bmkn , as compared with e mm for bmkb ). the activity of bmkn against strains of multidrug resistant n.gonorrhoeae has recently been analysed via an mtt assay with these strains having an mic value of between . and . mm (arpornsuwan et al., in press). this study also noted the c-terminally amidated residue was critical for function with a dramatic decrease in activity observed upon deletion. based on these findings, pcr primer sets were designed to detect bmk homologues in other venoms, for example mucroporin from lychas mucronatus (dai et al., ) and imcroporin from isometrus maculates . mucroporin shows % homology with bmkb and consists of a -amino acid pre-pro peptide which gives rise to a residue mature peptide amidated at the c-terminal. mucroporin (charge þ ) and a mucroporin-m analogue (charge þ ) were both active against gram-positive bacteria (mic values e mm), although the difference in charge did not have a consistent effect on bacterial susceptibility. in contrast, both peptides had much weaker activity against gram-negative bacteria, with mic values greater mm (dai et al., ) . because s. aureus was particularly sensitive to mucroporin m , this peptide was further tested against clinically important isolates of this particular bacteria. the data revealed that mucroporin-m can inhibit a range of antibiotic-resistant pathogens of both methicillin-and penicillin-resistant strains ( e mm), as well as penicillinsensitive strains ( e mm). scanning electron microscopy (sem) studies showed rapid lyses of bacterial cells after mucroporin or mucroporin-m treatment, suggesting a membrane disruptive mechanism. mucroporin m has been shown to specifically inhibit rna viruses, including sars-corona virus, influenza and measles viruses, presumably by targeting the viral membrane . dna viruses were not inhibited. the same groups have more recently shown (zhao et al., ) that the peptide inhibits hepatitis b virus replication, both in vitro and in vivo, by activating the mitogen-activated protein kinase (mapk) pathway, which resulted in the down-regulation of the nuclear hormone transcription factor, hnf a. imcroporin, the second peptide identified as a bmkb homologue through a pcr screen, has the same number of amino acids and charge, as mucroporin. secondary structure prediction and helical wheel analysis suggested that imcroporin has a highly amphipathic nature with a % alpha-helical conformation . the antimicrobial activity profile of imcroporin was very similar to mucroporin; growth of gram-negative organisms was weakly inhibited (mics typically > mm) whereas gram-positive organisms, including methicillinand penicillin-resistant strains of clinical isolates of s. aureus were more sensitive to the peptide (mics typically e mm). for comparison, the mics of all clinical isolates against vancomycin were mm. imcroporin was also tested in an in vivo mouse model . the peptide (single dose, mg/g, given one hour after intraperitoneal infection with s. aureus) was as effective as vancomycin ( mg/g) in curing mice; all mice survived, seven days after treatment. however imcroporin also exhibited significant cytolytic properties against mammalian cell lines and red blood cells, as evidenced by mtt assays and haemolysis assays respectively. imcroporin ( mm) killed between and % of kidney and hepatoma cells and haemolysed % of human erythrocytes. kill kinetics and enzyme activity assays of cell supernatants revealed, as with mucroporin, a rapid membrane disruptive mechanism of action. the great majority of scorpions are considered harmless and probably as a consequence, the characterisation of peptides found in their venom glands has been very limited. the asian scorpion, scorpiops tibetanus is one such example and cao, li and colleagues (yuan et al., ) have cloned a new antimicrobial peptide gene (stct ) from s. tibetanus by screening a venom gland cdna library with isct primers. the precursor of stct is characterized by a signal peptide ( amino acids) followed by a putative mature peptide of residues and finally, an unusual amino acid acidic propeptide at the c-terminus, suggesting that the mature peptide is c-terminally amidated. a synthetic amidated peptide corresponding to the putative mature peptide of stct showed preferential activity towards s. aureus with weak activity towards gramnegative organisms, in a similar manner to mucroporin and imcroporin. mic values against methicillin-resistant s. aureus strains ranged between and mm whilst penicillin-resistant strains were more sensitive (mic mm). it is interesting to note that although the amidated stc peptide has approx % homology with isct, the latter peptide (in contrast to stc ) has activity against both gram-positive and gram-negative organisms.using the same approach, a stct gene has also been cloned . a synthetic, amidated amino acid stct peptide, corresponding to the predicted mature peptide, also showed potent activity against s. aureus and methicillin resistant s. aureus (mics values e mm). both stct and stc peptides belong to the ndbp - family (see table ). two structurally divergent peptides (meucin- ; meucin- ) have been isolated from a venom gland library of the asian scorpion mesobuthus eupeus by a random sequencing strategy (gao et al., ) . based on mic values, most gram-positive organisms were e fold more susceptible to meucin- than meucin- , while gramnegative organisms were e fold more susceptible to the longer peptide. meucin- was also e fold more potent than meucin- against a range of fungi and yeast. both meucin- and meucin- were cytotoxic toward rabbit erythrocytes; however meucin- ( . mm) was twice as haemolytic as meucin- at the same concentration ( vs % hemolysis, respectively). whole cell patch clamp experiments identified a sharp decrease in ion currents followed by a rapid, partial recovery, when rat dorsal root ganglion (drg) cells were exposed to either peptide. these electrophysiological experiments were supported by morphological studies, when the surface of drg cells was transformed from a smooth to a rough state after incubation with peptides. evidence for the peptides causing membrane permeabilization in microbial cells (bacteria, yeast and fungi) was obtained with the fluorescent dnabinding dye, propidium iodide. taken together, these studies suggest that meucins cause permanent cell damage. gao et al. ( ) have then gone on to try and rationalize their biological data in terms of structural differences between meucin- and meucin- . both peptides belong to group (table ) . meucin- has an amidated cterminal in contrast to meucin- which does not. extensive structural studies have been carried out on both peptides using cd spectroscopy and nmr spectrometry. the cd spectrum of meucin- in % tfe characterized the peptide as being primarily a-helical. this was confirmed by nmr, which identified an a-helical structure between residues e with an unordered n-terminus. meucin- showed similar results to meucin- , although nmr revealed a more ordered n-terminal structure (fig. ) . this is thought to be due to the presence of alanine at position (meucin- ) instead of phenylalanine (meucin- ). a structurally ordered n-terminus is postulated to be important for biological activity. gao and colleagues have also suggested that differences in biological potency might be related to differences in the hydrophilic/ hydrophobic balance between the two meucins due to the more balanced amphipathicity of meucin- . the c-terminal extension of meucin- with a free (charged) carboxy terminus means that overall, meucin- is more hydrophobic. this relative increase in hydrophobicity may reduce the biological activity of the latter due to its increased self-association in aqueous solution (see jiang et al., for a useful summary of this proposal). gao and colleagues also noted that meucin- has a positivelycharged region (adjacent lys- and lys- residues) that could prove crucial for increased interaction with negatively-charged prokaryotic membranes. the pattern of a-helical peptides from scorpion venoms showing preferential activity towards gram-positive organisms has continued with the cloning of ctriporin by the random screening of a large number of clones from the venom gland cdna library of chaerilus tricostatus (fan et al., ) . ctriporin is a amino acid peptide (net charge: þ ) with, like many other previously described peptides, an amidated c-terminus that is produced by the post translational cleavage of a -residue acidic propeptide. secondary structure analysis and cd spectra of the peptide in and % tfe revealed an amphipathic a-helical structure. however, the ambiguity still remains as to whether the peptide is completely a-helical or contains a random coil region, due to the presence of gly- , gly- and pro- , residues known to disrupt a-helical structures. mic values (gram-positive organisms) ranged from to mm whereas gram-negative organisms had mics > mm). all clinical isolates of mrsa strains gave mic values of mm (c.f. vancomycin e mm). similarly the penicillin-resistant strain of s. epidermidis had an mic of mm, analogous to vancomycin. ctriporin also had antifungal activity against candida (mic mm) and in vivo studies with mice demonstrated that topical applications of the peptide were more effective than currently established topical treatments, pexiganan and omiganan pentahydrochloride in treating fungal and s. aureus infections. time kill kinetics and scanning electron microscope studies suggested that ctriporin caused a rapid disruption of cell membranes, analogous to other a-helical peptides. two further members of the ndbp- class of short antimicrobial peptide (aamap and aamap ) have been isolated from the venom of the north african scorpion androctonus amoreuxi (almaaytah et al., ) and the precursor-encoding cdnas have been cloned, using a shotgun approach. they both have amino acids and are amidated at their c-termini. aamap and differ by only residues with leucine being replaced by proline at position and phenylalanine being replaced by isoleucine at position . secondary structure prediction suggested that aamap has a far greater random coil region than aamap . however these structural differences did not manifest themselves in changes in biological activity. although neither peptide was particularly active (mics e mm) aamap was slightly more active against gram-negative organisms while the reverse was true for gram-positive organisms. both were equally effective against yeast and caused a significant haemolysis of red blood cells in the same concentration range. using a size-selective screening strategy, three new antimicrobial peptides (pantinin- , - and - ) were characterized from a venom gland cdna library of the african scorpion pandinus imperator . all three were predicted to be mature peptides of e amino acid residues with precursor peptides indicating the nowcharacteristic c-terminal post-translational processing, resulting in c-terminal amidation. peptides were cationic and secondary structure prediction suggested that pantinins are a-helical, amphipathic structures. amino acid sequence homology revealed that these peptides belong to the ever-growing th family of ndbps. pantinins are relatively potent against gram-positive bacteria (mics e mm), including vancomycin-and methicillin-resistant bacteria but are much weaker against gram-negative bacteria (mics e > mm). in addition to their antimicrobial activities, the three peptides showed haemolytic activity on human rbcs in a dose-dependent manner. two c-terminal amidated peptides (tsap- and - ) have been isolated from the venom of tityus serrulatus by shot gun cloning and lc-ms identification (guo et al., in press) . each peptide contained residues with a net charge of þ . tsap- had very weak antimicrobial activity against yeast and both gram-positive and gram-negative organisms (mics typically e mm) and showed very little haemolytic activity. tsap- had no effect against gramnegative organisms at the highest concentrations tested but was surprisingly effective in inhibiting the growth of both yeast and gram-positive organisms (mics e mm). tsap- had weak haemolytic activity ( %) at four times the mic for gram-positive bacteria. the a-helical content ( % tsap- vs. % tsap- ) and hydrophobicity ( . hydrophobic moment tsap- vs. . tsap- ) has been suggested for the divergence in biological activities of these two peptides. an n-terminal, di-helical region of the scorpine family of cysteine-constrained peptides has been postulated to be responsible for their antimicrobial activity (zhu and tytgat, ) . the first residues of opistoscorpine were noted to have similarity to the cecropins, a family of linear amp's from the insect hyalophora cecropia, which have antimicrobial activity and remarkably low haemolytic activity (holak et al., ; hu et al., ) . this idea (zhu and tytgat, ) is supported by the alignment (fig. ) of cecropin a with the n-terminal regions of other scorpine family peptides (heteroscorpine, panscorpine and opistoscorpine- ). these regions also align well with the two helices of cecropin a determined by nmr (holak et al., ) . mechanistic studies of cysteine constrained peptides such as the scorpines are limited, compared with that of their non-cysteine containing counterparts. to date, no comparable haemolytic or cytolytic studies of the n-terminal regions of scorpines have been published. because of their noted similarity and low haemolytic activity this should be an attractive and profitable area to revisit. because mechanistic studies have only been carried out on the long-chain peptide pin , it is logical to compare the structures of other long chain peptides to this. alignment of the sequences of the six biologically active, long-chain amps (opistoporins, hadrurin, bmkbpp and vejovine) with pin (fig. ) reveal a remarkably conserved region within the n-terminal helices, including the trp corresponding to position in pin . when compared with the helical regions of pin identified from nmr studies (nomura et al., ) (fig. a , highlighted in yellow), this conservation becomes apparent. there is also high conservation within the hinge region (fig. b ). solid state nmr data of pin interaction with phospholipid bilayers suggested that the two alphahelical regions of the peptide cause lipid bilayers to adopt a cubic phase (nomura et al., ) . since the long chain peptides identified in fig. also have two distinct alpha helices, then it is a likely proposition that these other peptides also promote a cubic phase in lipid bilayers. taken together, these alignments may provide us with an insight sight into a general mechanism of action of this class of amp; however studies comparable to that of pin clearly need to be undertaken before any firm conclusions can be made. although these long peptides undoubtedly cause membrane disruption, it is very unlikely that they do so by forming pores, simply on the basis of their size. we are therefore left at present with the carpet like model to describe their mechanism of action. a structural review of diverse antimicrobial peptides highlighted that most of these molecules exhibit imperfect amphipathicity (wimley, ) . they are characterized by the presence of polar as well as non-polar faces with the latter containing a "polar pocket". this has led to the proposal of a new interfacial model for amp action (wimley, ; marks et al., ) , which is a hybrid between the classical carpet model and the more recent toroidal model. the interfacial model consists of three main processes that take place consecutively: (i) an initial electrostatic interaction with the negatively charged bacterial membrane driven by the polar face; (ii) an interaction with the membrane hydrophobic core driven by the peptide hydrophobic face, causing the peptide to insert into the membrane, and (iii) the polar pocket interacts with the phospholipid head groups on the inner membrane leaflet causing negative curvature of this inner membrane (marks et al., ; bobone et al., ) . analysis of scorpion short chain antimicrobial peptides by helical wheel projection reveals the amphipathic nature of these peptides with no clear "polar pocket" within the structures. however it is well documented that c-terminal amidation increases the biological activity of these short chain cationic peptides and it has been proposed that this is a consequence of an increase in net positive charge (strandberg et al., ) . if the helical wheel structures of these peptides are examined (fig. ) , it can be clearly seen that amidation of the negatively charged c-terminal carboxy group lies within the hydrophobic section of the peptide, thus changing the charge distribution of the peptide. however, the slightly longer peptides (e.g imcroporin, mucroporin, ctriporin and bmkb ) have c-terminal amidated residues that lie within the hydrophilic face of the peptide thus changes in charge distribution are less pronounced (fig. ). this 'rebalancing' of charge between the amphipathic facets of the peptide clearly has implications for biological activity and it poses an interesting question as to what the precise role of this amidation site has on scorpion amp mechanism of action. whilst extensive mechanistic studies have yet to be carried out on these peptides with amidation sites within the hydrophilic face, it is instructive to compare them to pin a mid-chain amp which contains residues (fig. a) . sequence alignments of pin with the four short chain peptides revealed similar features when a central galk motif was removed from the middle of the pin sequence (gly- to lys- , fig. b) . a lips motif found within pin (leu- to ser- ), thought important for biological activity, is also found in all four peptides with the exception of a ser/ala replacement in ctriporin (fig. c) . finally, another interesting separate alignment of pin and meucin- revealed the same motif as part of a conserved k(l/i)ipslf sequence (fig. d) . at present there is little biophysical experimental data to verify whether these identified sequence alignments have any structural importance (and therefore biological significance). further examination at a biophysical level is clearly important if scorpion amps are to provide viable templates for therapeutically useful molecules. recent proteomic studies (calvete, ; durban et al., ; gibbs et al., ) have highlighted the power of collision-and electron transfer-induced dissociation ms/ ms techniques to profile the venoms of a number of snakes for the determination of peptide sequences. these techniques have also been extensively used to analyse the venoms of various scorpions (abdel-rahman et al., ) . recent studies have highlighted the abundance of amp-like molecules within the transcriptomes of numerous scorpion venoms. % of all expressed sequence tags (ests) from transcripts of the venom gland of tityus stigmurus, were related to amps (almeida et al., ) . similar results were obtained from an analysis of the venom gland glands of the egyptian scorpion scorpio maurus palmatus (abdel-rahman et al., ) and the chinese scorpion, lychas mucronatus (ruiming et al., ) . in this latter study, scorpions were collected from two different and geographically-isolated areas (yunnan province and hainan island) and the data revealed that the relative abundance of ests corresponding to amps from scorpions collected on hainan island ( % total est population) were double that of scorpions found in yunnan. this phenomenon has been highlighted previously in mass spectrometry studies where geographical distribution, climate, age and sex have all been integral to the determination of venom components of a number of different scorpion species (e.g. newton et al., ; abdel-rahman et al., ; ma et al., ) . a combination of transcriptomic and ms/ms approaches have been used to identify a number of amps from the venom of australian scorpion urodacus yaschenkoi (luna ramirez et al., a,b) . three short chain a-helical peptides (uyct , uyct and uyct ) were characterized, with amidated c-terminii and sharing e % sequence homology with isct. although all three peptides were equally potent against a range of both gram-positive and gram-negative bacteria (mics typically e mm), they were also cytolytic ( mm peptides causing > % haemolysis of human erythrocytes). interestingly, when the nterminal gly was removed from either uyct or uyct , the hemolytic activity dramatically declined to %, although the range of mics increased in concert. two scorpine-like peptides and another with homology to pin- were also identified, but no antimicrobial assays have been carried out to date. a more recent study (luna ramirez et al., in press) revealed the synergistic effect of these peptides with combinational treatment increasing the therapeutic index. against a. baumanni uyct has an mic of mm whilst zhu and tytgat, ( ) that the n-terminal region is responsible for the antimicrobial properties of these peptides. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) uyct has an mic of mm, however combined the mic decreases to mm for uyct and mm for uyct . interestingly, the haemolytic potency of each peptide is increased when the peptides are administered separately. structural studies were also carried out on these four peptides (uyct , , and ). all four peptides showed unordered structure in aqueous buffer using cd spectra however form helices when in the presence of vesicles mimicking red blood cells (popc/cholesterol), gramnegative e.coli (pope/popg) and gram-positive s. aureus (popg/tocl). helical wheel projections revealed the characteristic amphipathic nature of the peptides. the membrane activity of these peptides was also confirmed by analysing the d-isomeric form which also showed activity. dye release assays and isothermal titration calorimetry (itc) suggests that all the peptides have a greater affinity towards prokaryotic model membrane systems over eukaryotic ones with a k d in the range of e mm compared with e mm, however the dye release assays suggest that the peptides lyse neutral membrane systems with greater ease than anionic systems. this apparent contradiction is postulated by the authors to be due to the increased surface interaction of the peptides with ionic lipids thus inhibiting insertion. however there is contraction between the dye release assays and the peptides biological activity which is thought to be due to the relative simplicity of the actual membrane systems. finally, several putative amp-like peptides have been identified on the basis of sequence homologies, although their antimicrobial activities are still to be determined. these include disulphide-bridged, scorpine-like peptides from opisthacanthus cayaporum (ocyc ; silva et al., ) fig. . helical wheel projections of six short chain cytolytic peptides; each peptide exhibits clear amphipathicity with the amidated c-terminal residue laying within the hydrophobic region creating a 'polar pocket' that is essential for the interfacial model. thus this key amidation site is key for the cytolytic mechanism of short chain peptides from scorpions. . two of these peptides, and hp , both residues in length and c-terminally amidated have recently been examined for their anti-viral activity on herpes simplex virus type (hsv- ). they caused a % decrease in plaque forming ability at mm when incubated with african green monkey kidney cells. however cell viability assays on the same kidney cell line revealed around % viability at mm with hp , only % of the cells were viable at the same concentration with hp . both peptides had the same hemolytic activity ( %) at mm (hong et al., ) . whilst the field of antimicrobial peptides has developed rapidly over the last two decades, with a plethora of peptides been identified and numerous structural/functional relationship studies undertaken, one fundamental paradigm still remains e how to develop amps into the clinical setting? as discussed at the beginning of this review a number of amps have failed to get approval although these have been largely commercial rather than scientific considerations, although as previously noted regulatory bodies may be starting to relax their attitudes towards approval (fox, ) . despite these positive advances in amp drug development, stability and immunogenicity still remain major problems when considering systemic therapeutic applications (yount and yeaman, ) . several groups have examined the protease stability of amps. as an example, carmona et al. ( ) have examined the biological activity of the proteolytic-insensitive dconformer of the scorpion amp, pin . a e % reduction fig. . helical wheel projections of longer chain short cytolytic peptides; each peptide exhibits clear amphipathicity with the amidated c-terminal residue lying within the hydrophilic region. thus no polar pocket is present which indicates a mechanism of action different from the interfacial model. in hemolytic activity was observed with the d-conformer, which retained the antimicrobial profile of the native peptide. crucially, when incubated with either trypsin, elastase, whole human serum or proteases from p.aeruginosa, only the d conformer retained activity, suggesting a feasible strategy for increasing amp stability. these results are mirrored by previous studies on phyllogenically diverse amps including cecropin a (wade et al., ) , melittin and paradaxin (oren et al., ) , ll- (dean et al., ), magainin (guell et al., and mastoparan (jones and howl, ) . other strategies employed to increase both peptide stability and plasma half life have focussed on the delivery system, for example using liposome encapsulation (du and stenzel, ) or conjugation to human serum albumin (has) as a carrier protein (xie, ) . more recent studies have investigated the potential of inorganic nanostructures for amp delivery (brandeli, ) . interestingly, as well as increasing stability toward proteases, these strategies have also increased the therapeutic index of amps in vivo. the reduction of immunogenicity of a protein/peptide is of great importance not just to amps but to protein therapeutics as a whole and consequently a number of strategies has been proposed to achieve this. although no studies have been directed toward decreasing the immunogenicity of amps in particular, various general strategies have been examined and could provide useful areas for further amp research. for example pegaylation is an approved nontoxic and non-immunogenic procedure by interfering with antibody and/or hla epitope binding (jevsevar et al., ) . similarly peptide glycosylation also results in decreased immunogenicity through a similar mechanism (von delwig et al., ) . pegaylation has also been shown to increase the stability of amps in the presence of proteases (falciani et al., ) as well as decrease toxicity and increase biocompatibility (morris et al., ) . conjugation of peptides to igg has also been carried out to successfully evade the adult immune system (zambidas and scott ) and is a strategy employed within clinical practise for the delivery of anti-inflammatory drugs. finally modification of a peptide sequence to delete potential t-cell epitopes have also been examined (de groot ) . therefore while a number of different strategies are available to improve stability and reduce immunogenicity, it is important to emphasize that the central goal of any strategy should be to maintain beneficial biological activity. one approach in the development of amps into the clinical setting could be to use them in combinational therapy with conventional antibiotics (brouwer et al., ) . whilst this approach would not rely on the direct killing of bacteria by amps they would act as 'entry peptides' allowing the conventional antibiotic to gain entry and interact with its intracellular target where resistance has built up at the level of the cell wall. another attractive strategy would be to use amp cocktail therapy (luna-ramirez et al., ) . these authors increased the therapeutic index of several uyct scorpion venom peptides. however in light of the current failure to bring a single amp to market, this would suggest that any duel amp cocktail therapies are presently an idealistic goal. over the past two decades, scorpion venoms have proved a rich source of amps with a varying range of mechanisms of action. the increased use of proteomic and transcriptomic approaches has already begun to prove fruitful, with a massive acceleration of peptides discovered by these approaches in the last few years. a rhetorical and thought-provoking question was asked during the st oxford world symposium on venoms (september ) e what approach is to be taken within future years regarding venom compound research? are we gatherers or hunters? (calvete, ) . the use of venom transcripts, mass spectrometry and bioinformatics tools leads to the question whether this combined approach is now regarded as paradigm of choice for identifying low abundance, and (perhaps?) more potent, antimicrobial peptides? in terms of both speed and scope of peptide identification through ms/ms techniques and the establishment of dedicated databases of fragmentation patterns, to the prediction of biological activity via bioinformatics, it is certainly an approach to be given serious consideration. however when one considers the many hundreds (over a thousand?) of antimicrobial peptides characterised in the last twenty years, the lack of any really new therapeutic anti-microbial agents is singularly depressing. we must therefore recognize that the approach of the "gatherers", presently in vogue, has one crucial intellectual limitation-you only get what you are looking for and is in essence a recipe for identifying more of the same. the alternative approach of the "hunter" may be more time consuming but one can argue that an investment in functional assays is the only way to identify truly novel compounds. one particular striking example of this is the identification of the bactridines (diaz et al., ) whose proposed mechanism of action is far removed from membrane active amps, indeed would not be considered a conventional target in other streams of antibiotic development. such serendipitous breakthroughs should be a lesson to us all. whether one is a philosophical hunter or gatherer, it cannot be contested that a more detailed biophysical approach to understanding the mechanisms of action of these new peptides is essential if they are to have a positive impact on the clinical management of infection. techniques such as nmr (nomura et al., (nomura et al., , , atomic force microscopy (afm) (garcia-sa ez, ; fernandez et al., ; lam et al., ) , secondary ionisation mass spectrometry (sims) (rakowska et al., ) and x-ray photoemission electron microscopy (won et al., ) can enable us to re-examine and refine our present interpretations of models of peptide interaction with microbial membranes (bobone et al., ; marks et al., ) . such experimental methods, taken together with molecular techniques to determine potential intracellular targets (e.g. brogden, ) and not forgetting that amps also have antiviral activities (e.g. zhao et al., ) , are crucial if our ultimate goal of using antimicrobial peptides as next generation therapeutics is to be realised. all authors declare that there are no ethical issues involved in this work. the requirements approved by toxicon and the publisher for accepting manuscripts for publication have been followed. venom proteomic and venomous glands transcriptomic analysis of the egyptian scorpion scorpio maurus palmatus (arachnida: scorpionidae) snapshots of scorpion venomics intraspecific variation in the egyptian scorpion scorpio maurus palmatus venom collected from different biotopes 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induction with an engineered immunoglobulin antimicrobial peptides of multicellular organisms scorpion venom peptides without disulfide bridges cloning and characterization of a novel cdna sequence encoding the precursor of a novel venom peptide (bmkbpp) related to a bradykinin-potentiating peptide from chinese scorpion buthus martensii karsch characterization of bmkbpp, a multifunctional peptide from the chinese scorpion mesobuthus martensii karsch: gaining insight into a new mechanism for the functional diversification of scorpion venom peptides identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from buthus martensii karsch three new antimicrobial peptides from the scorpion pandinus imperator imcroporin, a new cationic antimicrobial peptide from the venom of the scorpion isometrus maculates mucroporin-m inhibits hepatitis b virus replication by activating the mitogen-activated protein kinase (mapk) pathway and down-regulating hnf a in vitro and in vivo evidence for the existence of insect defensin-like peptide in scorpion venom the scorpine family of defensins: gene structure, alternative polyadenylation and fold recognition diversity of long-chain toxins in tityus zulianus and tityus discrepans venoms (scorpiones, buthidae): molecular, immunological, and mass spectral analyses the tale of a resting gland: transcriptome of a replete venom gland from the scorpion hottentotta judaicus mass fingerprinting of the venom and transcriptome of venom gland of scorpion centruroides tecomanus an albumin-conjugated peptide exhibits potent anti-hiv activity and long in vivo half-life the authors declare that there are no conflicts of interest. transparency document related to this article can be found online at http://dx.doi.org/ . /j.toxicon. . . . supplementary data related to this article can be found at http://dx.doi.org/ . /j.toxicon. . . . key: cord- - vbxyns authors: ronda, luca; tonelli, alessandro; sogne, elisa; autiero, ida; spyrakis, francesca; pellegrino, sara; abbiati, giorgio; maffioli, elisa; schulte, carsten; piano, riccardo; cozzini, pietro; mozzarelli, andrea; bettati, stefano; clerici, francesca; milani, paolo; lenardi, cristina; tedeschi, gabriella; gelmi, maria luisa title: rational design of a user-friendly aptamer/peptide-based device for the detection of staphylococcus aureus date: - - journal: sensors (basel) doi: . /s sha: doc_id: cord_uid: vbxyns the urgent need to develop a detection system for staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. biosensors are promising systems to achieve this aim. we coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. we show that the displacement of fluorescent peptides upon the competitive binding of s. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. this approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment. staphylococcus aureus, a common commensal of skin and nares, is one of the most frequent causes of infections [ , ] , one of the five most common causes of infections after injury or surgery. moreover, it has become the second major bacterium in food poisoning since it is able to produce heat-resistant toxins [ ] . in addition, antibiotic-resistant strains of s. aureus (e.g., methicillin-resistant staphylococci) appeared not only in clinical settings such as in hospitals, but also in whole communities [ ] . these premises make the development of rapid and reliable methods to identify s. aureus in biological samples as well as in food an urgent need to avoid epidemics. pathogen detection mainly relies on microbiological and biochemical methods, but these are usually time-consuming, expensive and not suitable for integration in on-site diagnosis. in fact, conventional methods to detect s. aureus, such as cell cultures, require more than one day and cannot distinguish among strains belonging to other species such as s. intermedius, s. caprae, s. simulans and s. capitis [ ] . rapid s. aureus agglutination tests have been developed as an alternative for routine diagnosis, but their accuracy, specificity and predictive capacity have been questioned [ ] [ ] [ ] . techniques involving manual or automated biochemical methods exploiting colorimetric reactions are widely used in clinical laboratories. they are faster than cell cultures and comparative studies indicate significantly better results [ ] , but they require specific instrumentation and trained personnel. for these reasons, there is currently a huge demand for alternative, rapid methods to detect s. aureus overcoming these limitations [ , ] . the advent of real-time pcr led to the development of rapid methods (a few hours), not requiring the bacteria isolation [ ] [ ] [ ] , but this technique requires expensive equipment and trained personnel. hence is not suitable for bedside point-of-care use. moreover, pcr-based methods are based on nucleic acids amplification, hence suffer from false positive results coming from contaminants sequences amplification, as well as false negative results coming from template nucleic acid degradation. as an alternative to nucleotides, peptide nucleic acids (pnas), pseudopeptides mimicking dna, show improved binding behavior and higher stability, but their cost is still too high for their application in this field. maldi-tof ms-based identification of bacteria is a fast and accurate technology [ ] [ ] [ ] [ ] and could be an alternative to molecular tests if the test accuracy is proven [ ] , but requires an expensive instrument as well. beside their interest and popularity, the costs, required time and the need for trained personnel and fixed equipment make the applicability of these techniques to point-of-care pathogen monitoring tools challenging to realize. an attractive alternative is represented by biosensors, devices where a biological component, such as a protein or oligonucleotide, is coupled with a transducer for obtaining a readable signal. biosensors may represent novel and user-friendly devices to handle human, animal of food samples, allowing the rapid detection of bacteria without the need for expensive and fixed equipment. the recognition of a bacterial pathogen such as s. aureus has been approached in different ways by biosensors, exploiting proper transducers to give a readable signal (table ) . commonly reported recognition tools are: ( ) antibodies [ ] ; ( ) bacteriophages [ ] ; ( ) phage display peptides and phage receptor binding proteins and ( ) nucleic acids. while successful in immuno-analytic protocols, antibodies are still costly, and show a relatively short shelf life and poor stability towards changes in temperature, ph and ionic strength. moreover, ethical issues regarding the need for animal immunization for their production have to be considered. however, optical fibers-based biosensors conjugated to monoclonal antibodies that bind methicillin-resistant staphylococci have been recently reported as a potential alternative to cell cultures [ ] . phages, which have better stability, and are selective for single strains of bacteria, have still to be optimized in terms of immobilization density and their purification remains challenging [ ] . nucleic acids are exploited for their capability to complementary hybridize each other or to form more complex tridimensional structures able to selectively recognize epitopes, similarly to antibodies. as an example of the first case, an electrochemical dna sensor based on dna hybridization has been recently developed and tested on contaminated food [ ] . the second case is mainly represented by aptamers, a novel and highly performing analytical tool for diagnostic applications [ ] . aptamers are dna or rna segments acting as artificial recognition elements that are able to recognize conserved epitopes on the surface of a bacterium. aptamer libraries are continuously growing thanks to the recent progress in the aptamer selection procedure (selex). moreover, due to the relatively easy prediction of their secondary structure, the possibility to modulate affinity towards a target by a rational modification of the sequence has been demonstrated [ ] . while several aptamers are available for recognizing s. aureus [ ] [ ] [ ] [ ] [ ] [ ] [ ] and hence a set of molecular recognition probes is available, the development of a proper transducer for a biosensor is still calling for an intensive research in the field of the technological challenges in translating aptamer-based biosensors in clinical practice is the possibility to have substrate materials adequate for the high density and functional immobilization of aptamers. a first endeavor to overcome this limit relies in the use of carbon nanotubes (cnts), chosen for their capability to form hybrids with nucleic acids and transducing electrical signals with high efficiency, exploited in the development of electrochemical sensors [ , , ]. an ultra-sensitive method for bacterial identification based on the resonance light-scattering signal of aptamer-conjugated gold nanoparticles was used for detecting single s. aureus cells [ ] . other recent advances in the development of aptamer-based s. aureus biosensors are based on gold electrode piezoelectric sensors [ ] or silver nanoparticles [ ] . despite these encouraging results, the development of surfaces able to immobilize aptamers is still crucial and nanostructured surfaces are very promising for this kind of applications [ ] [ ] [ ] [ ] [ ] [ ] , because they can allow high-density immobilization of biorecognition elements while retaining their structure and functionality. in particular, it has been demonstrated that the surface nanoscale morphology promotes protein [ ] [ ] [ ] and aptamer [ ] adsorption. thus, producing nanostructured substrates with well controlled and scalable roughness represents a unique asset for the implementation of aptamers-based devices. we took advantage of aptamers already known to interact with s. aureus to improve their surface immobilization density, by adopting a nanostructured zirconium dioxide support to promote surface linking. the final aim is to produce a detection system kit suitable for point-of-care use with a user-friendly read-out. we proved the possibility of creating a detection kit by immobilizing on the nanostructured support one of the highest affinity reported aptamers, sa [ ] . the detection is based on a fluorescein-labeled peptide bound to the aptamer that is displaced in the presence of bacteria and released free in the solution, with a concomitant change of its fluorescence properties. pathogens can be detected by visual inspection, ideally simply by illuminating the device with a visible commercial blue led. the novelty of our biosensor development approach resides in coupling an already known biorecognition element obtained with a high throughput approach (i.e., aptamers) with a rationally designed element (specifically interacting peptides), to give an easily readable signal. to design the structure of peptides we introduced in silico design in the development of a transducer [ , ] , i.e., a peptide able to selectively bind an aptamer based on its sequence. the interacting peptide was designed in silico exploiting the energetic amino acid-base recognition code previously obtained by estimating the interaction energy of several protein-dna complexes with the hint force field [ ] . the latter, originally developed for evaluating protein-ligand interactions [ ] , has also been successfully applied to protein-protein [ ] , protein-water [ ] and protein-dna [ , ] systems. differently from complementary double stranded dna, aptamers do not have a well-defined three-dimensional structure and present a high level of flexibility [ ] . it is known that aptamers are predominantly unstructured molecules in solution, and fold upon association with the corresponding ligands into molecular architectures, in which the ligand becomes an intrinsic part of the nucleic acid structure [ ] . this property was favorably exploited to increase the energy of interaction of peptides specifically designed to bind aptamers. starting from the aptamer sequence, the aforementioned recognition code was used to predict the residues able to give stronger interactions, considering that contacts as arg and lys with g, asp and glu with c, and asn and gln with a have demonstrated to be conserved and to better stabilize protein-nucleic acid complexes [ ] . the designed peptides were then synthesized using stepwise solid phase synthesis (spps), exploiting the effectiveness of microwave irradiation, which previously demonstrated its potential allowing the synthesis of a mer peptide [ ] . we finally created a device where the fluorescein-labeled peptide, hybridized to the aptamer immobilized on the nanostructured surface, is displaced by s. aureus and released in the biological fluid, losing its fluorescence. the properties of nanostructured materials could also fit in microfluidic devices, that demonstrated to be useful in the concentration of pathogens, hence improving the detection limit [ ] . this approach can in principle be expanded towards any biological agent for which selective aptamers have been identified. as a perspective, this platform has the potential for the development of point-of-care detection avoiding to access the healthcare environment. the recent sars-cov- pandemic highlighted how this aspect would be critical in the management of such emergency conditions. two main criteria were followed during the aptamer design phases: (i) evaluation of the aminoacid-nucleotide pairing suitability according to the employed pseudo-code [ ] ; (ii) the selection, whenever possible, of residues bearing side chain with similar length with respect to the wild type ones. the three-dimensional structure of sa - aii dna single strand was generated using mc-fold-mc-sym pipeline [ ] . then, the protein-aptamer complex was obtained by manually placing the ssdna molecules close to the cro protein, according to the protein-dna complex reported in pdb cro [ ] . we reproduced the interactions hypothesized by the mentioned recognition code [ ] , residues - and - of cro protein, which should interact with nucleobases at position - and - of sa dna sequence. to optimize the molecules and the relative interaction, we submitted the sa - aii complex model to ns of md simulation using the gromacs . . package [ ] . the complex was solvated in an octahedron box using the tip p water model, maintaining a . nm distance from the molecule border [ ] . counter-ions were added to neutralize the whole system. temperature and pressure were controlled with the berendsen algorithm [ ] , following previous protocols [ ] [ ] [ ] [ ] . the ewald method was applied to model electrostatic interactions. before starting the equilibration, waters were minimized for ps at k, restraining protein and rna atomic positions with a harmonic potential. starting from k, the system was heated up gradually to k in six steps phases and the production run in npt standard conditions for ns, without restraints. the md trajectory was analyzed with gromacs, vmd [ , ] and pymol [ ] packages. the second part of the simulation was clustered to extract a representative conformation, with the gromos clustering method [ ] . the structure having the lowest rmsd, with respect to the other cluster members, was considered as the most representative, for each cluster. peptide synthesis was carried out on a microwave-assisted solid phase-based peptide synthesizer, followed by labeling with ( )-carboxyfluorescein [ , ] . circular dichroism (cd) spectra were recorded with a jasco j- spectropolarimeter (jasco international co. ltd., tokyo, japan) thermostatted with a peltier unit set at • c. secondary structure estimation was performed by using the dichroweb server [ , ] . fluorescence spectra of each peptide-aptamer complex were recorded on a jasco fp- spectrofluorometer. the excitation wavelength was nm and the emission wavelengths were in the range from to nm. peptides tagged with carboxyfluorescein were dissolved in µm phosphate buffer and titrated with different amounts of aptamer. the peptide-aptamer mixture was incubated at room temperature for min to allow the formation of the complexes before the registration of each fluorescence spectrum. nanostructured zirconia (ns-zro x ) thin films were grown on glass microscope slides by depositing supersonic beam seeded with zirconia clusters produced by a pulsed microplasma cluster source (pmcs) under high vacuum conditions [ ] [ ] [ ] . a zirconium rod is ablated by a pulsed argon plasma stream, ignited by an electric discharge. the removed materials thermalize in the quenching inert gas and condense to form clusters. the mix of cluster and inert gas is extracted through a nozzle, forming the seeded supersonic beam. the zirconia clusters are collected on substrates mounted on the manipulator perpendicularly to the beam trajectory. since the kinetic energy of the zirconia clusters is sufficiently low to avoid fragmentation a cluster assembled film is grown. the films are partially oxidized in the deposition apparatus due to the presence of oxygen traces; further oxidation occurs upon exposure to air. the surface roughness of the substrates is crucial for the molecules immobilization since it affects their adsorption density and functionality [ , , ] . thus, the films were grown with deposition parameters optimized for producing films with morphological properties suitable for the aptamers immobilization. the rms roughness was estimated by afm measurements, as extensively described elsewhere [ ] . the value chosen as optimal was nm. the validation and quantification of the aptamers immobilization on ns-zro have been achieved by using the protocol called protein surface interaction microarray (psim), which allows the high throughput study of biomolecules-surfaces interactions [ ] . the protocol applied takes advantage of the biotin-streptavidin pairing to enhance the adsorption of aptamers on ns-zro . initially, a small volume droplet ( pl) of streptavidin is spotted on slides with the zirconia nanostructured surfaces, on top of those subsequent pl droplets biotin-triethylene glycol (teg) fluorescent aptamers are spotted. all the spotting process has been performed using an automated sciflexarrayer s -scienion ag spotter (scienion ag, berlin, germany). after incubation for min at % controlled humidity, the slides were blocked in a solution with pbsmt (pbs + mm mgcl + . % (v/v) tween ) for min and washed times in pbsmt for min, times in pbsm (pbs + mm mgcl ) for min and finally in doubly-distilled h o for min. slides were then dried under gentle nitrogen flux. the amount of adsorbed biomolecules is evaluated by reading the fluorescent signal with a tecan microarray scanner (tecan group ltd., männedorf, switzerland), and images are analyzed using scan-array express software (perkinelmer inc., waltham, ma, usa). figure s shows a sketch of psim protocol applied to different fluorescent aptamers spotted on ns-zro substrate. sa has been purchased from sigma-aldrich (sigma-aldrich corp., st. louis, mo, usa), aptamer sa biotin-teg modified has been purchased from primmbiotech (primmbiotech, inc., cambridge, ma, usa). the iia fluorescent peptide is incubated in the aptamers microarray immediately after the blocking step. the concentration of the peptide is µm in pbs and incubation time is of min. the microscope slides have been imaged in the xy and xz planes using a leica tcs sp confocal microscope (leica microsystems, wetzlar, germany), using a diode ( nm) laser with % laser power, a × oil immersion objective, × image resolution and uv ( nm) at % laser power, × oil immersion objective and × image resolution ( figure s ). staphylococcus aureus subsp. aureus (atcc- ) and escherichia coli (atcc- ) are from lgc standards (lgc standards s.r.l., milan, italy). the bacteria are cultured in solution, using lb broth (miller) from sigma-aldrich as growth medium, in a thermoshaker at • c overnight. then the concentration of bacteria is measured using a spectrophotometer at fixed wavelength ( nm). before the incubation with the immobilized aptamers, the bacteria were stained with hoechst , from sigma-aldrich ( µl per ml of solution). in order to detect the activity of aptamers immobilized on nanostructured surfaces for bacterial recognition the following protocol has been established: incubation with µm streptavidin for min; washing with pbsm time for min; incubation with µm biotin-teg-aptamer-cy labelled for min; washing with pbsmt times for min; incubation with s. aureus or e. coli, bacteria/ml for min at rt; washing with pbsmt and pbsm, times for min each. the bacterial displacement experiments are based on the previously described procedures: round cover glass (Ø mm) coated with ns-zro (rms roughness nm), coverage of the surface with streptavidin ( . µm, µl), incubation at rt for min in % controlled humidity, washing in pbsm, removal of pbsm, spotting of aptamer sa biotin-teg modified ( . µm, µl), incubation at rt for min in % controlled humidity, washing in pbsm, removal of pbsm, spotting of peptide ( µm, µl), incubation at rt for min in % controlled humidity (kept dark), plate reading (tecan), washing in pbsm, bacteria plating incubation with s. aureus or e. coli, bacteria/ml for min at rt (kept dark), washing with pbs, plate reading (tecan) of the fluorescein signal (excitation wavelength nm, emission wavelength nm). the five aptamers presented by cao and coworkers [ ] were considered for selecting the candidate that could be better targeted by peptides designed in silico. sa (figure a ) was chosen among these aptamers firstly for a structural peculiarity. according to the predicted secondary structure, in fact, it presents a relatively long trait in double strand configuration (the hairpin made by nucleotides , with high gc content in the inner portion. this double strand dna region of the aptamer was then identified as the putative binding region of the peptides. moreover, the sa aptamer results as the one with the higher affinity towards s. aureus within this group. aptamer folding prediction obtained with rnastructure (https://rna.urmc.rochester.edu/rnastructureweb/) for (a). full lenght sa ; (b). sa short sequence; (c). sa short . here the predicted contacts with the mutated residues are also indicated. the color-code shows the level of prediction reliability, as indicated in the legend. once the proper aptamer was chosen, we identified among proteins able to interact with nucleic acids λ-cro as a suitable scaffold for the design of aptamer-interacting peptides [ ] . λ-cro is a aa protein that plays a pivotal role in the switch from lysogenic to lytic phase in the growth cycle of phage λ and represented a suitable scaffold for several reasons: (i) the availability of threedimensional structures both in the presence and absence of its cognate dna (pdb id: cro [ ] and cro [ ] , respectively) allows detailed structural evaluations; (ii) the helix-turn-helix motif of the dna binding domain is relatively small and all the interactions between the nucleobases of the consensus sequence and the peptidic backbone are well characterized; (iii) the minimum functional portion of the consensus sequence is quite short and made by contiguous nucleobases along the two strands of the dna target; (iv) previous works reported successful examples of cro reprogramming for binding consensus sequences that differ from the wild type [ ] . moreover, the helix-turn-helix peptide domain of the cro protein showed to well mimic full-length protein for binding [ ] . therefore, the sequence of the helix-turn-helix motif of cro was mutated, accordingly to the previously reported recognition code, to specifically bind sa double strand region. here the predicted contacts with the mutated residues are also indicated. the color-code shows the level of prediction reliability, as indicated in the legend. once the proper aptamer was chosen, we identified among proteins able to interact with nucleic acids λ-cro as a suitable scaffold for the design of aptamer-interacting peptides [ ] . λ-cro is a aa protein that plays a pivotal role in the switch from lysogenic to lytic phase in the growth cycle of phage λ and represented a suitable scaffold for several reasons: (i) the availability of three-dimensional structures both in the presence and absence of its cognate dna (pdb id: cro [ ] and cro [ ] , respectively) allows detailed structural evaluations; (ii) the helix-turn-helix motif of the dna binding domain is relatively small and all the interactions between the nucleobases of the consensus sequence and the peptidic backbone are well characterized; (iii) the minimum functional portion of the consensus sequence is quite short and made by contiguous nucleobases along the two strands of the dna target; (iv) previous works reported successful examples of cro reprogramming for binding consensus sequences that differ from the wild type [ ] . moreover, the helix-turn-helix peptide domain of the cro protein showed to well mimic full-length protein for binding [ ] . therefore, the sequence of the helix-turn-helix motif of cro was mutated, accordingly to the previously reported recognition code, to specifically bind sa double strand region. the selected portion of λ-cro sequence suitable for in silico evaluation and following mutation spans the residues - (wild type sequence: gqtktakdlgvyqsainkaihag), that includes almost the totality of the a-specific and pseudo-specific residues that interact with its cognate dna sequence, according to the complex reported by olhendorf et al. (pdb id: cro) [ ] . the design was performed using the complex as template and considering the peptides acting as a monomer, which was reported to bind its operator site, with a binding constant of around µm or above for the wild type. this binding constant is theoretically appropriate for a system where the labeled peptide has to be displaced by the aptamer that, on the contrary, shows binding constant in the nanomolar range. the secondary structure of sa was predicted by using rnastructure and is reported in figure a . as mentioned, the paired region was chosen as possible target. the interacting peptides have been designed substituting the original residues to possibly generate more stable interactions with the paired aptamer ( table ). the peptides were synthesized using microwave assisted fmoc-based solid phase peptide synthesis on rinkamide resin [ ] . labelled peptides were prepared on resin using , carboxyfluorescein as the fluorescent tag [ ] . circular dichroism experiments were carried out on λ-cro peptide mutants optimized to interact with the double strand portion of sa aptamer in mm phosphate buffer, ph . . circular dichroism was used to characterize the secondary structure of synthesized peptides, and hence to determine the mutation effect on the structure stability. it has already been demonstrated that it is possible to mimic the dna binding behavior of λ-cro by shorter, chemically synthesized peptides, representing the helix-turn-helix region of the protein [ ] . these peptides, also in dimeric form, showed significant helical content only when α-amino isobutyric acid is introduced in the sequence [ ] . to quantify the λ-cro mutant helical content, the spectra of the peptides were analyzed using dichroweb server (cdsstr algorithm-reference set ), yielding a low percentage of helical content (table ), highlighting that the isolated peptides in solution are unstructured in the absence of the target nucleotide sequences. to verify that increasing the concentration the peptides does not result in the formation of aggregates or oligomeric forms, cd spectra were collected as a function of peptide concentration ( - µm). the cd spectra in this range of concentration appeared identical (data not shown). peptide solutions demonstrated to be stable even after - freeze-thawing cycles. the use of circular dichroism for evaluating the interaction between sa aptamer and the peptides requires also to characterize sa cd spectra. since peptides were specifically designed to bind a specific region on the aptamer (the hairpin formed by nucleotides , shorter sequences were also characterized: sa short and sa short , the former being the short paired sequence on which the peptide is expected to bind, while the latter is the complete hairpin. cd spectra of sa , sa short (figure b) and sa short (figure c) were collected in the far-uv and near-uv regions. the peptide-aptamer interaction was first studied by near-uv cd spectroscopy. in this region, only aptamers and not peptides contribute to the spectra. the analysis of this spectral region allowed to monitor the peptide effect on the aptamer structure, without any interference from the peptide conformational change. solutions containing aptamers and peptides at the same concentration ( µm) were incubated before recording the spectra. a comparison between the spectra of the aptamer-peptide mixture and the arithmetic sum of the two separated components would highlight potential interactions. the comparison between arithmetic and experimental mixtures highlighted no differences with the short versions of sa , sa short and sa short , while a marked difference was observed for the full length aptamer sa with the four peptides iia , iaser, iia and ib . in figure the comparison between arithmetic and experimental mixtures for the iia peptide is shown. the absence of cd spectra difference between the mixtures and the arithmetic sums observed for the shorter versions of sa (sa short and sa short ) may derive from the lack of conformational adjustment upon peptide binding. however, the collected data showed that the aptamer sa interacts with four peptides, promoting a conformational change detectable by a cd band intensity decrease. the most evident effect was observed for peptide iia , as reported in figure where the ellipticity at nm is reported. to demonstrate that peptide binding is determined by the specific mutation inserted on λ-cro peptide mutants, we synthesized and checked sequence-scrambled peptides, and peptides where mutations causing unfavorable interaction were inserted. the comparison of the mixture of these peptides with sa with the arithmetic sum gave overlapping results, demonstrating the absence of interaction (data not shown). to demonstrate that peptide binding is determined by the specific mutation inserted on λ-cro peptide mutants, we synthesized and checked sequence-scrambled peptides, and peptides where mutations causing unfavorable interaction were inserted. the comparison of the mixture of these peptides with sa with the arithmetic sum gave overlapping results, demonstrating the absence of interaction (data not shown). based on cd results, further analyses were carried out by fluorescence spectroscopy, using the most promising iia peptide labeled with fluorescein, in order to: (i) confirm peptide-aptamer complex formation also after peptide labeling, and (ii) calculate the dissociation constants (kd) of the peptide-aptamer complex, which determination is prevented in circular dichroism measurements by experimental limits of acquiring cd spectra at low concentrations. the labeled peptide was titrated with increasing concentrations of aptamers and spectral perturbations were followed at nm emission wavelength, allowing to calculate the dissociation constants ( figure ). in accordance with the cd measurements, specific binding could be detected for the aptamer sa with peptide iia , with a measured kd of . ± . μm. differently form cd measurements, fluorescence analysis was able to detect peptide binding also to the shorter versions of sa , sa short and sa short , for which a kd of . ± . and . ± . μm was obtained, respectively. these data demonstrated that iia peptide is able to bind to sa with an affinity in a low micromolar range, and peptide binding is specific for the target sequence, as demonstrated by binding of sa short and sa short , that showed similar binding affinity. iia peptide affinity for sa aptamers, compared to the reported affinity of sa for s. aureus of . ± . nm, makes iia peptide a good candidate for a device where the displacement of the peptide from sa binding site in the presence of the pathogen bacterium easily allows the s. aureus detection. based on cd results, further analyses were carried out by fluorescence spectroscopy, using the most promising iia peptide labeled with fluorescein, in order to: (i) confirm peptide-aptamer complex formation also after peptide labeling, and (ii) calculate the dissociation constants (kd) of the peptide-aptamer complex, which determination is prevented in circular dichroism measurements by experimental limits of acquiring cd spectra at low concentrations. the labeled peptide was titrated with increasing concentrations of aptamers and spectral perturbations were followed at nm emission wavelength, allowing to calculate the dissociation constants ( figure ). in accordance with the cd measurements, specific binding could be detected for the aptamer sa with peptide iia , with a measured k d of . ± . µm. differently form cd measurements, fluorescence analysis was able to detect peptide binding also to the shorter versions of sa , sa short and sa short , for which a k d of . ± . and . ± . µm was obtained, respectively. these data demonstrated that iia peptide is able to bind to sa with an affinity in a low micromolar range, and peptide binding is specific for the target sequence, as demonstrated by binding of sa short and sa short , that showed similar binding affinity. iia peptide affinity for sa aptamers, compared to the reported affinity of sa for s. aureus of . ± . nm, makes iia peptide a good candidate for a device where the displacement of the peptide from sa binding site in the presence of the pathogen bacterium easily allows the s. aureus detection. the fluorescence behavior of fluorescein upon binding will allow an easy detection of aptamer:peptide complex displacement by s. aureus. in fact, fluorescein loses fluorescence upon release from the aptamer, and this can be appreciated by the loss of fluorescence of the nanostructured chip upon its incubation in the putative contaminated fluid. the fluorescence behavior of fluorescein upon binding will allow an easy detection of aptamer:peptide complex displacement by s. aureus. in fact, fluorescein loses fluorescence upon release from the aptamer, and this can be appreciated by the loss of fluorescence of the nanostructured chip upon its incubation in the putative contaminated fluid. to further investigate the interaction of sa with the most promising peptide iia , we modelled the aptamer-peptide complex, using as guide the coordinates of the cro protein cocrystallized with a dna duplex molecule (pdb id cro [ ] , see method section for details). to stabilize and investigate the dynamic and structural properties of the complex, molecular dynamic simulation (md) in explicit waters was performed for ns. although no major perturbations affect the complex, the root mean square deviation (rmsd) values exhibited by either the complex, the peptide or the aptamer, with respect to the initial state, reveal the system reaches a global equilibrium in the second half of md simulation ( figure s ). in figure (panel a) a representative structure of the equilibrated part of the trajectory is shown. even if changes occurred with respect to the starting modelled structure, a good protein-aptamer interface is established and preserved along the trajectory and the aptamer maintains the same loop arrangement dictated by the original folding. we have calculated the occurrence percentage of hydrogen bonds formed at the complex interface, finding that out of the significative hydrogen bonds (preserved for more than % of the trajectory frames) involve the aptamer backbone atoms ( figure s and table s ). the remaining connections involve nucleobase atoms of the aptamer. in detail, lys and thr (out from the peptide length) interact with the phosphate groups of guanine and (table ), lys and his make persistent bonds with the nucleobases of guanine (dg ) and timine (dt ). the last interactions are formed by the exposure of lys and his out of the double strain towards the peptide and are able to anchor the peptide with specific nucleobase interactions. likely, the iia peptide sequence is able to maintain the required secondary structure arrangement to bind the aptamer through non-specific and specific contacts. to further investigate the interaction of sa with the most promising peptide iia , we modelled the aptamer-peptide complex, using as guide the coordinates of the cro protein co-crystallized with a dna duplex molecule (pdb id cro [ ] , see method section for details). to stabilize and investigate the dynamic and structural properties of the complex, molecular dynamic simulation (md) in explicit waters was performed for ns. although no major perturbations affect the complex, the root mean square deviation (rmsd) values exhibited by either the complex, the peptide or the aptamer, with respect to the initial state, reveal the system reaches a global equilibrium in the second half of md simulation ( figure s ). in figure (panel a) a representative structure of the equilibrated part of the trajectory is shown. even if changes occurred with respect to the starting modelled structure, a good protein-aptamer interface is established and preserved along the trajectory and the aptamer maintains the same loop arrangement dictated by the original folding. we have calculated the occurrence percentage of hydrogen bonds formed at the complex interface, finding that out of the significative hydrogen bonds (preserved for more than % of the trajectory frames) involve the aptamer backbone atoms ( figure s and table s ). the remaining connections involve nucleobase atoms of the aptamer. in detail, lys and thr (out from the peptide length) interact with the phosphate groups of guanine and (table ), lys and his make persistent bonds with the nucleobases of guanine (dg ) and timine (dt ). the last interactions are formed by the exposure of lys and his out of the double strain towards the peptide and are able to anchor the peptide with specific nucleobase interactions. likely, the iia peptide sequence is able to maintain the required secondary structure arrangement to bind the aptamer through non-specific and specific contacts. the typical electrostatic profile of the protein-aptamer interface, showing positively charged patches on the peptide side in contact with the negatively charged nucleic acid backbone, is shown in figure (panel b and c) . the typical electrostatic profile of the protein-aptamer interface, showing positively charged patches on the peptide side in contact with the negatively charged nucleic acid backbone, is shown in figure (panel b and c) . to exploit the possibility to realize an aptamer-based biosensor for bacteria detection the microarray technique was applied to test different aptamers and different experimental conditions for their immobilization in one single experiment and to test bacteria-aptamer interactions. figure shows the image acquired by a scanner/reader of sa fluorescent aptamer spotted on a glass slide coated with ns-zro for various concentrations, namely , , and μm. the protein is shown in blue cartoon and the aptamer in yellow ribbon; the atoms involved in each connection are labeled. (b,c). frontal and lateral view of molecular dynamic representative structures of the protein-aptamer complex simulations derived using a rmsd based clustering approach. the cro protein is shown as surface and colored according to the electrostatic potential. the red color (negative potential) arises from an excess of negative charges near the surface and the blue color (positive potential) occurs when the surface is positively charged (± kt/e). the white regions correspond to fairly neutral potentials. the aptamer is colored yellow. to exploit the possibility to realize an aptamer-based biosensor for bacteria detection the microarray technique was applied to test different aptamers and different experimental conditions for their immobilization in one single experiment and to test bacteria-aptamer interactions. figure shows the image acquired by a scanner/reader of sa fluorescent aptamer spotted on a glass slide coated with ns-zro for various concentrations, namely , , and µm. the isotherms showing the adhesion of sa aptamer on different substrates are reported in figure . the promotion of the adhesion of bare aptamers on ns-zro substrate with respect to clean glass is mainly evident at high concentrations. however, the adhesion enhancement is of about a factor of two for all the concentrations, when the biotinylated aptamers pair with streptavidin deposited on the nanostructured surface. furthermore, no manifest hindering to aptamer adhesion appears to be induced by the teg spacers. the sa aptamer immobilized on the microarray was left to hybridize with fluorescein-labeled iia peptide. the total absence of fluorescein signal suggests that aptamers loose their functionality, possibly because of interactions with the microarray surface leading to incorrect aptamer folding, i.e., electrostatic interactions between the negatively charged dna phosphodiester backbone and the positively charged surface. to overcome this problem, biotin-teg modified aptamers were used since the atoms spacer should be able to avoid any possible interaction between the aptamers and the surface [ ] . thus, a new microarray with biotin-teg aptamers spotted on ns-zro coated with streptavidin was left to hybridize with the fluorescein-labelled peptide (iia ). the assays result is reported in figure and figure s . the expected signal originating from the hybridization between sa and iia was clearly appreciable, demonstrating that the sa aptamer was immobilized with the correct folding. the isotherms showing the adhesion of sa aptamer on different substrates are reported in figure . the promotion of the adhesion of bare aptamers on ns-zro substrate with respect to clean glass is mainly evident at high concentrations. however, the adhesion enhancement is of about a factor of two for all the concentrations, when the biotinylated aptamers pair with streptavidin deposited on the nanostructured surface. furthermore, no manifest hindering to aptamer adhesion appears to be induced by the teg spacers. the isotherms showing the adhesion of sa aptamer on different substrates are reported in figure . the promotion of the adhesion of bare aptamers on ns-zro substrate with respect to clean glass is mainly evident at high concentrations. however, the adhesion enhancement is of about a factor of two for all the concentrations, when the biotinylated aptamers pair with streptavidin deposited on the nanostructured surface. furthermore, no manifest hindering to aptamer adhesion appears to be induced by the teg spacers. the sa aptamer immobilized on the microarray was left to hybridize with fluorescein-labeled iia peptide. the total absence of fluorescein signal suggests that aptamers loose their functionality, possibly because of interactions with the microarray surface leading to incorrect aptamer folding, i.e., electrostatic interactions between the negatively charged dna phosphodiester backbone and the positively charged surface. to overcome this problem, biotin-teg modified aptamers were used since the atoms spacer should be able to avoid any possible interaction between the aptamers and the surface [ ] . thus, a new microarray with biotin-teg aptamers spotted on ns-zro coated with streptavidin was left to hybridize with the fluorescein-labelled peptide (iia ). the assays result is reported in figure and figure s . the expected signal originating from the hybridization between sa and iia was clearly appreciable, demonstrating that the sa aptamer was immobilized with the correct folding. the sa aptamer immobilized on the microarray was left to hybridize with fluorescein-labeled iia peptide. the total absence of fluorescein signal suggests that aptamers loose their functionality, possibly because of interactions with the microarray surface leading to incorrect aptamer folding, i.e., electrostatic interactions between the negatively charged dna phosphodiester backbone and the positively charged surface. to overcome this problem, biotin-teg modified aptamers were used since the atoms spacer should be able to avoid any possible interaction between the aptamers and the surface [ ] . thus, a new microarray with biotin-teg aptamers spotted on ns-zro coated with streptavidin was left to hybridize with the fluorescein-labelled peptide (iia ). the assays result is reported in figure and figure s . the expected signal originating from the hybridization between sa and iia was clearly appreciable, demonstrating that the sa aptamer was immobilized with the correct folding. to finally test the functionality of the proposed device, we investigated the capability of s. aureus to outcompete iia peptide binding to sa . the ns-zro substrate functionalized with sa and hybridized with iia peptide was subjected to bacterial incubation followed by a washing step; then fluorescence was measured. successful competition of bacteria with respect to the fluorescently labelled peptides for binding to sa is expected to bring about fluorescence loss upon washing. we compared the fluorescent peptide displacement by s. aureus and e. coli to evaluate the selectivity of peptide displacement by sa -s. aureus interaction. the residual fluorescence signal, averaged on samples using a plate reader, is related to the remaining amount of bound peptides, that is inversely related to their displacement by bacteria. the data shown in figure (panel a) and analyzed in figure indicate that the device plated with s. aureus exhibits a significantly lower signal with respect to that with e. coli. this result supports the effectiveness of the device for selective detection of s. aureus. to finally test the functionality of the proposed device, we investigated the capability of s. aureus to outcompete iia peptide binding to sa . the ns-zro substrate functionalized with sa and hybridized with iia peptide was subjected to bacterial incubation followed by a washing step; then fluorescence was measured. successful competition of bacteria with respect to the fluorescently labelled peptides for binding to sa is expected to bring about fluorescence loss upon washing. we compared the fluorescent peptide displacement by s. aureus and e. coli to evaluate the selectivity of peptide displacement by sa -s. aureus interaction. the residual fluorescence signal, averaged on samples using a plate reader, is related to the remaining amount of bound peptides, that is inversely related to their displacement by bacteria. the data shown in figure (panel a) and analyzed in figure indicate that the device plated with s. aureus exhibits a significantly lower signal with respect to that with e. coli. this result supports the effectiveness of the device for selective detection of s. aureus. we demonstrated that the rational design of a peptide able to interact with a nucleotide sequence (aptamer) selected for the recognition of a specific pathogen is a viable approach for the development of a biosensor. moreover, we show that the developed zirconia nanostructured substrate is a promising platform for generating biosensors based on the immobilization of receptors for the detection of pathogenic agents. important structural determinants related to the aptamer sequence and responsible for the specificity of the protein targeting have been revealed. md simulations performed on the modelled peptide-aptamer complex validated the interaction and identified key residues fundamental for the complex stabilization. even if the original interaction pattern, as predicted by the protein-dna recognition code [ ] , has not been totally preserved, these results are of significant relevance, considering that no x-ray structure of the peptide-aptamer is available and the complex has been hardly modelled on the structure of the λ-cro protein interacting with a double strand dna. this likely supports the use of the recognition code for the prediction of key residue-nucleobase contacts in protein-nucleic acids interaction. the microarray technique allows the development of biosensors to screen in parallel for more targets, given the possibility to immobilize multiple receptors on the same substrate, retaining their structure and functionality. moreover, the high surface-to-volume ratio that characterizes the porous materials used as substrates for the microarrays, allows the adsorption of a higher amount of molecules/receptors/aptamers with respect to a flat surface. we demonstrated that the rational design of a peptide able to interact with a nucleotide sequence (aptamer) selected for the recognition of a specific pathogen is a viable approach for the development of a biosensor. moreover, we show that the developed zirconia nanostructured substrate is a promising platform for generating biosensors based on the immobilization of receptors for the detection of pathogenic agents. important structural determinants related to the aptamer sequence and responsible for the specificity of the protein targeting have been revealed. md simulations performed on the modelled peptide-aptamer complex validated the interaction and identified key residues fundamental for the complex stabilization. even if the original interaction pattern, as predicted by the protein-dna recognition code [ ] , has not been totally preserved, these results are of significant relevance, considering that no x-ray structure of the peptide-aptamer is available and the complex has been hardly modelled on the structure of the λ-cro protein interacting with a double strand dna. this likely supports the use of the recognition code for the prediction of key residue-nucleobase contacts in protein-nucleic acids interaction. the microarray technique allows the development of biosensors to screen in parallel for more targets, given the possibility to immobilize multiple receptors on the same substrate, retaining their structure and functionality. moreover, the high surface-to-volume ratio that characterizes the porous materials used as substrates for the microarrays, allows the adsorption of a higher amount of molecules/receptors/aptamers with respect to a flat surface. the cluster-assembled zirconia developed in this work was used as reliable support for bioactive molecules' immobilization, particularly aptamers, for biosensing applications. the protocol developed for their immobilization preserves the functionality of the nucleotides. this immobilization technique well couples with the use of fluorescently labeled synthesized peptides able to selectively bind specific aptamers. we finally created a device where the fluorescent chip, loaded with the labeled peptide, looses fluorescence upon bacterium displacement and consequent peptide release in the fluid. a colorimetric switch as a detected signal could also be possible by preparing peptides labeled with solvatochromic fluorophores [ , ] . this device development platform can, in principle, be applied to any analyte for which selective aptamers have been identified, and whose rapid and specific point-of-care detection is desired. supplementary materials: the following materials are available online at http://www.mdpi.com/ - / / / /s : peptide synthesis and labeling, figure s : sketch of psim protocol applied to one fluorescent aptamer spotted on ns-zro at different concentrations. upper left: spotting; upper right: incubation in a controlled atmosphere ( % humidity), immersion in a blocking solution and rinsing; bottom left: drying and bottom right: scanned image, figure s : time evolution of the rmsd values with respect to the starting model. the rmsd values have been computed considering the c alpha and c ' atoms of the protein and aptamer, respectively. the following color code was used: overall complex (mean: . nm, sd: . nm): black line, peptide aii (mean: . nm, sd: . nm): red, aptamer (mean: . nm, sd: . nm): green, figure s : absolute number of protein-aptamer interfaces hydrogen bonds during the entire trajectory, figure s : sketch of the biotin-streptavidin paring strategy. the aptamers are functionalized with biotin-teg and the ns-zro is coated with streptavidin. the -atom tetraethylene glycol (teg) spacer is added for minimizing steric hindrance when conjugating the biotin with other molecules, table s : persistent hydrogen bonds computed for the last ns of simulation time. we kindly acknowledge the competence centre for scientific computing (c s) at the university of turin (c s.unito.it) for providing the computational time and resources, and molecular horizon srl for supporting i.a. the authors are grateful to alfonso zecconi of the department of biomedical, surgical and dental sciences (university of milan) for his support in bacterial culture, and to carla perego and alessandra galli of the department of pharmacological and biomolecular sciences (university of milan) for their contribution to performing measurements with plate reader useful to the determination of bacterial displacement. we thank the interdepartmental measure centre (cim) "giuseppe casnati" of the university of parma for the use of the jasco j- spectropolarimeter. the authors declare no conflict of interest. the following abbreviations (in order of appearance) are used in this manuscript: pcr polymerase chain reaction pna peptide nucleic acid maldi-tof matrix assisted laser desorption ionization time-of-flight ms mass spectrometry selex systematic evolution of ligands by exponential enrichment cnt carbon nanotubes sars-cov- severe acute respiratory syndrome coronavirus pei-ga glutaraldehyde pre-coated with polyethyleneimine cfu colony forming unit pdb protein data bank md molecular dynamics cd circular dirchoism ns-zrox nanostructured zirconia pmcs pulsed microplasma cluster source the following abbreviations (in order of appearance) are used in this manuscript: afm atomic force microscopy psim protein surface interaction microarray teg triethylene glycol pbs phosphate buffed saline rt room temperature rmsd root mean square deviation food handler-associated methicillin-resistant staphylococcus aureus in public hospitals in salvador, brazil staphylococcal and streptococcal infections enterotoxigenic staphylococci and their toxins in restaurant foods community-associated methicillin-resistant staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic micrococcus, and other catalase-positive cocci that grow aerobically rapid systems and instruments for the identification of bacteria rapid systems and instruments for antimicrobial susceptibility testing of bacteria evaluation of six agglutination tests for staphylococcus aureus identification depending upon local prevalence of meticillin-resistant s. aureus (mrsa) phoenix versus vitek in the identification of gram-positive and gram-negative bacteria: a comprehensive meta-analysis label-free detection of staphylococcus aureus in skin using real-time potentiometric biosensors based on carbon nanotubes and aptamers a smart dna sensing system for detecting methicillin-resistant staphylococcus aureus using modified nanoparticle probes multiplex real-time pcr assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures molecular laboratory tests for the diagnosis of respiratory tract infection due to staphylococcus aureus clinical impact of a real-time pcr assay for rapid identification of staphylococcal bacteremia mass spectrometry tools for the classification and identification of bacteria mass spectrometry in clinical microbiology and infectious diseases identification of molecularly defined staphylococcus aureus strains using matrix-assisted laser desorption/ionization time of flight mass spectrometry and the biotyper . database development of a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrum (maldi-tof-ms)-based typing method to identify meticillin-resistant staphylococcus aureus clones identification and discrimination of staphylococcus aureus strains using matrix-assisted laser desorption/ionization-time of flight mass spectrometry development of pei-ga modified antibody based sensor for the detection of s. aureus in food samples lytic phage as a specific and selective probe for detection of staphylococcus aureus-a surface plasmon resonance spectroscopic study detection of methicillin-resistant staphylococci by biosensor assay consisting of nanoscale films on optical fiber long-period gratings recent advances in bacteriophage based biosensors for food-borne pathogen detection rapid and sensitive detection of foodborne pathogenic bacteria (staphylococcus aureus) using an electrochemical dna genomic biosensor and its application in fresh beef analytical applications of aptamers rationally designing aptamer sequences with reduced affinity for controlled sensor performance combining use of a panel of ssdna aptamers in the detection of staphylococcus aureus comparison of whole-cell selex methods for theidentification of staphylococcus aureus-specific dna aptamers selection and characterization of dna aptamers against staphylococcus aureus enterotoxin c a new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of staphylococcus aureus aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of staphylococcus aureus capture and detection of staphylococcus aureus with dual labeled aptamers to cell surface components aptamer-assisted novel technologies for detecting bacterial pathogens aptamers: molecular tools for analytical applications solid-contact potentiometric aptasensor based on aptamer functionalized carbon nanotubes for the direct determination of proteins rapid single cell detection of staphylococcus aureus by aptamer-conjugated gold nanoparticles designing materials for biology and medicine functional dna nanotechnology: emerging applications of dnazymes and aptamers physical approaches to biomaterial design new approaches to biomaterials design aptamers in nanostructure-based electrochemical biosensors for cardiac biomarkers and cancer biomarkers: a review principles and applications of photoelectrochemical sensing strategies based on biofunctionalized nanostructures high-throughput tools for the study of protein-nanostructured surface interaction the effect of surface nanometre-scale morphology on protein adsorption an integrated plasmo-photoelectronic nanostructure biosensor detects an infection biomarker accompanying cell death in neutrophils high surface area electrodes generated via electrochemical roughening improve the signaling of electrochemical aptamer-based biosensors label-free fiber optic optrode for the detection of class c β-lactamases expressed by drug resistant bacteria long period fiber grating working in reflection mode as valuable biosensing platform for the detection of drug resistant bacteria energy-based prediction of amino acid-nucleotide base recognition simple, intuitive calculations of free energy of binding for protein-ligand complexes. . models without explicit constrained water bound water at protein-protein interfaces: partners, roles and hydrophobic bubbles as a conserved motif mapping the energetics of water-protein and water-ligand interactions with the "natural" hint forcefield: predictive tools for characterizing the roles of water in biomolecules energetics of the protein-dna-water interaction the intrinsic flexibility of the aptamer targeting the ribosomal protein s is a key factor for the molecular recognition adaptive recognition by nucleic acid aptamers expedient chemical synthesis of mer dna binding domain of mafa: an insight on its binding to insulin enhancer agarose-based microfluidic device for point-of-care concentration and detection of pathogen electrochemical dna biosensor with chitosan-co o nanorod-graphene composite for the sensitive detection of staphylococcus aureus nuc gene sequence the mc-fold and mc-sym pipeline infers rna structure from sequence data crystal structure of λ-cro bound to a consensus operator at . Å resolution algorithms for highly efficient, load-balanced, and scalable molecular simulation building water models: a different approach the missing term in effective pair potentials a combined experimental and computational study on peptide nucleic acid (pna) analogues of tumor suppressive mirna- a ligand migration in nonsymbiotic hemoglobin ahb from arabidopsis thaliana energetics and dynamics of the non-natural fluorescent ap:dap base pair in silico investigation and targeting of amyloid β oligomers of different size vmd: visual molecular dynamics peptide folding: when simulation meets experiment tuning pfkfb bisphosphatase activity through allosteric interference protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases dichroweb, an online server for protein secondary structure analyses from circular dichroism spectroscopic data manipulation of nanoparticles in supersonic beams for the production of nanostructured materials cluster-assembled cubic zirconia films with tunable and stable nanoscale morphology against thermal annealing cluster-assembled nanostructured titanium oxide films with tailored wettability adsorption and stability of streptavidin on cluster-assembled nanostructured tiox films nanoscale roughness affects the activity of enzymes adsorbed on cluster-assembled titania films a synthetic peptide mimic of λ-cro shows sequence-specific binding in vitro and in vivo refined structure of cro repressor protein from bacteriophage λ suggests both flexibility and plasticity edited by synthetic peptides containing a conserved sequence motif of the id protein family modulate vascular smooth muscle cell phenotype optimization of aptamer microarray technology for multiple protein targets based polarity-sensitive dyes synthesis and photophysical properties of isocoumarin-based d-π-a systems key: cord- - jgao w authors: conticello, vincent; hughes, spencer; modlin, charles title: biomaterials made from coiled-coil peptides date: - - journal: fibrous proteins: structures and mechanisms doi: . / - - - - _ sha: doc_id: cord_uid: jgao w the development of biomaterials designed for specific applications is an important objective in personalized medicine. while the breadth and prominence of biomaterials have increased exponentially over the past decades, critical challenges remain to be addressed, particularly in the development of biomaterials that exhibit highly specific functions. these functional properties are often encoded within the molecular structure of the component molecules. proteins, as a consequence of their structural specificity, represent useful substrates for the construction of functional biomaterials through rational design. this chapter provides an in-depth survey of biomaterials constructed from coiled-coils, one of the best-understood protein structural motifs. we discuss the utility of this structurally diverse and functionally tunable class of proteins for the creation of novel biomaterials. this discussion illustrates the progress that has been made in the development of coiled-coil biomaterials by showcasing studies that bridge the gap between the academic science and potential technological impact. phpma poly (n-( -hydroxypropyl))methylacrylamide mtx methotrexate rhcc right-handed coiled-coil epr enhanced permeability and retention nhl non-hodgkin's lymphoma rgd arginine-glycine-aspartate lz leucine zipper hpma n( -hydroxypropyl)methylacrylamide dama n',n'-(dicarboxymethylaminopropyl)methacrylamide mts -( , -dimethylthiazol- -yl)- -( -carboxymethoxyphenyl)- -( -sulphophenyl)- h-tetrazolium comp cartilage oligomeric matrix protein pegda polyethylene glycol diacrylate hsafs hydrogelating self-assembling fibres safs self-assembling fibres auc analytical ultra centrifugation sars severe acute respiratory syndrome smcc the n-hydroxysuccinimide ester of -(n-maleimidomethyl) cyclohexanecarboxylic acid biomaterials derived from designed peptide and protein sequences have become the focus of significant scientific research effort over the last two decades. rapid developments in genome sequencing, bioinformatic analysis, atomic resolution structural determination, and computational protein design, in combination with extant synthetic and analytical methods, present an unprecedented opportunity to create peptide-and protein-based biomaterials of defined structure and controllable function. progress toward the goal of creating tailorable biomaterials for applications in nanotechnology and human health has been impressive, although the practical promise has yet to be realized on a substantial scale. one strategy has involved the identification of stable structural motifs that can serve as flexible scaffolds for the introduction of function. this approach requires the identification of protein folds that can accommodate substantial sequence modification without compromising structure or function. ideally, these protein motifs would be highly designable and amenable to computational structural prediction. the α-helical coiled-coil motif is the most studied protein fold and fulfills many of the criteria that one would envision as a scaffold for biomaterials design. in recent years, the well-known coiled-coil motif has been widely employed in the generation of novel biomaterials. the canonical coiled-coil, comprised of two (or more) right-handed α-helices, is a left-handed super-helix wherein the constituent α-helices have a crossing angle (angle between two helices of a coiled-coil (lupas ) of roughly ° (lupas and gruber ) ). however, minute mutations to the primary sequence can produce profound changes that can cause the coiledcoil to deviate from the classical coiled-coil definition referenced above (zimenkov et al. ; wood et al. ) . such deviations, such as stutters and stammers, often alter the resultant biophysical properties of the coiled-coil (gruber and lupas ) . coiled-coils present multiple advantages as biomaterials scaffolds, including: . well-understood design rules (lupas and gruber ) . accessibility of a variety of oligomeric states (harbury et al. ) . dynamic responsiveness to environmental factors (zimenkov et al. ; dublin and conticello ; banwell et al. ) . versatility in sequence and structure . biocompatibility as discussed in previous chapters, coiled-coils can be designed de novo using a handful of simple rules that can be implemented computationally to select for oligomerization state with a great degree of reliability (thompson et al. ; huang et al. ; wood et al. ) . hydrophobic residues usually occupy a majority of the core a and d positions of the heptad repeats. charge complementarity can be employed at proximal positions to control helix alignment or promote the formation of heteromeric assemblies. sequence control of the residues that occupy the (a, d, e, g) positions, which define the extended helix-helix interface, can be employed to control the oligomerization state in the range from to helices. in contrast, design rules (grigoryan and degrado ) to specify the formation of specific super-secondary or tertiary structures are not as readily apparent for β-strand-based biomaterials, or, at this juncture, more complex structural motifs. by explicit design, coiled-coils have been developed with environmental "switches"; in which a ph drop might cause dissociation of a coiled-coil into its constituent helices, or a metal ion may induce a registry shift (fig. . ), leading to an exclusive population of one oligomeric structure (anzini et al. ) . as evidenced by their ubiquity in the proteomes of sequenced organisms, coiledcoil proteins comprise varied sequences, adopt a variety of native structures, and display a range of functions (testa et al. ). the scope of coiled-coil sequences and structures has been further expanded through the carefully applied use of rational design. non-canonical amino acids can be incorporated at specified sites in the sequences using either solid-phase peptide synthesis methods or supplementation of growth media used in in vitro or in vivo protein expression. the introduction of nonnative chemical functionality can further expand the material properties of this flexible scaffold. indeed, coiled-coils are a versatile and robust scaffold for generating a diverse range of functional biomaterials that can be tailored for specific applications through modification of the sequence at structurally permissive sites. a major problem facing modern medicine is the dearth of strategies for treating diseases that present differently in each patient, e.g. cancer. the need for easily customizable treatments has led to the development of a broad field known as nanotheranostics (a portmanteau derived from therapeutics and diagnostics), which seeks to use nano-scale materials to image and/or treat diseases. a dominant strategy in nano-theranostics is to specifically target diseased cells for the delivery of small doses of extremely cytotoxic low molecular weight drugs. a clear challenge in this field is thus developing site-specific drug delivery to reduce side effects from design of a metal-ion induced registry shift into a trimeric coiled-coil structure. briefly, cd + ions coordinate with cysteine sidechains in the coiled-coil's hydrophobic core, locking them into a trimeric state, rather than the off-register fibrillar structure seen in non-metallated samples. (a) single-letter amino acid sequence of the tz c peptide, with the a and d positions labelled to highlight the hydrophobic core of the coiled-coil structure. (b) helical-wheel representations of the out-of-register (left), metal-free peptide, and the in-register (right), metal-coordinated peptide. (c) fibre representation of the metal-ion oligomerization switch (reprinted with permission from anzini et al. ( ) copyright apoptosis of healthy cells. coiled-coil motifs have recently been explored as potential nano-theranostic agents, due to their lack of immunogenicity, exquisite sensitivity to biologically relevant environmental changes (such as ph), and well-known engineering rules. this inherent designability makes coiled-coils an attractive scaffold from which to develop materials for nano-scale treatments. coiled-coil-based therapeutics can be broken up into two main categories: coiled-coil drug carriers, and drug-free coiled-coil systems. the first of these categories is more familiar to those studying nano-medicine. essentially, a low molecular weight drug is encapsulated by a self-assembling coiled-coil, which dissociates upon some environmental change (typically ph, which can vary in a diseased state) (yu ) . the low molecular weight drug is generally highly cytotoxic and, as such, the design focus for this approach is towards target specificity (pola et al. ) . these systems also offer the potential for multi-valent drug binding, which improves the loading efficiency by minimizing the entropic penalty for binding. assuming one chain in a heterodimeric coiled-coil system contains a single binding site, its sequence may be doubled to introduce a second binding site (assal et al. ) . recently, klok and co-workers investigated a heterodimeric coiled-coil carrier comprising two peptides (k and e ), which self-assemble at ph . , but dissociate at ph . (endosomal ph), thereby releasing their cargo (apostolovic et al. (apostolovic et al. , . these peptides were bound to a poly (n-( -hydroxypropyl)methacrylamide (phpma) polymer backbone and designed to carry an anti-cancer drug, methotrexate (mtx) in one case, and a fluorescent dye in another. by combining these two technologies, it follows that one could monitor cellular uptake of the drug carrier while simultaneously treating the patient. though uptake of the drug into the endosomes was shown, the distribution of the drug throughout the organelles has not yet been obtained. in a more direct therapeutic solution, thanasupawat et al., recently reported a coiled-coil-based system with improved selectivity that targets gliomas (malignant tumours that bud from glial cells in either the brain or the spine) (thanasupawat et al. ) . briefly, they developed a right-handed coiled-coil (rhcc) comprising four identical alpha helices. each alpha helix presents a methionine residue on the solvent-exposed surface of the tube, through which it coordinates a platinum (iv) ion derived from tetrachloroplatinate ( fig. . ). the complexed pt(iv) displayed a stronger selectivity for the target tumour and less cytotoxicity than the more labile cis-platin derivative of the rhcc. the rhcc essentially serves as a carrier for four equivalents of the pt(iv) complex, which acts as a pro-drug that is activated via conversion to the therapeutic pt(ii) state in the reductive environment of the tumour cell. this phenomenon affords higher efficacy and fewer side effects, resulting in minimization of treatment cost and duration. the second approach involves grafting of coiled-coil peptides to a synthetic polymer backbone; upon self-assembly, the polymer-peptide complex induces a physiological change in the cell that triggers apoptosis (pechar et al. ) . although this approach has only recently emerged, it presents a strong advantage over drugbased therapies; namely, none of the components acting alone can induce apoptosis. assuming specific bio-recognition of the constituent peptides, apoptosis will only occur at the diseased cell. this represents a new paradigm in nanomedicine, and was pioneered by kopeček and co-workers (wu et al. ; chu and kopeček ) . these researchers developed the idea of attaching high molecular weight polymers to small coiled-coil-forming peptides. the biocompatible polymer is linked to one peptide to increase endocytosis by cancer cells, via the enhanced permeability and retention (epr) effect. this effect states that due to the increased vasculature and poor draining of tumour cells, high molecular weight polymers are more likely to be endocytosed than smaller species, and are also less likely to be drained through the lymphocytes. a second peptide, complementary to the first, is conjugated to a receptor recognition motif, which directs the peptide to a cell type of interest. the kopeček group explored the use of these materials for the treatment of non-hodgkin's lymphoma (nhl). in nhl, a specific surface receptor (cd ) is present on > % of tumourous cells. cd is also present on healthy b cells, but not on stem cells. it has been shown that the temporary b cell depletion is not deleterious. the recognition motif present on a specific antibody (fab') is used as the targeting moiety in this model system. fab' and its peptide partner bind to the cd receptors, and the peptide-polymer conjugates are effectively drawn towards the b cells. at this point, the peptides interact to form a heterodimeric coiled-coil, which induces cross-linking of the cd receptors. the cross-linking of cd receptors results in apoptosis, and effective remediation of the disease (fig. . ). hydrogels are formed from covalent or non-covalent cross-linking of individual polymer chains into a three-dimensional water-swollen network. hydrogel materials offer a wealth of potential applications (elisseeff ) , in that the biological, chemical, and mechanical properties may be tailored for particular applications through modification of the structure of the polymer and cross-linker. they have innate capacity to absorb and retain large volumes of water relative to the mass of the hydrogel itself (elisseeff ; xu and kopeček ; yao et al. ) . moreover, hydrogels are often highly biocompatible due to their capacity for water absorption, physical elasticity, and relative softness (kopeček , yao et al. . the aforementioned qualities make hydrogels a convincing substitute for native extracellular matrix (slaughter et al. ; yao et al. ) , as well as suitable candidates for cell culture applications (frampton et al. ; jung et al. ) , biomedical uses such as wound treatment (wong et al. ; lin et al. ) , and drug delivery (garbern et al. ; guziewicz et al. ) . unsurprisingly, hydrogels were the first biomaterials developed for medical uses in humans (kopeček fig. . schematic representation of the drug-free coiled-coil-based therapeutics employed by the kopeček group. the unfolded cce peptide (blue coil) is chemically conjugated to the fab' recognition domain (red), which directs it to the cd antigen (yellow). unfolded cck peptide (green coil) is directed to the cell surface via the epr effect and conjugation to the hpma copolymer backbone (black coil). coiled-coil formation by the two peptides results in cd antigen crosslinking and subsequent apoptosis (reprinted with permission from wu et al. ( ) copyright ; kopeček and yang ). as biomedical materials, coiled-coil-based hydrogels offer an alternative to the more commonly employed β-sheet hydrogels. one potential concern with the latter materials is that the β-sheet morphology is analogous to amyloid structures, such as those observed in prion and amyloid diseases. these toxic assemblies often develop via a misfolding of endogenous proteins and can be auto-catalytically propagated in the presence of a β-sheet template (fletcher et al. ). due to the risk of initiating protein misfolding disease states, implementation of β-sheet-based hydrogels must be carefully scrutinized for the manifestation of deleterious physiological effects (fletcher et al. ) . biomaterials based on coiled-coil structures do not display similar behaviour, as self-assembly in most cases is restricted to the formation of closed oligomers. this section will cover the past decade of advances within coiled-coiled-based hydrogels. hydrogels based on coiled-coil self-association can offer several notable advantages (kopeček ). the coiled-coil is an exceptionally well understood structural motif, when compared to other protein folding domains (vide supra) (lupas and gruber ) . due to the wealth of information regarding the coiled-coil's structure and properties (in comparison to other super-secondary and tertiary structures), the coiled-coil has the potential to serve as an ideal 'tuning knob' for hydrogel properties. though the coiled-coil is certainly not the only factor that governs hydrogel properties, it represents the most facile mechanistic handle. tuning coiledcoil properties can involve a procedure as simple as altering the sequence of amino acids within the constituent alpha helices, allowing the researcher to alter hydrogel properties with relative ease and efficiency. coiled-coils offer versatility in terms of oligomeric state as well (harbury et al. ). moreover, due to the widespread prevalence of coiled-coils within native proteins, coiled-coil-based biorecognition domains are also pervasive throughout the natural world. co-opting such domains may yield significant advantages in the engineering and design of hydrogels, as doing so may offer a conduit through which artificially engineered hydrogels may interact with natural systems with high specificity (kopeček ) . for the purpose of this review two primary categories for self-assembling hydrogels exist: those that consist only of peptide/protein and those that are based on hybrid materials (kopeček ). the latter incorporate non-peptidic moieties within the component molecules of the hydrogel. the most common protein hydrogels derived from coiled-coils are based on an aba triblock copolymer architecture. petka et al. developed the first aba triblock polymer system for the fabrication of hydrogels (petka et al. ). subsequently, xu and kopeček further developed these materials. these aba triblock copolymers comprise a sequence in which coiled-coil motifs occupy the endblock domains (a) while a hydrophilic random coil sequence (b) defines the midblock (fig. . ). self-association of alpha helical (a)-end-blocks initiates cross-linking of the polymers into a water-swollen network in which the hydrophilic (b) mid-block connects the coiled-coil domains (kopeček ) . the self-assembly of these aba block copolymers is driven by two interconnected forces: hydrophobic collapse of a ends and polar association of water with the b interior region. one advantage of hydrogel formation from these aba block copolymers is their reversibility, wherein virtual cross-links resultant from the coiled-coil interactions can be reversibly denatured, yielding a gel to sol transition. however, the a blocks retain the capacity to re-form the coiled-coil structure upon removal of the denaturing conditions. moreover, the interaction between helices is highly tunable; minute changes in sequence, especially to the alpha helical regions can produce discernible effects on the biophysical properties of the resultant morphologies (kopeček ). xu and kopeček noted that the physical characteristics of their aba triblock and ab diblock hydrogels depended on the relative lengths of the a block and b block, the sequence composition, and the introduction of specific electrostatic interactions. tirrell and co-workers programmed further specificity into these systems through diversification of the polypeptide sequence to create an abc triblock system. in this architecture, the a and c domains comprise different coiled-coil sequences that exhibit little tendency to associate heteromerically, that is, the a and c blocks are structurally orthogonal (shen et al. ) . the presence of these orthogonal coiled-coil sequences inhibits the formation of intramolecular loops, which were prevalent within hydrogels derived from the aba triblock systems, and, consequently, lowers the rate of erosion of free polymer from the surface of the resultant hydrogel. an additional advantage of the coiled-coil motif lies in its ability to accommodate mutations at specific positions while retaining its structural integrity (harbury et al. (harbury et al. , . as a consequence, sequences that encode biological functions can be introduced into the scaffold of the coiled-coil structural units. this structural robustness enabled huang et al. to implant the rgd domain between two hexaheptad sequences (a ) derived from a previously published leucine zipper (lz) coiled-coil peptide. the rgd domain was separated from each a sequence by a nine amino acid spacer (c ). a c-terminal tail (c ), containing cysteine, was also added, completing an rgd-containing system constructed as follows: a -c - rgds-c -a -c . the insertion of rgds imbued cell adhesion to the hydrogel (huang et al. ) . specifically, huang et al. found that rgd sequences introduced into lz hydrogels promoted the attachment (as determined via mts cell proliferation assay kit, a colorimetric analysis which measures the reduction of -( , -dimethylthiazol- -yl)- -( -carboxymethoxyphenyl)- -( -sulphophenyl)- h-tetrazolium, or mts) and proliferation of metabolically active cells at quantitatively higher levels than the control system: a -c -c -a -c , which lacked the rgd domain. these results were corroborated in vivo using human marrow stem cells in a murine host system, which demonstrated cell attachment, growth and even neovascularity, thus proving that a mutable, tunable, coiled-coil-based hydrogel has potential for tissue engineering applications (huang et al. ). this "customization" of a hydrogel is possible primarily due to the structural stability encoded within the coiled-coil lz domain, and its ability to incorporate mutations near it without loss of its structure/function relationship. hybrid hydrogels are derived from self-assembly of synthetic polymers to which coiled-coil sequences have been covalently attached, usually at the termini, to afford aba-type protein-polymer conjugates (kopeček ). these hybrid materials combine the specificity of coiled-coil interactions with the well-known mechanical properties and biocompatibility of synthetic biomaterials. kopecek and yang ( ) linked to create a copolymer that served as the main polymer component of the hydrogel. the hpma polymer, a hydrophilic molecule, promotes the water absorption and swelling of the hydrogel. dama, a metal-chelating agent, provided a mechanism to couple the polymer chain to the peptide moiety of the hydrogel via ni + chelation between dama and a his-tag domain within the peptide. the hpma-dama copolymer was conjugated to two differing coiled-coil-forming peptide sequences with differing lengths and amino acid compositions: cc and cc . cc was derived from a portion of the stalk domain of kinesin, adopting a tetrameric coiled-coil structure. in contrast, cc was designed by the kopeček group as a tetrameric coiled-coil, but comprised a shorter sequence than cc . importantly, it was observed that cc exhibited greater thermostability than cc . these investigators hypothesized that differences in amino acid sequence between the two coiled-coil motifs (cc and cc ) would produce measurable effects in subsequent downstream biophysical properties. the difference in sequence between cc and cc influences the thermodynamic stability of the resulting coiled-coils, which, in turn, would affect the properties of the resultant hydrogel. for example, the t m of a coiled-coil could be raised or lowered through sequence control, which enabled the formation of hydrogels of varying thermostability. when subjected to a temperature increase from ° to °c, the cc -based hydrogel volume decreased tenfold (wang et al. ) . the mid-point temperature of the gel-sol transition coincided with the t m of the constituent coiledcoil that was determined from its cd melting transition. conversely, cc did not exhibit a hydrogel volume transition within the same temperature range (wang et al. ) . kopeček asserted that this data indicated that "properties of a well-defined coiled-coil protein motif can be imposed onto a hybrid hydrogel" (kopeček ) . in the pursuit of enhanced stability and a dynamic mechanical response, yao et al. created a hybrid hydrogel that incorporated reversible physical cross-links and photo-polymerizable groups (yao et al. ) . a reactive cysteine residue was introduced into the sequence of a coiled-coil derived from the five-helix bundle of cartilage oligomeric matrix protein (comp). the comp peptide was conjugated to a macromer of polyethylene glycol diacrylate (pegda) through michael addition to one of the terminal acrylate groups. the resultant pentameric assembly contained peg chains that were terminally functionalized with the remaining acrylate group. photo-induced polymerization of the terminal acrylates afforded a network in which the pentameric coiled-coil acted as a physical cross-link between polymer chains. this process has the advantage of retaining the dynamic physical cross-links associated with the coiled-coil motif, while permitting rapid in situ fabrication of the hydrogel using photo-induced polymerization (yao et al. ) . fibroblast cells could be encapsulated within a d hydrogel matrix, which was generated in situ via photo-polymerization of an rdg-modified peptide-macromer. the reversibility of the coiled-coil self-association enabled self-healing behaviour within the hydrogel, which promoted cell spreading and migration in d culture (yao et al. ). these hydrogel systems demonstrate a key advantage in the use of coiled-coils as structural units within self-assembling biomaterials, namely the potential for customization of the selectivity and stability of the interaction. in the cases described above, the coiled-coil was employed primarily as a biorecognition motif in order to create the virtual cross-links that defined the hydrogel. the hydrophilic central block provided the flexibility to accommodate the swelling solvent. however, woolfson and co-workers demonstrated hydrogels could be created solely from α-helical peptide sequences, which they termed hydrogelating selfassembling fibres (hsafs) (banwell et al. ). the hsaf system is based on previous work performed by the woolfson group, wherein they engineered selfassembling fibres (safs) via two α-helices co-assembling such that the resultant structure was a dimer of the two helices. importantly, this dimer featured sticky ends, and resultant end-to-end assembly produced coiled-coil fibrils, which in turn aggregated to form large fibres. it was observed that these fibres demonstrated a high level of order, leading to their precipitation from solution (banwell et al. ). using the saf as a starting scaffold, the researchers inserted alanine residues (to encourage hydrophobic interactions between fibrils) and glutamine residues (to promote hydrogen bonding). these mutations were performed only at the b, c, and f positions of the heptad repeat, as these lie on the exposed surface of the helix, while other residue positions are more critical in driving dimerization of the two constituent helices to form a coiled-coil. differing levels of alanine and glutamine were introduced at the aforementioned positions to form hydrogels driven largely by hydrophobic interactions between fibrils or driven by hydrogen bonding. it was observed that the hydrophobically-driven hydrogels (those featuring proportionally large amounts of alanine at the variable positions) showed an increase in gel strength when heated. conversely, the hydrogel variants driven primarily by hydrogen bonding (those featuring proportionally large levels of glutamine at the variable positions) melted and lost their gel strength upon heating. a distinct advantage of this system is that each constituent helix has its own sequence identity, and hydrogelation only occurs upon mixing. lastly, woolfson and co-workers showed that these hsaf systems are compatible with, and support both cell growth and cell differentiation. a recurring theme regarding coiled-coil biomaterials is the flexibility of this protein fold as a structural platform. mehrban et al. demonstrated that these fibrillar coiled-coils could be decorated with the rgds cell-adhesive sequence. the resultant hydrogels could promote the growth, directional migration, and differentiation of murine embryonic neural stem cells in culture (banwell et al. ; mehrban et al. ) . an important factor to consider in the development of biomaterials for in vivo applications is immunogenic response. for tissue engineering applications, minimizing the immune response to the coiled-coil construct facilitates integration into the body with minimal risk of rejection. in contrast, immunotherapies rely on a strong immune response to the coiled-coil material (boato et al. ). it follows that it is of great interest to not only measure the immunogenic potential of coiled-coil assemblies under physiologically relevant conditions for in vivo applications, but also to design systems in which immunological response can be controlled. for a biomaterial to provoke an immune response, it must interact with the immune system either by being recognized as a foreign body or by breaking down the native b-cell tolerance (if it is sufficiently homologous to a protein in the host organism). in both cases, aggregated (maas et al. ) or oligomerized (rosenberg ) proteins have been shown to be more immunogenic than their monomeric counterparts. biomaterials that do not provoke an immune response are invisible to the host cell and function freely within the cell. because coiled-coil systems can access a variety of oligomeric states, they are ideal for tuning an immune response. recently the collier group explored the effect of pegylation on the immunogenicity of coiled-coil assemblies. pegylation generally reduces immune response, while aggregation increases the immune response (rudra et al. a ). the interesting question then, is how strong will the immune response be when a pegylated peptide aggregates? to address this question, they investigated the murine immune response to three peptides: a domain from mouse fibrinogen (which disfavours coiled-coil formation due to poor charge pairings and buried polar residues), a mutated version of the same with a coiled-coil optimized sequence (γkei), and a triblock peptide-polymer conjugate (γkei-peg-γkei) (rudra et al. b) . they found that the native peptide and its coiled-coil derivative elicited no immune response, while the triblock peptide copolymer caused a mild immune response. they attributed this change in immune response to the higher degree of oligomerization observed in the triblock copolymer, as determined from analysis using analytical ultracentrifugation (auc). these results suggested that the immunogenicity of the peptide was not based on the folding, as the native random coil peptide and the coiled-coil forming mutant elicited similarly low immune responses. in a particularly interesting study, the burkhard group developed a coiled-coil nanoparticle with antiviral properties against severe acute respiratory syndrome (sars) (pimentel et al. ). sars is a pneumonia-like disease that caused widespread concern in , when it rapidly spread among different countries and resulted in the deaths of people (marra et al. ; nicholls et al. ) . the epicentre of this outbreak was in southern china, where several domesticated animal species were found to be carrying the disease in live animal markets (guan et al. ) . the search for a sars vaccine resulted in the identification of the sars corona virus s protein. attempts to use the full-length s protein to vaccinate different model animals resulted in increased virulence of hepatitis in ferrets (czub et al. ) and infection with sars in cats (corapi et al. ). thus, the design of an s protein b cell epitope became an important research target. the key to designing an effective epitope is mimicking the native protein structure; because of this consideration, the coiled-coil forming region of the s protein was used as the vaccine nanoparticle scaffold (raman et al. ) . they were able to develop a monomer that would self-assemble into a viral capsid-mimicking icosahedral nanoparticle (fig. . ) . the coiled-coil assembly had the advantages of high stability, ease of expression, and lack of infectivity seen in the full s protein. in addition, the coiled-coil assembly was found to be highly immunogenic due to its size, mimicry of the native epitope, and repetitive epitope display, making it a reasonable first step to developing a sars vaccine. one-dimensional assemblies derived from proteins and peptides have been the focus of significant scientific research over the last two decades (scanlon and aggeli ) . a significant motivation for this research stems from the presence of extended helical assemblies in native biological systems, in which they display a variety of functional roles that would be desirable to replicate within synthetic biomaterials. the knobs-into-holes packing typically associated with coiled-coils has been observed to play a significant role in stabilizing the structures of helical assemblies such as intermediate filaments (chernyatina et al. ) , type iv pili (craig et al. ) , bacterial flagella (yonekura et al. ) , the type iii secretion system needle complex (loquet et al. ) , and filamentous phage capsids (morag et al. ) . significant research effort has been directed toward the de novo design of helical assemblies based on coiled-coil structural motifs, which could be employed for the creation of synthetic scaffolds for biomedical applications (see above, for example). the primary approach of this research focused on design of sticky-ended coiledcoils that could oligomerize to form infinite one-dimensional polymers through non-covalent interactions. the staggered alignment of helices was promoted through introduction of electrostatic interaction between helical protomers that enforced an out-of-register alignment across the helical interface. however, most designs focused on coiled-coils of lower oligomerization state (usually dimers and trimers), primarily due to the difficulty in controlling alignment between adjacent helices for coiled-coils in larger helical bundles. in addition, at that juncture, limited structural information was available on coiled-coils of higher oligomerization state. one attractive feature of the latter systems is that coiled-coils of oligomerization state ≥ helices display an interior channel in which the diameter depends on the bundle size. self-assembly of coiled-coils of higher oligomerization state affords the potential to create nanotube structures in which the channel residues can be tailored for encapsulation of small molecules and the outer surface can be functionalized to enhance applications such as selective targeting for controlled release. numerous strategies have been described to drive the formation of tubular assemblies from peptides and proteins, though few of these approaches are applicable to coiled-coil systems (burgess et al. ) . the most promising approach to nanotube fabrication involves the end-to-end stacking of coiled-coils of defined oligomerization state (n ≥ ) into extended structures through selective introduction of attractive non-covalent interactions at the interfaces between subunits. as coiled-coils are perhaps the best understood protein fold, with a wealth of available structural information, they represent a promising test bed to develop methods to reliably generate structurally-defined supramolecular assemblies. xu et al. ( ) described an approach to promote the end-to-end self-assembly of a seven-helix bundle into a helical nanotube. these researchers employed a structurally characterized -helix bundle, gcn -paa, as the starting point for the design. in this case, the -helix bundle has helical symmetry, in that successive peptides in the assembly are axially translated by an increment of one residue ( . Å rise per residue for the superhelix) between successive helices in the bundle. this translation results in a registry shift corresponding to a heptad repeat between the first and last helix in the bundle. the corresponding edges at the n-and c-termini are solvent-exposed and, upon interaction, could bury sufficient hydrophobic surface area to promote stacking of the -helix bundles. xu et al. ( ) used the analogy of a helical lock washer to describe the structure of the coiled-coil subunits. the sequence of gcn -paa was redesigned to afford a fibrillogenic peptide sequence, hsap . this sequence preserved the residues that were critical for formation of the -helix bundle, but introduced features that would promote stacking of the subunits . hydrophobically-driven association of complementary helix faces yielded a non-covalent assembly of helices with an inner lumen. in addition, the n-terminus and c-terminus of hsap were populated with positively charged residues and negatively charged residues, respectively. this design promoted charge complementary association of helical heptamers through head-to-tail stacking of the heptameric subunits. the resultant nanotubes defined an internal cavity of approximately Å in diameter, in analogy with the crystal structure of gcn -paa, which was capable of binding shape-appropriate small molecules such as the solvatochromic fluorophore prodan ). hume et al. constructed a fibrillar assembly using the pentameric coiled-coil region of cartilage oligomeric matrix protein (compcc) as the basis for design. a variant of compcc was constructed in which residues that were non-essential for pentameric assembly were deleted (hume et al. ). an n-terminal pocket, inherent to the wild type matrix protein and consisting of three leucines and a valine, was shifted with respect to the overall sequence, but preserved in identity and conserved with respect to its occurrence within the heptad repeat sequence. the presence of the n-terminal pocket was necessary to retain the preference for the pentameric assembly (over alternative oligomeric states or lack of assembly). moreover, in the re-designed peptide, q, charge density was introduced at positions on the outer (lateral) surface of the assembly in order to promote complementary interactions between oppositely charged regions on adjacent pentameric assemblies. fibre formation would be promoted through head-to-tail stacking of helical bundles, which would be reinforced through lateral association between fibrils mediated through electrostatic complementarity. peptide q was found to form nano-fibres having diameters as large as half a micron (hume et al. ) . the pentameric assembly also defined a central pore of sufficient size to accommodate small molecules, such as the therapeutic agent curcumin. curcumin was demonstrated not only to bind to the fibrillar assemblies, but also to promote further lateral association through a mechanism that has yet to be fully elucidated. using this approach, hume et al. succeeded in engineering the first documented experimentally-assembled proteinbased microfibre, observing diameters of up to sixteen microns via aggregation of nano-scale moieties, while preserving a central cavity for encapsulation (hume et al. ). burgess et al. recently described a more general approach to the design of fibrils, including nanotubes, derived from coiled-coil sequences having different oligomerization states (burgess et al. ) . helical bundles of defined oligomerization state ranging from three to seven were designed, in which residues at the n-and ctermini were chosen to be electrostatically complementary in charge (as described above). this design element was introduced to promote end-to-end association of coiled-coil bundles, affording one-dimensional assemblies from blunt-ended stacking of helical bundles. the resultant sequences (termed cc-tri through cc-hept), self-assembled into fibrils based on subunits having the corresponding oligomerization state, albeit with differing degrees of lateral association and order (fig. . ). one system, cc-hex-t, based on a hexameric helical bundle, exhibited a high degree of para-crystalline order after thermal annealing in pbs buffer. burgess et al. further demonstrated that the hydrophobic dye , -diphenylhexatriene was able to penetrate and bind within the inner lumen of tubular assemblies derived from cc-hex-t, but not to the fibres derived from the tetrameric bundle cc-tet -f (burgess et al. ) . subsequent experiments established that dye binding only occurred for helical bundles having an oligomerization state from - , as these larger coiled-coil subunits define an inner lumen of sufficient size to accommodate the guest molecule. the encapsulation of , -diphenylhexatriene validates the potential of these tubular materials for applications involving drug or small molecule binding and release. while larger coiled-coil bundles (> subunits) have been observed in native protein assemblies, the de novo design of larger diameter coiled-coils remains a challenge, which has thus far limited the fabrication of nanotubes with larger lumens. walshaw and woolfson (walshaw and woolfson ) described general principles for design of these types of assemblies based on an analysis of the -helix bundle observed in the structure of the tolc homotrimer (koronakis et al. ) . however, such large diameter tubular structures based on coiled-coils have yet to be realized. egelman et al. ( ) investigated the self-assembly of a pair of de novo designed coiled-coils based on the structural principles proposed by walshaw and woolfson. the sequences of these two peptides were based on a canonical heptad repeat motif in which hydrophobic residues populate the a/d and c/f positions, producing hydrophobic seams along the helix offset by two residue spacing (koronakis et al. ) . in contrast to the α-helical cylinder of the tolc sequence (calladine et al. ) hydrophobic residues at the outer a and f positions were substituted with alanines, which feature smaller sidechains than the residues of the native tolc. this substitution served to decrease steric interference between the flanking residues of the two hydrophobic pockets to encourage larger tube diameter (egelman et al. ) . large sidechains at the outer positions would create steric interactions between neigh-bouring residues, and in the relaxation and mitigation of this steric stress, force the tube to constrict towards a smaller diameter. this was negated in the synthetic peptide via mutations to alanine at the aforementioned heptad positions a and f to encourage large diameter growth. moreover, complementary pairs of charged residues populate the b and e positions in the synthetic peptide to promote antitypic helix-helix association (egelman et al. ) . the sole difference between the two sequences was the substitution of four arginine residues in the first peptide (form i) with four lysine residues in the second peptide (form ii). both peptides formed tom] transmission electron micrographs of tube assemblies (a) graphical representation of coiledcoil oligomers ranging from two to seven, indicating the overall structure of the coiled-coil subunit as seen side-on. (b) x-ray crystal-derived graphical representations of coiled-coil oligomers ranging from two to seven indicating the overall structure of the coiled-coil subunit as seen when viewed along the fibre axis. in the pentamer, hexamer, and heptamer assemblies, a putative inner lumen is observed. (c) putative method of assembly of coiled-coil subunits, wherein end-to-end association drives fibre formation. (d) transmission electron micrographs of negative stained cc-tet. (e) transmission electron micrographs of cc-pent (reprinted with permission from burgess et al. ( ) copyright extended tubular assemblies in aqueous buffer, but differed significantly in diameter ( nm for the form i assemblies and nm for the form ii assemblies). electron cryomicroscopy with direct electron detection was employed for the structural analysis of the form i and form ii assemblies. egelman et al. ( ) funnelled data through the iterative helical real space reconstruction algorithm (egelman ) to produce near atomic resolution structural data for forms i and ii. form i features a unilaminar helical motif, wherein helices are packed nearly perpendicular to the nanotube axis in which the cross-section displays near four-fold symmetry in the left-handed one-start helix. the form ii assemblies feature a bilaminar helical motif, wherein three bilayer stacks of helices are packed together and define a nearly circular cross-section in which the helices are slightly inclined with respect to the cross-sectional plane. in either structure, the nanotube lumen and outer surface are lined with functional groups from polar amino acid residues. one might envision that the inner and outer surfaces of the tubes could be selectively functionalized to promote applications in controlled encapsulation and release. notably, the structures of the form i and ii assemblies diverged significantly from the structural model proposed by walshaw and woolfson, as well as that of the tolc α-cylinder (koronakis et al. ; calladine et al. ; egelman et al. ) . more remarkably, the structurally-conservative substitution of arginine for lysine resulted in significant differences in structure between the two helical assemblies. these differences highlighted a potential problem in the design of such assemblies, namely, that quaternary structure may not be robust in sequence space. indeed, egelman et al. demonstrated that a single mutation (r k) was sufficient for conversion of the form i assembly to the form ii assembly (fig. . ) . conversely, a coupled pair of mutations (k r, k r) provides for conversion of form ii assembly to the form i assembly (egelman et al. ) . these structural differences are manifested as a consequence of different local interactions between subunits that are propagated hierarchically to form the two distinct assemblies. the form i assembly derives its stability from the interaction of the sidechains of arg and arg with the c-terminal residues of a helix in an adjacent stack. these interactions form the corners of the fourfold symmetric cross-section of the tubular assembly. in contrast, the helical stacks of form ii assembly, lacking these structural critical arginine residues, are held together weakly through interactions between the termini of helices in adjacent stacks. the structural changes that occur as a consequence of mutagenesis can be understood in terms of abrogation or introduction of these arginine-derived interactions (egelman et al. ) . the difference between the form i and form ii assemblies dramatically illustrates that variations in primary sequence can result in large changes in quaternary structures. atomic resolution structural information enables the identification of these critical interactions that underlie these differences, but the development of coiled-coils with high oligomerization state as biomaterials will require better methods to reliably design higher order structure. in addition to tubular assemblies, coiled-coils have been employed in the fabrication of capsular and film-like biomaterials, which have advantages as carriers for encapsulation and controlled release of drugs and other small molecules. utilizing a layer-by-layer (lbl) approach, gormley et al. have been able to construct capsules and films by subsequent addition of layers in a sequential manner (gormley et al. ) . traditionally, deposition of sequential layers was achieved via selfassembly that was mediated through electrostatic complementarity between layers (hammond ; ai et al. ) . since this discovery, alternative means for lbl deposition have been accomplished in an effort to imbue enhanced levels of customization and specificity with respect to biophysical properties of the assembly. the lbl toolbox contains other methodologies such as hydrogen bonding, covalent bonding, dna-based oligomerization, and streptavidin-biotin complementarity (mart et al. ) . stevens and co-workers have added a new methodology to the toolkit by using the specificity inherent in the self-assembly of coiled-coil moieties (gormley et al. ) . using helix-loop-helix motifs (jr ec and jr kc) that oligomerize as a coiled-coil tetramer, these researchers created a polymer-peptide hybrid by conjugation of cysteine to n-( -hydroxypropyl)methacrylamide-co-n-( aminopropyl)-methacrylamide [hpma-co-apma] polymer through a -(n-maleimidomethyl) cyclohexanecarboxylic acid n-hydroxysuccinimide ester [smcc] linker (gormley et al. ) . the linkage moiety was attached to c at the loop region of the helix-loop-helix. peptides jr ec and jr kc differ in sequence by a total of eight glutamates/lysines, which imbued a difference in net charge to each variant. this duality in charge promotes selective heteromerization of the two helix-loop-helix moieties, jr ec and jr kc, to form a tetrameric coiled-coil. as the peptide helices are conjugated to a polymer scaffold, coiled-coil formation results in sandwich-like constructions of iterative layers by sequential addition of polymer-peptide units with opposite charges. anchoring techniques could be implemented to grow these lbl assemblies on both planar and spherical, colloidal substrates, giving a degree of geometric variation to what can be accomplished via this approach (fig. . ) . lastly, as with many coiled-coil-based systems, a ph-induced conformational switch can be actuated. at ph , the dimer subunits stack against one another in the film. at ph , positive charges dominate the surface and the dimers repel one another, causing dissociation of the stacked units (gormley et al. ) . this review has largely focused largely on developments related to the potential of coiled-coils for applications in biomedicine, and, specifically, their interactions with other biological materials in vitro or in vivo. recently, however, significant research has been focused on integrating inorganic materials with coiled-coil biomaterials. in particular, the use of coiled-coils has been proposed for applications involving the templating of inorganic materials. for example, numerous studies have used genetically engineered viral capsids for directing the deposition of inorganic materials, leading to the formation of highly ordered inorganic structures (lee et al. ; huang et al. ). these strategies have produced very efficient electron-capture devices (dang et al. ) , as well as highly functioning lithium batteries (lee et al. ). while using a phage capsid is an elegant solution to the selection problem, a coiled-coil scaffold would be preferable due to its ease of production, robust structure, and well-known design rules. several examples have been recently published in which coiled-coils, or assemblies derived from their self-association, have been employed to direct nanoparticle synthesis and assembly (ryadnov ; slocik et al. ; wagner et al. ; hume et al. ; abram et al. ). these materials might ultimately find use as imaging agents or therapeutics in medical applications, especially as they could leverage the favourable electronic, optical, or magnetic properties of the inorganic nanoparticle and the structural specificity of the coiled-coil interaction (seo et al. ) . this objective was realized in a particularly innovative biomaterial design based on a coiled-coil composite. martelli et al. developed a hybrid system that consisted of a coiled-coil heterodimer, fluorescein cargo, and mesoporous silica nanoparticles (fig. . ) (martelli et al. ) . briefly, mesoporous silica nanoparticles encapsulating large quantities of fluorescein were fabricated and conjugated to an acidic fig. . a schematic representation of the mesoporous silica nanoparticle (msn) system. the msn construct is depicted in blue, with the small pores indicated by light blue. spacers (green squares) of varying length are conjugated to the "e" peptide (blue ribbon), allowing it the flexibility needed to cover the pores. addition of peptide "k" (red ribbon) leads to the sealing of the pore upon heterodimerization. temperature increase leads to the unwinding of the coiled-coil, resulting in cargo (yellow spheres) delivery (reprinted from martelli et al. ) coiled-coil peptide e. these complexes were then incubated overnight with the complementary basic coiled-coil peptide k, which was free in solution. the e and k peptides underwent selective heterodimerization, which resulted in de facto closure of the silica pores. the coiled-coil dimer could essentially perform as a valve, which could be actuated through denaturation of the dimer. for example, thermolysis of these complexes induced denaturation of the coiled-coil dimer. dissociation of the k peptide actuated the valves and resulted in release of the cargo. fluorescein was selected as a model cargo due to its fluorescent properties and ease of imaging. these materials have considerable potential for in vivo applications, especially if this approach proves to be more general. for example, ferromagnetic iron oxide nanoparticles would enable targeting to a specific location, e.g., a tumour, using an externally applied magnetic field. once in the correct location, the cargo, e.g., a cytotoxic drug molecule could be released via localized heating in which the temperature leads to thermal denaturation of the heterodimeric coiled-coil. recent technological advances, such as structurally predictive computational algorithms and higher resolution characterization techniques, are expanding our ability to design and structurally characterize novel coiled-coil assemblies. the availability of this information has facilitated the development of novel biomaterials scaffolds based on coiled-coils. the natural abundance and structural diversity of coiledcoils, as well as their well-understood design rules, make them an obvious choice for biomaterials scaffolding. in addition, the versatility of the coiled-coil motif allows for the incorporation of artificial amino acids, which enhances the ability to do site-specific chemistry and generate more complex materials. as a result, coiledcoils have been used to develop a variety of structures, including hydrogels, nanotubes, nanosheets, and even composite materials with synthetic polymers or inorganic nanoparticles. given the ever-increasing need for tailored materials, coiled-coils are emerging as a highly modular scaffold capable of a wide variety of functions. coiled-coil forming peptides for the induction of silver nanoparticles biomedical applications of electrostatic layer-by-layer nanoassembly of polymers, enzymes, and nanoparticles controlling self-assembly of a peptide-based material via metal-ion induced registry shift hybrid polymer therapeutics incorporating bioresponsive, coiled coil peptide linkers cell uptake and trafficking behavior of non-covalent, coiled-coil based polymer-drug conjugates growth factor tethering to protein 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alpha-cylinders hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains engineered pullalan-collagen composite dermal hydrogels improve early cutaneous wound healing ccbuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies coiled-coil based drug-free macromolecular therapeutics: in vivo efficacy genetically engineered block copolymers: influence of the length and structure of the coiled-coil blocks on hydrogel self-assembly rational design of helical nanotubes from selfassembly of coiled-coil lock washers polypeptideengineered physical hydrogels designed from the coiled-coil region of cartilage oligomeric matrix protein for three-dimensional cell culture complete atomic model of the bacterial flagellar filament by electron cryomicroscopy coiled-coils: stability, specificity, and drug delivery potential rational design of a reversible ph-responsive switch for peptide self-assembly key: cord- -xvfl ycj authors: robson, b. title: covid- coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles’ heel conserved region to minimize probability of escape mutations and drug resistance date: - - journal: comput biol med doi: . /j.compbiomed. . sha: doc_id: cord_uid: xvfl ycj abstract this paper continues a recent study of the spike protein sequence of the covid- virus (sars-cov- ). it is also in part an introductory review to relevant computational techniques for tackling viral threats, using covid- as an example. q-uel tools for facilitating access to knowledge and bioinformatics tools were again used for efficiency, but the focus in this paper is even more on the virus. subsequence krsfiedllfnkv of the s ′ spike glycoprotein proteolytic cleavage site continues to appear important. here it is shown to be recognizable in the common cold coronaviruses, avian coronaviruses and possibly as traces in the nidoviruses of reptiles and fish. its function or functions thus seem important to the coronaviruses. it might represent sars-cov- achilles’ heel, less likely to acquire resistance by mutation, as has happened in some early sars vaccine studies discussed in the previous paper. preliminary conformational analysis of the receptor (ace ) binding site of the spike protein is carried suggesting that while it is somewhat conserved, it appears to be more variable than krsfiedllfnkv. however compounds like emodin that inhibit sars entry, apparently by binding ace , might also have functions at several different human protein binding studies. the enzyme β-hydroxysteroid dehydrogenase type is again argued to be a convenient model pharmacophore perhaps representing an ensemble of targets, and it is noted that it occurs both in lung and alimentary tract. perhaps it benefits the virus to block an inflammatory response by inhibiting the dehydrogenase, but a fairly complex web involves several possible targets. coronaviruses have been known to medicine for some time [ ] , but it is of course only very recently that the coronavirus sars-cov- , the covid- virus new and dangerous to humans, was identified. it is believed to be related to an initial cluster of pneumonia cases associated with a seafood and fresh meat market in wuhan, china, [ ] . current case rates at the time of writing are close to one million with close to , deaths. the genomic relationships to other coronaviruses were quickly examined by lu et al. to shed light on the origins, epidemiology, and receptor binding of the virus [ ] . on january th , , the wuhan isolate genbank entry mn . replaced mn . , and mn . probably represents an adequate stable description of the sequence for research into that strain isolate, and was immediately investigated by the present author [ , ] . originally, it was seen by authorities as a coronavirus outbreak but not as sars (severe acute respiratory syndrome). however, its genomic relationships examined in refs [ , ] also showed many fairly close correlations with the genomes of sars-cov in the previous human (but not pandemic) outbreaks and in pigs, bats and civets, and the emphasis was on finding subsequences that are well conserved across coronavirus strains and species. the earliest patients suffering from what is now called covid- had overall · % genome sequence identity to the above wuhan isolate, so that one may reasonably say that it is the origin of covid- , and its virus sars-cov- [ ] . the earlier wuhan isolates also related (with % identity) to two bat-derived severe acute respiratory syndrome (sars)-like coronaviruses collected in in zhoushan, china, but differed more from sars-cov (at about %) and mers-cov (at about %) [ ] . the wuhan and related isolates revealed a coronavirus that resides in the subgenus sarbecovirus of the genus betacoronavirus [ ] , and although genetically distinct from its predecessor sars-cov it appeared to have similar external binding proteins, meaning here the spike glycoprotein discussed extensively in the present paper. see section . below for introduction to this protein, which also discusses some further early identified genomic correlations. in addition, the rest of this present paper discusses many other genomic relationships relevant to the design of synthetic vaccines and therapeutic antagonists againstcovid- . one problem is that covid- is a new pathogen posing a global threat and so presents new challenges both in primary prevention, where a vaccine is required, and in secondary prevention, where a therapeutic compound (ideally, "in a pill") is required to treat patients who are infected. it might also present challenges for tertiary prevention, which seeks to remedy a persistent level of infection, or to prevent recurrence even to essentially the same strain, as discussed in section . . a main problem of concern, and a point of the present paper, is the likely appearance of new strains with resistance to vaccines and therapeutic agents. at the time of writing, confirmed cases double globally every days, and undetected cases are expected to be much higher (the current plateauing of reported cases in china offers a glimpse that this this should attenuate soon, but estimates of how and when are varied). with a significant portion of humanity already infected, there is enhanced probability of successful "escape mutations" in the genome of the virus. development of vaccines and perhaps particularly therapeutics that could, but do not, take account of this by targeting less variable protein regions could be a huge waste of resource and a dangerous delay. covid- is, of course, by far the most serious, but not the first sars outbreak of concern to humans, and coronaviruses have for decades been of veterinary concern [ ] . however, it still remains true that zoonotic coronaviruses have only rather recently seriously impacted humans, as far as is known. they include sars-cov ( , betacoronavirus, subgenus sarbecovirus), and mers-cov ( , betacoronavirus, subgenus merbecovirus). although the idea that sars-cov- was distinct from sars-cov was originally discouraged, distinction is here a matter of degree. by usual criteria they are fairly closely related, genetically clustering within betacoronavirus subgenus sarbecovirus. until very recently, sars-cov, effectively sars-cov- , was the primary reference point and model regarding molecular and functional details, and it remains important. shortly after the appearance in genbank of the apparently final version of the wuhan seafood market isolate mn . [ ] , the present author compared a variety of coronavirus genomes [ , ] . the krsfiedllfnkv protein subsequence was seen as a potential achilles' heel because it is exposed or potentially exposable, being required for proteolytic activation cleavage, and importantly is also a well-conserved feature on the surface of the virus [ , ] . being well conserved suggests that mutations are much less easily "accepted", meaning that the virus is less likely to survive more than one or two generations. as discussed below, the conservation is in a region of protein on the virus surface concerned with at least one step of lung cell entry, interesting because coronaviruses seem to be able readily adjust to alternative means of entry, possibly hinting at additional roles for the subsequence. whether or not that is the case, the above motif seems a likely primary target for synthetic vaccines and a basis for drug discovery, and was proposed as such [ , ] . it is a motif that was found to be quite well conserved even in more distantly related coronaviruses [ , ] , and the present paper also explores how far that seems to extend. it includes the common cold coronaviruses. another potential subsequence of interest popular with researchers is also examined (the ace binding domain discussed below), but the above remains popular with the present author because of its relatively high degree of conservation. present authors' opinion, however, it relates to a specter that recently haunted covid- vaccine research, and which might still cause some concerns. this is the question of why there is no significant immunity acquired by the body to prevent recurrence of common cold, of which up to roughly % of cases are believed to be due to coronaviruses. fortunately, at time of final writing of this paper, news reports indicate that neutralizing antibodies can be found in patients who have had covid- . however, with the risks of escape mutations of the virus in mind, it remains worthwhile considering whether the subsequence krsfiedllfnkv, again, found to be well conserved [ , ] across many coronaviruses [ , ] , is still present in common cold coronavirus. this is in order to force better immune response by targeting using synthetic or cloned vaccines with this epitope. most common cold strains fall into one of two coronavirus serotypes: oc -like and e-like, which are the main examples discussed below. while the common cold is generally considered as mainly an upper respiratory tract infection and a mundane inconvenience, common human coronaviruses betacoronavirus hcov-oc and hcov-hku , as well as alphacoronavirus hcov- e, also cause severe lower respiratory tract infections in children and the elderly. some discussion is also given to hcov-hku in this paper. the above motif krsfiedllfnkv occurs in the spike glycoprotein [ ] responsible for initial binding of previous sars coronaviruses to lung cells and their activation of the spike protein by a proteolytic cleavage [ ] [ ] [ ] . the spike glycoprotein (or just "spike protein") is the familiar spike that studs the surface of the coronavirus, giving it the appearance of a crown to electron microscopy, hence "corona" (latin: crown). after the completion of the first version of the previous paper [ ] , a bat virus with . % identity of the amino acid sequence of the spike protein discussed extensively in the present paper, was entered into genbank as entry qhr . . as of the time of final writing this on april nd , there is % match of this protein with entry yp_ . that appears to be a same or similar to the above wuhan isolate. the top hundred nonredundant matching entries found using mn . by blastp at https://blast.ncbi.nlm.nih.gov/blast.cgi (see below) for mn . spike protein used here vary from the above % match down to . %, such as aau . , which is a civet isolate. in viruses, proteins of a similar protruding nature, e.g. the hemagglutinin of influenza a, are primary targets for vaccine development, and important targets for development of therapeutic drugs that seek to block the virus from infecting host cells. at the time of the current project, only the three dimensional structures of the sars-cov spike proteins of the earlier sars outbreak was known (e.g. ref [ ] ), which has only %- % sequence match to sars-cov- [ ] . note that it is customary to write sars-cov rather than sars-cov- . rna viruses mutate with high frequency but so far the differences in spike proteins in emergent sars-cov- variants are much less. at the time of the study in late february and early march , the amino acid residue sequences of the spike proteins of covid- isolates from different states and countries, such as california, brazil, taiwan, and india, remain identical or almost so. for example, with respect to the original wuhan isolate [ ] , phenylalanine (f) is replaced by cysteine (c) as residue in a swedish isolate, and alanine (a) is replaced by valine (v) as residue in an indian isolate. as of st march , largest variants in the sars-cov- genome as a whole show . % nucleotide sequence match, which for a genome of , rna bases, suggests approximately base changes, and of the order of in the spike glycoprotein gene of nucleotides. that then suggests roughly to amino acid differences in the spike protein of current (march ) sars-cov- variants, consistent with the above more specific observations of isolates from california etc. a single amino acid change can, of course, sometimes have significant effect, e.g. on the aggressive character of a coronavirus, and so be considered as creating a new strain. some new strains are being reported at the time of writing, but to the author's knowledge none of them are spike protein variations, and more specifically none are as yet in the krsfiedllfnkv subsequence. the left hand side of fig. shows the sars-cov (previous sars) s spike glycoprotein within the trimer that makes up the spike. the right hand side shows sars-cov- , the sars of current concern. all human sars coronaviruses (and indeed the spike proteins of many other related coronaviruses) appear similar in overall conformation, and the variations seen in experimental structures are probably more to do with crystallization or other preparation methods, particularly regarding solvent details and ligands. sars-cov, on the left, has been well studied and still serves as the reference model. in order to fuse with and infect cells, the spike protein needs to be in an open state; presumably the closed state makes it less vulnerable to antibodies. on the left, fig. also shows the approximate positions of the cleavage points superimposed on the protein data bank (pdb) entry xlr for sars-cov. reading from the n-terminus of s , the important functional elements of sars coronaviruses deduced from sars-cov studies [ , ] and applicable to sars-cov- are the s nterminal domain (s -ntd), the s c-terminal domain (s -ctd), the s /s site as the first protease cleavage site as a loop between a pleated sheet and a-helix, the fusion peptide (fp) associated with a highly disordered loop between two a-helices which contains the second cleavage site s ', and a heptad repeat (hr). the arginine (r) in the conserved motif krsfiedllfnkv that was of interest in the previous study [ ] is the cleavage point in s '. recall that the krsfiedllfnkv subsequence associated with s ' is potentially important, not least because it must be exposed or exposable (because it permits proteolytic cleavage) and therefore the site cannot be well shielded. the experimental three dimensional structures of coronavirus spike proteins do not for the most part reveal the large amount of glycosylation that protects most of the spike protein surface. possibly the major problem, however, is not so much in the selection of accessible surface regions as a basis for design entry inhibitor and vaccine design [ , ] but that the coronavirus readily escapes from such agents by mutation, including in the spike protein [ , ] . this is the further importance of being a highly conserved motif, i.e. a subsequence that does not readily change from strain to strain except for a conservative sidechain replacement in more remote strains. of course, as one carries the study forward to more distantly related viruses, one expects the motif to differ at some stage, and this is investigated later below. in contrast, nonetheless, the pigag motif "associate with the s /s cleavage site disappears in coronaviruses that are not too distantly related [ , ] . as noted above, a high degree of conservation of krsfiedllfnkv in the face of genetic indicates that it is in some way important to the virus, presumably for the proteolytic activation cleavage, and/or initial binding to lung cells, but there could be other interactions with other proteins, i.e. to reduce an inflammatory response, as discussed later below. agents. modern computer-driven strategies, and the kind of chemical products that they help produce, differ substantially from the earlier and more familiar approaches in which the computer played little if any role. in large part this is due to the invention of automatable peptide synthesis by merrifield in , who used solid phase peptide synthesis based on crosslinked polystyrene beads [ ] . traditional vaccines are purely biological, being composed of dead or attenuated strains of pathogen (meaning mainly, viruses and bacteria). in contrast, a synthetic vaccine is a vaccine consisting mainly of synthetic peptides but also sometimes carbohydrates, often linked to a carrier protein to render it immunogenic. such vaccines produced via chemical synthesis are safer because they do not involve cell-derived material or biological processes for production. their purity can be controlled as in the case of classical drugs. the world's first synthetic vaccine was created in from diphtheria toxin by louis chedid (scientist) from the pasteur institute and michael sela from the weizmann institute. in , manuel elkin patarroyo created spf , the first version of a synthetic vaccine for malaria. primarily applications so far have been veterinary. many early vaccines used dead samples of foot and mouth disease virus to inoculate animals, but they caused real outbreaks. scientists discovered that a vaccine could be made using only a single key protein from the virus, and later also found that loops from the surface proteins could be cloned or used in cloned or synthetic constructs. novartis vaccine and diagnostics, among other companies, developed a synthetic approach that very rapidly generates vaccine viruses from amino acid sequence data in order to be able to administer vaccinations early in a pandemic outbreak. traditional vaccines have so far remained the popular choice, but during the h n outbreak in , they only became available in large quantities after the peak of human infections. this was a learning experience for vaccine companies. creating vaccines synthetically would be currently more expensive but has the ability to increase the speed of production and to retune and fine tune the solution to combat new variations in pathogens. this is all especially important in the event of a pandemic. synthetic vaccines are also considered to be safer by researchers than vaccines grown from e.g. eggs or from bacterial cultures (in the latter case there may even be other viruses present). cloned proteins can however reflect the same desirable principles; regions of pathogen amino acid sequence acting as epitopes (see below) can be presented as loops at the surface of a cloned protein. the general idea is that synthetic vaccines are freer of contaminants and focus on the essential features of the required immune response. they can also be developed in a more logical step by step approach. for example, sometimes diagnostics are considered as a useful early step on the way to a vaccine, since they are only required to raise antibodies in animals such as sheep for diagnostic kit production, not to be safe in humans and also raise immune system memory and a cellular as well as antibody response. synthetic vaccines also have the advantage that they can be seen as cartridge vaccines, meaning that they contain bits and pieces that can readily be replaced by others to update the vaccine in order to combat new strains of pathogen. a synthetic vaccine thus has several functional components, looking somewhat like a swiss army knife under the electron microscope. the key component reproduces the essential features of a pathogen protein that the immune system sees. it is an epitope that typically means a patch of some to amino acid residues. reproduced as a short peptide, epitopes can be considered as haptens. haptens are substances with a low molecular weight such as peptides, small proteins and drug molecules that are generally not immunogenic and require the aid of a carrier protein to stimulate a response from the immune system in the form of antibody production. there are two main types of epitope, b and t, discussed in theory section . a synthetic vaccine consists of t-epitopes as haptens (for cell response and immune system memory), molecular adjuvant (e.g. muramyl dipeptide), and possibly excitatory or anti-inhibitory peptides. b-epitopes are good for raising antibodies in e.g. sheep to use in diagnostics/biosensors, all attached to, or cloned into, a carrier protein. the latter must be safe but at the same time sufficiently different from any human protein to avoid autoimmune disease. used extensively as a carrier protein in the production of antibodies for research, biotechnology and therapeutic applications, keyhole limpet hemocyanin (klh) is the most widely employed carrier protein, and least for studies using laboratory animals. for humans the food and drug authorities may have other preferences for carrier protein, but klh illustrates the desired features. its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues for coupling haptens allows a high hapten:carrier protein ratio, increasing the likelihood of generating hapten-specific antibodies. because klh is derived from the limpet, a gastropod, it is phylogenetically distant from mammalian proteins, thus reducing false positives in immunologically-based research techniques in mammalian model organisms, and clinically avoiding autoimmune effects. so far, the food and drug authorities do not seem to have favored synthetic vaccines for human use, but this may be more to do with the peptides themselves than the carrier proteins available. the earlier methods of peptide synthesis did not achieve high levels of purity. however, this has changed and quite elaborate peptides as well as proteins can be made, facilitated by making peptide synthesizers run fast to avoid the slower side reactions, and by methods that join shorter synthetic peptides into longer chains [ ] [ ] [ ] [ ] . one of the original motivations for the present study was to capture experience and design strategies from vaccine, diagnostic and antagonist design [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . methods by the author and colleagues ranged from the expert system approach to automated bioinformatics and protein modeling [ ] [ ] [ ] and automated drug design (e.g. refs [ ] [ ] [ ] [ ] ). see also ref [ ] . more recently there has been an automated approach based on the proposed q-uel language [ ] [ ] [ ] [ ] . the more fine-grained principles for the design of synthetic peptide vaccines, and antagonist peptides made of d-amino acids, are discussed in some detail in the previous paper [ ] . a variety of bioinformatics techniques are available to help in development of these solutions (e.g. ref [ ] [ ] [ ] [ ] [ ] ), as well as computational (e.g. refs [ ] ) and synthetic techniques (e.g. ref [ ] ). the q-uel language [ ] [ ] [ ] [ ] [ ] used in the preceding work [ ] is also a means of gathering relevant information from the world wide web efficiently when encountering a new problem such as an epidemic caused by a new virus, or at least a problem new to the researcher [ , , ] . it also enables more automated interaction with websites for publically available bioinformatics tools. the motivation for this was all the stronger because the popular highly integrated approach to bioinformatics' called the biology workbench at the university of san diego supercomputer center has been no longer available for some time [ ] . however, the standard bioinformatics tools (e.g. refs [ ] [ ] [ ] ) used in the present study can of course be used readily by researchers reasonably experienced in bioinformatics. although peptidomimetics (containing amino acids that would not occur in normal ribosome-based biosynthesis) have been considered by authors as a basis for haptens in synthetic vaccines, they are in the author's opinion probably best considered as potential therapeutic antagonists. in the present study, the specific aims include design of molecules to impede binding and activation of the spike glycoprotein at the surface of lung cells [ ] [ ] [ ] . synthetic peptides copied from subsequences in the spike protein could be used directly for such clinical purposes, but then an important design step would be to render them resistant to biodegradation by human proteases. this is typically by inclusion of d-amino acid residues [ ] [ ] [ ] [ ] . previously, in the author's personal opinion, peptides and peptidiomimetics have been currently best considered as first steps in the research and development of small organic "in a pill molecules" of the traditional kind favored the by the pharmaceutical industry. their role there nonetheless is a powerful one, linking amino acid sequences seen in nature, conveniently already "designed" by millions of years of evolution, to (typically) smaller novel organic molecules designed to have van-der-waal's and electrostatic features in the binding site. however, the author's reticence has been largely based on cost, including cost in changing traditional production strategy, and in the reservations of food and drug authorities, but fairly recently all that appears to be changing. thpdb (http://crdd.osdd.net/raghava/thpdb/) includes an example of a manually curated repository of peptides and related molecules approved by the us food and drug administration (fda). over some peptide drugs are approved in the us and other major markets, and those in pipelines and in or approaching clinical trials may now be exceeding entries. as natural compounds, peptide drugs are typically less toxic than more traditional chemical candidates. although d-amino acids are not natural features of ribosomal production of peptides and proteins, human metabolism can handle them. they occur in gut microbes and ingested material and in human proteins they form spontaneously in a kind of aging process from some amino acids in situ in protein sequences (e.g. l to d-aspartate). diverse d-amino acids such as d-serine, d-aspartate, d-alanine, and d-cysteine are found as free amino acids and small peptides as well as in some proteins, and quite commonly in mammals. they are often found having playing important roles in the nervous system. for example, n-methyl-daspartate (nmda) receptors are associated with learning and memory and d-serine, daspartate, and d-alanine bind to those receptors. hydrogen sulfide generated from dcysteine reduces disulfide bonds in receptors and potentiates their activity. peptides made of d-amino acids resist not only normal proteolytic degradation but also resist an immune response (unless attached to a carry protein) [ , ] . they persist some - days in the body, which is an ideal time period for pharmaceutical action, and are ultimately degraded to safe products (probably mainly in the peroxisomes and by enzymes in the kidneys) [ , ] . the negative aspect is that they do have higher entropy to overcome than many drugs of more traditional form, but in practice this appears to be more a barrier to computer simulation of binding than to the real molecule, as extensively discussed below. studying the binding of synthetic peptides or small organic molecules to human proteins benefits from computer simulations of the solute-solvent system, and it was early found that these should ideally include water molecules in a detailed way because there are hydrogen bonding options between water molecules and amino acid residues which are not particularly intuitive [ , ] . in most cases, the spatial locations of hydrogen atoms are deduced rather than seen in experimental protein and peptide three dimensional structures. this is likely to impact considerations of docking ligands to protein targets. in the present author's opinion, this provides a beneficial possibility for retroinverso designs [ ] made by reversing the sequence and using d-amino acids that has the unfortunate or complicating effect of interchanging the c=o and n-h groups in the backbone of the synthetic peptide [ ] . the beneficial possibility is that, for example, a repulsive c=o…o=c electrostatic interaction between a synthetic peptide and the spike protein could be ameliorated in the manner c=o…h…o=c where the h is a water, serine or threonine hydrogen atom, or by c=o…h-o-h…o=c, albeit that in practice the water molecule likely lies more to the side of the o..o interaction vector. somewhat similar considerations apply to any n-h…h-n interactions that can ameliorated by the lone pair orbitals of an oxygen atom. both could also involve tautomerization and/or rearrangement of internal hydrogen bonding networks (e.g. in the manner …o-h…o-h…. to …h-o..h-o…). today, to take care of such matters, researchers consider docking of ligand to protein and high grade molecular dynamics simulations of the overall solute-solvent system by molecular dynamics, at least as the final refinement step [ ] , but even the awareness that the above compensations and others can take place can make it worthwhile to synthesize and test a proposal. somewhat similarly, design of peptide synthetic vaccines and diagnostics can make direct use of peptides duplicating sequence motifs in the pathogen protein found by bioinformatics and relatively simple computational tools. after that, researchers often go straight to synthesis and experimental immunological testing of the constructs rather than using complex simulations [ ] [ ] [ ] . epitope predictions for sars-cov- (simply meaning the choice of amino acid residue subsequences to synthesize for synthetic vaccines, but also for peptidomimetic antagonists) have been made by several authors (e.g. ref [ ] ). they have typically made use of extensive historical experimental data about the amino acid residue sequences of epitopes such as the epitope database and analysis resource (iedb) and the virus pathogen resource (vipr) (e.g. ref [ ] ). the immune system by its nature can make its own adjustments to recognize pathogens and vaccines, but designing some kind of therapeutic antagonist against virus binding to the lung cells requires rather more consideration about what human protein the spike protein is binding. bioinformatics as the study of biosequences is a powerful tool, but it is well known that having the detailed three dimensional structure of the human protein target for a potential new pharmaceutical agent, or to which a virus attaches, is a great benefit to rational computer-aided design. studies specifically investigating human protein binding and activation of previously known sars viruses have for some years been carried out by several groups (e.g. [ ] [ ] [ ] [ ] ). it seems reasonably well agreed that angiotensin converting enzyme type (ace ) is responsible for binding the sars associated with the outbreak, combined with a proteolytic cleavage to activate the spike protein, for which type ii transmembrane serine protease (tmprss ) is the current popular candidate [ ] . several three dimensional structures are known for ace complexed with sars spike protein e.g. protein data bank (pdb) entry ( acg) and of variants of the latter (e.g. tmprss protein data bank entry oq ). however, the full story involving human cell surface proteins (with which sars-cov- interacts in order to infect and replicate) is possibly not quite as firmly established at the time of this present study as some summaries would suggest. the origin of the general problem for a more detailed conformational chemistry approach is that diversity of genome and means of infecting cells are readily generated in nature in the case of different virus hosts, virus strains, and species jumps, and it is long established that the binding shows variation in the receptors used that correspond to viral groups. there have been alternative proposed candidates for initial binding receptors, e.g. carcinoembryonic antigen-related cell adhesion molecule (ceacam ), and various dipeptidyl peptidases. highly virulent coronaviruses that form syncytia between cells can even spread in a receptor-independent fashion. even when an initial binding receptor such as ace is identified for a coronavirus, initial uncertainty or enduring complexity for the rest of the entry process may be the norm. many other human proteases present in the lung seem capable of cleaving various sites on the spike protein and which could cause its activation. for example, a variety of proteases such as trypsin, tryptase clara, mini-plasmin, human airway trypsin-like protease (hat), and tmprss (transmembrane protease, serine ) are known to cleave the glycoprotein hemagglutinin (ha) of influenza a viruses as prerequisite for the fusion between viral and host cell membranes and viral cell entry. human airway trypsin like protease (hat), tmprss , tmprss , tmprss have also all been considered by sars researchers at various stages. other human proteins that might have similar involvement to the above in the sars-cov- case, and that are also affected by the same antagonists against the sars-cov- targets in the preceding paragraph, have also attracted the attention of researchers. the trypsin-like serine protease hepsin which has a fairly broad action and which is significantly inhibited by a diverse set of ligands, a particular example of one such binding is represented by protein data bank entry ce . even intracellular proteases could be released on cell damage resulting from the first wave of lung infection or from other disease or tissue trauma. some variants and strains may use other, as yet unknown, proteins, or sugars, to assist entry. it is also plausible the spike protein might be activated by other proteases on exit from the epithelial lung cells, so allowing it efficiently to infect other cells. the spike glycoprotein of sars-cov- also has the so-called furin cleavage sequence (prrars or prrars), which is an extension to the so-called pigag motif of ref [ ] . consistent with the present author's preferred choice of krsfiedllfnkv motif, coronaviruses with high sequence homology (such as that isolated from a bat in yunnan in ), lack the furin cleavage sequence. nonetheless, because furin proteases are abundant in the respiratory tract, sars-cov- spike glycoprotein might be cleaved on exit from cells. even if the means of binding, activation and entry is well established for a viral strain, recall that a single rna base difference resulting in a single amino acid residue difference could alter all that, and there also appear to be several other possibilities that the virus can exploit in parallel. indeed, somewhat similarly, potential inhibitors of sars entry and/or activation proposed by researchers (e.g. refs [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) may work by several routes in parallel, and significantly at least three mechanisms were reported in one relevant study [ ] . once a target protein and its relevant binding site are clearly understood, methods are available for screening available ligands (binding molecules) to bind to those sites as potential antagonists, or even for "growing" or evolving antagonist molecules in those sites, whether smaller organic molecules [ ] [ ] [ ] or peptides [ ] . pharmaceutical chemists have long used evidence and hunches to deduce a pharmacophore, i.e. an abstract description of recurrent molecular features that are necessary for molecular recognition of the ligand by the protein [ ] . a pharmacophore ultimately implies at least a schematic model of the interfacial surface between ligand and protein, but in practice, a pharmacophore tends to be either considered from the perception of the ligand (one compares similar inhibitors etc.) or from the perception of the binding site (one compares positions of key residues in the binding site). the choice depends on the quality of each kind of data, but could involve both. historically, drug design was frequently based only on indirect deduction of binding site features using the chemical features of the ligands which successfully inhibit (or in a few instances excite) a response. this is essentially the use of quantitative structure activity relationships (qsar). in effect the perception of the binding site was indirect and typically based on the chemist's expertise and hunches, and so often extremely "fuzzy". subsequent elucidation of many protein structures with clear pictures of their binding sites led to a crisper physical perspective, exemplified by a study [ ] that included many ligand molecules in the present investigation, and so faced some similar issues. in the approach which may now be considered traditional, docking calculations are fast, using grid maps that consist of a three dimensional lattice of regularly spaced points, centered on some region of interest of the protein target under study. as discussed above, ace and tmprss are very likely correct targets, but again they are not necessarily the only targets even for cell entry of current sars-cov- , and the mechanisms used by each new coronavirus strain can differ, as the result of even a single amino acid residue change. in such circumstances, the conservation of the krsfiedllfnkv motif might be considered suspicious. the activation cleavage is at the arginine (r) and workers tend to conclude that this site is more essential for action than s /s , and mutation of the arginine (r) specifically inhibits trypsindependent fusion in both cell-cell fusion and laboratory assays. but also, with the arginine retained, many other proteases can active the spike protein as above, and others can do so in laboratory conditions. because of the conservation, one might therefore hold the seemingly reasonable hypothesis that this site is not also susceptible to cleavage and activation by other extracellular proteolytic enzymes, but also doing something else. whether or not this is so, all this complexity makes detailed interaction models of spike protein binding and activation difficult, and while the "best bet" for ranking the choices of target protein may currently seem obvious, making a reasonable, currently conventional, choice which is actually an incorrect assumption can delay productive research into therapeutic agents. in the case of the hunt for prevention and cure of virus diseases, and particularly covid- , there seems to be increased justification for a "fuzzier" set-theoretic picture of a pharmacophore as an ensemble of different binding sites, or of ligands in a ligand-oriented perspective, as follows. many of these, and perhaps all, suggest that even if one is using an incorrect picture of the mechanisms of entry and replication, even using the "wrong" or less important protein target, one might achieve some success. in brief summary, the justifications for the ensemble pharmacophore in the coronavirus case, i.e. the contributions to "fuzziness", include parsimony, that proteins and parts of proteins sometimes have more than one function [ ] encouraged by limited numbers of accessible sites (due to e.g. glycosylation) and exemplified by parallel alternative mechanisms of cell entry, multiple methods of drug action, escape from scientific defense measures by virus mutation, polymorphism of human proteins involved, different expression levels of human proteins involved, and the potential problem of the "specter of vaccine development" (concerns about missing the appropriate region of the virus that allows common cold viruses to escape the appropriate immune response). to the above may be of course added the fact that even if an experimental researcher is convinced of the value a specific protein as appropriate target, the picture for the computational chemist is a fuzzy one. the system itself, real and simulated, is to be seen as a statistical mechanical ensemble of multiple states, sampled over the population of molecules and across their conformational behavior in time. not least, protein binding sites are often partially disordered before binding, and in any case there may be several binding modes. picking the right one can be difficult because there is a fine balance between solvent and conformational entropy, and entropy is notoriously hard to compute [ ] . given this argued uncertainty as to the nature of the target protein and its binding site, a broader initial net as an ensemble pharmacophore can help. docking approaches are continually being improved by researchers, and recently include ways of combining features that could ultimately relate to different protein binding sites. while many authors of these studies include the word "ensemble" in their discussion of pharmacophores, they appeared to be significantly different to the particular means of combining multiple pharmacophores that was explored here. however, the present author has had his attention drawn to some that are rather similar and the approach of kumar [ ] appears to particularly akin, especially in regard to distributions of expected values and use of weighting. kumar's description [ ] thus suffices, and briefly stated, it explores the ability of an ensemble of selected protein-ligand complexes to populate pharmacophore space in the ligand binding site, assesses the importance of pharmacophore features using poisson statistical and information-theoretic entropy calculations, and generates the pharmacophore models with high probabilities. a scoring function then combines all the resultant high-scoring pharmacophore models. there is one significant operational difference between kumar's approach and that used here. recall that in the more traditional docking approach, it is the ligand as candidate drug that is typically seen as the variable and constantly changing and in many studies "evolving" the ligand chemistry with the pharmacophore is the basis of drug design [ ] [ ] [ ] [ ] . ref [ ] , related to the present study, has aspects of that applied in a different way. kumar's approach can, however, combine the perspective of both pharmacophore and ligand as conceptual variables. despite that, the present author's approach, as used in the present overall project, considers one candidate ligand at a time. this seems less efficient from the point of view of designing candidates, and not even as smart as the older single, non-probabilistic pharmacophore approaches [ ] [ ] [ ] [ ] . nonetheless, a single, simpler one-ligand-at-a-time strategy is both adequate and appropriate in the present case. this is because there is already a data collection of candidate antagonists to build on [ ] , as discussed in section . . approaches of the ensemble pharmacophore kind are currently highly recommended for investigation of sars-cov- and for the spike protein in particular, again because of some uncertainties and the likely multiple functions of some spike protein features. however, it has not as yet had significant impact in the present study. the approach actually taken remains consistent in the sense that inclusion of one particular source for a pharmacophore, an enzyme considered by the author, was evidently going to dominate the ensemble because of certain similarities in the antagonists of sars virus entry and inhibitors of the enzyme [ ] , given the knowledge available at this time. that choice is not, however, obvious, as follows. pharmacophore. what may be more controversial is the case when there is a representative choice and it is a protein that is not obviously relevant to the target protein, or simply not "on the radar" of coronavirus researchers. what makes it a candidate is not necessarily that it is relevant to viral infection and not necessarily that it has an evolutionary relationship to proteins that are considered relevant, although this is a question addressed briefly in this paper. rather, it may simply be based on the pragmatic notion that there may be ligands, potential binding molecules as antagonists, which are common to both more popular choices human target proteins and a less obvious candidate. the small organic molecule emodin has been found to inhibit sars coronavirus entry [ , ] , as also so have other compounds some of which have emodin-like features [ , ] . similar molecules, and importantly emodin itself, are also inhibitors of β-hydroxysteroid dehydrogenase type , an example of a steroid binding enzyme [ ] . it is normally anchored within the endoplasmic reticulum through an nterminal transmembrane domain. its involvement as a protein target is here based on a chemical justification. a biological justification might be that this enzyme is involved in the inflammation response which a coronavirus might also benefit by inhibiting. if so, the goal is not, of course, to help the virus by inhibiting at the same target which it would also gain by inhibiting, but rather to inhibit protein targets more crucial to it, i.e. for cell entry and possibly replication which are even more crucial to the virus. some inhibition of β-hydroxysteroid dehydrogenase type might even be a desirable thing because excessive or prolonged inflammation (including in response to pathogens) is well known to be potentially damaging to the host. an excessive inappropriate immune response may also include the basis of allergic reactions and even of autoimmune diseases. a pragmatic reason for this choice of protein as pharmacophore is that was also one of those protein-ligand interaction systems that have been well studied by the present author and collaborators [ ] . such studies pursued the idea of using a more rigid molecular framework, including the steroid framework and fragments of it, as a more rigid scaffold for active drug groups [ ] . importantly, that study and subsequent work has already established data base of compounds that bind to β-hydroxysteroid dehydrogenase type , and it includes many molecules including some discussed in this paper that again have some of the features of emodin. it also includes many weak binders that could also be much stronger binders at what turns out to be a more obviously relevant protein target. these issues can be addressed quickly in the laboratory and certainly seem worthy of investigation before addressing the more popular targets. there was a further implication in the previous paper [ ] , though not a requirement for its main arguments, that the peptides designed on the basis of the krsfiedllfnkv motif bind the same krsfiedllfnkv site as do emodin-like molecules. that seems currently to be an even less reliable assumption than the assumption that the above steroid dehydrogenase enzyme is relevant to coronavirus biology, and it is not of course an assumption that even matters if either a peptidomimetic and/or small organic molecule is found effective. however, again keeping in mind that there are a limited number of accessible, conserved sites in the spike protein, and that these may be involved in multiple mechanisms as discussed above, common targets for action of both peptides and smaller organics like emodin seems plausible. partially the problem is extensive glycosylation. it is well known that glycosylation plays an important role in receptor-ligand recognition but also have structural influence in receptor-ligand recognition because of its bulky shape caused by branched side chains. for that and other reasons it may be that the krsfiedllfnkv site is, with just a little variation, almost the only site on the spike protein that is persistently recognizable in coronavirus strains, and so presumably carrying out an important function and accessible, as also discussed in this paper. angiotensin converting enzyme (ace ) binding is however also considered in this paper. a number of ideas and principles, borrowed in established and recent design of synthetic vaccines and petidomimetics, were used (see ref [ ] for discussion and e.g. refs [ ] [ ] [ ] [ ] [ ] [ ] [ ] ), as well as some of the ideas that lie behind the popular zinc data base [ ] . as discussed in refs [ , ] , the present investigation started as a use case for the hyperbolic dirac net (hdn) and particularly the associated q-uel language for automated inference [ ] [ ] [ ] [ ] . the theory has been discussed elsewhere, e.g. in refs [ ] [ ] [ ] [ ] , which relate more to the practical and general uses of q-uel. these considerations are less important here because present studies can be reproduced by standard bioinformatics and molecular modeling means. nonetheless, it is doubtful that the research for refs [ , ] could have been done and written up so rapidly without the aid of q-uel to interact with websites of the world wide web, gather knowledge, and facilitate use of the publically available bioinformatics tools [ ] . the challenge is ultimately one of molecular recognition but in practice many key principles for hapten design relate to distinguishing types of naturally occurring epitope. by the term "epitope" in this paper is meant "continuous epitope", though several smaller epitopes may be joined to represent a discontinuous epitope in which conformation and relative position in space can sometimes be important. while a synthetic construct implies the use of synthetic chemistry typically combined with a judicious carrier protein to which the peptide is linked chemically, constructs can also be obtained by cloning, using protein engineering principles [ ] . the terms b-epitope and t-epitope relate to the traditional picture of a bone marrow b or thymus t response. b cell epitopes occur at the surface of the protein against which an immune system response is required. they are recognized by b cell receptors or antibodies in their native structure, and are concerned with the bone marrow response and antibody production. t epitopes may be buried inside protein structures and released by proteolysis, and are traditionally considered as concerned with a cellular response and immune system memory, i.e. active immunity. continuous b cell epitope prediction is very similar to t cell epitope prediction. the focus is on b-epitopes here, though a bepitope can also be (or overlap with) a t-epitope especially if it has a significant content of hydrophobic residues. prediction of these has traditionally been based has mainly been based on the amino acid properties such as hydrophilicity, charge, exposed surface area and secondary structure. there are many predictive algorithms available, but the present author prefers a more "expert system" kind of approach that incudes experimental data, though the above biophysical considerations certainly still play a strong role (see below). infection. the previous paper [ ] focused primarily on design of synthetic peptides as infection antagonists. however, partly for the reason of greater conformational flexibility discussed below, smaller less flexible organic molecules (i.e. with fewer rotatable bonds) are the traditional province of the synthetic chemist rather than use of an automated peptide synthesizer, are preferred for pharmaceutical application. consideration of peptides is more often considered as merely a useful intermediate step in more traditional pharmaceutical compound design. biodegradability per se of peptides is not the main concern, since including d-amino acids in the design prevents proteolysis. in preliminary docking and simulation studies, the peptides do bind to βhydroxysteroid dehydrogenase type , but less strongly and with several binding modes [ ] . this weaker binding is not in itself a contraindication of the idea that these peptides bind at the same site as the more rigid non-peptide molecules, because it is an expected consequence of the much greater flexibility of peptides compared with molecules with, for example, multiple aromatic ring scaffolds. conventional wisdom (e.g. ref [ ] ) frequently uses the rule-of-thumb that the total change in intramolecular (bond rotational) entropy of a peptide ligand is roughly t∆s total = . kcal⋅mol − per residue at k, corresponding approximately to a -fold reduction in conformational freedom per residue on binding. because van der wall's and hydrogen bonding tend to be very roughly equivalent for peptides in water and in well bound forms, the water entropy effects known as hydrophobic effects (along with electrostatic forces) play an important role in determining the balance of energies and final outcome. krsfiedllfnkv would thus cost about + . kcal/mole entropic contribution to bind rigidly, primarily compensated by hydrophobic contacts at up to about - . kcal/mole in going from an aqueous to a non-polar environment, i.e. - . kcal/mole for a residue peptide or analogue of krsfiedllfnkv. that example would not favor binding, but the proper calculation is in the details which of should show balance that favors good binding if that is found to be the case experimentally. despite the above comments, the flexibility of peptides does provide more opportunities to fit a specific binding site, i.e. they can show some accommodation and they are more tolerant to imperfections in the design process. however, this is also an argument for their importance as an intermediate step in the design of more conventional pharmaceutical agents. the main methods are essentially standard bioinformatics approaches as used in refs [ , ] . some methods, e.g. rules for epitope prediction, are best discussed in context in results section . the q-uel methods specifically for bioinformatics are discussed in [ ] , and those for computational chemistry and docking of compounds are those using krunch as described in ref [ ] and the appendix to ref [ ] . they are somewhat unorthodox by focusing on heuristics to handle the multiple energy minimum problem, but the end effect is probably similar to that of long runs using high grade molecular dynamics calculations, given opportunities for calibration [ ] . epitope predictions lie in more traditional "one dimensional" bioinformatics, and in this paper and the previous paper depended on predictions using a gor secondary structure prediction of α-helix (h), extended chain or β-sheet (e), and coil or loop (c). the reason for this and the particular use of gor is discussed in ref [ ] , but briefly, it is in part because sections predicted by runs of c tend to be immunogenic even if they are incorrect as structure predictions [ ] . however, charged residues in α-helices and βsheets are believed to be occasionally b-epitopes, and short sections extended chain can effectively imply loops. the core and initial rules for b-epitope prediction used in the present study consider (i) surface exposure when a three dimension structure is known, but allowing for conformational adjustment to expose residue when in a likely disordered or flexible loop, scores + , (ii) known exposure based on other kind experiment, which also recognizes the possibility that a partially buried site by the above criteria can be brought to the surface on binding, notably for proteolytic cleavage [ ] , (iii) runs of amino acid from the set [stnqy] score + , from the set [dekhr] they score + , and from the set [livfcm] they score - , (iv) runs of secondary structure prediction as coil or loop c, though runs of three or less e and the first and last three of helix h can be considered as c for this purpose, score + , and (v) the motif nx(s/t)x of asparagine (n) serine (s) or threonine (t), where x means "not a proline" (p) scores + . however, this will not permit a corresponding peptidomimetic or vaccine without considering glycopeptide synthesis technology. see discussion below, which would justify a negative score, depending on the technology available. in addition, these may be combined with predictions based on significant homology with proven epitopes in data bases, which has already been done by several groups for sars-cov- (e.g. ref [ ] ). for sources of data concerning covid- virus spike proteins, genbank and the protein data bank were the main sources. there was some use of in-house collections of data, e.g. of typical b-epitopes and t-epitopes, although publically available collections would probably serve the same function. there was also use also of a data base of non-peptide ligand molecules of potential interest already generated during and since the work described in ref [ ] that was used where appropriate. many of these molecules (including emodin) are also found on the public zinc data base [ ] as indicated in results section below, but several, including derivatives of carbenoxelone, are not, and these derivatives are of interest as potential coronavirus antagonists. to look up an entry on the zinc data base by the codes used in this and other papers, one can go to http://zinc .docking.org/ substances/ and enter zinc . in an automated approach such as that favored by q-uel, a variable (such as a perl variable $mol) to zinc and is set an the q-uel application goes to http://zinc .docking.org/substances/search/?q=$mol. any references to experimental binding results concern data from cited papers, and see for example ref [ ] for typical methods used for natural herbal compounds. as discussed in ref [ ] , q-uel helped gather these in the form of q-uel knowledge representation tags, so they become part of the growing knowledge representation store. in regard to peptides and proteins, table used in ref [ ] shows the standard iupac one-letter codes used for amino acid residues in sequences throughout this paper. table . one letter amino acid codes used in the text. conservative replacements are those common substitutions from a peptide design perspective, but for example phenylalanine (f), isoleucine (i), and alanine (a) are seen as natural substitutions that appear in discussion of spike protein sequence motifs later below. these amino acid residues have hydrophobic sidechains but they are not conservative replacements but rather substantially different size. a reasonable explanation is of course that sidechain size conservation matters less when the sidechains are at exposed at the surface of the protein. similar notions underlie the idea that what can readily replace what is not always an equal probability in each direction. in that respect, table tends to reflect the changes that are used in the present project for design, when starting from epitopes. the previous paper [ ] should not give the impression that the specific motifs discussed (and particularly krsfiedllfnkv) are the only sections likely of the sars-cov- spike protein to be of interest in the above respect. the preference for one choice was based on (a) conservation across many strains, suggesting that the site has an important function and is likely at the spike surface, and (b) avoiding the shielding of the spike protein by extensive glycosylation. the dramatic effect of relaxing these restrictions is a major point of this section, in which a large number of candidates are found. over-prediction is not necessarily a bad thing, because once a laboratory has a peptide synthesizer and other tools for constructing and testing designs, it is relatively easy and cheap to test and reject ideas, and more problematic to miss opportunities. the intention here is also to cover most possibilities, to enable index numbers to be assigned to them according to their order in the sequence (putative epitope etc). consequently, in future one may then readily refer to the index number, or speak of a new proposal or experimental epitope extending, overlapping, or even lying between two of these epitopes. they are primarily to be seen as b-epitope predictions, though they are favored if some t-epitopic character is also expected. an initial step is based on adding up weights as described in methods section . . in practice, there was also some judicious use of expertise and an epitope data base in an attempt to refine assignments. recall that the trimeric sars coronavirus (sars-cov) spike glycoprotein consists of three s -s heterodimers. some of these will be shielded by that configuration during most of the life cycle of the virus, but not necessarily in every s protein monomer, and also shielded by glycosylation. the higher scoring predicted epitopes in the sequence below are underlined and in bold, and are primarily to be considered as b-epitopes but with some extension to include t-epitope character where possible. also included in these predictions are those using the immune epitope database and analysis resource (iedb) and the virus pathogen resource (vipr) which have already been made [ ] (see later below). these are shown in underlined, bold, and in italics in the following, and since some are contiguous sections that look like a single long representation in the following, they are also stated separately below. it is apparent that while focus was on just krsfiedllfnkv, if strain variation and glycosylation are ignored then much of the spike protein sequence contains epitope candidates. many of the epitopes predicted in the present study overlap with prediction made using the immune epitope database and analysis resource (iedb) and the virus pathogen resource (vipr) [ ] , and these comprised the following. recall that one of the reasons for the original single preferred candidate krsfiedllfnkv was that many of predicted epitopes contain evidence of glycosylation, reflecting the last of the "rules" (v) in methods section . above. that rule has, however, a special status, and the present author has tended to consider them undesirable for synthetic vaccine or diagnostic development. it indicates likely glycosylation of the protein. the bulky oligosaccharides so attached can be immunogenic, but they are rather difficult to work with synthetically, traditionally expected to make bulk production expensive, and may be variable in structure which cannot typically be seen in detail in experimental three dimensional protein structures (typically as obtained by x-ray crystallography or high grade electron microscopy). antibodies that are raised against the glycosylated surface patch of the protein or corresponding synthetic glycopeptides may be specific for their carbohydrate units. these can be recognized irrespective of the peptides, or in the context of the adjacent amino acid residues. conformation and exposure of b-peptide epitopes of glycoproteins may be modulated by glycosylation because of intramolecular carbohydrate-protein interactions. the beneficial versus undesirable effects of glycosylation in synthetic vaccines is also a complex matter. glycosylation may be essential for reactivity with the antibody, but conversely it may in effect inactivate the capabilities of a section of amino acid sequence to function as a b-epitope, which seems to be a very good reason for giving the glycosylation motif a strong negative rather than positive score. unfortunately this will depend on the structure of the antigenic site and antibody fine specificity, and the recognition mechanisms involved are not fully clear. there is a (usually) positive aspect, however, in the current view that similar effects of glycosylation apply to t-celldependent cellular immune and igg antibody responses, and that glycosylated peptides can elicit glycopeptide-specific t cell clones after being bound and presented by mhc class i or ii molecules. it is of course only a positive aspect if the intended effect is obtained by the synthetic construct. the overall spike glycoprotein protein sequence shown above changes across the coronaviruses, but the krsfiedllfnkv subsequence is most notable amongst the exceptions. it extends to the common cold coronaviruses with minor variation, and may imply a better targeted approach to stimulate immunity. for common colds caused by the rhinovirus, recent research suggests misdirection of antibody responses against a non-protective epitope as a mechanism how the virus escapes immunity and so permits recurrent infections [ ] . a clearer understanding of conserved subsequences in coronaviruses may also help tune the action of toll-like receptors to initiate the appropriate response. these are a class of proteins that play a key role in the innate immune system. they are single-pass membrane-spanning receptors usually expressed on sentinel cells (e.g. macrophages and dendritic cells) that recognize structurally conserved molecular features of pathogens [ ] . despite concerns about two or more strains of covid- virus appearing, these are not big changes for present purposes. it is sufficient to consider the sequence of the original wuhan isolate as reference in comparisons for present purposes, i.e. for comparing the spike protein sequences of other coronaviruses. recall that as discussed in introduction section . , at the time of the study in late february and early march , the sequences of the spike proteins of covid- isolates from different states and countries, such as california, brazil, taiwan, and india, remain identical or almost so. for example, with respect to the original wuhan isolate [ ] , phenylalanine (f) is replaced by cysteine (c) as residue in a swedish isolate, and alanine (a) is replaced by valine (v) as residue in an indian isolate. neither of these relate to the sequence motif krsfiedllfnkv of particular interest here. in the initial studies [ , ] , the genome of the common cold coronavirus, and particularly the sequence of the spike protein, was considered sufficiently far from that of the covid- virus so as to be less relevant to that problem. while looking at differing sequences is essential for detection of conserved motifs, very different and less relevant pathogens are unlikely to preserve them, except perhaps as pattern matches involving quite complex substitution rules. however, the appearance of the covid- krsfiedllfnkv motif does appear in common cold coronaviruses and with typically at most two relatively conservative substitutions. that is in the sense of preserving hydrophobic sidechain as discussed above in methods section . the conservative aspartate (d) and asparagine (n) replacement is also fairly common in the motif in the sequences examined. an example shown below is a clustal omega alignment of the covid- virus spike protein original wuhan seafood market isolate (genbank entry mn . ) with spike proteins representatives members of the two major common cold coronaviruses strains e and oc (genbank entries np_ . and aiv . ). despite radical sequence differences for the spike protein sequences overall (only . % identity, well within the range for a random match), the underlined sequence motif krsfiedllfnkv of covid- virus is essentially retained as that sequence, except that alanine (a) replaces phenylalanine (f) in the common cold coronavirus (which is moderately conservative at the surface of a protein) and a conservative leucine for valine substation in one case. in the sequence (not shown) of hcov-hku which is often associated with more serious cases of cold-like diseases the above motif is still noticeable as rsffedllfdkv in which the isoleucine (i) is replaced by phenylalanine (f). the "a for f" modified motif rsaiedllfdkv is also found in the coronaviruses of dogs, cats, rodents, pigs, rabbits, camels, ferret badgers, raccoon dogs, amongst others. all of these might be eaten by humans in certain countries and notably they are, for the most part, species that live in close proximity to humans. the "pigag" motif does not show up in the above alignment, as is also the case in many other distantly related coronaviruses [ , ] . however there is a subsequence pigtnyrscestt in the hcov-hku spike protein that appears to relate to pigagicasyqtq in the covid- virus (recall that hcov-hku is a common cold virus, albeit usually associated with more severe, lower respiratory tract cases). in contrast, not only does the krsfiedllfnkv motif stand out as potentially important to the covid- virus by virtue of such comparisons, but also a match with that motif is almost the only continuous stretch of amino acid residues in most alignments like that above. the subsequence kwpwyiwl is an exception that is of interest and a characteristic feature of many sars coronaviruses. it is not, however, considered further in the present paper, except to note that it does not appear to be associated with a covid- virus spike protein proteolytic cleavage site. these sites are most prominently trypsin: s /s htvsllrstsqksivaytmsl, s ' lpdplkptkrsfiedllfnkv; cathepsin: s /s htvsllrstsqksivaytmsl; elastase: s ' lpdplkptkrsfiedllfnkv, plasmin: s /s htvsllrstsqksivaytmsl, s ' lpdplkptkrsfiedllfnkv, tmprss : s /s htvsllrstsqksivaytmsl; tmprss : multiple sites; tmprss a: s /s htvsllrstsqksivaytmsl, s ' lpdplkptkrsfiedllfnkv. as one looks out to more distant relatives, there are a number of variations in the krsfiedllfnkv motif which, despite large variations in spike protein sequence as a whole, are still recognizable in the spike proteins of coronaviruses of diverse various host species, as shown for some examples in table . the most noticeable variation is the occasional substitution of the cleavage point arginine (r) by a g. rather than disrupt the possibility of cleavage, however, it is seemingly displacing that role to a arginine (r) or lysine (k) that lies to the n-terminal (left) side of the motif. it is interesting that this commonly retains firmly the iedllf core of the motif. the notion that the krsfiedllfnkv motif overall plays an important role, and presumably a common or similar function across at least a very large number of known coronaviruses, still seems a reasonable one. most important of course is that it is at least the case for the sars-cov- virus and its near relatives. at this time, no match with a coronavirus in genebank has been detected by the author by blast-p using queries with no phenylalanine (f), e.g. rsaiedllldkv, rsaiedllidkv, rsaiedlladkv, rsaiedllmdkv, rsaiedllwdkv, and rsaiedllydkv as queries, but the search has not been exhaustive because it would not be too contradictory to any of the current hypotheses if some were found. in the group with the inserted glycine (g) replacement of initial argine (r) by the similar positively charged lysine (k) is common. however, as long as the motif is significantly recognizable, no histidine (h) as opposed to initial arginine (r) has been found. the motif cannot extend to other strains indefinitely as recognizable because at some point the evolutionary tree will bring up virus and hosts subject to quite different selective pressures, and the motif is not the definition of coronaviruses. however, it still persists as recognizable in birds such as duck (e.g genbank kx , kc white-eye bird cov hku (nc ), magpie-robin (shama) cov hku (nc )) strains, a selction which spans a large range of coronavirus genome sizes. see the alignments below compared with the wuhan seafood market isolate genbank mn . , showing the motif underlined and in bold. :.: .: : : eeyihklnatlvdldwlnrvetyikwpwwvwllitlaivafvvilvtiflctgccggcfg : . ** ::::*: *. : *:****::** : .: : : : *.**. some indication of the limit of the survival of the motif rsfiedllfnkv as the researcher departs from sars-cov- might be given by the nidoviruses other than coronaviruses. somewhat coronavirus-like nidoviruses are common as e.g. reptile viruses. the order nidovirales contains enveloped, positive-strand rna viruses with the largest known rna genomes. nidoviruses have been identified in snakes. they appear to be most closely related to coronavirus subfamily torovirinae, and might be best represented as a genus in this subfamily. sequences suggestive of rsfiedllfnkv, e.g. knfidlllagf do occur in genomes such as the ball python genome, but these really lie beyond the limit of serious detection. for example, clustal omega gives % exact match between the wuhan isolate and spike protein nidovirus of the reptile shingleback, but the motif is barely recognizable. including fish nidovirus of the pacific salmon (genbank qeg . ) is notable here because it supports the above alignment because it is preserved, but gtlywldy of the salmon nidovirus is far from krsfiedl and the nearest preceding plausible cleavage point is an arginine (r) residues in the n-terminal direction (to the left). however, a similar occurs in some mammalian coronaviruses and so that residue may still play a similar role as an activation cleavage. looking for similar motifs in human proteins has a somewhat different motive. it makes sense in that, if there is significant match with subsequences, they might represent features of proteins to which both the spike protein and other human proteins may bind, irrespective of any other justification for commonality. even if coincidental, as epitopes similar to those in a proposed synthetic vaccine they are always of possible interest in assessing the risk of cross-reaction and inducing autoimmunity in synthetic vaccine designs, and on certain occasion with peptidomimetics that induce an immune response, perhaps by a binding strongly to a human protein that the designer did not intend. as discussed in ref [ ] , there is a motif match at % identity with % coverage is with tumor protein d isoform [homo sapiens], id: np_ . , and similarly with tumor protein d -like [homo sapiens] id: aah . . next match is in regard to neprilsyn entries at only % match and % coverage. none of these are sufficient close of concern regarding induction of an autoimmune response. some fairly close matches of krsfiedllfnkv and of the "a for f" modified motif rsaiedllfdkv have come to light that might plausibly have a biological significance if supported by biological relevance, but are more likely to be random matches. selecting only for human proteins, hits vary from % cover with % identity to % cover with % identity. these hits cannot be considered significant for peptides of this length in isolation from other evidence. however, a few seem worth recording for future reference in regard a potential biological function for the virus. as already noted [ ] , rrsfidelafgrg a section of a human semaphorin (genbank np_ . ) produced in response to lung disorders. running rsaiedllfdkv itself in blastp generates coronavirus hits. rnareellfd is found in human mhc class ii antigen, genbank axn . . rnareellfd is found in human immunoglobulin heavy chain junction region genbank mcg . . dllfekv is found in human tubulin, gamma complex associated protein , isoform cra_d genbank eaw . . e is of interest with % identity % matches for sfleellfin khksfleellf in ubiquitin. the cellular e ubiquitin ligase ring-finger and chy zinc-finger domain-containing (rchy ) have been identified as interacting partners of the viral sars-unique domain (sud) and papain-like protease (pl pro ), with the involvement of cellular p as antagonist of coronaviral replication. down-regulation of p is a major player in antiviral innate immunity [ ] . again, however, these matches remain tenuous. genbank has of the order of . billion nucleic acid sequences but a residue peptide can have , , billion sequences. while the krsfiedllfnkv motif remains favored by the author as a target at this time, identifying the amino acid residues in ace and the spike protein is important. it may for example involve conserved residues that are not together in a continuous sequence. while a conserved run of amino acid residues is sufficient to be on the list of candidates for an important site, important sites are not necessarily conserved runs of amino acid residues. here is shown that there is some conservation, but significant variation compared with rsfiedllfnkv. subsequences rsfiedllfnkv and pigagicasy…r discussed in ref [ ] as motifs associated with activation cleavage sites do not lie in the receptor (ace ) binding domain of the sars-cov- spike glycoprotein. the relationships between the whole spike protein and the receptor binding domains in pdb entries m and pdb vw are shown in the alignment below. note that the above receptor binding domain precedes the above motifs in the sequence. a three dimensional perspective is required for an appreciation of the important sequence features. in fig. , the pdb vw binding domain is on the right, bound to ace on the left. fig. of course, not all the receptor binding domain is interacting intimately with ace . the sections of the receptor binding domain that do interact with ace are also shown (underlined). to facilitate deeper analysis, the loops on the spike protein receptor binding domain were initially classified as loops a,b,c,d,e, and f in order of visual perspective, then joined into three subsequences , , that contain these loops. the part of the spike glycoprotein sequence that represents the receptor (ace ) binding domain can be shown by considering the proteins used in the two structural determinations m and vw in the protein data bank, shown below in an alignment made using clustal omega alignment. note that the above receptor binding domain precedes the above motifs in the sequence. the amino acids residues in bold and underlined font are the subsequences of ace that interact with the above spike protein ace binding domain loops, which are indicated above each subsequence. these include some longer range electrostatic interactions and potential solvent effects. those also in italics dkfnheaedlfy, dkfnheaedlfy and kgdfr have particularly strong interactions. as a reference perspective, the full sequence for ace as angiotensin-converting enzyme isoform x [homo sapiens] genbank entry xp_ . , is as follows. the part in the three dimensional structure above is in bold underlined font. note that at least in these particular experimental structures there is an involvement of "glycosylation-like" molecules. for example, in vw there are well localized n-acetyl-d-glucosamine, β-d-mannose, and , -ethanediol molecules that make significant interactions in a glue-like manner, and essentially "glue around the edges". however there no obvious indication of involvement of glycosylation in the main interior interaction face of the complex. the intimate interactions are proteinprotein, i.e. between amino acid residues. primarily, but not solely, ace and spike glycoprotein association involves interaction between the bent α-helix residues - (stiee…nyntn) of ace and an extended chain configuration, effectively a stretched loop, that runs from residue - (gfncy….ygqpt) of the spike glycoprotein and involves or ends in loops a, b, and f. in the case of ace interacting with the ace binding domain of the spike glycoprotein, one could in principle imagine blocking the ace as receptor with a mimic of the spike protein surface, or blocking the receptor binding site of the spike glycoprotein protein with a mimic of the ace receptor surface. in other kinds of infection the former is usually considered more plausible, but the latter would not interfere with normal function of ace and it is of course essentially the way in which immune system, and notably antibodies, work. possibly the main argument against this second choice it is that it is essentially equivalent to using antibodies raised against the spike protein, i.e. in effect, passive immunization. at the time of final writing, various news articles are drawing attention to potential use of the upper sequence or parts of it, which is that of the α-helix of ace (for example https://scitechdaily.com/mit-chemists-have-developed-a-peptide-that-could-block-covid- /). in the above "alignment", the helix contains residues and the extended chain below contains . they have similar length as is as expected for such structures. an αhelix has a rise of . Å per residue along its axis and there are in typical protein helices with turn variations that imply up to . Å. the β-strand or a similar extended chain in the spike glycoprotein that interacts with it has a rise of circa . Å per residue. this general geometry naturally puts the two sequences above in roughly the one-to-one spatial correspondence shown. note that this is not intended to be a sequence match representation; these chains have to interact. in that respect, there is a lack of charged residues (acidic and basic sidechains) in the extended chain of the spike glycoprotein in structure vw , although an aspartic acid (d) replaces the serine (s) in some strains, arginine (r) replaces asparagine (n) in others, and so on (e.g. see blastp alignments later below). a detailed backbone view is confusingly cluttered, but one may identify residues that interact at the boundary between ace and spike protein. all sidechains in the above spike protein subsequence gfncyfplqsygfqpt either make close contact or are likely to have some influence at the interface. it is perhaps useful to have the initial mental picture that, very roughly speaking, the planes of the peptide groups are tangential to the above α-helix surface, rather than constituting an extended chain that makes an edge-on approach. as even fig. suffice to makes clear, however, the extended chain, like any so-called extended chain in proteins in practice, is essentially a visible helix of larger pitch, resembling a very stretched-out α-helix, and is itself slightly supercoiled to wrap around the ace α-helix. in this case, this tends to follow the elbow or bend in the α-helix, staying roughly parallel to the local axis of the α-helix, so as to make intimate contact overall. if gfncyfplqsygfqpt is to be use as an epitope analogue, the cysteine (c) may be tested as a convenient linker to a carrier protein, otherwise replaced by serine (s) as a close analogue. as far as peptide antagonists are concerned, the difficulty with using the above sequences stiee…. and gfncy…. is that they are readily degraded by host proteases. this would not occur if the peptide is made entirely of d-amino-acid restudies. a retro-inverso peptide [ , ] is made up of d-amino acids in a reversed sequence to the subsequence which is seeks to mimic, and in the extended conformation assumes a side chain topology similar to that of the original native peptide, but with backbone n-h and carbonyl c=o groups interchanged. these are peptidomimetics of the subsequences sequences stiee…. in ace and gfncy….. in the spike glycoprotein respectively. the cysteine (c) in ….cnfg in the second molecule may be a convenient linker for an epitope for a vaccine but should be replaced by serine (s) in an antagonist. recall that the problem of having the backbone amide n-h and carbonyl c=o groups interchanged is that if, in the original section of backbone being mimicked, any n-h and c=o groups form a hydrogen bond with recipient and donor groups in the protein, those hydrogen bonds are now disrupted in the intended competitive antagonsist, e.g. they would be unstable n-h…h-n or c=o…o=c interactions. it would thus seem a significant advantage in using the ace mimic, because that is essentially an α-helix which uses up its backbone amide and carbonyl groups. however, retro-inverso α-helices are not typically found in the areas that have shown some degree of success [ ] , such as antigenic mimicry. it would nonetheless seem to be of value to test both of the retro-inverso peptides in laboratory studies. as to developing the above further both as the basis of a synthetic vaccine, or as a peptidomimetic, and as to the worth of extending the studies to small organic drug molecules, everything in the above depends on the extent to which gfncyfplqsygfqpt can produce escape mutations which might soon rend such solutions useless. as in the previous paper, we can relate this to variations of the above sequence bother in closer and much more distant relatives. as the following shows, using blastp we do not have to go very far from sars-cov- to find matches with only part of this sequence (coverage) and differences within that area of partial match: in the original wuhan seafood market pneumonia virus isolate wuhan-hu- , genbank id mn . , the subsequence in this regions is fncyfplqsygf, and the following are examples of coverage as found by blastp. the last of the above blastp at https://blast.ncbi.nlm.nih.gov/blast.cgi match results differs in total alignment by clustal omega at https://www.ebi.ac.uk/tools/msa/clustalo/, as follows, but of course this illustrates the high degree of variation that occurs as one proceeds on to coronaviruses less related to the wuhan seafood market isolate that is believed to be associated with the origin of covid- . phenylalanine (f) commonly immediately precedes many of these matching subsequences ncyfp… ncywp… etc., and the conservative substitution tryptophan (w) substitution for the second phenylalanine (f) is also very common, so it may be worth noting that fnctwp is a subsequence in the mammalian vomeronasal type- receptor on sensory cells within the main nasal chamber that detects heavy moistureborne odor particles, and fnctwp is also found in in dynein. many viruses require the minus-end-directed dynein motor complex transport on microtubules from cell surface toward the nucleus, and dynein in addition to kinesins for the transport toward the plasma membrane. however a direct connection to viral infection, while tempting, is far from obvious as to any mechanistic or evolutionary explanation. also, dynein nuclear shuttle transport may be less relevant to the coronavirus (an rna virus), but certainly rna viruses can rely on the dynein system (e.g. hanta virus uses it for endoplasmic reticulum-golgi intermediate compartment). at very least, the above illustrates the kinds of further, perhaps immediately less obvious, functions that the above ace binding domain of the spike glycoprotein, and the above motif, might have. within the coronaviruses, there is some degree of conservation that suggests that ncywplndygf is a segment for the virus to conserve, and a hint that fnctwpgf is the key part, but there are soon very clearly significant variations across coronaviruses of different hosts as we depart from the wuhan seafood market isolate compared with the rsfiedllfnkv motif in the s ' cleavage regions [ ] . small organic drugs design to mimic this section, or simply designed to designed to antagonize ace binding, are thus potentially susceptible to escape mutations, i.e. rapid appearance of drug resistance. binding studies with β-hydroxysteroid dehydrogenase type as model pharmacaphore. β-hydroxysteroid dehydrogenase type , which is inhibited by emodin, was an interim model pharmacophore of choice [ ] . at this point in the development of the argument for optimal targets for vaccines and therapeutic antagonists, the above target fits in as follows. while the above regarding ace binding must be kept in mind for antagonist development, as noted above the motif is not well conserved, and so could be prone to development of escape mutations, i.e. acquired resistance to vaccines and therapeutic antagonists. because of the dominant theme of an ace α-helix interacting with an extended chain loop of the spike glycoprotein, the structure of the interaction region is fairly easy to deduce for various sars strains, and there was as yet no obvious strongly recurrent theme of significant conserved residues that are discontinuous (i.e. not together in the same subsequence) that could be interacting closely with ace . at the same time, while emodin appears to act at the ace binding site [ ] , it remains of interest because there are complexities [ , ] as discussed in introduction section . . notably, the ace binding domain of the spike protein and the binding sequence discussed above might bind other human proteins and might have other functions that emodin and related compounds, related in the sense that they are at least consistent with pharmacophore features, might inhibit. a priori, the binding properties of emodin and the choice of β-hydroxysteroid dehydrogenase type as model pharmacophore could equally relate to the rsfiedllfnkv site, or some other site, or a mix of several. the case for interaction vomeronasal type- receptor and dynein discussed above was at best marginal, but these examples illustrated the diversity other kinds of functions, important to the virus, that might apply. in any event, any relations between emodin and similar and potentially related molecules remains of interest to impeding sars-cov- entry and the worse casualty would be the continuity of the story developed above, which is intended to illustrate a flow of reasoning in using the standard tools of bioinformatics. β-hydroxysteroid dehydrogenase type is interesting as accommodating a great variety of ligands at the steroid binding site, but not without a degree of specificity as to general features of the ligands, and so far these resemble those of potential sars-cov- therapeutics. keeping in mind the refutation principle [ ] that a pharmacophore (or contribution to a pharmacophore ensemble) the dehydrogenase is worthy of use until a new ligand or other information proves otherwise. so far pharmacophore validation here, i.e. a demonstration that it is a suitable pharmacophore model until proven otherwise, has been based circumstantially on emodin and compounds looking chemically similar to it, that are known in practice or argued theoretically to interact with sars virus entry in some way and bind at least weakly, experimentally or computationally, to β-hydroxysteroid dehydrogenase type [ ] . a review of compounds that are known experimentally to inhibit the dehydrogenase and known experimentally inhibit coronavirus entry, replication and maturation is being prepared. however, validation is also extensively based on a weaker but larger body of preliminary binding studies involving a variety of antagonists of coronavirus infection and very often other kinds of virus infection, that also bind at least very weakly to the dehydrogenase (see discussion on "very weakly" below). most of these, emodin-like and otherwise, were first found by q-uel knowledge gathering tools as used in the initial coronavirus study [ ] combined with "very early candidate selection rules" based on estimates of the mean binding strength of groups when binding well. note that a hydrogen bond worth about - kcal/mole is nonetheless effectively zero when binding well, because it is relative to binding to water. in contrast aromatic and large aliphatic and are worth circa - kcal/mole due to hydrophobic interactions which depend on being considered relative to water. there are more complex electrostatic and intramolecular entropic considerations beyond present scope, noting that at least preliminary study of the interaction with β-hydroxysteroid dehydrogenase type is the arbitrator. weak and very weak candidates are also considered because there may be multiple binding modes that will take a great deal of computer time to explore but which could yield lower binding fre energies. this produces a fairly "mixed bag" of compounds, based on the argument that viruses and coronavirus in particular may use each of its limited number of exposed or exposable sites for several purposes, and the coronavirus seems to be able to readily adjust to new mechanisms under the selective pressure of drugs and vaccines. the details of these molecules and studies are the subject of a further paper that will also discuss some interesting unifying themes. briefly, they include many names as hopedfor drugs against the coronavirus that appear in the news and internet discussion. it is convenient to see them as dividing into three classes (i) quinone-like. a "quinone" is any of a class of aromatic compounds having two carbonyl or ketone c=o functional groups in the same six-membered ring, though in "quinone-like" the author includes include many compounds resembling steroid fragments that may have many or just one carbonyl groups and several rings. this group includes , anthraquinone and derivatives that relate to many important drugs some with suggestive laxative and antiinflammatory functions, collectively called anthracenediones. they include ubiquinone as coenzyme q, and various shorter aliphatic chain forms hydroxyl-decylubiquinone and shorter aliphatic chain forms, laxatives such as dantron, emodin, and aloe emodin, and some of the senna glycosides, antimalarials such as rufigallol, antineoplastics used in the treatment of cancer, such as mitoxantrone, pixantrone, and the anthracyclines. caution is reuired in reading this list as a list of potential therapeutics, because anthraquinone derivatives rhein, aloe emodin or anthrone that lacks the methyl group, parietin (physcion), to some extent emodin itself, and chrysophanol extracted from cassia occidentalis are toxic and known to cause hepatomyoencephalopathy in children. it is a medical term effectively defined to cover lethargy, jaundice, and altered senses of children in india after consumption of cassia seeds. (ii) steroid-like. this group includes some plant steroid-like compounds such as carbenoxolone itself from liquorish (licorice) and others found in soy and sprouts. βestradiol (the endogenous ligand responsible for the growth and development of many tissues) diethylstilbestrol (a synthetic estrogen); -methyl-benz[a]anthracene- , -diol (a possible natural product from a common polyaromatic hydrocarbon) is also of interest. this group resembles group (i), but the concern for this group (ii) is that molecules like emodin that are known to antagonize viral or other infections are generally smaller, so it possible that a more relevant pharmacophore would sterically exclude a large steroid-like ring. (iii) quinine-like. these should not be confused with "quinone-like". quinine is an alkaloid derived from cinchona bark, used to treat malaria and as an ingredient of tonic water. a common feature is, nonetheless the abundance of aromatic and other rings that in the quinine-like case include nitrogen, so variously resembling pyrimidines, purines, histidine and tryptophan. this group is of current considerable interest as potential thereputics for covid- . of particular interest are chloroquine, theophylline, tavipiravir, baloxavir marboxil. some ace and ace inhibitors can be classified in this group. they are weak but not very weak binders as discussed later below. camostat, a serine protease inhibitor that has been considered as a potential therapeutic for covi- is convenient to place in this class because of its analogues but it does not itself include a nitrogen atom within a ring. there are several possible intriguing biological connections that will be discussed elsewhere. one might be briefly mentioned when considering combined therapeutic use of a member of each set. ubiquinone-like compounds can inhibit ubiquinone sites that work in concert with nadh and nadph cofactor sites. the latter in turn are often inhibited by the quinine-like members. many other above compounds generally bind "very weakly", though steroid-like compounds are strong binders and many quinine-like compounds are medium binders: these are discussed below. binding strength is of course a matter of degree. rt (where r is the gas constant and t the absolute temperature) is . at o k, i.e. circa . at biological temperatures, so kcal/mole is not significant above thermal noise. free energies of , , , and correspond to binding association constants of , , , and . the above free energies are usually expressed as negative, for the perspective from the associated system. considering that absolute values are much less reliable than relative values in this field, one might conservatively consider a binding energy of - . kcal/mole as worthy justification for keeping a compound on a list, if one does not wish to reject prematurely, and this seems reasonable if one still has in mind the refutation principle. this includes the mental picture that a model pharmacophore such as β-hydroxysteroid dehydrogenase type has a fairly large cavity which does not provide strong steric inhibition to the candidate ligands, but new evidence might show that a large ligand such as a steroid might be too big to fit the real target which the experimental data is describing. in other words, deficiencies in the pharmacophore model will start to show up when considering larger potential drugs. there is also the benefit of using β-hydroxysteroid dehydrogenase type as model pharmacophore that the present author has a data base of experimental and computational studies on compounds that bind to it. it should be stated, nonetheless, that any case for any common evolutionary relationship between this dehydrogenase and the spike protein binding receptor ace would be, at best, marginal. βhydroxysteroid dehydrogenase type has residues and ace has . there is a % identity match of amino acid residues in the region of best possible match of the dehydrogenase. there is also further % conservative substitution (clustal ':', i.e. conservation between groups of strongly similar properties with a score greater than . on the pam matrix). if taken alone, this would provide some basis for further exploring a relationship. admittedly, the conventional rule of thumb is that any two sequences are considered homologous if they are more than % exact amino acid residue matches, and strictly speaking this should apply over their entire lengths (this is discussed in chapter of ref [ ] and a brief review of standard tools is given in refs [ ] and [ ] ). nonetheless, caution is required because the % exact match criterion is well known to miss many easily detected homologs and - % is sometime found supported by evidence of evolutionary and functional relationship. for example, alignments between common cold and sars-cov- spike proteins already discussed above are in this range, but there is every good reason to believe a common ancestry, there is an overall conformational similarity, and essential features of some sequence motifs are preserved. there is some sense of comparable fold motifs with ace comprising two β-hydroxysteroid dehydrogenase type folds. the dehydrogenase is a bundle of some well defined, roughly parallel and antiparallel α-helices of up to about residues, interspersed by short β-pleated sheet strands. ace has some well defined, predominantly and very roughly parallel and antiparallel α-helices of up to about residues, interspersed by short β-pleated sheet strands. if there is a common evolutionary origin of β-hydroxysteroid dehydrogenase type and ace domains, it is distant, but it remains marginally possible and more extensive conformational analysis is underway. there is even less evidence of homology between β-hydroxysteroid dehydrogenase type and tmprss , although a serine residue is highly conserved in the catalytic site in both cases, which arguably makes it worthy of some initial exploration. tmprss comprises distinct cystine rich scavenger domain (residues - ) and a serine protease domain (residues - ). clustal o( . . ) multiple sequence alignment gives an exact match of amino acid of only . %. there are some grounds for further investigation in the future. there is also further . % conservative substitution (clustal ':', see above). for tmprss there are some suggestive short section matches in same order of appearance, e.g. aqyyys with ayyyys, vvshc with vvshc, lyhsd with lfhdd, and gilrqs with galrqe, which by some arguments slightly increase statistical significance. no significant conformational homology is apparent, so it is even more likely to be a chance match, and any argument for similarity between the proteins would be on the basis of some kind of convergent evolution based on certain common ligands, recalling again that the coronavirus might benefit from inhibiting an inflammatory response. preliminary studies on the panel of ligands discussed below suggest some degree of binding (- . kcal/mole and better, i.e. more negative) to both the above and β-hydroxysteroid dehydrogenase type , but these studies are still not fully complete and low energies may yet be obtained. the most substantial data base of results that can reasonably be considered final is in large part from the original studies [ ] . there carbenoxolone was automatically evolved (by automatic editing of its chemical structure) under the combined selective pressure of improve binding to βhydroxysteroid dehydrogenase type while avoiding significant match with compounds covered by all us patents [ ] , and subsequent docking and high grade molecular dynamic simulations were carried out on ibm's blue gene [ ] . many subsequent studies have, however been carried out on using krunch on a personal computer, because in the initial study it predicted well the blue gene results providing that the krunch binding energies obtained were corrected (or refined) to fit the blue gene results by a linear regression formula [ ] . recall again that it is on the basis of similarities between some compounds that antagonize sars virus entry and bind the steroid dehydrogenase, plus a notable commonality in the case of emodin (i.e. it binds both), that this model pharmacophore was chosen. since emodin and many other compounds of interest contain two or three or more aromatic rings, it is reasonable, at least as an initial tactic, that one may regard them as pieces of the steroid ring system and start them in the steroid binding cavity in the same "plane" as the steroid ring. in such a case involving minor variations as sidechains on the original steroid core, the way to make initial fit to using carbenoxolone as guide is obvious. however, the flat view of a steroid is misleading. the steroid ring system can "buckle" in various cis-trans combinations of bonds in the rings, and the longer sidechain conformations preferred on the basis of intramolecular energy are perhaps not obvious. although the rotation barriers for most of the transitions are clearly above the thermal energy (kt) energy conformations ( . kcal/mole), the associated energy demands for buckling of parts of the steroid ring system of variously and roughly . to . kcal/mole are less that the ligand-receptor binding the associated energy demands are below the gain in energy from ligand-receptor binding to the protein target. this is shown in the high grade quantum mechanical hartree-fock gamess calculations on blue gene in the original study [ ] but which have not been described in the literature. minimized energy conformers of steroid-like compounds considered are shown in fig. . calculations. such calculations in vacuo are less reliable for the charged species, but one may obtain a qualitative assessment from relative values and comparative uncharged species. these compounds are also shown more clearly from the chemist's perspective in two dimensional formula format, later below. fig. shows one of early analogues of carbenoxolone (the thioketone derivative cbos discussed later below) in the βhydroxysteroid dehydrogenase type steroid binding site. the particular interest in this compound is as follows. since in the original study [ ] krunch judged this as the strongest binder at - . kcal/mole, this compound was frequently used as a starting template for initial docking configurations when using krunch. this is even though (a) it is probably an unlikely choice for a chemist to use in practice because of likely oligimerization of the thioketone groups, and even though (b) corphos (also known as cortisol -phosphate, cortisol, phosphate, hydrocortisone- -phosphate or -hydrocortisonephosphoric acid) was the strongest at - . kcal mole when using instead the amber force field for molecular dynamics on ibm's blue gene [ ] . the thioketone still retained a reasonable binding energy of - . kcal/mole in the latter study, however, i.e. effectively the same binding strength within the state of the art. fig. does not of itself give details of any ligand-protein interactions (but see discussion in refs [ , ] ), although it does illustrate the tightest of fit. that is, except to the lower right of the thioketone ring of the ligand, which does appear to relate to genuine opportunities for additional groups to be added to carbenoxolone at that position. carbenoxolone and initial closely related derivatives derived in that study [ ] are shown in fig. , binding in the range, - to - kcal/mole. accuracy and limited realism of such methods does not really justify more precise statements on binding energy, and the classification of binding below is as strong, medium, and weak, but see ref [ ] for more detail on some of the compounds. authors variously consider binding energies - to - as a safe requirement for significant binding, but again this is subject to considerations of accuracy and almost all agree that it is only the relative values that are significant. note that while they are often interpreteted as estimates of binding free energy, the entropy component, particularly of the aqueous solvent and solute-solvent intercations, is difficult to estimate. experimental binding values of ligands in general in biological systems typically range from - to - kcal/mole, though over % lie in the range - to - kcal/mole. the thiioketone derivatives are more of theoretical interest in binding studies because in practice they may cause oligmerization organic compounds binding the pharmacophore. strong binders. from the original study [ ] . the estimated binding energy is in the range - to - kcal/mole. these were deigned from carbenoxolone with the intent to have a stronger or comparable strong binding (- kcal/mole). corphos, cbonring, and cbos bind at - kcal/mole. recall that the two peptide analogues of features of the spike protein of interest [ ] are as follows. so far, simulations only show these to be binding relatively weakly at - and - kcal/mole respectively, but these compounds are highly flexible with a theoretical internal in vacuo conformational entropy corresponding to about - . kcal/mole as discussed in theory section , show multiple binding modes and conformers on binding, and may not yet be complete. a high performance computer like ibm's blue gene used in the earlier drug design study [ ] would certainly help. in fig. is shown a set of compounds from the zinc data base [ ] , and most were identified from the original β-hydroxysteroid dehydrogenase type study [ ] and subsequent studies. these bind significantly by usual criteria, but more weakly. they are in the range found for the synthetic peptides of interest, but have much less conformational freedom. a small few not shown here did appear in the original higher grade studies, but the reasonable binding energies could not be reproduced for reasons that are not as yet clear. organic compounds binding the pharmacophore. medium binders. from the zinc data base. the estimated binding energy is in the range - to - kcal/mole. fig. shows some weaker binding results using krunch [ ] . great caution is required in drawing conclusions from the compounds in fig. . camostat is definitely of interest as an inhibitor of the ace protein to which the spike protein initially binds for cell entry and does seem effective in blocking entry [ ] , and similarly hepsin results are of related interest, e.g. as it is a potential alternative entry point. the ace inhibitors also looked initially interesting by virtue of certain similarities to the other potential ligands, and of course because of their binding in this theoretical study, but most of the traditional ace inhibitors are commonly viewed as not inhibiting ace . taking a drug such as valsartan that acts on ace might up-regulate ace , so facilitating virus entry, [ ] but emerging information is revealing a complicated picture: see discussion and conclusions. there are possible explanations that would still allow for competing with spike protein binding, but these seem somewhat unlikely. most probably, the binding is sufficiently weak that the normal substrate, and also the spike protein, displace it. aromatic group interactions may be important here [ ] . organic compounds binding the pharmacophore. weak binders. selected known drugs or proposals (caution: use of ace inhibitors might be counterproductive, see text). the estimated binding energy is in the range - to - kcal/mole. some consideration has been given to prediction of ligand binding site motifs, but so far these have proven essentially negative as regards interesting results that might shed any further light on the above, although some clues may well have been missed. binding sites are often comprised of conserved residues that are not contiguous (continuous in a sequence), which will require further and more detailed study, although subsequences of to amino acid residues in length are worthy of a quick preliminary study because they are commonly involved in ligand interactions. the matches involved in here as judged by blastp and clustal omega are not statistically significant, but one might think of weak matches as ligand binding site predictions in much the same way that one thinks of epitope predictions. in much of the present paper, the structure of emodin, carbenoxolone and related compounds have involved discussion of aromatic rings and hence phenylananine (f), tyrosine (y) and tryptophan (w) and more generally amino acid residues with hydrophobic character. very polar subsequences are also strong binders of charged ligands, or have a role for charged molecules or inorganic ions in some way. as far as such subsequences in the coronavirus spike protein are concerned, very polar charge-pattern motifs such drets and dreds are common in ligand binding some of the molecules that may be of interest as antagonizing sars entry, activation or replication in some way, in the present author's experience. specifically, motifs like this were of initial interest in the present project because sars-cov) nonstructural proteins have zinc finger motifs, and ret and especially red are common in prosite motifs at https://prosite.expasy.org/cgibin/prosite/prosite_search_full.pl, including zinc-finger motifs. this is not considered directly relevant to the spike protein but, for example respectively in genbank entry aia . and dreds and drets align with srldkv in three-way clustal omega alignment with srldk of the original wuhan spike protein sequence mn . and drldt of np_ . spike protein. however, these and many similar alignments also illustrate considerable sequence variation, and the weak matches are not close in the sequence to the subsequences of interest neither for coronavirus spike protein nor human proteins of potential discussed above. as far as ace is concerned, the closes match with drets and dregs is drkkps, but this weak match again lays well away from regions of current interest, e.g. in the sequence from the region that interacts with the spike glycoprotein. dteta and drfin do occur in the c-terminal half of human β-hydroxysteroid dehydrogenase type , but again these are expected to be coincidental matches. like the first vaccines [ ] therapeutics too have, of course, been drawn directly or almost directly from nature, until the late th century when chemical synthesis became a science, and only in the s did use begin to made of computers for rational drug design. the advantages of still seriously considering herbal remedies is that they tend to be tolerated by cells because they are produced in cells, they are already subjected to hundreds of years of human trial, are often economic solutions for bulk production, and are leads for further drug development and discovery. the principal non-peptide compounds considered above as possible therapeutics have such convenient and herbal sources. as reviewed previously [ ] , the herbal extract emodin is a convenient product extracted from rhubarb, buckthorn, and japanese knotweed, and several fungi. the previous paper [ ] also noted that emodin had certain molecular similarities with anti-inflammatory drugs such as carbenoxolone, derived from an extract, glycyrrhizic acid, from liquorish (licorice), that variously inhibit or are believed to inhibit human β-hydroxysteroid dehydrogenase type . the above does not guarantee efficacy of emodin carbenoxolone against sars-cov- , not least because even the emodin studies concerned sars-cov not sars-cov- , and the case for the dehydrogenase is circumstantial, but these and related substances are worthy of investigation. indeed, this paper has described a number of compounds that bind the dehydrogenase. importantly, however, recall that the weak binders that are also ace inhibitors may be more dangerous and promote infection because they upregulate ace [ ] . nonetheless, that situation is not resolved, as follows. the above considerations as to the action and possible usefulness are empirical observations that are largely independent of bioinformatics and molecular computation and they are even independent of whether the correct human protein targets discussed here are correct and relevant; however, it would be valuable to know what might relate ace and β-hydroxysteroid dehydrogenase type , and ideally also understand why both enzymes might "benefit" the coronavirus by interaction with them. also, this paper could not provide any evidence of an evolutionary relationship between these proteins, despite certain similarities, or with tmprss . so far, there is no obvious relationship with the dehydrogenase, and some other studies by the author on other transmembrane serine proteases do not, as yet, suggest any relationship. without such connections, the dehydrogenase can only be considered as a rather arbitrary model pharmacophore. as such, it is possibly meritorious as correctly representing an ensemble of multiple targets, but that may be fortuitous, and hence only to be used until refuted by evidence. nonetheless, possible clues as to mutual relevance of these human protein targets might come noting their tissue distribution and considering how this may relate to their biological role. as regards ace , its mrna is known to be present in virtually all organs. studying sars entry into human cells, hamming et al. [ ] considered their most remarkable finding to be the substantial surface expression of ace protein not only on lung alveolar epithelial cells but also enterocytes of the small intestine, as in arterial and venous endothelial cells and arterial smooth muscle cells in all organ studied (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain). there is the attractive prospect that several many herbal remedies considered as laxatives interact with ace and inhibit sars-covid- entry. recall that emodin is an antagonist of both ace [ ] [ ] [ ] and β-hydroxysteroid dehydrogenase type [ ] . in the past, βhydroxysteroid dehydrogenase type has been considered to be distributed mainly in the human liver, with no detectable levels in the intestine or kidney, mostly membranebound and retained in the liver microsomal fraction [ ] . this was not however the finding of bruley et al. ] . they found it to be highly expressed in glucocorticoid target tissues including liver and notably the lung, and modest levels in the brain. it was also found in modest levels in adipose tissue where it is of medical interest that selective increase expression occurs in obese humans and rodents and is likely to be of pathogenic importance in the metabolic syndrome [ ] . lung expression appears to be managed differently: a new promotor that the authors discovered and called p predominated in lung while the previously known promotor predominated in liver, adipose tissue, and brain [ ] . researchers therefore need to sort out an intriguing web of information. it is possible that a complex web of laxative and anti-inflammatory effect may provide clues by somehow relating to the body's attempts to reject and eject viruses of this kind, and the virus's attempts to resist. it is well known that some covid- patients complain of stomach upsets and diarrhea. to recapitulate the essential themes in terms of action in the alimentary tract, recall again that emodin had earlier been shown by several groups of researchers (e.g. ref [ ] ) to inhibit sars-cov entry into cells (apparently initially by binding ace ), and emodin is taken as a herbal laxative. licorice has, conversely, been sometimes taken as a soother for alimentary disorders, and carbenoxolone has been used commercially in the past specifically to treat peptic ulcers. intriguingly, carbenoxolone is also known to influence the renin-angiotensin system involving ace , so at least there appears to be a connection in terms of networks of physiological control. as noted above, while traditionally β-hydroxysteroid dehydrogenase type has been thought of as a liver enzyme, many researchers have indicated that both ace and the dehydrogenase are available in both lung and intestinal tract (e.g. refs [ ] [ ] [ ] ). this all hints also that some of the other laxatives that work in a similar stimulatory way might block viral entry on ace and perhaps other targets, and should be explored. of course, absolutely nothing should be done by patients without physician direction, because dosages are difficult matters (not least in herbal products) and there are potentially serious side effects on such as salt balance and blood pressure, and some might cause birth defects, all potentially worse than covid- would be, for most people. but more worryingly still, the situation is not settled, and physicians and patients could take action in the wrong direction. gurwitz [ ] has emphasized that the picture is even more complex. he examined reports from china suggesting that a mechanism of production of lung injury during the viral infection may be due to excess free angiotensin-ii, which might be displaced from ace by the sars virus particles. if so, then increasing the amount of ace- could be desirable and administering angiotensin receptor antagonists could beneficially upregulate the production of ace- . it now becomes important to examine medical records of patients who have, and who have not, been infected by sars-cov- , with a particular eye on who is, and who is not, taking ace inhibitors. as noted in section . , merrifield developed first solid phase peptide synthesis on crosslinked polystyrene beads in [ ] . somewhat like natural compounds discussed above, peptides and petidomimetics are potentially important first steps in more detailed rational design of small organic molecules convenient as traditional "in a pill" drugs. however, as in the present paper, the ability to propose specific peptides and peptidomimetics does depend on bioinformatics, and benefits from some computational chemistry. note that in this case, one is thinking largely not of screening natural products, but now considering truly novel molecules using theoretical methods because they do not yet exist. the variations in the krsfiedllfnkv motif that might be appropriate to synthetic vaccine and peptidomimetic antagonist design suggest the following where the amino acid residues in square brackets [ ] represent alternatives. (g?) means an optional glycine insertion. the above is also valid as a regular expression, i.e. a match query in operating systems and software. more generally and colloquially, (positive charge)-(optional glycine)-serine-hydrophobic-hydrophobic-glutamate-aspartatehydrophobic-leucine-phenylalanine-(hydrophilic or alanine)-lysine-valine considerations at the n-terminus and c-terminus to design a synthetic vaccine, and the retro-inverso approach for a peptidomimetic agonist, are described in ref [ ] . other variations appear as the strain becomes more distant; there is not a universal clear indication of any sharp point of departure, although the above glycine (g) insertion is evidence that a significant jump can happen. one may therefore ask what variations should be included. with the emphasis on sars-cov- , only closely and medium distance relatives are of interest, with the purpose of prevent mutations that escape from vaccines and antagonists, and elude diagnostics. as far as sars-cov- is concerned, krsfiedllfnkv is a satisfactory basis because a large number of coronaviruses significantly different from sars-cov- preserve it, or in a few cases have very conservative substitutions. it may well be that the fact that residues are, for example, hydrophobic or positively charged is sufficient to for the approach to be applicable to other mammalian coronavirus diseases, if successful for the above basic motif form. attempting to tackle the common cold is not a priority. in other words, it may well be that an immune response against krsfiedllfnkv will also illicit a response against the motif variants, providing of course that krsfiedllfnkv elicits a response itself. it remains that this motif is one of very few subsequences that still recognizable when moving on to rather distantly related coronaviruses such as those of the common cold. one feature of both figs. to is of course the constant appearance of aromatic rings, and this is also noticeable in many of the studies of antagonist's against sars virus binding and activation. of course, the aromatic (i.e. benzene) ring makes copious appearance in many pharmaceutical agents in any event, because they provide rigid scaffolds for added groups supported by many long-established recipes for synthesis. the compounds in figs. to should be distinguished from those such as lopinavar, ritonar, promazine and particularly niclosamide that have been explored for sars viruses in the past, because these are targeted by drug designers against the sars virus own protease required for maturation of the assembling virus. nonetheless, some of these do have a visual similarity to the compounds in fig. , particularly niclosamide (which is normally a medication used to treat tapeworm infestation). also, in the present case, prevalence of aromatic rings in figs to is hardly surprising, since carbenoxolone and derivatives shown in fig. were the starting point for their evolution or selection from the zinc data base. nonetheless, there is, in principle, nothing to constrain the evolution to aromatic chemistry [ ] and later unpublished studies did produce molecules departing from aromatic chemistry. however, these bound relatively weakly. with the possible importance of aromatic rings and avoidance of escape mutations by the coronavirus in mind, a question is whether occasional loss of phenylalanine (f) from the krsfiedllfnkv motif discussed above contests the tentative hypothesis that the peptidomimetic candidates derived from krsfiedllfnkv bind to a similar site as the smaller organic ligands considered here, because of two phenylalanine residues (f) in the original motif and a tendency to several benzene rings in the case of organic ligands. the answer is: perhaps. there seems to be a need to have one aromatic ring present in the motif, and no match with a coronavirus in genebank was detected by the author by blast-p using queries with no phenylalanine (f), e.g. rsaiedllldkv, rsaiedllidkv, rsaiedlladkv, rsaiedllmdkv, rsaiedllwdkv, and rsaiedllydkv as queries, though as also noted above, the search has not been exhaustive. it would not be too contradictory to any of the current main hypotheses if some examples were found. the fact that tyrosine (y) does not seem to readily substitute here for phenylalanine (f) (from which it differs only by a hydroxyl -oh, i.e. phenolic group) suggests an important hydrophobic feature of the pharmacophore at that point. of course, many or most drug-like molecules contain at least one aromatic ring and this is almost certainly because they can form especially strong stacking interactions in the binding site. one very relevant report in the same month of writing the present paper emphasizes that the use of protein and other fragments to characterize binding pocket and determine the strengths of ligand-protein interactions is common in both a computational and experimental approach, and that aromatic interactions are both strong and need special attention [ ] . because of resonance and the special nature of the π orbitals, the strength of stacking is best calculated using high level quantum mechanical approaches, not empirical force fields [ ] . however, as these calculations are performed in vacuum, solvation properties are neglected, and this led to the proposal of a grid inhomogeneous solvation theory (gist) to describe the properties of individual heteroaromatics and complexes; this gave good correlation for the estimated desolvation penalty and the experimental binding free energy, and prediction of binding sites [ ] . a main conclusion is that peptide krsfiedllfnkv remains of special interest as well conserved across coronaviruses. other sites and other proteins of the virus may, of course, emerge as the solutions to this formidable problem. all aspects of the virus must be considered. however, even the ace binding domain is significantly more prone to accepted mutations. the recurrence of the core features of the krsfiedllfnkv motif over so many diverse species reminds us of zoonotic origins, and it might be recalled that jenner, the inventor of vaccination, consider that many and perhaps all plagues of mankind might ultimately be of animal origin [ ] . the molecular biology of coronaviruses genomic characterization and epidemiology of novel coronavirus: implications for virus origins and receptor binding, www.thelancet preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the sars-cov- ( -ncov, covid- ) coronavirus preliminary bioinformatics studies on the design of synthetic vaccines and preventative peptidomimetic antagonists against the wuhan seafood market coronavirus. possible importance of the krsfiedllfnkv motif structure, function, and evolution of coronavirus spike proteins cleavage of the sars coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro published activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites entity containing chain a, b, c sars-cov spike glycoprotein peptides corresponding to the predicted heptad repeat domain of the feline coronavirus spike protein are potent inhibitors of viral infection the heptad repeat region is a major selection target in mers-cov and related coronaviruses coronavirus escape from heptad repeat (hr )-derived peptide entry inhibition as a result of mutations in the hr domain of the spike fusion protein peptides as 'drugs': the journey so far prediction of hiv vaccine synthetic peptides related to hiv-env proteins, patent patent: ep a synthetic polypeptides derived from the hiv envelope glycoprotein.patent : eu fragments of prion proteins, patent ep a fragments of prion proteins from zika to flu and back again. cavirc (report of the caribbean anti-virus informatics research center computer aided peptide and protein engineering the epsitron concept of peptide and protein engineering. applications of computer-aided molecular design an expert system for protein engineering. its application in the study of chloramphenicol acetyltransferase and avian pancreatic polypeptide studies on rationales for an expert system approach to the analysis of protein sequence data -preliminary analysis of the human epidermal growth factor receptor modélisation des polypeptides: application aux oligopeptides vaccinants" inra/euc rapport programming environment for the chemical pharmaceutical and biotechnology industries computer aided design of biomolecules: the big hammer approach pro_ligand: an approach to de novo molecular design. . application to the design of organic molecules pro_ligand: an approach to de novo design. . design of novel molecules from molecular field analysis (mfa) models and pharmacophores pro_ligand: an approach to de novo molecular design. . a genetic algorithm for structure refinement pro_ligand: an approach to de novo molecular design. . application to the design of peptides receptor pharmacophore ensemble (repharmble): a probabilistic pharmacophore modeling approach using multiple protein-ligand complexes suggestions for a web based universal exchange and inference language for medicine implementation of a web based universal exchange and inference language for medicine. sparse data, probabilities and inference in data mining of clinical data repositories studies in using a universal exchange and inference language for evidence based medicine. semi-automated learning and reasoning for pico methodology, systematic review, and environmental epidemiology studies in the extensively automatic construction of large odds-based inference networks from structured data. examples from medical, bioinformatics, and health insurance claims data extension of the quantum universal exchange language to precision medicine and drug lead discovery. preliminary example studies using the mitochondrial genome the gor method for predicting secondary structure in proteins"in 'prediction of protein structure and the principles of protein conformation synthesis of angiotensin-converting enzyme (ace) inhibitors: an important class of antihypertensive drugs doppelganger proteins as drug leads chemical synthesis and activity of d, superoxide dismutase pseudoproteins: non-protein protein-like machines peptide and protein mimetics by retro and retroinverso analogs simulation of water behaviour around the dipeptide n-acetylalanyl-n'methylamide some views of solvation effects in the light of a monte carlo simulation drug discovery using very large numbers of patents: general strategy with extensive use of match and edit operations an overview of bioinformatics tools for epitope prediction: implications on vaccine development advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for pseudomonas aeruginosa candidate targets for immune responses to -novel coronavirus (ncov): sequence homology-and bioinformatic-based predictions receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars , jvi accepted manuscript posted online dissecting and designing inhibitor selectivity determinants at the s site using an artificial ala protease (ala upa) different residues in the sars-cov spike protein determine cleavage and activation by the host cell protease tmprss cleavage specificity analysis of six type ii transmembrane serine proteases (ttsps) using pics with proteome-derived peptide libraries emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction emodin inhibits current through sarsassociated coronavirus a protein novel inhibitors of severe acute respiratory syndrome coronavirus entry that act by three distinct mechanisms emodin, a natural product, selectively inhibits β-hydroxysteroid dehydrogenase type and ameliorates metabolic disorder in diet-induced obese mice hydrophobicity and hydrophilicity of steroid binding sites exploring the papillomaviral proteome to identify potential candidates for a chimeric vaccine against cervix papilloma using immunomics and computational structural vaccinology prediction and validation of potent peptides against herpes simplex virus type via immunoinformatic and systems biology approach a cascade deep forest model towards the prediction of drug-target interactions based on hybrid features cytomegalovirus infection database: a public omics database for systematic and comparable information of cmv camp: a tool for anti-cancer and antimicrobial peptide generation mechanism & inhibition kinetics of bioassay-guided fractions of indian medicinal plants and foods as ace inhibitors zinc database misdirected antibody responses against an n-terminal epitope on human rhinovirus vp as explanation for recurrent rv infections cooperative molecular and cellular networks regulate toll-like receptor-dependent inflammatory responses p down-regulates sars coronavirus replication and is targeted by the sars-unique domain and pl pro via e ubiquitin ligase rchy sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor rapid response (comment): sars-cov- , hypertension and ace inhibitors stacked -solvation theory of aromatic complexes as key for estimating drug binding the jenneration of disease: vaccination, romanticism, and revolution tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis β-hydroxysteroid dehydrogenase human tissue distribution, selective inhibitor, and role in doxorubicin drug metabolism and disposition a novel promoter for the β-hydroxysteroid dehydrogenase type gene is active in lung and is c/ebpα independent angiotensin receptor blockers as tentative sars-cov- therapeutics drug discovery using very large numbers of patents. general strategy with extensive use of match and edit operations towards automated reasoning for drug discovery and pharmaceutical business intelligence towards new tools for pharmacoepidemiology the concept of novel compositions of matter. a theoretical analysis suggestions for a web based universal exchange and inference language for medicine hyperbolic dirac nets for medical decision support. theory, methods, and comparison with bayes nets popper, a simple programming language for probabilistic semantic inference in medicine suggestions for a web based universal exchange and inference language for medicine. continuity of patient care with pcast disaggregation split-complex numbers and dirac bra-kets implementation of a web based universal exchange and inference language for medicine. sparse data, probabilities and inference in data mining of clinical data repositories interesting things for computer systems to do: keeping and data mining millions of patient records, guiding patients and physicians, and passing medical licensing exams data-mining to build a knowledge representation store for clinical decision support. studies on curation and validation based on machine performance in multiple choice medical licensing examinations studies of the role of a smart web for precision medicine supported by biobanking studies in using a universal exchange and inference language for evidence based medicine. semi-automated learning and reasoning for pico methodology, systematic review, and environmental epidemiology studies in the extensively automatic construction of large odds-based inference networks from structured data. examples from medical, bioinformatics, and health insurance claims data • this paper "drills down" into the studies of the author's previous covid- paper.• designing vaccine and drugs must seek to avoid escape mutations.• subsequence krsfiedllfnkv seems recognizable across many coronaviruses.• the ace binding domain is a target, but shows variation.• a steroid dehydrogenase is argued to remain an interesting model pharmacophore.this paper is provided to the community to promote the more general applications of the thinking of professor paul a. m. dirac in human and animal medicine in accordance with the charter of the dirac foundation , to emphasize the advantages and simplicity of the basic form of the hyperbolic dirac net, to encourage its use, and to propose at least some of the principles of the associated q-uel, a universal exchange language for medicine, as a basis for a standard for interoperability. these mathematical and engineering principles are used, amongst many others in an integrated way, in the algorithms and internal architectural features of the bioingine.com, a distributed system developed by ingine inc. cleveland, ohio, for the mining of, and inference from, very big data for commercial purposes. immediately prior to joining ibm in he was hired as principal scientist at mdl information systems in california to help put together the technology for the multimillion sale of a bioinformatics system to the holding company forming craig venter's celera genomics that produced the first draft of the human genome. prior to that, he was cso of gryphon sciences (later gryphon pharmaceuticals) in south san francisco, california, a bio-nanotechnology ultrastructural chemistry start-up largely held and then acquired by smithkline beecham. before moving to the us, barry was the scientific founder of proteus international plc in the uk, designing and leading the development of the prometheus expert system and its underlying global expert system, bioinformatics and simulation language for drug, vaccine, and diagnostic discovery. it sold for the equivalent of $ . million to the pharmaceutical industry in the mid- s. at proteus, he also led the team that used the above expert system to invent and patent several diagnostics and vaccines including the mad cow disease diagnostic subsequently marketed worldwide by abbott. he has some scientific publications including some patents and two books: "the engines of hippocrates. from the dawn of medicine to medical and pharmaceutical informatics" robson and baek, , wiley, pages)" and "introduction to proteins and protein engineering" (b. robson and j. garnier, garnier, , , elsevier, pages). he has contributed to several reports to governments including panels of the national innovation initiative including "innovate america" published by the council on competitiveness, washington d.c. ( ) as a whitepaper to the president of the united states. for five years, barry was a nature "news and views" correspondent on biomolecules. key: cord- -gf asni authors: galdiero, stefania; falanga, annarita; morelli, giancarlo; galdiero, massimiliano title: gh : a milestone in understanding the many roles of membranotropic peptides date: - - journal: biochim biophys acta biomembr doi: . /j.bbamem. . . sha: doc_id: cord_uid: gf asni here, we review the current knowledge about viral derived membranotropic peptides, and we discuss how they may be used for many therapeutic applications. while they have been initially discovered in viral fusion proteins and have been involved in the mechanism of viral entry, it is now clear that their features and their mode of interaction with membrane bilayers can be exploited to design viral inhibitors as well as to favor delivery of cargos across the cell membrane and across the blood–brain barrier. the peptide gh has been extensively used for all these purposes and provides a significant contribution to the field. we describe the roles of this sequence in order to close the gap between the many functions that are now emerging for membranotropic peptides. over the past few decades peptides have progressively achieved increased value in drug design and pharmaceutical delivery. moreover, great interest has been dedicated to the identification of peptides as drug candidates. the number of peptides in the pharmaceutical industry is continuously growing and about % of the entire drug market is represented by peptide based drugs [ , ] . bioactive peptides can be derived from natural sources or can be discovered through rational engineering, high-throughput screening, or structure-based design starting from defined protein regions [ ] . among the many peptides playing a relevant role in biology, some show a high propensity for binding to lipid membranes due to their simultaneous hydrophobic and amphipathic nature. this class of hydrophobic peptides is characterized by the presence of unusual conspicuous amounts of alanine and glycine residues and sometimes also prolines. such a degree of ala/gly content is uncommon for hydrophobic domains such as signal sequences and transmembrane anchors; in fact, their presence may account for the intrinsic conformational flexibility which is a typical feature of membrane interacting peptides. also aromatic residues are generally present and dominate the interactions that take place at this unique physical-chemical environment of the water-membrane interface [ ] . the favorable interactions of aromatic side chains with phospholipid moieties located at the membrane interface contribute to the insertion of the peptide into the bilayer. amphipathicity is a key feature of these peptides. the term amphipathicity generally refers to molecules with both hydrophilic and hydrophobic faces [ ] . peptides can be amphipathic in their primary structure or secondary structure. primary amphipathic peptides correspond to the sequential assembly of a domain of hydrophobic residues with a domain of hydrophilic residues divided by a spacer domain; while secondary amphipathic peptides are generated by the conformational state which allows positioning of hydrophobic and hydrophilic residues on opposite sides of the same molecule. in particular, amphipathic, hydrophobic peptides present one face with large and aromatic residues and the other with small residues such as ala/gly. this distribution of amino acid residues facilitates the membrane interaction and peptide insertion into the bilayer [ ] . conformational polymorphism plays a key role; in fact, the ability to shift from random to α/β conformations as a consequence of membrane composition and peptide concentration has emerged as a common structural pattern for this class of peptides [ ] . there are several types of membrane active peptides which can be roughly divided in antimicrobial peptides [ ] , viral peptides [ ] and cell penetrating peptides [ ] . although very different in primary sequence one from the other, it may be hypothesized that their common physical features could result in a shared mechanism of action and essentially determines the many roles that they can play in nature. among the hydrophobic peptides with a propensity for membrane binding, characterized by a high interfacial hydrophobicity or amphipathicity, the ones derived from enveloped virus glycoproteins are attracting considerable attention. these peptides can interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on membranes and/or fusion proteins and are, thus, critical for both fusion and entry. viral glycoproteins undergo conformational changes as a consequence of either low endosomal ph or receptor binding which leads to the exposure of hydrophobic peptides, loops or patches, which then interact with and destabilize one or both the opposing membranes. crystallographic data on the post-fusion structures of viral fusion proteins have allowed the identification and characterization of three different classes [ , ] . class i fusion proteins are characterized by trimers of hairpins with a central α-helical coiled-coil structure and have been identified in orthomyxoviruses, paramyxoviruses, retroviruses, filoviruses and coronaviruses [ ] [ ] [ ] [ ] [ ] . class ii fusion proteins are present on viral envelopes as pre-fusion dimers which convert into post-fusion trimers of hairpins composed of β structures and have main representatives in the flaviviridae and togaviridae families [ , ] . class iii fusion proteins are characterized by a central α-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated β-sheet similar to class ii fusion proteins and members are present in herpesviridae and rhabdoviridae families [ ] . despite several differences in the mechanism of entry elicited by the three classes of fusion glycoproteins, they all induce membrane fusion in a similar manner through the formation of an analogous hairpin structure which allows fusion peptides to insert into cell membranes and to drive membrane destabilization. thus, during the viral entry process, the hydrophobic surfaces that become exposed are characterized by somewhat variable but at the same time detailed physical characteristics which include the size, shape and secondary structure of exposed hydrophobic patches, as well as the nature of the neighboring polar or charged residues. the wimley-white interfacial hydrophobicity scale (wwihs) is an experimentally-determined free energy scale that calculates the propensity of individual amino acids in peptide sequences to partition from water into a phosphatidylcholine interface [ , ] and has been effectively used to identify fusion peptides in viral glycoproteins. the hydropathy analysis allows calculating a hydrophobicity score along the sequence of a protein, identifying segments with a propensity to interact with membrane interfaces. wwihs values are calculated assuming random coil peptides partitioned into the bilayer interface; the values are minimum possible values and the Δg may get more favorable if peptide binding also promotes an increase in secondary structure [ , ] . the interfacial helical hydrophobic moment (ihhm) is a further physico-chemical factor that is important for membrane interactions and secondary structure formation of peptides bound to membrane interfaces. the ihhm describes the degree to which a peptide sequence would have segregated hydrophobic and hydrophilic faces if it folded into an α-helix [ ] . a peptide with a large ihhm can interact strongly with membranes as a helix due to partitioning-folding coupling [ , ] even with a wwihs score that is not positive overall. the presence of the fusion peptide within the ectodomain exposed to the aqueous phase is a feature shared by all viral fusion proteins and constitutes an absolute requirement for their fusogenic activity. fusion peptides are typically - residues long and potentially fold into amphipathic helices and are rich in glycines and alanines, providing them a high degree of conformational flexibility. thus, their structure is polymorphic and strongly dependent on the environment. the fusion peptide of influenza virus, for example, has been observed in random coil, α-helical and β-sheet conformations in different environments [ ] (fig. ) . it has been proposed that all three forms have some physiological relevance; the peptide may be unstructured in solution on the way to the target membrane; it may be helical at low concentrations but may self-associate in β-sheets at higher concentrations in the membrane interface. the fusion peptide of hiv also undergoes conformational transitions; it adopts α helical or β sheet structures depending on concentration, lipids and ionic conditions [ ] . the helical form of the influenza fusion peptide is probably a key determinant to promote fusion. the structure of the peptide in membrane shows a kink which separates the n terminal and the short c terminal helices which together form a boomerang shaped structure [ ] . both helical arms are amphipathic with bulky hydrophobic residues facing the membrane interior. the conserved n-terminal glycine residue is critical for fusion and for the correct structure of the peptide inside the membrane; in fact, when it is mutated to a valine the n-terminal helix is partially unwound and the fusion peptide is inactive [ ] . actually, there are numerous studies demonstrating that a delicate balance between α and β structures, is essential for membrane fusion and is influenced by environmental conditions such as ph, ionic strength, peptide sequence, presence or absence of divalent cations, cholesterol content and also by the lipid/peptide ratio. for instance, studies performed on the ebola fusion peptide, show that the conformational transition from an α-helix to a β-sheet is induced by a change in the peptide to lipid ratio in the membrane [ ] . at low peptide concentration in lipids, it is essentially an α-helix; while as the local peptide concentration increases in the membrane, the proportion of α-helix drops off in favor of a mainly antiparallel β-sheet structure. this concentration dependent effect on peptide conformation might be of biological relevance [ ] . the functional meaning of the conformational polymorphism is unclear, although it is believed to be fundamental to enable backbone reorientation of the fusion protein; therefore, the ability to insert at various levels might be required for the evolving of final stages of the fusion cascade [ ] . independently from the principal conformational organization, the degree of insertion plays a key role for inducing membrane fusion. it has also been hypothesized that [ ] fusion domains first assemble as β-sheets on the surface of the membrane and later convert into α-helices to complete fusion. aromatic residues are generally present in fusion peptides and may help in overcoming the energy cost of peptide bond partitioning into membranes. the interactions with phospholipid moieties located at the membrane interfaces may also help in stabilizing the insertion into just one leaflet of the bilayer. the initial interaction with the external leaflet is thought to generate elastic stresses which drive to bilayer fusion, helping to overcome the hydration repulsion forces between approaching bilayers by orienting the poorly solvated face toward the external medium [ ] . the asymmetric insertion into one membrane monolayer may promote expansion of the polar head region and determine a curvature stress onto the overall lipid bilayer; the created bulges that protrude from the membrane can facilitate the formation of lipid contacts between fusing bilayers [ ] . particular attention has also been devoted to the effect of additional membranotropic sequences on the overall fusogenicity. the presence of additional fusogenic sequences was evidenced in sendai f [ ] , measles f [ ] , sars-cov s [ ] , hepatitic c virus e and e [ ] , dengue e [ , ] and herpes virus gb and gh [ ] [ ] [ ] . the idea that a single fusion peptide is the solely responsible for the complete membrane fusion event has been substituted by the assumption that a concerted action of different membranotropic regions is necessary for membrane interacting/perturbing activity. as a matter of fact, also membrane proximal regions (pre-tm) play a key role in fusion [ , [ ] [ ] [ ] . the pre-tm domains are particularly rich in aromatic residues which enable them to insert into the membrane interface. effective therapeutics against enveloped viruses are still scarcely represented. a few drugs have been developed against hiv, influenza virus, hepatitis virus and a few other viruses but they are still not ideal and in some cases have proved to induce resistance [ ] . as a consequence, for most enveloped viruses, there are no effective therapies and entry inhibitors represent an interesting and underutilized target. peptides with a propensity for membrane binding can also interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on cell membranes and/or fusion proteins. as recently reviewed by badani et al. [ ] , there are many peptide inhibitors that are somewhat hydrophobic and/or amphipathic with a propensity to bind to bilayer membrane interfaces and other hydrophobic surfaces. it is not known whether membrane binding directly affects viral fusion, or whether interaction with the fusion protein itself is an absolute requirement for entry inhibition. it is widely accepted that membrane binding of an inhibitory peptide will greatly increase the effective concentration of the peptide close to the fusion protein, indicating that the interaction with membrane and the interaction with the fusion protein may be effectively coupled [ ] . as a matter of fact, the potential of numerous fusion peptides and/or membranotropic peptides derived from proteins of enveloped viruses as entry inhibitors has been widely described in literature [ ] [ ] [ ] [ ] . the accepted view is that the inhibition of infectivity may be due to the formation of inactive aggregates between the fusogenic stretches present in both the viral protein and the synthetic peptides. these aggregates are formed as a consequence of their ability to oligomerize or to mimic the modes of binding of their original domains in their partner protein. it has been hypothesized that they stabilize a pre-fusion intermediate and prevent merging of the bilayers. it is now evident that several domains are essential for membrane fusion and thus peptides involved in the fusion mechanism may all interfere with the intramolecular interactions between the several domains and may represent interesting targets for the design of entry inhibitors. thus, membrane physical properties could be critically important to the events that drive viral entry and it is conceivable that peptides interfere with the function of viral fusion proteins by changing the physical chemistry of the membrane itself by direct interaction. many laboratories are working to unravel the mechanism of action of viral membranotropic peptides and several hypotheses have been proposed. many studies suggest that multiple mechanisms may take place simultaneously. indirect ways in which interfacial binding peptides can affect viral entry have also been hypothesized. selfoligomerization of membrane embedded fusion peptides has been proposed to be responsible of inhibition [ , ] . the most studied case is that of hiv, where the inhibition has been attributed to the formation of structurally defined oligomeric complexes [ , ] ; while mutants with a lower helical content and tendency to self-associate into β-sheets [ ] are able to inhibit membrane fusion at various stages. it is interesting to note that there are clinical studies on a peptide called virip, which is designed as an inhibitor of the hiv fusion peptide [ , ] . this sequence was able to block hiv- infection by targeting gp fusion peptide [ ] and optimized versions of this sequence proved to be as potent as inhibitors targeting the coiled coil sequences and moreover were devoided of cellular toxicity. a -day monotherapy clinical trial enrolling hiv- infected patients [ ] showed that the drug can be well tolerated by patients and reduces their plasma viral load. its identification and clinical evaluation represent the first proof of concept that membranotropic sequences could suppress viral replication in infected individuals and have potential clinical effectiveness. the hypothesis that peptide entry inhibitors act by a physicalchemical interaction with hydrophobic surfaces exposed during the fusion process suggests that this novel approach may be a general rule; moreover, instead of focussing on the structure-based design, it would be possible to design novel hydrophobic/amphipathic inhibitors which could be easily made protease resistant by the introduction of nonnatural or d-amino acids. the membrane bilayer represents a semi-permeable barrier, defining the interior of an individual cell; its existence confers cells their potential to survive and function properly. nevertheless, crossing of the cellular membranes remains one of the major obstacles for the proper delivery of therapeutics [ , ] . the lipophilic nature of biological membranes restricts the direct intracellular delivery of most compounds; whereas small molecules and ions can diffuse across the bilayer, larger molecules are generally excluded from simple diffusion into the cell. the differing hydrophobicity/hydrophilicity of the lipid membrane renders the transfer across this barrier extremely difficult due to differences in solubility. notwithstanding the therapeutic potential of a number of novel molecules, their pharmaco-distribution properties hamper the possibility to reach the stage of pharmaceutical preparations and stimulate industrial interest; in fact, these molecules need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm or onto individual organelles. it is, thus, evident that the therapeutic potential of a drug is largely dependent on the development of delivery tools able to selectively and efficiently carry it to target cells with minimal toxicity. the translocation across the membrane is by far less well understood than the binding step. there is a significant similarity in the physico-chemical parameters between membrane partitioning peptides and membrane translocating peptides [ ] . a novel intriguing hypothesis is that hydrophobic peptides that partition into membranes may also be able to cross cell membranes and enter cells. therefore, these peptides may also cross endothelial layers in vivo, including the blood-brain barrier [ , ] . delivery across cellular membranes involves several membrane reorganization processes such as transient permeabilization of the cell membrane, which are similar to the ones involved in the entry of viruses. membrane fusion and its disruption are related processes, although leakage and fusion capacities of peptides do not always correlate and the features/activities of membranotropic peptides may depend on particular environmental and temporal conditions. since not all membranotropic peptides are able to cross the membrane bilayer, it is essential to identify structural characteristics of hydrophobic peptides know to enter the cell membrane to highlight any feature that is involved in the penetration which may help in the design of novel delivery tools. thus, an important feature to consider is the structural requirements for cellular uptake and the ability of membranotropic peptides to interact with the cell surface and lipid moieties of the cell membrane. a very complete review describing the binding and translocation of membrane active peptides has been recently published [ ] which highlights the fact that peptide translocation is not coupled with dye flux. graded dye flux would occur concomitant with peptide translocation, which would explain incomplete dye release; whereas all-or-none flux would occur with peptides unable to translocate, and therefore these peptides accumulate on the membrane until a rupture point is reached, resulting in complete dye release [ ] . cell-penetrating peptides (cpps) have been widely used due to their capability to transport several kinds of macromolecules across the membrane bilayer in vitro and in vivo [ ] [ ] [ ] . cpps are short and usually basic amino acid rich peptides originating from proteins that are able to cross biological barriers, such as the viral tat protein. although the uptake mechanism of cpps is still debated, it seems to involve mainly the endocytic pathway, trapping the conjugated cargo in endosomes eventually ending in lysosomes where common enzymatic degradation mechanisms take place, therefore leading to a limited delivery of therapeutic agents to the intracellular target. hydrophobic peptides that efficiently traverse biological membranes, promoting lipid-membrane reorganizing processes, such as fusion or pore formation and involving temporary membrane destabilization and subsequent reorganization [ , ] , may be able to circumvent the endosomal entrapment either favoring the escape from the endosome or by translocating a cargo through the plasma membrane directly into the cytosol. this idea has been exploited to design the drug delivery tool called mpg. mpg is an amphipathic peptide whose primary sequence is composed of the hydrophobic amino acids of the hiv- fusion peptide (galflgflgaagstmga) associated to a hydrophilic domain derived from the nuclear localization sequence (nls) of simian virus (sv ) large t antigen (pkkkrkv). these hydrophilic and hydrophobic segments are separated by a three amino-acid spacer (wsq) [ , ] . this peptide exploits the known properties of the glycine-rich hiv fusion peptide essential for membrane fusion activity and the nls of the sv large t antigen to improve the nuclear addressing of the peptide [ , ] . at the moment this is the only viral fusion peptide that has been widely exploited for applications in drug delivery. a milestone in understanding the role of hydrophobic viral peptides is represented by the sequence "gh " derived from glycoprotein h of herpes simplex virus type i. herpes simplex virus (hsv) is an important human pathogen, responsible for significant morbidity and mortality worldwide and is characterized by a complex multi-component entry machinery. hsv enters host cells by fusion of the viral envelope with either the plasma membrane or an endosomal membrane, and the entry pathway is likely determined by both virus and host cell factors and involves multiple viral glycoproteins and cellular receptors in a cascade of molecular interactions [ ] [ ] [ ] [ ] . the envelope glycoproteins gh/gl, gb and gd are all essential for the entry process and their expression is able to induce the fusion of cellular membranes in a virus-free system [ , ] . both gh/gl and gb constitute the core fusion machinery and cooperate to induce the initial lipid destabilization that ends in fusion [ ] and both gb and gh contain several membranotropic sequences [ ] [ ] [ ] , , [ ] [ ] [ ] . although it has recently become available the crystal structure of the gh-gl complex [ ] , it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process and many studies suggest that the gh-gl complex may undergo dynamic rearrangements [ , ] . in particular, some peptides derived from the gh ectodomain block virus entry, while others have the ability to bind and disrupt model membranes. gb is considered a canonical class iii fusion protein and has been demonstrated to be involved in virus attachment, penetration and cell-to-cell spread. the crystal structure of gb is a trimer in which multiple contacts between protomers throughout the molecule contribute to its stability [ ] . it has been hypothesized [ ] that gb refolds similarly to class i fusion proteins and that the packing of the c-terminal arm against the coiled-coil provides the driving force for gb refolding from the prefusion to the post-fusion conformation. the gb structure corresponds to a post fusion conformation and it is now widely accepted that gb undergoes conformational changes upon variations of ph in order to bring about fusion [ ] [ ] [ ] [ ] . several synthetic gb peptides induced the fusion of large unilamellar vesicles and inhibited herpes virus infection [ , ] . when the crystal structures of gb and of gh/gl were not yet available, we reported the identification of several sequences in gh and gb with the ability to interact with the membrane and among these sequences there was also the canonical fusion peptide of gb [ , , , ] . although it is not yet well understood the role played by the other membranotropic sequences and in particular by the glycoprotein gh in the whole fusion process, it is now widely accepted that several regions in the fusion glycoproteins are involved in the local destabilization of the membrane bilayer which ends in the fusion of the viral envelope with the host cell membrane. gh was first selected and characterized by our group in [ ] and still is the hsv- peptide with the highest fusion capability and the most widely studied. it was initially identified using the wwih scale and subsequent works allowed determining the many applications of this sequence from membrane fusion, to viral inhibition and drug delivery [ , , ] . the twenty residue peptide gh (from aa to aa ) is a membrane-perturbing domain, (fig. ) which interacts with biological membranes and is implicated in the merging of the viral envelope and the cellular membrane [ , ] . the peptide contains residues crucial for its capacity to interact and destabilize target lipid membranes. it is rich in hydrophobic residues including glycines, leucines, alanines, and aromatic residues such as tryptophan and tyrosines, which are known to be located preferentially at the membrane interface. the peptidelipid interactions are initiated by the arginine residue located at the c-terminus; in fact, when the arginine is mutated, the fusogenic activity of the peptide is strongly impaired. the hydrophobic domain is also crucial for its insertion into the membrane and further supports the view that hydrophobic interactions between fusion proteins and cellmembrane phospholipids initiate membrane perturbation in the early stages of viral fusion. the many biophysical experiments performed on gh have shown that the peptide interacts with model membranes, penetrates the bilayer from its n-terminal side, has a tryptophan residue buried inside the bilayer, and adopts a helical conformation with its hydrophobic residues on one face of the helix and polar or charged residues on the opposite face [ , , ] . the analysis of peptides with longer and shorter sequences derived from this region and of their interactions with membranes clearly demonstrated that the activity of this region depends on the amino acid sequence and on its length. the presence of a histidine residue at the n-terminus of the native sequence strongly increases the fusion activity [ ] . the importance of a single histidine residue as a switch for triggering viral fusion was also reported for other viruses [ ] such as paramyxoviruses, therefore supporting the importance and specificity of the histidine moiety in activating fusion. furthermore, a conserved histidine in one of the fusion loops of semliki forest virus e protein was found to be fundamental [ ] . the histidine in gh both helps the initial interactions with the membrane and the oligomerization process [ ] . this hypothesis is further supported by the fact that the histidine is located at the n-terminus and correct configuration of the n-terminal fusion peptide appears to be crucial for the fusogenic function of several fusion proteins as well as its location in the membrane core after peptide-bilayer interaction. the addition of one histidine at the n-terminus of gh is sufficient to make the peptide approximatively -fold more active. in particular, the addition of any other residue at the n-terminus impaired the fusion ability of the sequence (data not published). gh strongly interacts and spontaneously penetrates the lipid-phase and inserts into membranes with a α-helical structure [ , , ] . both the tryptophan and tyrosine are on the same side of the helix in the three-dimensional structure, forming an amphiphilic helix in which one side is constituted by aromatic and hydrophobic residues, whereas the other side is formed by hydrophilic or small residues. the interaction between the aromatic ring of tryptophan and the side chain of tyrosine is important for maintenance of structural stability during the interaction with the membrane. an amphipathic α-helix is believed to be an important feature of membranotropic peptides playing a crucial role for mediating lipid-protein interactions during the binding of proteins to membranes and once bound, the hydrophobic face of the amphipathic peptide would allow the peptide to enter the membrane interior, thereby triggering local fusion of the gh has the ability to penetrate deep into the bilayer as a helix without causing significant bilayer perturbations which may help explaining its ability to perform several different roles. gh showed a significant inhibitory effect and this effect appears conditioned by its ability to partition into membranes and aggregate within them. since the peptide self-associates in aqueous and lipid solutions, it is possible that it binds to its counterpart in the gh protein. gh may also interact with the host cell membrane, therefore its ability to moderately inhibit viral entry when cells are treated first, is dependent on the possibility that the virus will find a modified cell membrane still exhibiting on its surface the peptide [ ] . moreover, gh does not have any activity in virus preincubation experiments, indicating that an eventual binding partner site on the pre-fusion gh protein is probably hidden and not available to interactions with free peptides, as also demonstrated by the analysis of the gh crystallographic structure. while the n-terminal histidine residue was proven to be fundamental for the interaction with the membrane bilayer and for translocation across the membrane, the absence of this residue induced similar levels of viral inhibition when compared with the full length peptide. the substitution of leu with a valine residue does not alter the hydrophobicity of the peptide, and does not influence its infectivity inhibition properties while its substitution with a polar residue (serine) substantially reduces its inhibitory activity [ ] . recently, poly(amide)-based dendrimers functionalized at their termini with gh were shown to inhibit both hsv- and hsv- at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite for viral internalization [ ] . the % inhibitory concentration was and nm against hsv- and hsv- respectively, with no evidence of cell toxicity at these concentrations, indicating that the functionalization of a dendrimer with a membranotropic peptide represents a promising strategy for inhibition of viruses of the herpesviridae family. the multivalent display of gh on the dendrimer scaffold results in an almost six fold increase of antiviral activity for hsv- and two fold for hsv- in comparison to the activity of the dendrimer itself, and more than -fold increase in the activity of the unsupported peptide. the cytotoxicity profile measured by the mtt assay showed that the peptidodendrimer is not toxic to vero cells up to the highest concentration investigated in antiviral testing, while some toxicity was observed for the unfunctionalized dendrimer, especially at higher concentrations, demonstrating another advantage of the peptide functionalization [ ] . any inhibitory activity was excluded when the compounds were added at a post-entry step and also when cells were pre-treated with the dendrimer derivatives, indicating that both the peptidodendrimer and the dendrimer are not able to interfere with viral replication once the virus has gained access to the cellular milieu. the peptidodendrimer might sterically hinder the gh relative domain, either in a pre-fusogenic or in an intermediate conformation, preventing a complete and functional interaction between gh and the membrane to fuse. the mechanism of inhibition may involve binding to gh itself through oligomerization of the gh domain present on the glycoprotein or interaction with other glycoproteins present on the virion envelope, such as gb or gd. the modification of a dendrimer scaffold with membranotropic peptides represents an attractive strategy for the design of a new class of antiviral drugs that exert their effect, coupling the intrinsic anti-viral properties of the dendrimer with the activity of membranotropic peptides and have the potential of being developed as multifunctionalized scaffolds to provide a therapeutic molecule to directly deliver to its target [ ] . the inhibition of membrane fusion represents an attractive target for drug design and although further studies are needed to better define the exact mechanism of inhibition by hydrophobic peptides and the specific nature or location of their interactions with viral targets, the data obtained for gh suggest that hydrophobic domains play a significant role in membrane fusion and provide an alternative approach to the development of viral peptide inhibitors outside of the classical inhibitory heptad repeat regions. gh cellular uptake is associated with its hydrophobic and amphipathic characters which provide the necessary ability to interact with membrane lipids and to form a transient helical structure that temporarily affects membrane organization, thereby facilitating insertion into the membrane and translocation [ ] . compared to tat peptide (a positively charged cpp) which mainly exploits the endocytic pathway, gh crosses membrane bilayers mainly through a translocation mechanism. a one amino acid shorter version of this fusogenic peptide was also found to improve the endosomal release of dna/lipofectamine lipoplexes and transgene expression up to -fold in human cell lines [ ] . it has been recently demonstrated that gh is able to traverse the membrane bilayer and to transport into the cytosol several compounds, such as qds [ ] , liposomes [ ] , nps [ ] , dendrimers [ ] , and proteins [ ] . examples of using gh as an intracellular delivery enhancer are provided in the remaining part of the paragraph (fig. ) . qds are fluorescent probes under intense research and development for broad applications in molecular, cellular and in vivo imaging [ ] . although considerable success has been achieved in using qds for labeling fixed cells and for imaging cell membrane proteins, only limited progress has been made for molecular imaging inside living cells because of their insufficient ability to traverse cell membranes. several authors have recently reported on the functionalization of qds with positively charged cpps and established that the main route of entrance is via endosomal uptake, therefore, escape from the endosomal system is of paramount importance [ ] [ ] [ ] . gh -qd internalization was demonstrated to be highly successful and to involve the endocytic pathway only to a minor extent [ ] . liposomal aggregates have also attracted great attention due to their success as in vivo carriers of drugs [ ] . to enhance the antitumor efficacy of liposomal drugs, the efforts of many research groups are directed toward the improvement of cellular internalization of liposomes through the addition of surface ligands and cell penetrating peptides. liposomes decorated with gh and loaded with doxorubicin (dox) [ ] , were able to penetrate inside living hela cells. the results obtained suggest that the functionalization of liposomes with gh could affect the uptake mechanism of liposomes and their intracellular distribution and dox release. this evidence could be useful in the design of carriers for a controlled delivery and release of dox in order to avoid side effects associated to dox itself. dendrimers [ , ] also represent a very promising tool for drug delivery, combining the advantageous features of nanoparticles (ideal size as in vivo carriers, multivalency), of polymeric materials (low cost, tunable properties, biocompatibility) and of small molecules (monodispersity and detailed control of their properties) [ , ] . their surface modification by means of conjugation or adsorption of a biospecific ligand, may allow their delivery to specific sites and modulation of drug release minimizing toxic effects and increasing intracellular bioavailability [ ] . thus, the dendrimeric scaffolds may be a promising tool for an efficient drug delivery engine. little information is available on the mechanism of dendrimer uptake and intracellular trafficking [ ] . studies performed on pamam dendrimers [ ] and pamam dendrimers functionalized with the tat [ ] indicate that endocytosis mechanisms contribute to the internalization and intracellular trafficking and that adding the tat failed to enhance delivery efficiency. the attachment of gh to the termini of a poly(amide)-based dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. the peptide-functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism [ ] . the combination of the benefits of dendrimers and peptides chemistry could be useful for the development of a selective carrier which could cross the membrane and be efficiently internalized into the cellular targets. many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain-barrier (bbb) [ ] . the development of new strategies for enhancing drug delivery to the brain is fundamental in diagnostics and therapeutics of central nervous diseases (cns). most strategies to transport drugs inside the cns cause disruption of the anatomical texture of the bbb, therefore impairing its natural function; as a consequence, effective delivery approaches should be cautiously assessed considering their impact on the overall protective function of the bbb [ ] . targeted delivery of a therapeutic cargo to the intended site of action in the brain appears to be one of the most promising non-invasive approach to overcome the bbb, combining the advantages of brain targeting, high incorporation capacity, reduction of side effects and circumvention of the multidrug efflux system [ ] [ ] [ ] [ ] . polystyrene nanoparticles (nps) decorated on their surface by gh showed that the uptake of nps with gh by brain endothelial cells was greater than that of the nps without the peptide and functionalized nps were free to move intracellularly [ ] . most importantly, gh decreased np intracellular accumulation as large aggregates and enhanced the np bbb crossing. the surface functionalization with gh may change nps fate and provides a good strategy for the design of promising carriers to deliver drugs across the bbb for the treatment of brain diseases. whether multifunctional nanosystems, designed and tested in vitro, are able to properly work in vivo into mammalian hosts, is not fully granted. to address this issue, in vivo studies are necessary, thus validating design strategies and facilitating optimization and further functionalization. although numerous studies showed that gh is an efficient carrier for bioactive cargoes in vitro [ ] , these results did not guarantee that it can be developed into a useful pharmaceutical delivery platform. the ability of gh to cross the bbb in vivo was also recently evaluated [ ] . gh was administered in vivo to rats and its presence in the liver and in the brain was detected. within . h from its i.v. administration, gh can be found beyond the bbb in proximity fig. . the many applications of gh to drug delivery. confocal microscopy images showing the internalization of gh functionalized: a) proteins [ ] ; b) liposomes [ ] ; c) qdots [ ] ; d) dendrimers [ ] . e) scanning electron microscopy images of functionalized polystyrene nanoparticles [ ] . of cell neurites. gh has no toxic effect in vivo, since it does not affect brain maximal oxidative capacity and mitochondrial respiration rate. the data suggest that gh , for its ability to cross the bbb, represents a novel nano-carrier system for drug delivery to the central nervous system. these results open new possibilities for direct delivery of drugs into patients in the context of theranostics and might address the treatment of several human diseases. other peptides have been proposed as a drug delivery system; it was demonstrated that tat was able to enter tissues in vivo in mice [ ] ; antp was able to activate endogenous t cells in mice [ ] . these peptides are highly positively charged, and absorptive-mediated transcytosis has been proposed for their transport across the bbb. a bradykinin analogue has also been reported to increase the penetration of small molecules by transitory opening of the bbb [ ] . gh is the first viral membranotropic peptide which was shown to be a potential delivery system for macromolecules in vivo; these results coupled with previous in vitro data support the view that gh enters the bbb without involving endocytic processes. hence, the eventual cargo may be immediately and completely available [ ] . the presence of multiple metabolic barriers may restrict the application of such peptide-based ligand for targeted drug delivery in vivo. peptides alone or conjugated on the surface of nanocarriers are subject to proteolysis in the blood after systemic administration. in addition, the bbb is also a metabolic barrier due to the presence of various enzymes in brain capillary endothelial cells. gh starts to be degraded after h of incubation but the intact peptide is still present after . h of incubation and thus holds the potential for extending brain targeting efficiency due to its resistance to proteolysis for . h [ ] . it is still under-recognized that some amino acid sequences in virtue of their specific features can play many different roles in nature. membranotropic viral peptides derived from fusion glycoproteins are widely studied especially for their ability to fuse membranes but there are many literature data also describing other roles besides membrane fusion. gh is an example of how these sequences can be employed for completely different purposes: fusion of membranes, viral inhibition and drug delivery. till now, gh is the only membranotropic peptide that has been extensively used for many applications and among them as a drug delivery system for the brain. in the development of new therapies to treat brain pathologies, the bbb represents a major obstacle against the use of potential drugs for treating disorders of the cns due to the impermeable nature of the cell membranes of this compartment to several molecules [ , ] . the data reported on the in vivo application of gh for brain delivery, support the novel view that synthetic peptides derived from viral membranotropic sequences can be used successfully to deliver biologically active substances inside the bbb. the exact molecular mechanism of gh entry remains to be established but it appears to be a general feature of membranotropic peptides, which may be used for mediating delivery to virtually any tissue and in particular across the bbb, conveying a wide variety of cargoes with intact bioactivity into virtually any tissue or organ. other sequences have been found to be useful for drug delivery which happens to have the same features of the viral fusion peptide, indicating that it may be possible to design novel sequences with pre-determined characteristics which can be useful to treat many diseases. however, still much work has to be done on this type of peptides; we should not forget that their mechanism of perturbation of membrane bilayers may also allow the design of new membranotropic peptides with the ability to denature the membrane bilayer of bacteria and thus we may add to their many roles also the antibacterial activity which may represent an alternative to classical antibiotics in order to combat the antibiotic resistance problem. much of the vast literature on membranotropic peptides is devoted to single activities of these sequences; yet compelling structure-function relationship studies bridging the gap among all these activities are necessary. the future of peptide-based drugs synthetic therapeutic peptides: science and market therapeutic peptides: technological advances driving peptides into development the preference of tryptophan for membrane interfaces use of hydrophobic moment plot methodology to aid the identification of oblique orientated α-helices antimicrobial peptides and viral fusion peptides: how different they are? antimicrobial peptides: promising compounds against pathogenic microorganisms membrane fusion and fission: enveloped viruses recent progress of cellpenetrating peptides as new carriers for intracellular cargo delivery mechanism of membrane fusion by viral envelope proteins viral membrane fusion structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution retrovirus envelope domain at . angstrom resolution crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain structure of the parainfluenza virus f protein in its metastable, prefusion conformation structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core the envelope glycoprotein from tick-borne encephalitis virus at Å resolution the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph class iii viral membrane fusion proteins hydrophobic interactions of peptides with membrane interfaces experimentally determined hydrophobicity scale for proteins at membrane interfaces folding of β-sheet membrane proteins: a hydrophobic hexapeptide model folding of amphipathic α-helices on membranes: energetics of helix formation by melittin amphipathic helix motif: classes and properties ph-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers conformational transitions of membrane-bound hiv- fusion peptide membrane structure and fusiontriggering conformational change of the fusion domain from influenza hemagglutinin structure and function of membrane fusion peptides structure and orientation study of ebola fusion peptide inserted in lipid membrane models the three lives of viral fusion 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single n-terminal histidine residue enhances the fusogenic properties of a membranotropic peptide derived from herpes simplex virus type glycoprotein h computational identification of self-inhibitory peptides from envelope proteins self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: effects of positional difference of glutamic acids on side chain ionization constant and intra-and inter-peptide interactions deduced from nmr and gel electrophoresis measurements oligomerization of fusogenic peptides promotes membrane fusion by enhancing membrane destabilization fusion peptides derived from the hiv type glycoprotein associate within phospholipid membranes and inhibit cell-cell fusion. structure-function study a synthetic all d-amino acid peptide corresponding to the n-terminal sequence of hiv- gp recognizes the wildtype fusion peptide in the membrane and inhibits hiv- envelope glycoproteinmediated cell fusion thermodynamics of fusion peptide-membrane interactions short-term monotherapy in hiv-infected patients with a virus entry inhibitor against the gp fusion peptide discovery and optimization of a natural hiv- entry inhibitor targeting the gp fusion peptide multifunctional nanocarriers endocytic mechanisms for targeted drug delivery direct cytosolic delivery of polar cargo to cells by spontaneous membranetranslocating peptides shuttle-mediated nanoparticle delivery to the blood-brain barrier exploitation of viral properties for intracellular delivery membrane-active peptides: binding, translocation, and flux in lipid vesicles twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics truncated hiv- tat protein basic domain rapidly translocates through the plasma membrane generation of endosomolytic reagents by branching of cell-penetrating peptides intracellular delivery: exploiting viral membranotropic peptides a novel potent strategy for gene delivery using a single peptide vector as a carrier a new peptide vector for efficient delivery of oligonucleotides into mammalian cells detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus in vitro mutagenesis of a putative dna binding domain of sv large-t nectin- -mediated entry of a syncytial strain of herpes simplex virus via ph-independent fusion with the plasma membrane of chinese hamster ovary cells structure-function analysis of herpes simplex virus glycoprotein b with fusion-from-without activity entry of herpes simplex virus and other alphaherpesviruses via the paired immunoglobulin-like type receptor alpha glycoprotein d receptor-dependent, low-ph-independent endocytic entry of herpes simplex virus type fusing structure and function: a structural view of the herpesvirus entry machinery glycoproteins gb, gd, and ghgl of herpes simplex virus type are necessary and sufficient to mediate membrane fusion in a cos cell transfection system herpes simplex virus glycoproteins gb and gh function in fusion between the virion envelope and the outer nuclear membrane biophysical characterization and membrane interaction of the two fusion loops of glycoprotein b from herpes simplex type i virus role of membranotropic sequences from herpes simplex virus type i glycoproteins b and h in the fusion process structure and orientation of the gh - membrane interacting region of herpes simplex virus type in a membrane mimetic system crystal structure of the conserved herpesvirus fusion regulator complex gh-gl crystal structure of the epstein-barr virus (ebv) glycoprotein h/glycoprotein l (gh/gl) complex crystal structure of glycoprotein b from herpes simplex virus residues within the c-terminal arm of the herpes simplex virus glycoprotein b ectodomain contribute to its refolding during the fusion step of virus entry conformational modifications of gb from herpes simplex virus type analyzed by synthetic peptides structure of a trimeric variant 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translocation and applications to the delivery of quantum dots a fusogenic segment of glycoprotein h from herpes simplex virus enhances transfection efficiency of cationic liposomes clickable functionalization of liposomes with the gh peptide from herpes simplex virus type i for intracellular drug delivery dendrimer functionalization with a membrane-interacting domain of herpes simplex virus type : towards intracellular delivery pedone, gh is a viral derived peptide for effective delivery of intrinsically disordered proteins probing cellular events, one quantum dot at a time intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides intracellular trafficking and unpacking of sirna/quantum dot-pei complexes modified with and without cell penetrating peptide: confocal and flow cytometric fret analysis spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques the benefits and challenges associated with the use of drug delivery systems in cancer therapy dendrimers and dendrons: concepts, synthesis, applications designing dendrimers for biological applications dendrimers and dendritic polymers in drug delivery surface modifications of nanocarriers for effective intracellular delivery of anti-hiv drugs crossing cellular barriers using dendrimer nanotechnologies comparison of the endocytic properties of linear and branched peis, and cationic pamam dendrimers in b f melanoma cells tat-conjugated pamam dendrimers as delivery agents for antisense and sirna oligonucleotides current approaches to enhance cns delivery of drugs across the brain barriers astrocyte-endothelial interactions at the blood-brain barrier nanoparticle-mediated targeted delivery of antiretrovirals to the brain nanobiotechnology-based strategies for crossing the blood-brain barrier biomaterial-based technologies for brain anti-cancer therapeutics and imaging receptor-mediated delivery of magnetic nanoparticles across the blood-brain barrier review of a viral peptide nanosystem for intracellular delivery the peptide gh enters into neuron and astrocyte cell lines and crosses the blood brain barrier in rats tat-mediated delivery of heterologous proteins into cells introduction of exogenous antigens into the mhc class i processing and presentation pathway by drosophila antennapedia homeodomain primes cytotoxic t cells in vivo facilitation of drug entry into the cns via transient permeation of blood brain barrier: laboratory and preliminary clinical evidence from bradykinin receptor agonist, cereport getting into the brain: approaches to enhance brain drug delivery the authors thank luca de luca for excellent technical assistance. key: cord- - esdjy authors: delhalle, sylvie; schmit, jean-claude; chevigné, andy title: phages and hiv- : from display to interplay date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: esdjy the complex hide-and-seek game between hiv- and the host immune system has impaired the development of an efficient vaccine. in addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. for more than years, phage display technology has been intensively used in the field of hiv- to explore the epitope landscape recognized by monoclonal and polyclonal hiv- -specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. in parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify hiv- inhibitors. besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the hiv- protease. phage particles also represent valuable alternative carriers displaying various hiv- antigens to the immune system and eliciting antiviral responses. this review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures. in , the human immunodeficiency virus (hiv- ) was identified as the causative agent of the acquired immunodeficiency syndrome (aids) [ , ] . in years of pandemic, hiv- has infected more than million individuals and killed million. thirty-three million individuals are currently living with hiv- making this disease a major worldwide public health problem (unaids ). natural sterilizing immune response against hiv- has never been described and despite decades of intensive research, a vaccine against hiv- is still lacking, mainly due to the high ability of the virus to escape from the immune response. in the absence of a vaccine, combinations of small antiviral molecules are intensively used to control hiv- infection. the majority of these drugs are reverse transcriptase and protease inhibitors [ ] . more recently, new molecules targeting the fusion step, ccr or integrase were licensed for clinical use [ ] [ ] [ ] . despite the increased life expectancy observed with the advent of these therapies, severe side effects, lack of adherence and emergence of drug-resistant virus strains still limit the long-term control of the infection [ ] . hiv- is an enveloped virus whose genetic material consists of two identical rna strands coding for the structural genes gag, pol and env as well as the accessory genes tat, rev, nef, vif, vpr and vpu. the gag gene codes for structural proteins p and p , while pol codes for viral enzymes (reverse transcriptase, integrase and protease) and env for the gp envelope protein precursor that is subsequently cleaved into gp and gp . gp and gp proteins assemble at the surface of hiv- into trimeric spikes composed of three monomers of membrane-embedded gp complexed to free gp . these two proteins are involved in virus entry and represent the principal targets for the humoral response. upon cd receptor binding, glycoprotein gp undergoes conformational changes exposing the v loop, a region that further interacts with the chemokine receptors ccr or cxcr thereby promoting viral entry [ ] (figure ). coreceptor binding leads to the insertion of the gp fusion peptide into the cell membrane, the creation of a hairpin loop intermediate and finally the fusion of both viral and cell membranes. the viral capsid then enters the cell and the genetic material is released in the cytoplasm. most viral strains use only one coreceptor to enter host cells and are classified accordingly as ccr -(r strains) or cxcr -tropic (x strains), although viruses with broadened coreceptor usage (dual-tropic) have also been described. r viruses infect macrophages and ccr -expressing t lymphocytes, and are mainly associated with transmission. in contrast, x viruses infect cxcr -expressing t-cells and t-cell lines, and often appear at the later stages of infection. the envelope glycoprotein gp is composed of variable and more constant regions. several studies demonstrated that the elicitation or binding of effective neutralizing antibodies are impaired by the gp glycan shield or steric hindrance of its constant regions [ ] . moreover, variable immunodominant domains were shown to be recognized by non-neutralizing antibodies. nonetheless, it is estimated that % to % of hiv- -positive subjects develop neutralizing antibodies (ntabs) appearing at least year after infection. only % of infected patients develop a broad neutralizing response against heterologous virus strains [ ] . among hiv- -infected patients, such antibodies arise only rarely and tardily, thus inefficiently controlling viral replication. however, the recent identification of broadly neutralizing antibodies (bntabs) and mapping of their epitopes fueled interest in the humoral immune response against hiv- (reviewed by overbaugh [ ] ). to better understand the reasons underlying the persistance of viral infection despite the strong and sustained immune response on the one hand, and to identify new protective immunogens, numerous studies were conducted to map the epitope landscape of both hiv- -neutralizing and non-neutralizing antibodies isolated from infected patients. in parallel, the development of new molecules or antibody fragments capable of blocking either viral proteins or host receptors has been widely investigated. to serve this purpose, the phage display technology has been extensively exploited in the field of hiv- as it represents one of the most powerful technologies for epitope mapping as well as for the identification of ligand binding to many types of targets. bacteriophages (phages) are bacteria-infecting viruses whose dna or rna genome is packed in a capsid composed exclusively of surface proteins. the principle of phage display relies on cloning of exogenous dna in fusion with the phage genetic material allowing the display of foreign peptides in an immunologically and biologically competent form at the surface of phage capsid proteins [ ] . the significance of phage display was first demonstrated for filamentous phages such as m , fd or related phagemids and later extended to lytic bacteriophages λ, t and t (reviewed by beghetto [ ] ). the phage biopanning process consists of iterative cycles of binding, washing and elution steps leading to the progressive selection of phages displaying peptides/proteins binding to the target of interest [ ] . the target is usually immobilized on a solid support which can be plastic, beads or even cells. a significant advantage of this technology is that phages may be used to display a collection of sequences (phage library), reaching up to billions of distinct sequences. phage libraries can be constructed to express combinatorial peptides or proteins/immunoglobulin fragments/variants that may be further screened for many different purposes including drug discovery, epitope mapping, diagnosis as well as identification of therapeutic antibodies. the phage display technology is versatile as it can be applied to different domains of research; it allows the easy handling and high-throughput screening of billions of sequences. it is affordable and enables the identification of linear as well as conformational epitopes when applied to an antibody target. numerous studies have described the use of the phage display technology in the field of hiv- were reported. they can be classified in four main applications ( figure ): i. epitope mapping, which relies on the screening of random peptide libraries on immobilized monoclonal or polyclonal antibodies to determine the linear and/or conformational epitopes recognized by these antibodies (linear epitope: sequence of continuous amino acids recognized by the paratope of a given antibody; conformational/discontinuous epitope: group of amino acids scattered along a protein sequence which come together in the folded protein and are recognized by the paratope of a given antibody). such screening usually results in the identification of sequences mimicking the natural epitope (mimotopes) and provides precise information about the location of residues forming the natural epitope. these mimotopes may in turn be used as valuable immunogens to elicit antibodies targeting the original epitope, an approach referred to as -reverse vaccinology‖. ii. inhibitor discovery, based on screening of phage libraries displaying random peptides or antibody fragments against viral or host proteins critical for viral replication. iii. -phage substrate‖ approach in which potential substrate sequences are displayed at the phage surface to enzymes such as proteases. this approach not only allows for proteolysis specificity profiling, but provides information and data to develop specific inhibitors. iv. carrier phage, in which the phage functions as a -carrier‖, -vehicle‖ or -virus-like particle‖ to display exogenous peptides such as mimotopes or even full size antigens to the immune system, to elicit specific humoral and/or cytotoxic t-cells responses. this review is intended as an overview of the different studies conducted using phages in the field of hiv- , laying special emphasis on the nature of the phage libraries used, the target display mode, the biopanning procedure as well as the results obtained. these studies are classified according to the applications described above and the main results are presented in tables. monoclonal antibodies (mabs) or polyclonal antibodies (pabs) epitopes can be identified through screening either combinatorial or antigen-fragment libraries displayed at the surface of phages. antibodies may be derived from infected/immunized animals or from hiv seropositive patients with peculiar immunological profiles, such as the long term non progressors (ltnp) [ ] , leading to the identification and characterization of hiv mimotopes. the seminal paper characterizing the epitope recognized by a mab directed against hiv- using the phage display technology was published in . keller et al. screened a -mer random peptide library (rpl) against the bntab - d which targets the v loop of gp (krkrihigpgrafy) [ ] (figure ) and identified clones presenting a gpxr consensus sequence [ ] (table ). these mimotopes were further used in rabbit immunization experiments and elicited neutralizing responses. boots et al. later investigated the linear epitope recognized by the mab - d by combining gp competition and panning of v -region biased/constrained libraries. such a set-up favors the selection of mimotopes in which residues surrounding the gpgr crown motif are similar to those present in the gp used for competition, suggesting that the use of strain-specific competitors with a mab of broad specificity can select for strain-specific mimotopes [ ] . [ , ] . in their study, a -mer rpl was constructed and panned against mab . according to two different protocols, either streptavidin capture of phages mixed with biotin-labeled mab . (sa-bio) or panning against mab . immobilized on microwells (micropan) [ ] . phages selected with the sa-bio protocol shared a consensus sequence (y/l)(v/l/i)gpgrxf homologous to the v loop. the micropan protocol allowed for the identification of sequences sharing the same motif, of which two were also identified in the sa-bio panning. biopanning results were further validated in peptide array hybridization assays. hybridization of mab . to -mer peptides containing all possible point substitutions within the v loop sequence demonstrated that both phage display and peptide array experiments identified the same critical amino acids, thereby confirming the quality of the -mer rpl and the validity of the screenings performed. epitope mapping was also performed on a monoclonal antibody (mab b) isolated from an asymptomatic hiv- -infected patient and recognizing the xxix pgrafytt motif within the v loop sequence (krihigpgrafytt) [ ] . binding of mab b to viral isolates presenting mutations in this sequence revealed that not all residues within this recognition motif were crucial for reactivity [ ] . biopanning with a -mer rpl resulted in the selection of sequences compatible with the minimal binding site (-i----g--fy-t) inferred from gp sequence alignment from clades a to f which bound mab b. taken together, data from binding assays as well as phage biopanning experiments demonstrated that the mab b epitope spans both sides of the v loop. substitutions within the residues located at the crown of the loop are however tolerated, provided that the formation of a β-turn induced by the gpgr crown motif is allowed. however, one exception was reported by boots et al. who reported that the phe to trp substitution may be tolerated in the absence of a β-turn [ ] . in parallel, grihalde et al. constructed and panned a -mer rpl against mab , which recognizes a constrained linear epitope on the v loop [ ] . several clones were obtained and presented the common motif (r/k/h)xgr mimicking the crown of the v loop sequence, thereby confirming the epitope sequence of mab . to assess the reactivity of peptides deprived of the phage scaffold, the mimotope with the highest affinity for the mab was expressed in fusion with the e. coli alkaline phosphatase. binding of the phage and fusion protein to the mab was assessed by elisa, western blot and spr assays and highlighted that binding was independent from the scaffold, although interactions were weaker when the peptide was displayed in the fusion protein format than in the phage scaffold. in another study, laisney et al. investigated the minimal epitopes recognized by two mabs interacting with the v loop, -a and . . , whose specificity is strictly restricted to the x -tropic lai isolate [ ] . the screening of a -mer rpl on the mab -a allows the selection of numerous sequences with a consensus motif. binding assays with synthetic peptides further showed that both mabs reacted with residues - of the lai gp . in this narrow region, the minimal epitope deduced for the mab -a was hyxrgp, whereas the mab . . recognized the xq(r/k)gp motif (hy: non-aromatic aa, underlined: aa tolerating substitutions). interestingly, the essential qr residues located at positions - correspond to a qr insertion located upstream of the v loop gpgr crown motif that is characteristic of the lai isolate and may thus explain the restricted specificity of the two mabs. the same authors screened a -mer rpl on the mab , specific to the v loop of the mn isolate, and identified two groups of sequences [ ] . a representative sequence from the first group ( . , hlgpgr), corresponded to the crown of the v loop, a linear epitope, while two sequences of the second group ( . , kaihri and . , kslhrh), showed no homology to linear hiv- epitopes. both peptides . and . nevertheless inhibited the interaction of mab with gp , and were even able to compete with each other for binding to the antibody, indicating that peptide . was also a mimotope of peptide . . when conjugated to klh and injected separately into rabbits, both peptides - and - were able to elicit gp -reacting antibodies that partially competed with the homologous peptide, confirming that . and . peptides are both antigenic and immunogenic mimics of the gp mn v loop. the isolation of the bntab f , which interacts with an epitope (eldkwa) located on the gp mper was reported in [ ] . conley et al. further characterized this epitope by biopanning a -mer rpl on immobilized f ab. different sequences were obtained and classified in four groups, whose consensus motifs, dkw, ldxw, ed(k/r)w and eldkw, revealed information on the residues involved in f ab recognition [ ] . immunization attempts with eldkwa peptides failed to elicit f -like ntabs, suggesting that the epitope necessitates additional residues in order to be immunogenic. therefore, menendez et al. screened a panel of libraries of linear and constrained peptides against the mab f and deduced that residues flanking the dkw core at the c-terminal side region were important for high-affinity binding to the mab [ ] . they subsequently constructed and screened two phage sublibraries displaying random residues either upstream or downstream of the dkw core (x -aadkw and aadkw-x ) and isolated three peptides displaying high affinity for f from the aadkw-x library. ala substitution and deletion studies revealed that each clone bound f according to a different mechanism. this data led the authors to postulate that the f paratope was composed of two binding domains either recognizing the dkw core with strong specificity or multispecifically binding to the residues located at its c-terminus. based on this study, additional investigations were recently conducted on the bntab f epitope to assess the importance of structural constraints for mab f recognition [ ] . a linear -mer rpl and a constrained -mer rpl were screened against this antibody and all the sequences selected from the -mer rpl contained the d(k/r)w core motif, with flanking residues l, a and s present at different frequencies. analysis of the sequence representation compared to their estimated probability of occurrence indicated a trend towards enrichment for sequences such as dkwa or ldkwa throughout panning of the -mer library, while all sequences selected from the constrained library contained dkwa or ldkwa. these results demonstrated that the strong epitope specificity postulated by menendez [ ] is only displayed when the epitope sequence is presented in a certain structural context provided in the constrained -mer peptides. immunization studies performed with both linear and constrained forms of the peptide in mice and rabbits resulted in the inhibition of cell fusion only with sera of rabbits immunized with the linear peptide. rpl screening may also contribute to the elucidation of the antigen structure. to that purpose, stern et al. used a -mer rpl to analyze two different mouse mabs (gv a and gv d ) recognizing non-overlapping sequences between residues and of gp [ , ] . biopanning performed on gv a allowed for identification of mimotopes sharing a (l/i)w motif identical to residues - of the gp c domain and highlighted a hxxixxlw motif compatible with two turns of an α-helix. computer modeling confirmed that such a structure placed the residues recognized by gv a contiguously on one face of the helix while other secondary structures did not. similarly, biopanning on mab gv d yielded sequences with a trend towards an nx wxxd motif. the epitope maps to the fnmwknd sequence satisfying the helical motif fxxwxxd. in this study, the use of phage display not only predicted the α-helix structure of the c domain of gp , but also pinpointed the contact residues defining the surface of the helix. phage display was also applied to epitope mapping of antibody-dependent cellular cytotoxicity (adcc)-inducing mabs since hiv- infected cells may be targets for fc receptor-bearing effector cells interacting with hiv- -specific abs. screening of -mer, -mer-c and -mer rpls against the adcc-inducing mab id resulted in the identification of phages with txxfxxwxxd ( -mer rpl) and fxdwxf ( -mer and -mer-c rpls) motifs homologous to the c domain of gp [ ] . competition assays showed that binding of mab id to gp or gp was abrogated in the presence of -mer mimotopes. in contrast, heptapeptide mimics only slightly impaired this binding, supporting the hypothesis that the mab-id epitope probably encompasses residues additional to the fxdwxf motif. as this epitope is highly conserved among circulating hiv- subtypes, it might be useful to induce mab id -like antibodies. the bntab igg b was the first neutralizing mab selected from a phage-displayed fab (antibody fragment composed of one constant and one variable domain of the heavy (ch and vh) and the light (cl and vl) chains linked together) library derived from an hiv- -infected donor (see section . . . . .) [ ] . this antibody recognizes a conformational epitope overlapping the cd -binding site of gp [ ] . attempts to precisely map the residues interacting with the igg b mab with -mer and -mer rpls provided no consensus sequence [ ] . as previous screening of cysteine-enriched peptide libraries resulted in the identification of two sequences bearing an sdl motif flanked by one or two cysteine residues (rekrwifsdlthtci and tclwsdlraqci) [ ] , zwick et al. constructed two sublibraries (x sdlx ci and xcxxsdlx ci) sharing the sdl motif and reflecting the cysteine content of the two clones [ ] . a b . peptide (hersymfsdlenrci) containing a unique cysteine bound b in fab as well as igg formats with a much higher affinity than the other clones. moreover, the phage-borne b . peptide was used to screen the fab library from which b was identified. this -reverse panning‖ experiment showed that b . was able to select only the fab sequence corresponding to b , confirming the specificity of the b . mimotope towards the b ab. b . peptide was immunogenic in mice and rabbits but did not elicit significant anti-gp cross-reactive abs titers. dorgham et al. attempted to map the b epitope with a rpl of two random -mers joined through an allry spacer (x allryx ) [ ] . selection resulted in the identification of clones sharing a m/varsd consensus motif (ar standing for any aromatic residue) as previously observed [ ] . a second-and a third-generation of semi-rpl containing fixed consensus motifs identified in the previous panning surrounded by randomized residues were constructed (x (m/v)wsdx and xlxvwxdexx). phagotopes (phage particle displaying a particular peptide sequence selected on a given target) obtained from the first, second and third generation libraries showed increasing binding affinity for b , respectively. phagotopes were able to compete with gp for b binding and triggered the production of abs capable of recognizing at least five distinct, unrelated hiv- strains. in contrast, the corresponding peptides were not able to compete for b binding and did not elicit anti-gp mabs. such discrepancies between phagotopes and peptides might be explained by constraints imposed by the phage scaffold. detailed characterization of the bntab b was conducted with the mapitope algorithm developed by enshell-seijffers et al. to facilitate the identification of discontinuous epitopes. this approach is based on the assumption that the collection of mimotopes recognized by a given antibody must in some manners reflect the antibody's paratope [ ] . a constrained -mer rpl was screened against b and selected sequences were compared to those obtained from previous panning experiments performed against b [ , , , ] . although no similarity was observed with the mimotopes selected by boots et al. [ ] , a consensus wsdl motif was observed in the newly identified mimotopes and the sequences isolated by bonnycastle et al. [ , ] . mapitope analysis conducted on these sequences as well as on the peptide sets isolated by boots and bonnycastle resulted for each of the three panels in the prediction of two clusters located at the periphery of the cd binding site. at the same time, both a linear -mer rpl and a constrained -mer rpl were used in panning experiments against another gp cd binding site mab ( a) [ ] . screening of the -mer rpl resulted in selection of a single sequence (wkpvvidfe), while screening of the -mer-c rpl on a allowed for identification of a gpxepxgxwxc consensus motif. peptides were synthesized as peptide-piii fusion proteins and their affinity for a was assessed in phage/mab and gp /mab binding inhibition assays. the two most affine peptides (aecgpaeprgawvc and aecgpyeprgdwtcc) were used to immunize rabbits and elicited antibodies binding to recombinant monomeric gp . nevertheless, generated abs seemed to target a different epitope since they were unable to compete with the a cd -binding site specific mab. the extreme c-terminus of gp forms a pocket which may interact with gp and was suggested to undergo conformational changes weakening the interaction between gp and gp upon cd binding. in the absence of available crystallographic information, ferrer et al. utilized the mouse mab - . to analyze an epitope overlapping with this pocket region [ ] . epitope mapping of mab - . achieved by cross-blocking experiments on gp suggested that the ab recognized residues - while the screening of an heptapeptide rpl against mab - . preincubated with gp allowed for the recovery of phages presenting an axxkxrh motif homologous to residues - . affinity studies confirmed that ala was the n-terminal residue of the mab - . epitope and showed that affinity increased when c-terminal residues were added. the mapitope algorithm designed by enshell-seijffers et al. was initially developed to elucidate the cd -induced epitope recognized by the mab b [ ] . screening of a -mer-c rpl yielded sequences with no homology to gp . comparison of the mimotopes to the gp structure in complex with mab b and scd predicted candidate epitopes that were in agreement with the actual b contact residues. for further validation of the algorithm, rpl libraries were screened against the p -specific mab b and analysis of the selected sequences predicted four clusters, the largest of which corresponded to the genuine epitope. the algorithm was finally applied to the mab cg , an ab with an unknown epitope competing with the mab b for the binding to the cd /gp complex. mimotopes sequences were analyzed and produced seven clusters, one of them being in accordance with previous mutation analysis impeding mab cg binding [ ] . noteworthingly, when reconstituted in a phage scaffold, the epitope was capable of binding mab cg . after having successfully identified linear or nearly linear epitopes [ , , ] , boots et al. extended the use of the phage display technology to the identification of epitopes recognized by mabs binding to discontinuous sequences [ ] . one of these abs (mab a ) binds to a cd -induced discontinuous epitope involving residues within the c , c and c regions of isolates from clades b, c, d, e and f [ , ] . panning of a -mer rpl yielded several phages which only shared a trp residue. in the same study, panning of a -mer rpl against mab - , which reacts with the id gklic region of gp , resulted in the identification of sequences sharing a common trp within motifs wgcx(k/r)xlxc and fgxwfxmp. the selected consensus sequences were however not further characterized. the bntab g presents the typical feature of recognizing a cluster of high-mannose oligosaccharides of gp [ ] [ ] [ ] . in an attempt to identify peptidic immunogens capable of eliciting g -like abs, menendez et al. screened a set of previously described rpls [ ] against g and identified one phagotope specifically binding to g ( g . ) [ ] . the crystal structure of mab g complexed to the synthetic g . peptide was compared to structures of g -oligomannose epitopes and revealed that interactions with the abs were different for the two ligands. these results showed that the peptide selected from rpl panning experiments is not a structural mimic of the g oligomannose epitope. the phagotope g . was used in rabbit immunization experiments and elicited high titers of peptide-specific antibodies, but no cross-reactivity with gp was obtained, further supporting that peptide g . is not an immunogenic mimic of the mab g epitope. the first attempt at identifying epitopes recognized by hiv-specific pabs was performed in on plasma igg from two ltnp patients (table ). using linear and constrained -mer rpls, scala et al. identified mimotopes of the linear immunodominant (id) gklic region of gp or the v and c domains of gp [ ] . these mimotopes were immunogenic when injected to mice and elicited an ntab response against hiv- . moreover, the same mimotopes reduced viraemia to undetectable levels in immunized monkeys as shown in a subsequent study [ ] . the same year, a similar study conducted on one ltnp with an rpl library of cysteine-constrained -mers selected for peptides defining the gp id epitope csgklic. the levels of reactivity of these phagotopes were further assessed against a panel of hiv positive plasma to evaluate the plasticity and polyclonality of the immune response mounted by infected individuals [ ] . later, palacios-rodriguez et al. evaluated the impact of factors such as highly active antiretroviral treatment (haart) or ab titers on a selection of peptides mimicking the id epitope csgklic [ ] . in their study, a mix of linear -mer as well as linear and constrained -mer rpl was screened against the individual plasma samples of four hiv- infected patients initiating haart and presenting different titers of anti-gklic antibodies. a consensus motif cxxkxxc was obtained from the -mer linear rpl, and the percentage of occurrence of the motif in the selected sequences was proportional to the anti-gklic ab titers of each sample, indicating that these abs are involved in selection of the consensus motif. mice immunization experiments with the two mimotopes resembling most to the gp id parental epitope as well as with pools of phages eluted from the panning experiments showed that all phages elicited reactivity, and that immunization with the phage eluates induced the strongest recognition. these findings indicate that the immunogenic properties of mimotopes are different and additive, opening the possibility of immunizing animals with different mimotope combinations (see section ). in , humbert et al. investigated the immune response of eight ltnp patients presenting bntabs. by using linear and constrained rpls they identified epitopes recognized by plasma iggs captured on tosylactivated beads [ ] . each panning round consisted of a positive selection performed on ltnp iggs followed by a negative selection on the iggs of healthy donors. homologies of some selected sequences to immunodominant regions such as the gp v loop or the gp gklic region were observed, as reported in previous studies [ , , ] . further homologies to linear motifs located near the v loop (nnnt), downstream of the id gklic region (avpw motif) and overlapping with the f bntab epitope (ppwx w motif) were also identified. additionally, the authors applied the dex software to compare the phage insert sequences to hiv- protein structure files from the rcsb protein data bank (www.pdb.org) [ , ] . phage pools corresponding to the linear v loop, gklic domain and wxxxw motif, as well as pools representing potential conformational epitopes, were selected for mice immunization assays, and elicited plasma-associated neutralizing activity against primary hiv- strains. the highest neutralizing ability was obtained with mice immunized with the v mimotopes, although immunization with potential conformational epitopes also provided a modest neutralizing response. a similar approach was used by the same authors on a rhesus macaque infected with an shiv chimera encoding the env of a clade c hiv- strain (shiv ip) and presenting a broad neutralizing response against homologous shiv-c as well as heterologous hiv- strains of different subtypes [ ] . biopanning yielded clones similar to gp (v and v loops or c-terminal domain) or to regions of gp (id gklic region, other id regions and mper domain) [ ] . remaining clones showed no significant homology to linear hiv- regions and were analyzed with the dex software, which allowed the identification of a discontinuous mimotope located near the v loop crown. the antibodies binding to this phagotope were affinity-purified and subsequent assays demonstrated that recognition was conformation-dependent. an immunofocused immunization of mice primed with a dna vector coding for the gp shiv ip and boosted with pools of phage particles corresponding to the v loop, the gp c-terminus, the gp id region, the gklic region and the mper domain was set up. almost all mice developed anti-env abs and % of them presented a neutralizing activity. in , dieltjens et al. applied the phage display technology to identify the epitopes potentially involved in the bntabs response of an hiv- crf ag-infected individual (itm ) and to monitor the evolution of humoral response and viral escape through the course of infection [ ] . biopanning of a -mer rpl against plasma samples from itm resulted in the identification of different peptide sequences. half of these sequences were homologous to linear epitopes on gp , i.e., the e epitope region in the mper domain (nwfnltqtlmpr) or the lentivirus lytic peptide (llp ) (slxxlrl) while the other peptides shared homologies with the c domain (kxwwxa) and the crown of the v loop (kx igphxxy) of gp . further analysis of the levels of reactivity of the phage groups against itm six-year follow-up samples revealed different temporal patterns of recognition, confirming the dynamic nature of the immune response. interestingly, the mper region was the only epitope retaining immunogenic properties during this period. in a more recent study, the same group investigated the antigenic landscape of an hiv- subtype a-infected individual with bntabs by screening an rpl library against a pool of sequential samples drawn from to [ ] . the biopanning procedure yielded sequences predicted to represent autologous v sequence (kx hx y), v loop (kxxhxgpx f) and gp id domain (cxgxlxctxnxp). again, follow-up sample recognition of the four phage groups showed different patterns. antibody reactivity towards gp id region fluctuated slightly in all plasma samples. reactivity against the v loop-like phages decreased over time. in contrast, the v loop mimotopes were not recognized before , but once emerged, reactivity persisted until . env sequence analysis of the follow-up samples showed that a tyr to his mutation in the v loop sequence coincided with the emerging antibody response against this sequence. additionally, the authors highlighted that the neutralizing activity observed in the samples was partially due to antibodies recognizing the v mimotopes. besides the multiple reports on the use of rpl to characterize the humoral response against hiv- env proteins, gupta et al. evaluated the reliability of using targeted antigen gene fragment libraries for the identification of epitopes recognized by antibodies elicited in rabbits immunized with p . to this end, they constructed a phage library composed of dnase-digested fragments of gag dna [ ] . phagotopes obtained after the first panning round displayed mainly - -mer peptides, % of which mapped to of the n-terminus of p ( - of gag) and % corresponded to the c-terminal region of p ( - of gag). only one phagotope mapped to the central region of gag ( - ). at the end of the second round, selected phages displaying longer inserts of to aa corresponding to the n-and c-terminal regions of gag were identified, revealing the presence of two distinct antigenic regions in gag. this study demonstrated that gene-fragment phage display could be used to identify epitopes targeted by polyclonal abs. although they occur at a very low frequency in humans, antibodies targeting host proteins involved in hiv- infection have been reported in immunized animals. given their potential value for viral entry inhibition and the general understanding of this mechanism, rpls were screened on these mabs to gain better knowledge of their epitopes (table ) . na: data not available. the murine mabs a and c were raised against cells transfected with the seven transmembrane-spanning domains chemokine receptor ccr , one of the main coreceptors for hiv- . they recognize a common epitope located near the ccr n-terminus [ , ] . both mabs were used to screen a constrained -mer rpl [ ] . phagotopes selected on a displayed the sequence chasiydfgsc while cphwlrdlrvc was the most prevalent sequence isolated on c . these sequences showed homologies to residues located at the n-terminus but also within the first or third extracellular loop (ecl) of ccr . both reacted against the targeted mab either in phage, cyclic peptide or linear peptide formats. moreover, they were able to bind to gp and the peptide selected on a inhibited binding of the mab to a cell line expressing ccr . to further characterize the conformational epitope recognized by a , additional screening rounds of -mer, -mer and -mer-c rpls were performed [ ] . sequences with an hw motif homologous to the cphwlrdlrvc motif selected on the mab c were identified, and ala-scanning confirmed the importance of the hw motif and siyd motifs previously identified for a binding [ ] . another murine antibody (mab d ) recognizing a conformational epitope on the second ecl of ccr [ ] was explored by screening a linear -mer rpl [ ] . three phagotopes (m , m and m ) were isolated and one of them (m ) was able to inhibit cell infection by the hiv- sf isolate. the corresponding peptide (fcaldgdfgwlapac) fused to the piii phage coat protein neutralized infection mediated by the jr-fl but not the iiib strain. the fusion protein specifically bound d and was recognized in a dose-dependent manner by three ccr chemokine ligands, i.e., ccl (rantes), ccl (mip α) and ccl (mip ß), confirming its ccr mimicry. six years later, another screening campaign was conducted with a linear -mer rpl on d and the ewqkeglvtlwl sequence of a high-affinity binding peptide was obtained [ ] , revealing that this peptide presented homologies to the n-terminal ( -qkegl- ) and c-terminal regions of the ccr ecl . ala substitutions of the tl residues confirmed their crucial role in d binding. the selected peptide was used in rabbit immunization studies and elicited abs with d -like biological functions, i.e., which inhibited hiv- -mediated cell fusion and pbmc infection. the cd cell surface molecule, a part of the lfa- molecule, is involved in the syncytia formation of hiv- -infected lymphocytes [ ] . as mab mhm , a cd binder, inhibits hiv- -mediated cell fusion, poloni et al. applied the phage display technology to map the mhm epitope and thereby identify the cd domains which account for syncytia formation [ ] . linear and constrained -mer rpl were panned on the mhm mab, to allow for the selection of linear and constrained sequences. a ppfxyrk consensus motif was inferred by sequence comparison, assigning the epitope recognized by mhm to residues - of cd . two phagotopes inhibited in vitro hiv- -induced syncytia formation and one of them retained this ability in the peptide format, confirming its role in syncytia formation and highlighting that mimics of this epitope could prevent cell-mediated viral propagation. as summarized in the first section of this review, phage-displayed rpls are powerful tools to determine or to characterize mabs as well as pabs epitopes. besides epitope mapping the phage display technology was also widely applied to the identification of hiv- inhibitors. screening of phage-displayed rpls, antibody-fragment or ligand libraries on viral or host targets contributed to the discovery of molecules interacting with the key players of hiv- infection. antibody libraries were particularly investigated, and repertoires of fab, scfv (antibody fragment corresponding to variable regions of the heavy (vh) and light (vl) chains of antibody connected by a short peptide linker), v hh /nanobodies (single domain antibody fragment (sdab) corresponding to the variable heavy-chain domain of a camelid heavy-chain only antibody (hcabs)) or cdr fragments from naï ve or hiv- infected subjects as well as from immunized animals were displayed at the surface of phages ( figure ). most of the hiv- inhibitors selected with the help of phage display were identified by targeting viral proteins (table ). ( ) -helix, izn ( ) -helix ( ) pie -trimer ( ) tat ( ) cyclin t [ ] [ ] ( ) ncp ( ) psi rna the cd binding site represents one of the main achille's heels of the virus since it is involved in the earliest step of hiv- entry and is conserved in almost all hiv- strains [ , ] . numerous phage display biopannings were performed on gp and are classified here according to the type of antibody libraries used. burton et al. were the first to report the construction of a phage-displayed fab library from the bone marrow of an asymptomatic hiv- -infected patient with high titers of gp -specific abs [ ] . this library was screened against recombinant gp from the iiib isolate and clones displaying high affinities (< nm) for gp were selected [ ] . one fab (b ) (see section . . . .) was able to neutralize the mn and iiib strains in different set-ups. this ab is the most potent neutralizing ab isolated to date, featuring neutralizing activity against % of primary isolates of hiv- tested at concentrations that could be achieved by passive immunization [ ] . to improve its affinity, the b fab was submitted to cdr walking, a procedure involving randomization of its cdr and expression of the derived libraries expressed on phages, followed by screening against on gp [ ] . sequential cdr walking of the hcdr and hcdr domains was performed and four clones were chosen for detailed analysis of their binding affinity and neutralization potency against the iiib and mn isolates. a pro glu mutation of the hcdr was identified in the clone with highest affinity, b , which bound iiib gp with an -fold improved affinity ( . nm) compared to the parental b fab. similarly, fab b was able to neutralize four isolates that were insensitive to the parental b fab. the cdr walking mutagenesis strategy was pursued in a subsequent study and a further -fold improvement of the binding affinity of b for gp was achieved, reaching pm [ ] . these studies were the first to demonstrate that recombinant fabs (devoid of the typical igg contamination residual of calpain cleavage) featured neutralizing activities similar to those of whole igg. as the fabs fragments were easier to produce and their smaller size allowed them to target binding sites that were not accessible to full-length igs, this led to the construction of many fab libraries to elucidate the immune response to hiv- and to identify therapeutic antibodies. the extremely high binding affinity of b was also applied to develop an immunotoxin which could specifically kill hiv- -infected lymphocytes [ ] . the authors engineered b scfv fused to a truncated form of pseudomonas exotoxin a. the b (fv)-pe fusion immunotoxin bound to the mn strain of gp with the same affinity as the parental fab antibody and specifically killed a gp -expressing cell line and a chronically hiv-infected lymphocytic cell line. this study provided the proof-of-concept that high affinity anti-hiv- antibodies have a dual application since they may be used for their neutralizing potency but also as carriers for antiviral compounds. most antibodies obtained through screening fab libraries against monomeric gp targeted epitopes related to the cd -binding domain of gp , pointing to it as an immunodominant epitope [ ] . to expedite the identification of ntabs directed against weakly immunogenic epitopes, a strategy called epitope-masking was applied in several studies. this biopanning approach is designed to mask a particular epitope with antibodies or ligands directed against the region of interest prior to addition of the phage library. ditzel et al. panned a fab library from an asymptomatic hiv- -infected patient on immobilized recombinant gp and identified two dominant clones targeting the cd -binding site [ ] . these two fabs were then incubated with gp to mask their respective epitopes and the panning of the library was repeated, highlighting (based on sequence similarities) four groups of fabs recognizing gp with affinities in the range of to nm. epitope mapping of one representative fab for each group showed that gp binding of three clones was influenced by the v loop and the cd -binding site and was not affected by the glycosylation status of gp . furthermore, one of these fabs (l ) featured a broad neutralization spectrum against various hiv- strains. the authors performed further epitope masking by using different selection strategies with the same fab phage library [ ] . the first strategy involved masking the cd -binding site (cd bs) epitopes either with soluble cd or with a cd bs ab. all fabs selected on scd -bound gp recognized the c region, while the fabs isolated from the cd -bs ab-captured gp were classified in four different groups: (i) fabs targeting the c region, similarly to fabs isolated on scd -bound gp ; (ii) fabs directed against the c -c -region; (iii) fabs recognizing the v loop; and (iv) fabs directed against a cd bs/v loop region, similar to the neutralizing fab isolated by ditzel et al. [ ] . multiple epitope masking was then conducted by masking the cd bs-mab-captured gp with one of the c -specific fabs selected on the scd -bound gp prior to phage addition, leading to the identification of two c /c -dependent fabs. all isolated fabs bound their targets with affinities ranging from to nm. however, these fabs targeting weakly immunogenic regions were not or poorly neutralizing. more recently, koefoed et al. investigated the anti-gp ab repertoire of the circulating gp -binding igg-bearing b cells of hiv- -infected patients by constructing phage displayed fabs libraries from unselected cells or from cells preselected with immobilized gp [ ] . panning against gp selected for a higher number of phagotopes from the preselected library. clones from the unselected library recognized the v loop, while clones from the preselected library targeted the cd bs or a cd -induced epitope encompassing the c region. these fabs displayed no significant differences with respect to epitope specificity, affinity and neutralization ability compared to fabs obtained from bone marrow libraries, and most of them were unable to neutralize hiv- . these results were in accordance with previous findings by parren et al. ( ) concluding that the majority of the circulating hiv- specific antibodies were elicited by viral debris and were therefore devoid of neutralizing activity [ ] . antibodies recognizing the amino acids - of the gp cd bs were isolated from patients suffering from the systemic lupus erythematosus autoimmune disease [ , ] . however, whether these antibodies neutralized hiv- was not known, which prompted karle et al. to quantify gp -recognizing abs in an existing scfv phage-displayed library from the pbmcs of lupus-suffering patients [ ] . biopanning selected for clones binding both gp and the - region of the gp -cd -binding site. one of these clones (jl ) neutralized r and x -tropic hiv- primary isolates from clades b, c and d with ic ranging from . to . µg/ml. a subset of gp -binding antibodies was shown to hydrolyze gp by a mechanism analogous to serine protease [ ] , as the nucleophilic region responsible for this activity was localized in the light chain [ , ] , a library of light chains prepared from three lupus patients [ ] was screened with an electrophilic analogue of gp residues - to isolate antibodies capable of binding and hydrolyzing gp [ ] . one of the light chain clones selected (skl ) cleaved a gp - -reporter substrate as well as full-length gp . engineering of abs composed of such a light chain coupled to a gp -binding heavy chain might provide abs with anti-viral proteolytic activities. the advantages of reduced antibody formats were also explored with naturally occurring smaller antibodies. in addition to conventional antibodies, camelids also produce antibodies devoid of light chains ( figure d ). these heavy-chain only antibodies (hcabs) lack the c h domain and their binding specificity is provided by the variable heavy-chain domains of hcabs (v hh or nanobodies) [ ] . the cdr regions of nanobodies are on average longer cdr than those of conventional abs and display a protruding conformation, thereby more easily binding clefts and active sites. nanobodies were successfully used for panning against various pathogens (reviewed by vanlandschoot [ ] ). forsman et al. immunized llamas with a recombinant gp of subtype b/c (cn ) [ ] . three different panning strategies against various gp resulted in the selection of three nanobodies (a , d and c ) with neutralizing activity against a limited panel of clade b and c strains (ic values ranging from . to µg/ml). these nanobodies bound gp with affinities from . to nm and inhibited its binding to cd as well as to mabs known to recognize the gp cd binding site and, last but not least, competed with each other for gp binding. in a follow-up study, koh et al. described a family-based approach to produce nanobodies similar to a and d [ ] . they used a degenerated oligonucleotide annealing to the last six codons of cdr and framework (fr) and an fr -specific primer to amplify a sublibrary of related v hh clones with properties similar to the parental a or d v hh . more than phagotopes were tested for their ability to neutralize clade b and clade c strains. all tested v hh displayed similar neutralizing activity against the clade b viruses. three different neutralization profiles (broad a -like, intermediate and narrow d -like potency) were observed against the clade c type strains and the breadth of neutralization potency appeared to be correlated to the presence of an yyd motif in the c terminus of a cdr . when the cdr ydd motif from a was introduced within d , gp binding affinity increased by -fold and neutralization of clade c strains increased by -fold. these studies were the first to demonstrate bntabs elicited in immunized animals. as v hh are stable and can be produced at a relatively low cost, they are promising hiv- inhibitors. the cd receptor recognizes gp through residues located within its v region, and engineering of this cell receptor was applied to identify cd variants with a better affinity for gp , thereby displaying hiv- inhibitory properties. in , krykbaev et al. constructed a phage-displayed library of cd v and v -v variants generated by error-prone pcr and screened it against gp [ ] . five clones with increased affinity for gp and presenting mutations within the cd v domain were identified. all of these clones inhibited hiv- entry with ic ranging from . to µg/ml. the phage display technology was used to improve the affinity of the mab - d for its v loop epitope, which had also been identified through phage display [ , ] . in , thompson et al. expressed mab - d as scfv on phages and combined its v h with λ and κ chains from a non-immunized pbl repertoire prior to panning on a peptide containing the v loop sequence [ ] . additional shuffling of the hcdr and hcdr regions was combined to hcdr -spiking‖, i.e., the introduction of random mutations, resulting in the identification of four key residues that could be mutated to improve ab affinity. a sublibrary in which all four codons were simultaneously mutated was constructed and biopanning allowed to select for one scfv ( p h ) with improved k d against the mab - d epitope. two neutralizing fabs (fab loop and fab do - ) were obtained by screening a fab library against recombinant gp reaching ic ranging from . to µg/ml [ , ] . in , ferrer and harrison screened -mer, -mer and constrained -mer rpl against gp and identified two peptides from the -mer rpl [ ] . the first sequence rinnipwseamm ( p ) inhibited cd as well as ntmab b binding while the second peptide, tspyedwqtylm ( p ) did not affect the cd interaction and rather enhanced b binding. the p peptide was further investigated and shown to inhibit binding of monomeric yu gp to both scd and b with ic values of . and . µm respectively. the p peptide also inhibited binding of these ligands to trimeric envelope glycoproteins, blocked binding of gp to the native coreceptor ccr , and specifically inhibited hiv- infection of target cells in vitro [ ] . hiv- entry is a multi-step process requiring the successive binding of gp to cd and to a coreceptor, ccr or cxcr , and triggers successive conformational changes that expose transient epitopes. targeting of these epitopes with ntabs could therefore prevent hiv- infection, as has been proven with the clinically approved fusion inhibitor enfuvirtide. to identify such receptor-induced epitopes, moulard et al. screened a fab library constructed from an hiv- infected patient (fda- ) with high ntab titers against gp -cd -ccr complexes [ ] . one fab clone, x , bound gp from several strains with a low nanomolar affinity. furthermore, binding affinity was significantly increased in the presence of cd and slightly enhanced by ccr . competition assays with a panel of antibodies targeting different hiv- epitopes revealed that x recognized an epitope located in close vicinity to the cd and coreceptor binding sites. neutralization assays with isolates from clades a, b, c, d, f and g demonstrated that x neutralized all isolates with potency comparable to that of b . x is the first bntab recognizing a receptor-induced epitope identified to date. to select for ligands for cd -induced epitopes, murine leukemia virus particles carrying the env protein of the dual-tropic . strain pre-incubated with scd were recently used to screen -mer, -mer-c and -mer rpls [ ] . one of the selected phagotopes (xd : hkqpwydywllr) displaying sequence similarities (in bold) with the n-terminal extracellular part of the ccr and cxcr coreceptors was identified, suggesting novel potential leads for tyrosine sulfation. both the xd phagotope and xd peptides strongly and specifically bound to . gp regardless of cd and xd competed with mab b for binding to a cd -induced epitope. the sulfated form of the xd peptide recognized x , r and dual-tropic strains and inhibited hiv- entry in the high micromolar range. the salp salivary protein of ixodes scapularis inhibits cd + t cells activation by binding to the cd molecule in a region that may overlap with the gp binding site. this inhibition is mediated by the c-terminal - gpngqtcaeknkcvghipgc sequence [ ] . juncadella et al. thus analyzed salp as a potential hiv- inhibitor and demonstrated that salp inhibited gp -cd interaction and subsequent cell fusion and that the gpngqtcaeknkcvghipgc peptide also interacted with gp [ ] . to identify which gp amino acids interacted with peptide - of salp , the authors screened a -mer rpl against salp and isolated a hvitplw sequence homologous to an (i/l)tpl motif of the gp c /v domain which is highly conserved across hiv- isolates. finally, they mapped the interaction site of full-length salp protein or of its - c-terminal domain were able to bind to the pcvkltplcvtlnct peptide within the gp c /v region. in addition to epitope mapping and inhibitor identification, phage display was also widely applied to elucidating the determinants of the initial response to hiv- antigens and more particularly the importance and different roles of igm and igg during the establishment of infection. indeed, all known bntabs are iggs that are somatically hypermutated and are thus more difficult to elicit. in contrast, igms are closer to germline antibodies and the identification of hiv- specific igm could be relevant for the development of vaccine immunogens. the studies listed in this section explored and emphasized the importance of the initial igm response against hiv- and viral strategies to skew it towards non-neutralizing or infection-enhancing antibodies. screening against gp with igm and igg fab repertoires constructed from a healthy donor demonstrated that only fabs isolated from igm were able to recognize gp , although they were polyreactive, displayed low affinities and no neutralizing properties [ ] . sequence analysis evidenced that selected gp -binding fabs originated from different v h germline genes. several studies reported that gp displays superantigenic properties giving it the ability to bind and stimulate non-immune b cells to secrete v h ig in vitro [ ] . interestingly, the v h antibody family is the most represented immunoglobulin gene family in healthy adults ( % of peripheral repertoire) and hiv- infection leads to altered v h production through selective depletion of the anti-hiv- v h antibodies [ ] . toran et al. further applied the phage display technology to examine and compare the human v h genes involved in igm and igg responses to gp to identify the correlates of long-term non progression. two igm and igg phage-displayed fab libraries from an hiv- -infected ltnp with high gp -specific igm and igg titers were constructed and screened [ ] . several clones were selected from the igm library (m , m , m and m ) and three clones from the igg library (s , s and s ). all igm fabs were polyreactive and had a binding affinity for gp in the micromolar range while the igg fabs were specific and bound gp with affinities in the nanomolar range, as expected. sequence analysis showed that igg fabs originated from the same germline ab. the igm fab m displayed the same v h region nucleotide substitutions as those of igg fab s and used similar d h and j h segments, suggesting that s arose from m by isotype switching. in addition, a four aminoacid difference in the hcdr sequence of m (tgqwe) and s (rggsi) was proposed to be associated with the -fold affinity increase for gp and to the higher neutralizing activity of igg fab s (id = ng/ml) than of igm m (id = µg/ml). in a follow-up study conducted two years later, the three igg fabs were submitted to reverse mutations to reconstitute the germline amino acid residues [ ] . the higher affinity and neutralizing ability of s were due to the ala arg and ala asp somatic mutations in the hcdr region of the germline gene sequence, providing clues for rational modifications of cdr in human antibodies to improve affinity and hiv- neutralization capacity. the igm to igg isotypic switch generating high affinity neutralizing or non-neutralizing antibodies is triggered by the activation of igm-producing b cells. to characterize the epitopes recognized by hiv- -specific igm and to assess the effects of these abs on hiv- infection, chen et al. constructed a fab library from blood, lymph nodes and spleen from healthy donors [ ] . the library was panned against gp (env ectodomain containing both gp and a truncated gp lacking transmembrane domain and cytoplasmic tail) of a clade b isolate and allowed for the selection of one fab clone (r h m) with a relatively high binding affinity for gp from different strains. a sublibrary derived from this clone was panned against gp from different isolates, resulting in the selection of clones (m , m a, m b, m c and m d) binding with high affinity to clade b and f gp (ec ranging from nm to nm). while these antibodies had weak neutralizing properties against x -tropic isolates, they did not inhibit and in some cases even enhanced infection with r -tropic isolates. the m ab, whose sequence is relatively similar to the germline ab, targeted highly conserved epitopes located near the cd binding site or the coreceptor binding site. the high immunogenic capacity of the conserved non-neutralizing epitopes of such antibodies could divert the immune system from actually neutralizing epitopes. the authors suggested that these newly identified mabs could be used as probes to further characterize conserved non-neutralizing or enhancing epitopes and to modify or remove them from candidate vaccine immunogens. the epitope masking strategy (section . . . .) might be applied to these epitopes to redirect the immune system to elicitation of antibodies targeting neutralizing epitopes. to select for antibodies closely resembling the germline antibodies as a candidate source of bntabs, the same authors constructed a cord-blood-derived igm library which they submitted to parallel screening against hiv- env, sars coronavirus protein binding domain (rbd) and soluble hendra virus g protein (sg) [ ] . although rbd and sg antigens provided enriched igm, the library could not be enriched in hiv env-binding phagotopes. these results are in accordance with the hypothesis that hiv- could have evolved strategies based on weak or absent binding to antibodies of the germline repertoire [ ] . presenting epitopes unsuitable for binding of somatically hypermutated antibodies would enable the virus to escape from strong immune responses. . this library was screened on the mn peptide encompassing the f epitope eldkwa, and led to the identification of the z mab [ ] . epitope mapping experiments based on synthetic peptides and recombinant proteins showed that the epitope targeted by mab z was located downstream of the f epitope and centered on the nfwdit sequence. mab z neutralized primary isolates from hiv- b, c and e subtypes. to enhance binding potency, fabs sublibraries of z variants were engineered and screened against an mper peptide and gp [ ] . the selected z e variant displayed an over -fold increase in neutralization breadth and potency compared to the parental z mab. binding experiments coupled to competition assays revealed that the z e mab bound to a waslwnwfditn minimal epitope overlapping the f epitope and that the asn and asp residues were essential for peptide recognition as well as hiv- neutralization. very recently, phage-and yeast-displayed abs libraries constructed from an hiv- -infected patient with f -like bntabs were panned against peptides containing the f epitope and against the hiv- jr-fl gp [ ] . two mabs (m and m . ) were identified and the most mutated variant (m . ) neutralized hiv- with a higher potency than m . ala substitutions indicated that both abs recognized the dkw core of the f epitope and two additional leucine residues located upstream (l( , )). fusion of viral and host membranes, the last step of hiv- entry, requires the initiated by the insertion of the gp -encoded fusion peptide into the host cell membrane and the formation of an extended prehairpin intermediate (phi). the gp n-and c-terminal heptad repeats (nhr, chr) then collapse to form a six-helix bundle ( -hb) in which the nhr form a trimeric coiled-coil, creating grooves where the chr bind. the viral and cellular membranes are thereby brought into close proximity, enabling fusion ( figure ). during phi formation, the nhr and chr do not interact and may thus be transiently targeted by compounds which prevent the formation of the six-helix bundle in a dominant-negative manner. one such compound is the synthetic chr mimic enfuvirtide (t ) [ , ] . a hydrophobic pocket located on the n-terminal peptide trimer groove of the -hb is highly conserved among hiv- sequences and plays a critical role in membrane fusion, and therefore represents a select target for inhibitors. eckert et al. used the particular approach called -mirror-image phage display‖ to identify d-peptides targeting the gp hydrophobic pocket [ ] . in this approach, a phage library of natural l peptides is screened against the mirror image of a target synthesized as d-peptide [ ] . by symmetry, the selected d-peptide phagotope sequences will bind the natural l-form of the target. the main advantage of d-peptides over l-peptide inhibitors is their resistance to natural proteases which enhances their oral bioavailability and serum half-life. in a first study, the authors screened a constrained -mer rpl against the d-peptide sequence of the gp hydrophobic pocket fused to a soluble trimeric coiled-coil (izn ). pocket-specific binders with a consensus motif cx ewxwlc were identified and inhibited cell fusion or hiv- entry into cells with an ic in the micromolar range when synthesized using d-amino acids. in a second study, the same authors constructed a sublibrary based on the consensus sequence identified in their first study [ ] which allowed the selection of sequences with a fourfold potency increase [ ] . surprisingly, the most potent peptide was a -mer with a cx ewxwlc motif which was probably selected from the -mer sublibrary because its smaller size favored a more compact hydrophobic core upon binding to the gp hydrophobic pocket. screening of second generation d-peptides from a -mer cx wxwlc library led to the selection of pocket-specific inhibitor of entry (pie) with an ic of nm. dimeric and trimeric forms of pie had respective ic values of . nm and pm. in a third study, the same authors constructed a phage library based on the pie core sequence flanked by two randomized amino acids (xxcdypewqwlcxx) and obtained phages with the h(a/p)-[pie core]-(r/k/e)l consensus sequence [ ] . a peptide (pie , hpcdypewqwlcel) exhibited a broad neutralizing spectrum and was even more efficient than t , reaching an ic of . nm, when trimerized. moreover, this third generation pie -trimer displays broadened inhibitory potency and resistance to viral variants, as escape mutants required over weeks of selection in vitro to emerge. the pie trimer is thus a promising entry inhibitor and may be used as a topical microbicide in its d conformation. in , there was no evidence of abs capable of binding the highly conserved nhr region targeted by the t inhibitor. to determine whether antibody fragments could target this determinant on the gp protein, miller et al. constructed a phage-displayed naï ve scfv library and screened it against a synthetic protein mimicking the -hb [ ] . this construct, named -helix, lacks one of the three chrs and the nhr trimer is partially exposed, presenting a single binding site for a chr mimic [ ] . the scfv library was also panned against the izn compound, a homotrimerized form of nhr amino acids fused to a coiled-coil peptide, therefore representing a -hb mimic devoid of the chr trimer [ ] . the authors identified a scfv (d ), which blocks hiv- entry and inhibits infection in a single-cycle infectivity assay. this scfv retained its properties when produced as a whole igg . the antibody was found to bind the hydrophobic pocket of the nhr trimer and ala scan experiments revealed the crucial role of residues l , w , and k located in the hydrophobic pocket for this interaction. igg d was able to neutralize at least five hiv- isolates with ic ranging from to nm, thereby demonstrating that the hydrophobic pocket of the nhr trimer is accessible for binding of hiv- inhibitors as large as iggs. the same year, another study demonstrated that antibodies binding with high affinity and specificity to heptad repeats can be isolated from synthetic fab minimalist libraries presenting tyr/ser randomization of their cdr [ ] . very recently, liu et al. took advantage of these results and constructed a minimalist fab library where the lcdr and hcdr - domains were randomized with tyr and ser [ ] . screening of this library on the -helix mimic selected for fabs with affinity and specificity values comparable to those obtained with the scfv of the previously described d ab [ , ] . huang et al. used the n (l )c polypeptide mimicking the -hb [ ] to screen -mer and -mer rpls and selected sequences bearing a hxx(n/d)pf motif [ ] . this consensus motif synthesized as peptide (jch- ) inhibited hiv- -mediated syncytia formation [ ] . fusion inhibitors were also identified by screening non-immune human fab libraries. louis et al. screened such a library [ ] against antigens comprising the trimeric coiled-coil nhr fused or not to the gp six-helix bundle (n ccg-n and nccg-gp , respectively) [ , ] . they identified fabs targeting (i) the -hb; (ii) the nhr trimeric coiled-coil or (iii) both -hb and trimeric coiled-coil [ ] . these antibodies were tested in a cell fusion inhibition assay and the two more potent mabs, belonging to the third group, featured an ic of - µg/ml. two years later, the same library was screened against nccg-gp and -hb antigens [ ] . two clones, fabs and , inhibited cell fusion while one fab clone selected against nccg-gp was also effective in infection neutralization assays. fab bound the -hb as well as stable nhr trimers, and recognized an epitope that partially overlapped the hydrophobic pocket targeted by the d ab. the same authors subsequently demonstrated that the n mut(e,g) peptide presenting mutations within the th and th aa residues of the heptad repeat [ ] increased the temporal window of viral sensitivity to fab and thereby synergistically enhanced the neutralizing activity of fab as well as of the bntabs f and e [ ] . a fab sublibrary was created by affinity maturation of the fab hcdr loop and screened against nccg-gp , selecting for three fabs (fabs , and ) with enhanced potency (average -fold decrease in ic ) and neutralization breadth [ ] . to follow-up a study demonstrating that affinity-purified iggs from rabbits immunized with n ccg n inhibited hiv- -mediated fusion [ ] , nelson et al. rescued a scfv antibody library from these animals [ ] . three n ccg n binders were selected, and one of them, k , displayed neutralizing activity against hxb . in parallel a more complex fab library was constructed from the fda- hiv- positive patient from whom z ab had previously been isolated [ ] . screening this library against n ccg n allowed for the isolation of fab dn [ ] . both scfv k and fab dn neutralized hiv- infection with a panel of viral strains with ic ranging from to nm and targeted the nhr trimeric coiled-coil, presumably close to the hydrophobic pocket. three additional gp -specific abs (m , m and m ) were obtained by screening antibody phage libraries from asymptomatic seropositive patients [ ] against gp [ ] [ ] [ ] . a recombinant gp (gp r ) isolated from an asymptomatic seropositive patient with bntabs was reported to elicit bntabs in monkeys, further demonstrating that immunogenic epitopes were exposed on this recombinant antigen [ ] . competitive antigen panning (cap) (biopanning approach designed to outcompete phagotopes binding to an immunodominant region of a multi-domain target through concomitant addition of an excess of soluble forms of this immunodominant domain) using a mixture of gp r as antigen and gp r as competitor resulted in the selection of a gp -specific m ab [ ] . m displayed broad neutralization properties and recognized a conformational epitope and bound weakly to -helix antigen but not to the trimeric nhr nor to -hb. in two other studies, the same libraries were panned against gp / from three different isolates ( . , cm and r ), which led to the identification of the m ab recognizing a conformational epitope of gp [ ] and the m ab, which binds gp , -helix and -hb but not to the nhr trimeric coiled-coil. m recognized a conserved conformational epitope and neutralized isolates from different clades with a significantly higher potency than e or z [ ] . the competitive antigen panning approach against gp / thus allows the selection of abs recognizing conformational epitopes on gp , which are not properly folded when gp is used as a target. most of the studies found in the literature that apply the phage display technology to the discovery of hiv- inhibitors target the env protein. however, reports about the identification of peptides directed against other hiv- proteins involved in viral replication as well as interfering with rna sequences have been published and are summarized in this section. vpr is involved in the nuclear import of the viral preintegration complex (pic) as well as in the induction of apoptosis after cell cycle arrest and can be packaged into virions in quantities similar to the structural proteins [ ] [ ] [ ] [ ] . vpr was also reported to be associated with numerous cellular proteins such as glucocorticoid receptors, transcription factors or the uracyl dna glycosylase (udg) [ , [ ] [ ] [ ] . to investigate whether vpr may be used as a docking protein to deliver anti-viral compounds into virions, bouhamdan et al. used a -mer rpl to determine a common motif involved in the interaction between vpr and its various ligands [ ] . screenings rounds against vpr fusion proteins pinpointed sequences sharing a wxxf motif. since udg contains a wxxf sequence, mutants were constructed and confirmed the importance of this motif for vpr binding. cotransfection experiments indicated that the wxxf motif might be used to deliver a fusion protein into the hiv- virion through a new docking strategy. in , krichevsky et al. conducted a study to elucidate the exact role of vpr and its contribution to the nuclear import process of the hiv- pic [ ] . to that aim, a semi-synthetic scfv library [ ] was screened against the n-terminal (aa - ) part of vpr (vprn) conjugated to bsa (vprn-bsa). purified scfvs fragments featuring their strong and specific binding to the vprn sequence recognized full-length vpr and inhibited vpr-mediated nuclear import, indicating that targeting vpr may lead to the development of new peptides to fight viral infection. the phage display technology has also been applied to the identification of the hiv- integrase inhibitors. in , desjobert et al. screened a -mer rpl against recombinant hiv- integrase and identified a high affinity phagotope displaying the fhnhgkq sequence [ ] . in peptide format, this sequence inhibited the strand transfer activity of in by competing with the target dna, providing the proof-of-concept that in is also a valuable target for phage display. interaction of the viral transcription activator tat with the human cyclint subunit of the positive transcription elongation factor (p-tefb) complex and the cooperative binding of this complex to the transactivation response element (tar) rna are prerequisites of hiv- transcription [ ] . screening rpls or fab libraries against tat, cyclint or tar elements using the phage display technology identified peptides impairing tat-mediated hiv- replication. the first study was conducted in , when pilkington et al. screened a fab library constructed from the ab repertoire of an hiv- -infected asymptomatic patient and selected fabs recognizing a region comprised between amino acids to of the tat protein in a conformation-dependent manner [ ] . many years later, a non-immune human scfv phage-displayed library was explored to identify peptides binding to cyclint [ ] . clones recognizing the cyclin box domain of cyclint or interacting with the tat/tar recognition motif (trm) were isolated after panning against the n-terminal amino acids of cyclint . when expressed as intrabodies (antibody or antibody fragment expressed intracellularly), one of these scfvs inhibited tat-mediated transactivation without impairing cellular basal transcription or inducing apoptosis and partially inhibited hiv- replication in cultured cells. the tar rna sequence adopts a specific structure recognized by the basic arginine rich motif (arm) of tat and thus represents a potential target for phage display screening. in , kolb and boiziau screened a -mer rpl against tar rna molecules and selected -mer and unexpectedly -mer sequences from the library [ ] . the latter were proposed to arise from incomplete enzyme restriction during the construction of the initial library. clones were further characterized in peptide format and displayed tar-specific binding. the authors suggested that the surprisingly long peptides might have been selectively retrieved from the library because they presented a conformation that shorter -mer peptides were unable to adopt. the hiv- nucleocapsid protein p (ncp ) is processed from the gag precursor and is involved in the protection and encapsidation of viral rna leading to viral assembly through interaction with a specific secondary structure of the -base long psi rna [ ] [ ] [ ] [ ] . lener et al. screened a constrained -mer rpl against ncp and selected phagotopes sharing a ppx(d/e)r consensus motif [ ] . further binding experiments suggested that the ncp -phage interactions involved amino acids to of ncp , encompassing a zinc finger domain. studies to identify inhibitors of viral packaging were also conducted. rpls were screened on the psi rna immobilized onto a streptavidin-coated surface by annealing its '-end to a biotinylated oligomer, leading to the selection of peptides characterized by an hwwpww motif [ ] . peptide variants presenting this motif were subsequently synthesized and the most efficient binder was shown to strongly reduce virus release by infected cells, suggesting that it could serve as a lead compound to develop new anti-hiv- drugs [ ] . a similar screening campaign was conducted, where the '-end of the psi rna was covalently immobilized, leaving the secondary structure intact and fully accessible [ ] . screening of a -mer rpl selected for four clones with either whxt or hssxy motifs which were assessed for specific and dose-dependent binding to psi rna. the most prevalent sequence (syqwwwhspqtl) was expressed in fusion with the maltose-binding protein and was able to compete with ncp for binding to psi rna, confirming the value of the peptide as a potential hiv- inhibiting compound. hiv- accessory proteins nef and vif have an important role in hiv- viral replication and infectivity and, as such, represent as such interesting targets for inhibitors. in , yang et al. demonstrated that vif was able to multimerize and that its -aalikpkqikpplp- domain was critical for multimerization pointing to it as an interesting target to impair vif-mediated viral replication [ ] . the authors therefore, screened a -mer rpl library against vif and selected phages sharing a common pxp motif [ ] . four of these sequences synthesized as peptides bound the c-terminus of vif with high affinity and were able to inhibit vif-vif as well as vif-hck tyrosine kinase interactions. moreover, these peptides inhibited hiv- replication in cultured cells. besides the identification of inhibitors from rpl, the discovery of abs targeting nef and vif applicable to intrabody-based therapy may represent an alternative way to impede viral replication [ ] . in a very recent study, yoshikawa et al. evaluated the effect of two different schedules of nef and vif administration for mice immunization prior to the construction of scfvs phage-displayed libraries [ ] . results demonstrated that the immunization protocol influenced the complexity of the elicited ab repertoire and thus the successful identification of abs specifically recognizing the target. a nanobody (sdab ) recognizing a conformational epitope and reacting with a high affinity (k d : nm) with nef proteins from a panel of hiv- m, n, o and p groups was isolated through phage displaying the v hh repertoire of a llama immunized with a purified recombinant nef protein (fragment - ) [ ] . when expressed as an intrabody, this anti-nef sdab inhibited important biologic functions of nef both in vitro and in vivo in cd c/hiv- nef transgenic mice. the first step in cytotoxic t lymphocytes (ctl) activation is the recognition by a t cell receptor (tcr) of the antigen-derived peptide/mhc class i complex (pmhc). to date, few studies were undertaken to examine antigen presentation at a cellular and molecular level. nunoya et al. screened pooled scfv libraries [ ] on an immunodominant hla-a* (a ) restricted ctl epitope within the nef protein (nef - ; rypltfgwcf) [ ] . the panning procedure yielded clones binding specifically to nef - /a . clones scfv and scfv were able to bind to nef - /a expressed at the cell surface and retained this specificity when expressed as reconstituted whole iggs. this recent study was the first to address the identification of monoclonal antibodies binding specifically to an immunodominant hiv- ctl epitope loaded on an hla class i molecule. reverse transcriptase is a valuable target for anti-hiv- compounds, as illustrated by the success of the multiple small compounds used in haart. in , gargano et al. panned a phage-displayed library of synthetic combinatorial human fab fragments against recombinant hiv- rt [ ] . two ab fragments that specifically inhibited the rna-dependent dna polymerase (rddp) activity of rt were identified. both fragments also inhibited the activities of avian and murine retroviral rts as well as the human dna polymerase α and prokaryotic dna polymerases. because of their lack of specificity, these abs fragments were not exploited further as anti-hiv- molecules. to develop a panel of recombinant mabs reacting with different epitopes of the rt, ohba et al. immunized mice with recombinant rt expressed in a vaccinia virus vector and constructed a phage-displayed mice fab fragments library [ ] . biopanning against recombinant rt led to the identification of two fab fragments ( f and g) able to strongly inhibit the rddp activity of hiv- rt. epitope mapping and competitive elisa showed that f and g recognized an epitope similar or closely related to the epitope targeted by the mouse mab ( c ) previously described by the same authors [ ] . two years later, a semisynthetic phage display library of human scfvs with randomized heavy and light chain cdr was screened against recombinant rt [ ] . five different scfv abs directed against rt were isolated, of which three (f- , e , b ) inhibited the rddp activity of rt; of note, (f- ) also inhibited rt dna-dependent dna polymerase (dddp) activity. synthesis of the peptides corresponding to the cdr regions of the heavy and light chains showed that the heavy chain cdr inhibited rddp activity while the light chain peptide had no effect. these hcdr peptides represent the smallest antibody fragments inhibiting the rt identified to date and demonstrated that hcdr repertoire is a potential source of bioactive molecules (see section . . . .). rev is a key regulatory protein. oligomerized rev binds to unspliced or singly spliced viral mrna and ensures its transport to the cytoplasm, thereby allowing the translation of viral gene products. despite considerable efforts, the structure of rev is poorly characterized since rev is refractory to crystallization, mainly because of its tendency to form insoluble aggregates [ ] . in the absence of structural information, the phage display technology was used by different authors to map the domains involved in the interaction of rev with its network of partners. pilkington et al. identified two rev-specific fabs from a fab library derived from the ab repertoire of an hiv- -infected asymptomatic patient [ ] . these fabs were directed against sites adjacent to the rev basic nuclear localization signal (nls) (residues - ) and to the activation domain (residues - ). two years later, jensen et al. screened a -mer rpl to identify potential rev peptidic antagonists [ ] . three groups of sequences sharing a srlxg(x) - r motif (group i), sharing a rvv(x) - rg/a motif (group ii) or featuring no sequence similarity (group iii), were obtained. three clones were selected based on their high frequency of occurrence (p and p , group i) or on their strong binding affinity for rev (p , group iii). they were synthesized as peptides and were shown to retain rev binding specificity. more recently, llama nanobody libraries from animals immunized with recombinant rev allowed the identification of rev-binding nanobodies [ ] . one of them (nb ) prevented or disrupted rev multimerization by interacting with lys and tyr of the rev n-terminal α-helix [ ] . besides inhibitor discovery, fabs were recently proposed as -crystal chaperones‖ to support crystallization of their partners by locking them in specific conformations and blocking aggregation [ ] . stahl et al. described the preparation, characterization, and crystallization of an equimolar complex formed between rev and a chimeric rabbit/human fab (sjs-r ) selected through phage display [ ] . the rev/sjs-r fab complex was successfully crystallized and the fab sjs-r was shown to recognize a conformational epitope in the n-terminal half of rev. structural characterization of the crystallized fab/rev complex is ongoing and a corresponding scfv has been engineered and may have anti-hiv- properties. to identify peptides interfering with hiv- capsid assembly, sticht et al. screened a -mer rpl against the capsid (ca) protein generated by the proteolysis of the gag precursor and identified phagotopes whose sequences could be classified in four groups [ ] . one of these sequences (cai, capsid assembly inhibitor) competed with phagotopes for binding to ca and inhibiting capsid assembly in vitro. interaction with cai was mapped to ca amino acids , with additional contacts in helix . cai did not inhibit capsid assembly in vivo, but may nevertheless serve as tool for drug screening and as a starting point for drug design based on its ca-binding properties. peptides and antibody fragments selected by means of phage display may also be used for diagnostic purposes or to assess the diversity of the immune response against hiv- -specific antigens. de haard et al. constructed a scfv library from pbls of an hiv- positive patient presenting antibodies against gp , gp and p and screened the library against gp and p [ ] . one phagotope recognizing an epitope within the f and e bntabs epitopes on gp (ab# ) with affinities in the nanomolar range was isolated. importantly, it was shown to compete with out of gp -reactive plasma samples from north-american and african hiv- positive patients, indicating that this antibody recognizes an epitope conserved in a large panel of isolates and might be suitable for diagnostic applications. since all vaccine candidates elicited antibodies reacting positively in hiv tests, khurana et al. applied the phage display technology to identify hiv- epitopes susceptible to help discriminate between successfully immunized vaccinees and seroconverters [ ] . they constructed a phage library displaying the full hiv- genome and screened it against the sera of newly seroconverted hiv- positive individuals. they identified conserved epitopes present in gp and in gag p that were not part of the vaccine used at that moment and established a new detection test, named selectest, that demonstrated over % selectivity and sensitivity for the early detection of seroconversion. hiv selectest was able to detect antibodies against these epitopes in newly infected patients as early as to weeks after infection. in parallel to the targeting of viral proteins, many efforts were undertaken to identify peptides, antibody fragments or modified ligands binding to the hiv- host proteins and impairing their interactions with the viral proteins (table ). the hiv- host receptors cd , ccr and cxcr are involved in the early steps of hiv- infection and thus represent valuable targets for the identification of antiviral peptides or neutralizing abs. moreover, these receptors display very low variability compared to the viral env proteins facilitating the identification of neutralizing antibodies. although the cd receptor plays a crucial role in the entry process, the only phage display biopanning assays reported to date targeted the chemokine receptors ccr and cxcr . however, to circumvent the difficulties of purifying and immobilizing such complex receptors on a solid support without losing their native structure, biopanning procedures had to be adapted. in this regard, screening strategies using biopanning on living cells [ , ] , proteoliposomes [ ] or peptides derived from the receptors extracellular parts [ , ] were particularly successful. the cc chemokine receptor (ccr ) is one of the two major hiv- coreceptors and binds three different endogenous chemokines ccl (rantes), ccl (mip- β) and ccl (mip- α) which were reported to prevent r -tropic hiv- entry. interestingly, inhibition of ccr binding to hiv- provides an almost complete protection against r -tropic viruses with only minor effects on the normal physiological functions of the cells [ ] . the first biopanning experiment targeting ccr was performed using receptor embedded in paramagnetic proteoliposomes [ ] . to create such proteoliposomes, magnetic beads were added to a mixture of synthetic lipids, a detergent-solubilized c -tagged ccr receptor and a capture antibody, reconstituting membrane bilayers containing pure, native and properly oriented ccr receptor. these proteoliposomes were used in biopanning experiments with a human scfv antibody library and several antibody fragments specifically binding to ccr -expressing cells were identified. the same year, steinberger et al. used the phage display technology to select and to humanize rabbit anti-ccr antibodies preventing the export of ccr to the cell surface [ ] . following rabbit immunization with a gst-nterm ccr fusion protein, the authors constructed a phage displayed fab library that was screened against the antigen initially used for the immunization. a phagotope (st ) binding strongly and specifically to the immobilized antigen as well as to ccr -positive cells was identified, expressed as a scfv and humanized by successive replacements of the rabbit light and heavy chains by their human counterparts. one humanized antibody fragment, st / , that retained the strong ccr -binding capacity of the parental st antibody was isolated from the screening of the intermediate libraries. when expressed as intrabody the st / scfv efficiently blocked the ccr expression at the cell surface. [ ] (reviewed in chevigné et al. [ ] . in this strategy, a phage library displaying randomly mutated and n-terminally extended ccl chemokine variants (xs#xssx###-ccl , where # represents either a, p, s or t) was constructed and screened on ccr -expressing cells. only intracellular phagotopes that had induced ccr receptor internalization were recovered. two ccl variants (p = lspvssqssa-ccl and p = fsplssqssa-ccl ) were identified. these variants displayed a higher selectivity for ccr and had more potent hiv- inhibitory abilities than the wild-type ccl chemokine. further characterization demonstrated that p acted as a ccr superagonist and potently induced intracellular ccr sequestration. p was less potent but significantly reduced ccr -dependent intracellular calcium signaling. in a subsequent study, p and p variants were optimized by phage display biopanning to select for variants that retained the high anti hiv- potency of p and p but reduced ccr -agonist activity [ ] . three successive generations of libraries (xxpx q#tp-ccl , qgpplmx -ccl and qgpΨ$x -ccl where Ψ represents g, l or p and $ represents g, l or m) were constructed and screened as previously described [ ] . the three most interesting candidates ( p -ccl , p -ccl and p -ccl ) produced as soluble proteins displayed highly potent antiviral activities. analogue p -ccl acted as an agonist and sequestered ccr , p -ccl induced no signaling or receptor sequestration while p -ccl induced ccr internalization without triggering g-protein signaling. altogether these data demonstrated that antiviral activities of similar molecules identified through phage display screening can rely on various mechanisms of action. shortly after the identification of the p and p variants, zhang et al. reported an alternative biopanning methodology relying on the use of small cyclic biotinylated peptides mimicking ccr extracellular loops (ecl , ecl and ecl ) for the identification of ccr -binding scfvs [ ] . a mouse phage-displayed scfv library was incubated with biotinylated-ecl peptides and ecl-binding phages were specifically recovered using streptavidin-coated beads. among the ccr -binding scfv identified, three clones (a , b and l ) selected on cyclic ecl or ecl peptides inhibited r -tropic hiv- . screening of rpl was also applied to the discovery of small ccr -blocking peptides through the targeting of receptor-expressing cells [ , ] . vyroubalova et al. screened a partially randomized -mer phage library (cdx kpcallryx -piii) using competitive elution with a ccl analogue (nny-ccl , nm) and selected a unique peptide (allrynpfyylsfsp). this peptide was further optimized through n-terminal extension, exon shuffling and biopanning. by applying successive treatments consisting of a preselection using a low amount of nny-ccl ( pm) to discard low-affinity binding phages followed by classical alkaline elution using tea and a competitive elution containing nny-ccl ( µm) they identified an extended peptide (lldstfftadallrynpfyyls-fsp) inhibiting r -tropic hiv- cell fusion with an ic of μm. in parallel, wang et al. screened a fully randomized -mer rpl using acidic elution and identified phagotopes binding specifically to ccr -expressing cells and sharing the afdwtfvpslil sequence [ ] . in peptide and phage formats this sequence blocked the binding of the anti-ccr neutralizing d mab and completely inhibited binding of the chemokine ccl to the receptor [ ] . the cxc chemokine receptor (cxcr ) is the second major hiv- coreceptor. cxcr binds only to one endogenous chemokine ligand (cxcl ) and is also expressed at the surface of numerous cancer cell types underlining its high value as therapeutic target [ , ] . despite this importance and the relative success of phage biopanning on ccr , only two recent studies reported the use of phage display to search cxcr inhibitors [ , ] . in , jahnichen et al. isolated llama-derived v hh binding specifically to cxcr and inhibiting the entry of x -tropic virus [ ] . to select v hh binding exclusively to functional and properly folded receptor, llamas were immunized with cxcr -expressing hek t cells. a phage library was subsequently constructed from the pbmcs of immunized camelids and several phage clones inhibiting the binding of labeled cxcl chemokine to the receptor were identified. in particular, two v hhs ( d and d ) showed low nanomolar affinity for the receptor and inhibited entry of x and x /r -viruses into different cxcr + cell types with ic values ranging from to nm. dimerization of d and d to form biparatopic proteins increased their antiviral properties to ic values in the picomolar range. epitope mapping revealed that the two v hh s inhibited cxcr mainly through binding to the second extracellular loop. very recently, we used a peptide corresponding to this particular extracellular loop (ecl ) as target to identify short cxcr antagonists [ ] . by screening a non-immune phage library displaying the human hcdr peptide repertoire [ ] , several small peptides binding to the ecl peptide that specifically recognized cxcr -expressing cells were identified. notably, one of these hcdr peptides (typgry) acted as a cxcr antagonist with potency in the micromolar range. in addition to targeting host receptors, a few studies reported the identification of peptides or antibody fragments directed against other host proteins such as cell surface determinants (cd) or intracellular enzymes [ , , , ] . the cd receptor, a member of the tumor necrosis factor receptor superfamily, and its cd l ligand are involved in several biological processes including cell proliferation, activation and production of cytokines and chemokines. during hiv- infection, viruses were proposed to selectively downregulate or even deplete the pool of cd l-expressing cd + t cells. in this context, antibodies binding to cd and restoring the hiv- -induced cd l downregulation might be of interest. in , ellmark et al. identified a set of anti-cd antibody fragments through biopanning of a human scfv phage library against a biotinylated cd antigen [ ] . when expressed as full-length igg one antibody (clone b ) suppressed hiv- infection by a r -tropic virus, most probably through induction of cc chemokine production [ ] . more recently, the ddx protein, a cellular rna helicase involved in rna unwinding was shown to play important roles in hiv- replication. this protein presents a unique region (alramkeng) responsible for high affinity binding to the hiv- rna. garbelli et al. carried out a biopanning experiment with a mix of linear -mer and linear and constrained -mer rpl against a peptide mimicking the ddx region interacting with hiv- . they identified a -mer peptide (sdvptqv) blocking the replication of an x -tropic virus with an ic of μm [ ] . besides therapeutic purposes, the phage display technology has also been applied for fundamental studies of host proteins. in particular, a study focusing on the roles and the diversity of the anti-cd autoimmune repertoire in the myelosuppression appearing in hiv- infected individuals was reported by rubinstein et al. [ ] . using a substractive biopanning procedure from the immune repertoire of a hiv- seropositive patient, the authors selected fab fragments binding specifically to the cd receptor. sequencing and binding analyses of these antibody fragments demonstrated the heterogeneous origin of the anti-cd autoimmune repertoire and suggested that these autoantibodies might be generated through antigen-specific driven processes. the use of phages for protease cleavage specificity profiling was first described by matthews and wells in [ ] . this -phage substrate‖ approach relies on the use of phage particles to screen for enzyme substrates instead of classical binder selection. protease cleavage profiling using phage takes advantage of the natural resistance of phage particles to proteolysis. phage particles displaying random peptides are immobilized on a solid support and submitted to proteolytic elution to specifically liberate phages presenting peptides corresponding to the protease cleavage site. the phage substrate approach allows thus to rapidly determine the cleavage profile of a given protease and provides optimized substrate candidates which can be further used as leads for the development of specific inhibitors. over the last two decades, phage substrate has been applied to a large variety of proteases including the hiv- protease (pr) [ , ] . the hiv- pr is a homodimeric aspartic protease responsible for nine critical cleavage steps within both the structural (gag) and the non-structural (gag/pol) polyproteins. the hiv- protease recognizes substrate residues encompassing p to p ' positions (schechter and berger's nomenclature), with the primary determinants from positions p to p ' positions [ , ] . interestingly, alignment of the nine natural substrate cleavage sites of the hiv- pr shows a high sequence diversity suggesting a broad proteolytic specificity. in , beck et al. reported the use of hexapeptide phage library to unravel the hiv- pr specificity and develop new protease inhibitors (table ) [ ] . this library was constructed by fusing the mab -e epitope upstream of the randomized sequences. phages were first incubated with the hiv- pr and uncleaved phages were removed by addition of pansorbin cells. biopanning selected for highly diverse sequences consistent with the suggested broad substrate specificity of the hiv- protease. however, none of the selected peptides corresponded to the hiv- polyproteins cleavage sites. nonetheless, several phage-displayed peptides were very efficiently cleaved by the hiv- protease. in peptide format, most of them displayed a k m value lower than the one determined for a peptide mimicking the natural substrate at the matrix/capsid junction (irkil↓fldg) ( table , italic). the most potent selected substrate (gsgif↓letsl) was cleaved times more efficiently and had a k m value of μm i.e., times lower than the natural substrate (k m = μm). interestingly, the gsgif↓letsl substrate displayed only two residues (in bold) of the optimal cleavage site model designed based on the most frequently selected residues for each position (sgvy↓fvts) ( table ) . table . phage substrate analysis of the hiv- protease cleavage specificity. arrow denotes cleavage sites in the natural or the selected substrates sequences. underlined substrates correspond to sequences used to derived inhibitors. italic sequences correspond to natural substrate (matrix/capsid junction) used as reference for the assessment of the cleavage efficiently. Ψ: amid-reduced bound. table . hiv- protease specificity model. the model was build based on the most efficiently cleaved phage substrates. underscript numbers represent the frequency of appearance of the amino acid at a given position. sequences corresponding to the four most potent substrates were synthesized as peptidic transition state analogues and presented inhibitory activity in the nanomolar range (table , underlined sequences). in , the same authors applied the phage substrate approach to compare the relative specificities of the human (hiv- ) and feline (fiv) immunodeficiency virus prs [ ] . hexapeptides specifically processed by each of the two proteases as well as peptides cleaved by both enzymes were identified. further mutational analysis of synthetic peptides derived from a sequence processed with the same efficiency by the two proteases (ksgvf↓vvng) was performed to assess the influence of amino acid substitutions on the catalytic process of each pr (table , underlined sequence). they showed that substitutions for a val at position p or p ' increased the cleavage by the hiv- protease whereas the introduction of a val at position p was more favorable to fiv protease activity. in particular, the gsgvfΨ(ch nh)vvngl inhibitor identified in the first study was active against both prs with different potencies and replacement of its amide group by a hydroxyethylene group resulted in a peptide with equivalent inhibitory activity towards both the hiv- and the fiv proteases. in parallel to epitope mapping, inhibitor discovery and enzyme profiling applications, bacteriophage particles were exploited as carriers for the development of anti-hiv- vaccines. for this particular purpose, phages are not used as affinity selection tools but rather to display antigens to the immune system, aiming to elicit specific neutralizing antibodies or/and efficient cytotoxic t-cell responses. indeed, phages can also be considered as biologically inert particles characterized by a dense and repetitive organization capable of displaying a wide range of exogenous proteins at a precise valency and in a controlled manner. moreover, phages were shown to be naturally immuno-stimulatory [ , ] and are particularly affordable, easy and rapid to produce and to administer in many animal models [ ] . altogether these characteristics make bacteriophages a suitable and valuable carrier for hiv- vaccine development. anti-hiv- vaccination trials to elicit humoral and cellular responses against various hiv- proteins such as the envelope proteins gp and gp as well as the rt and the p proteins [ ] [ ] [ ] [ ] [ ] were conducted with a large diversity of bacteriophages including m , t , ms and lambda phages [ , [ ] [ ] [ ] [ ] ( table ) . numerous phage-vaccination trials using mimotopes identified on anti-hiv- antibodies were reported (see tables - ). all these attempts aimed mainly at eliciting antibodies directed against the genuine epitope of an existing antibody. besides these descriptions, a few studies were exclusively dedicated to the use of phage particles as vehicles to display fragments or complete hiv- proteins to the immune system to prime new immune responses. the pioneer attempt was reported by minenkova et al. in [ ] . immunization of rabbits with filamentous m phage particles displaying a small peptide (gedrw) derived from the p gag protein in fusion with piii minor coat protein elicited the production of specific iggs reacting with the natural p antigen as well as with the gag precursor protein. shortly afterwards, two studies reported that mice immunization with fd phages displaying the gp v loop fused to the piii protein induced high titers of antibodies cross-reacting with v loops of different strains and featuring neutralizing activity [ , ] . the major coat protein pviii of the fd phage ( copies per particles) was also investigated as scaffold protein for vaccination. immunization with phages displaying a pviii-fused peptide corresponding to residues - of the hiv- rt resulted in the induction of a specific cytotoxic t-cell response (ctl) against the displayed peptide. priming nevertheless required a co-immunization with a t-helper epitope from the same protein (kdswtvndiqklvgk) provided by either the same or a separate phage particle [ ] . more recently attempts to prime ctl response were conducted with m phages displaying a mixture of thousands of variants of ctl epitopes (rgpgxax or xgxgxaxvxi) derived from the gp v loop (residues - ; rgpgrafvti) presented in an immunoglobulin v h domain scaffold [ ] . mice immunization provided potent and broad epitope-specific long lasting response ( months) and effector memory t cells were induced. moreover, recent studies demonstrated that mice immunized with these variable epitope libraries are capable of neutralizing half of the subtype b viral isolates used for challenge, including hiv- isolates which are known to be resistant to neutralization by several potent monoclonal antibodies [ ] . although they are versatile and easy to manipulate, filamentous phages present limitations for vaccination, and namely low display level and the limited size of the foreign peptides that can be incorporated without interfering with the natural functions of the phage coat proteins. therefore, alternative bacteriophage models were explored. t phage and its two accessory capsid-decorating proteins soc and hoc were demonstrated to be particularly efficient for high copy display of various large hiv- antigens [ , , ] . in contrast to piii and pviii proteins, soc and hoc present the major advantage of being dispensable for phage assembly yet of being able to strongly bind to the outer surface of the capsid, if added subsequently [ ] . soc and hoc self-associate to the capsid at a display level of and copies per capsid, a far higher density than that of filamentous minor coat protein ( to copies). hoc protein provides high-density display of single as well as multiple antigens and immunization of mice with the p -hoc-t elicits strong humoral and cellular responses. moreover, fusion of different antigens to soc and hoc offer the possibility to develop multicomponent hiv- vaccine particles [ ] t phage displaying v loop of the gp protein fused to soc protein was reported to elicit antibodies capable of recognizing the native antigen [ ] . further study demonstrated that hoc protein can also be used to display various hiv- antigens including the p , nef or a trimeric peptide derived from the c-terminus of gp [ ] . more recently, lambda phage and its decorating capsid protein were used for dense display of glycosylated mammalian cell-derived trimers of gp protein [ ] . lambda phage provided a display level of copies of gp trimer per particle, a -fold higher display than observed on native hiv- virions ( ± spikes per virion) [ ] . rabbit immunization trials were rather disappointing and higher antibody titers were elicited in animals receiving soluble oligomeric gp . these results were proposed to be due to the sequential immunization process and to the strong and immunodominant response against phage capsid proteins, which is most probably boosted upon sequential immunization, and could lead to a decrease of the humoral immune response to the displayed antigen. virus like particles (vlp) derived from the ms bacteriophage coat protein displaying the v loop of gp and devoid of phage genome were also reported [ ] . although characterized by a low display level ( copies per vlp) these particles were nevertheless able to elicit high titers of specific antibodies when injected in mice and provided neutralizing activity at / sera dilution. more recently, pp phage-derived vlps displaying the v loop of gp were used to immunize mice providing high-titer antibody response [ ] . aids was first described in [ ] , and two years later hiv- , its causative agent, was isolated [ ] . the phage display technology was first published almost concomitantly in [ ] . both protagonists lived parallel lives until , when burton et al. identified a human fab recognizing the cd -binding site of hiv- gp through phage display [ ] . this first antibody, b , turned out to be one of the rare bntabs characterized to date, and many studies are still ongoing to set up scaffolds presenting its epitope in a conformation capable of eliciting abs with b -like hiv- inhibiting properties. this article was the starting point of a dense literature exploiting the different applications of the phage display technology to gain as much knowledge as possible on the hiv- infection process. the complex hide-and-seek game between hiv and the host immune system and the absence of an efficient vaccine candidate emphasizes the need to better comprehend the hiv- -related immune response and to identify antiviral molecules. hiv- is to the best of our knowledge the only pathogen to which all four applications of the phage display technology described in this review-i.e., epitope mapping, inhibitor discovery, substrate presentation and carrier phage-have been applied. parallel improvements in the technology on one hand, and crucial discoveries in the hiv- field on the other hand, most likely account for such a tight interplay. the phage display technology applied to hiv- has provided precious knowledge concerning the regions recognized by the ab repertoire of immune patients, and allowed to identify immunodominant regions. it has also allowed to map the epitopes of numerous monoclonal antibodies directed against viral and host proteins, and, most importantly, discontinuous epitopes. alternative phage display biopanning substractive procedures such as epitope masking or competitive antigen panning also precisely highlighted non-immunodominant regions of the hiv- antigen, which had remained occult using classical biopanning approaches. although the antibodies identified through epitope-masking are mostly devoid of neutralizing activity, this strategy led to the identification of fabs binding with high affinity to specific targets. taking advantage of this affinity, approaches aiming at engineering bispecific antibodies, coupling these inhibitors to toxins, or multimerizing them to increase their binding avidity could lead to the development of antiviral compounds or more efficient vaccine candidates. although highly informative, immunization trials performed with mimotopes/phagotopes selected through phage display remained altogether rather disappointing as no long-lasting neutralizing response was reported, and provided yet further proof that antigenicity does not necessarily imply immunogenicity [ ] . among the bottlenecks in the field of hiv- vaccine research and development are the weak immunogenic properties of the identified mimotopes or native antigens. whether the difficulty of eliciting and/or isolating strong or broad neutralizing antibodies from hiv- -infected patients, be they normal progressors or ltnps, is a caveat of phage display or a real reflect of the restricted humoral response in fighting hiv, or to both, remains to be established. nevertheless, these discrepancies are not exclusive of hiv- mimotopes and were observed with other antigens. from a fundamental prospect, the use of phage-displayed igm libraries to model abs close to the germline abs allowed to elegantly expore the poorly characterized determinants of the initial antiviral immune response. phage display also contributed to a better knowledge on the structure of the diverse hiv- proteins as well as a gain of insight on non-structural proteins involved in replication mechanisms. a highlight example would be the phage substrate approach which allowed the precise characterization of the hiv- protease cleavage specificity and thereby provided valuable inhibitor candidates. unraveling viral replication steps or protein interactions led to exploit phage display to engineer various types of inhibitors targeting either viral or host proteins. in parallel, phage display allowed the identification of various types of inhibitors targeting either viral or host proteins. nevertheless, only a few strong antiviral candidates with broad neutralizing activities and/or potencies in the nanomolar or even picomolar range were identified with the phage display technology while most of them presented inhibiting activities that ranged at the best in the micromolar range and could not be further exploited. some of the most potent inhibitors originated from fab libraries derived from asymptomatic hiv- -infected patients whose reactivity against hiv- was previously assessed (b , x , z ) or from immunized camelids (v hh anti cxcr ). other inhibitors were identified from semi-synthetic (ligand analogues of ccl ) or randomized peptide libraries (d-peptides: pie ) and their affinity was improved through secondary libraries. these inhibitors were selected against different targets (env proteins, coreceptors) by biopanning carried out using different types of support (immobilized proteins, cells, peptides), illustrating the power and versatility of the phage display technology [ , , ] (figure ). inhibitors blocking key steps in the entry process were identified using the phage display technology. these inhibitors target: the cd binding site (fab b and z ), the coreceptors ccr (ccl variants) or cxcr (v hh d and d ), the cd -induced epitope of gp (fab x ) or the heptad repeat region of gp (peptide pie ). despite the variable success of the phage display technology per se in developing a direct antiviral agent or immune response, this technology continues to contribute precious and otherwise inaccessible developments in the field of hiv- research, mainly owing to the ability of phages to display a high density of either single antigens or libraries of antigens/mimotopes. to this regard, phages are being extensively explored to increase immune responsiveness. the latest developments include exploiting the phage as dna vaccine carrier or hybrid phage and pursuing the symbiotic interplay between phage display and hiv- . pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) running a tightrope: regulatory challenges in the development of antiretrovirals potent suppression of hiv- replication in humans by t- , a peptide inhibitor of gp -mediated virus entry maraviroc (uk- , ), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor ccr with broad-spectrum anti-human immunodeficiency virus type activity -dihydroxypyrimidine carboxamides and n-alkyl- -hydroxypyrimidinone carboxamides are potent, selective hiv integrase inhibitors with good pharmacokinetic profiles in preclinical species plasma hiv rna decline and emergence of drug resistance mutations among patients with multiple virologic failures receiving resistance testing-guided haart an overview of the determinants of ccr and cxcr co-receptor function the human immunodeficiency virus type envelope spike of primary viruses can suppress antibody access to variable regions continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers the antibody response against hiv- . cold spring harb. perspect filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface lambda-display: a powerful tool for antigen discovery antibody-selectable filamentous fd phage vectors: affinity purification of target genes human immunodeficiency virus type long-term non-progressors: the viral, genetic and immunological basis for disease non-progression production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type envelope glycoprotein identification of hiv vaccine candidate peptides by screening random phage epitope libraries anti-human immunodeficiency virus type human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries defining critical residues in the epitope for a hiv-neutralizing monoclonal antibody using phage display and peptide array technologies a human monoclonal antibody to a complex epitope in the v region of gp of human immunodeficiency virus type has broad reactivity within and outside clade b epitope mapping of anti-hiv and anti-hcv monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library permissive residues within the minimal epitopes of neutralizing monoclonal antibodies to the v loop of hiv- neutralization of divergent human immunodeficiency virus type variants and primary isolates by iam- - f , an anti-gp human monoclonal antibody human immunodeficiency virus type -neutralizing monoclonal antibody f is multispecific for sequences flanking the dkw core epitope constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-hiv- mab f helical epitopes determined by low-stringency antibody screening of a combinatorial peptide library phage-displayed mimotopes recognizing a biologically active anti-hiv- gp murine monoclonal antibody identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type antibody b probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage immunogenicity of hiv type gp cd binding site phage mimotopes the mapping and reconstitution of a conformational discontinuous b-cell epitope of hiv- peptides selected from a phage display library with an hiv-neutralizing antibody elicit antibodies to hiv gp in rabbits, but not to the same epitope structural and functional characterization of an epitope in the conserved c-terminal region of hiv- gp a peptide inhibitor of hiv- neutralizing antibody g is not a structural mimic of the natural carbohydrate epitope on gp antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a -amino acid sequence of the viral envelope, gp principal neutralizing domain of the human immunodeficiency virus type envelope protein human monoclonal antibody that recognizes the v region of human immunodeficiency virus gp and neutralizes the human t-lymphotropic virus type iiimn strain a conserved neutralizing epitope on gp of human immunodeficiency virus type humoral immune response to immunocomplexed hiv envelope glycoprotein a large array of human monoclonal antibodies to type human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals recombinant human fab fragments neutralize human type immunodeficiency virus in vitro computational prediction of the cross-reactive neutralizing epitope corresponding to the [corrected] monclonal [corrected] antibody b specific for hiv- gp a conserved hiv gp glycoprotein structure involved in chemokine receptor binding exploration of antigenic variation in gp from clades a through f of human immunodeficiency virus type by using monoclonal antibodies characterization of conserved human immunodeficiency virus type gp neutralization epitopes exposed upon gp -cd binding the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of α → mannose residues on the outer face of gp human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type the mannose-dependent epitope for neutralizing antibody g on human immunodeficiency virus type glycoprotein gp selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv- -positive sera protection of rhesus macaques against disease progression from pathogenic shiv- . pd by vaccination with phage-displayed hiv- epitopes dissection of the humoral immune response toward an immunodominant epitope of hiv: a model for the analysis of antibody diversity in hiv+ individuals collection of phage-peptide probes for hiv- immunodominant loop-epitope mimotopes selected with antibodies from hiv- -neutralizing long-term non-progressor plasma inducing cross-clade neutralizing antibodies against hiv- by immunofocusing evolution of antibody landscape and viral envelope escape in an hiv- crf _ag infected patient with e -like antibodies unravelling the antigenic landscape of the hiv- subtype a envelope of an individual with broad cross-neutralizing antibodies using phage display peptide libraries mapping of hiv- gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system the protein data bank u. d-epitope-explorer ( dex): localization of conformational epitopes within three-dimensional structures of proteins molecularly cloned shiv- ipd n : a highly replication-competent, mucosally transmissible r simian-human immunodeficiency virus encoding hiv clade c env requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on ccr monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor ccr : implications for the tertiary structure of the receptor and for an n-terminal binding site for hiv- gp inhibition of m-tropic hiv- infection by the fd phage-gene protein with mip- α-binding activity identification of a linear peptide recognized by monoclonal antibody d capable of generating ccr -specific antibodies with human immunodeficiency virus-neutralizing activity identification of a lfa- region involved in the hiv- -induced syncytia formation through phage-display technology interaction of chemokine receptor ccr with its ligands: multiple domains for hiv- gp binding and a single domain for chemokine binding ccr levels and expression pattern correlate with infectability by macrophage-tropic hiv- , in vitro human immunodeficiency virus (hiv) infection in cd + t lymphocytes genetically deficient in lfa- : lfa- is required for hiv-mediated cell fusion but not for viral transmission molecular profile of an antibody response to hiv- as probed by combinatorial libraries specific killing of hiv-infected lymphocytes by a recombinant immunotoxin directed against the hiv- envelope glycoprotein cdr walking mutagenesis for the affinity maturation of a potent human anti-hiv- antibody into the picomolar range in vitro evolution of a neutralizing human antibody to human immunodeficiency virus type to enhance affinity and broaden strain cross-reactivity neutralizing recombinant human antibodies to a conformational v -and cd -binding site-sensitive epitope of hiv- gp isolated by using an epitope-masking procedure antibodies to the superantigenic site of hiv- gp : hydrolytic and binding activities of the light chain subunit cross-clade hiv- neutralization by an antibody fragment from a lupus phage display library generation of a family-specific phage library of llama single chain antibody fragments that neutralize hiv- llama antibody fragments with cross-subtype human immunodeficiency virus type (hiv- )-neutralizing properties and high affinity for hiv- gp jones, i. mutant cd molecules with improved binding to hiv envelope protein gp selected by phage display affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity mapping the protein surface of human immunodeficiency virus type gp using human monoclonal antibodies from phage display libraries peptide ligands to human immunodeficiency virus type gp identified from phage display libraries broadly cross-reactive hiv- -neutralizing human monoclonal fab selected for binding to gp -cd -ccr complexes peptide ligands selected with cd -induced epitopes on native dualtropic hiv- envelope proteins mimic extracellular coreceptor domains and bind to hiv- gp independently of coreceptor usage broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type glycoprotein gp an affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type gp recognizes an epitope between those of f and e potent d-peptide inhibitors of hiv- entry design of a potent d-peptide hiv- entry inhibitor with a strong barrier to resistance inhibiting hiv- entry: discovery of d-peptide inhibitors that target the gp coiled-coil pocket a human monoclonal antibody neutralizes diverse hiv- isolates by binding a critical gp epitope design of potent inhibitors of hiv- entry from the gp n-peptide region synthetic fab fragments that bind the hiv- gp heptad repeat regions identification of the hiv- gp core-binding motif-hxxnpf the mechanism by which molecules containing the hiv gp core-binding motif hxxnpf inhibit hiv- envelope glycoprotein-mediated syncytium formation characterization and hiv- fusion inhibitory properties of monoclonal fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of hiv- gp a monoclonal fab derived from a human nonimmune phage library reveals a new epitope on gp and neutralizes diverse human immunodeficiency virus type strains affinity maturation by targeted diversification of the cdr-h loop of a monoclonal fab derived from a synthetic naive human antibody library and directed against the internal trimeric coiled-coil of gp yields a set of fabs with improved hiv- neutralization potency and breadth sequestering of the prehairpin intermediate of gp by peptide n mut(e,g) potentiates the human immunodeficiency virus type neutralizing activity of monoclonal antibodies directed against the n-terminal helical repeat of gp antibody elicited against the gp n-heptad repeat (nhr) coiled-coil can neutralize hiv- with modest potency but non-neutralizing antibodies also bind to nhr mimetics cross-reactive human immunodeficiency virus type -neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp and lacks reactivity against self-antigens cross-reactive hiv- neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp ) isolated from a patient (r ) with broadly hiv- neutralizing antibodies selection of a novel gp -specific hiv- neutralizing human antibody by competitive antigen panning diversity of hiv- vpr interactions involves usage of the wxxf motif of host cell proteins antibody fragments selected by phage display against the nuclear localization signal of the hiv- vpr protein inhibit nuclear import in permeabilized and intact cultured cells identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type integrase recombinant human fab antibody fragments to hiv- rev and tat regulatory proteins: direct selection from a combinatorial phage display library inhibition of tat-mediated transactivation and hiv- replication by human anti-hcyclint intrabodies selection by phage display of peptides targeting the hiv- tar element use of a constrain phage displayed-peptide library for the isolation of peptides binding to hiv- nucleocapsid protein (ncp ) identification of peptide ligands for target rna structures derived from the hiv- packaging signal ψ by screening phage-displayed peptide libraries inhibition of hiv- by a peptide ligand of the genomic rna packaging signal ψ selection and characterization of peptides specifically binding to hiv- ψ (ψ) rna modifying the antigen-immunization schedule improves the variety of monoclonal antibodies obtained from immune-phage antibody libraries against hiv- nef and vif inhibition of the nef regulatory protein of hiv- by a single-domain antibody short communication: generation of recombinant monoclonal antibodies against an immunodominant hla-a* -restricted hiv type ctl epitope the multimerization of human immunodeficiency virus type i vif protein: a requirement for vif function in the viral life cycle potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type (hiv- ) vif proteins an immunodominant neutralization epitope on the -thumb‖ subdomain of human immunodeficiency virus type reverse transcriptase revealed by phage display antibodies a novel neutralization epitope on the -thumb‖ subdomain of human immunodeficiency virus type reverse transcriptase revealed by a monoclonal antibody recombinant human antibodies against the reverse transcriptase of human immunodeficiency virus type- hiv- rev nuclear export signal binding peptides isolated by phage display measuring cooperative rev protein-protein interactions on rev responsive rna by fluorescence resonance energy transfer an intrabody based on a llama single-domain antibody targeting the n-terminal α-helical multimerization domain of hiv- rev prevents viral production generation and characterization of a chimeric rabbit/human fab for co-crystallization of hiv- rev a peptide inhibitor of hiv- assembly in vitro potential drug targets on the hiv- envelope glycoproteins, gp and gp small-molecule hiv- gp inhibitors to prevent hiv- entry: an emerging opportunity for drug development molecular characterization of the circulating anti-hiv- gp -specific b cell repertoire using antibody phage display libraries generated from pre-selected hiv- gp binding pbls relevance of the antibody response against human immunodeficiency virus type envelope to vaccine design binding of glycoprotein and peptides from the hiv- envelope by autoantibodies in mice with experimentally induced systemic lupus erythematosus and in patients with the disease prospects for immunotherapeutic proteolytic antibodies phosphonate ester probes for proteolytic antibodies naturally occurring proteolytic antibodies: selective immunoglobulin m-catalyzed hydrolysis of hiv gp theory of proteolytic antibody occurrence site-directed mutagenesis of proteolytic antibody light chain naturally occurring antibodies devoid of light chains nanobodies(r): new ammunition to battle viruses mode of action for linear peptide inhibitors of hiv- gp interactions cutting edge: cd is the receptor for the tick saliva immunosuppressor, salp the ixodes scapularis salivary protein, salp , prevents the association of hiv- gp and cd natural human antibodies retrieved by phage display libraries from healthy donors: polyreactivity and recognition of human immunodeficiency virus type gp epitopes a vh clonal deficit in human immunodeficiency virus-positive individuals reflects a b-cell maturational arrest selective variations in vivo of vh and vh gene family expression in peripheral b cell igm, igd and igg during hiv infection molecular analysis of hiv- gp antibody response using isotype igm and igg phage display libraries from a long-term non-progressor hiv- -infected individual improvement in affinity and hiv- neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by hiv- infection cross-reactive human igm-derived monoclonal antibodies that bind to hiv- envelope glycoproteins characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: implications for mechanisms of immune evasion and design of vaccine immunogens cross-reactive hiv- -neutralizing human monoclonal antibodies identified from a patient with f -like antibodies enfuvirtide: the first therapy to inhibit the entry of hiv- into host cd lymphocytes identification of d-peptide ligands through mirror-image phage display protein design of an hiv- entry inhibitor molecular recognition by a binary code structural basis for hiv- neutralization by a gp fusion intermediate-directed antibody subdomain folding and biological activity of the core structure from human immunodeficiency virus type gp : implications for viral membrane fusion fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides covalent trimers of the internal n-terminal trimeric coiled-coil of gp and antibodies directed against them are potent inhibitors of hiv envelope-mediated cell fusion design and properties of n(ccg)-gp , a chimeric gp molecule with nanomolar hiv fusion inhibitory activity design of a novel peptide inhibitor of hiv fusion that disrupts the internal trimeric coiled-coil of gp neutralizing and infection-enhancing antibody responses to human immunodeficiency virus type in long-term nonprogressors the human immunodeficiency virus type vpr transactivator: cooperation with promoter-bound activator domains and binding to tfiib human immunodeficiency virus type vpr induces apoptosis following cell cycle arrest human immunodeficiency virus vpr product is a virion-associated regulatory protein identification of hiv- vpr product and function human immunodeficiency virus type vpr protein binds to the uracil dna glycosylase dna repair enzyme the glucocorticoid receptor type ii complex is a target of the hiv- vpr gene product interaction of virion protein vpr of human immunodeficiency virus type with cellular transcription factor sp and trans-activation of viral long terminal repeat antibody fragments from a -single pot‖ phage display library as immunochemical reagents hiv- tat interacts with cyclin t to direct the p-tefb ctd kinase complex to tar rna a mimic of hiv- nucleocapsid protein impairs reverse transcription and displays antiviral activity evidence of interactions between the nucleocapsid protein ncp and the reverse transcriptase of hiv- hiv- : fifteen proteins and an rna basic residues in human immunodeficiency virus type nucleocapsid promote virion assembly via interaction with rna human fc epsilon rialpha-specific human single-chain fv (scfv) antibody with antagonistic activity toward ige/fc epsilon rialpha-binding human recombinant antibody fragments neutralizing human immunodeficiency virus type reverse transcriptase provide an experimental basis for the structural classification of the dna polymerase family human immunodeficiency virus type regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich -bubble‖ located within the rev-responsive region of viral mrnas selection of human anti-human immunodeficiency virus type envelope single-chain antibodies from a peripheral blood cell-based phage repertoire human immunodeficiency virus (hiv) vaccine trials: a novel assay for differential diagnosis of hiv infections in the face of vaccine-generated antibodies identification of peptide ligands to the chemokine receptor ccr and their maturation by gene shuffling selection of cc chemokine receptor -binding peptide from a phage display peptide library paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, ccr selection of active scfv to g-protein-coupled receptor ccr using surface antigen-mimicking peptides selection of a cxcr antagonist from a human heavy chain cdr -derived phage library the role of a mutant ccr allele in hiv- transmission and disease progression generation and characterization of a recombinant human ccr -specific antibody. a phage display approach for rabbit antibody humanization human immunodeficiency virus type entry inhibitors selected on living cells from a library of phage chemokines highly potent, fully recombinant anti-hiv chemokines: reengineering a low-cost microbicide cxcr nanobodies (vhh-based single variable domains) potently inhibit chemotaxis and hiv- replication and mobilize stem cells identification of a strongly activating human anti-cd antibody that suppresses hiv type infection modulation of the cd -cd ligand interaction using human anti-cd single-chain antibody fragments obtained from the n-coder phage display library a motif unique to the human dead-box protein ddx is important for nucleic acid binding, atp hydrolysis, rna/dna unwinding and hiv- replication engineering and screening the n-terminus of chemokines for drug discovery the therapeutic potential of cxcr antagonists in the treatment of hiv infection, cancer metastasis and rheumatoid arthritis targeting cxcr in hiv cell-entry inhibition non-immunized natural human heavy chain cdr repertoires allow the isolation of high affinity peptides mimicking a human influenza hemagglutinin epitope anti-cd + fabs generated against hematopoietic stem cells in hiv-derived combinatorial immunoglobulin library suggest antigen-selected autoantibodies sensitivity of hiv type primary isolates to human anti-cd antibody-mediated suppression is related to coreceptor use substrate phage: selection of protease substrates by monovalent phage display molecular basis for the relative substrate specificity of human immunodeficiency virus type and feline immunodeficiency virus proteases identification of efficiently cleaved substrates for hiv- protease using a phage display library and use in inhibitor development on the size of the active site in proteases. i. papain kinetic and modeling studies of s -s ' subsites of hiv proteinases interferon production by t coliphage bacterial viruses as human vaccines? bacteriophage lambda is a highly stable dna vaccine delivery vehicle design of specific immunogens using filamentous phage as the carrier phage display of peptide epitopes from hiv- elicits strong cytolytic responses structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage. the v loop of hiv- gp antigenic properties of phage displayed peptides comprising disulfide-bonded loop of the immunodominant region of hiv- gp phage display of functional, full-length human and viral membrane proteins engineering a peptide epitope display system on filamentous bacteriophage phage t soc and hoc display of biologically active, full-length proteins on the viral capsid immunogenic display of diverse peptides on virus-like particles of rna phage ms dense display of hiv- envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to hiv- env phage display of intact domains at high copy number: a system based on soc, the small outer capsid protein of bacteriophage t variable epitope library-based vaccines: shooting moving targets assembly of human immunodeficiency virus (hiv) antigens on bacteriophage t : a novel in vitro approach to construct multicomponent hiv vaccines immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus l epitope, on virus-like particles of the rna bacteriophage pp variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type -neutralizing antibody response the two dispensable structural proteins (soc and hoc) of the t phage capsid; their purification and properties, isolation and characterization of the defective mutants, and their binding with the defective heads in vitro distribution and three-dimensional structure of aids virus envelope spikes antigenic and immunogenic phage displayed mimotopes as substitute antigens: applications and limitations this manuscript is supported by the -centre de recherche public-santé‖, luxembourg (grants gpcr and eumimo). the authors are grateful to danielle perez-bercoff for thorough reading and helpful discussions. the authors also want to thank carole devaux, virginie fievez, marie-eve dumez and martyna szpakowska for critically reading the manuscript. key: cord- - vykwml authors: hainline, kelly m.; fries, chelsea n.; collier, joel h. title: progress towards the clinical translation of bio-inspired peptide and protein assemblies date: - - journal: advanced healthcare materials doi: . /adhm. sha: doc_id: cord_uid: vykwml supramolecular materials composed of proteins and peptides have been receiving considerable attention towards a range of diseases and conditions from vaccines to drug delivery. owing to the relative newness of this class of materials, the bulk of work to date has been preclinical. however, examples of approved treatments particularly in vaccines, dentistry, and hemostasis are demonstrating the translational potential of supramolecular polypeptides. here we describe critical milestones in the clinical development of this class of materials and describe currently approved supramolecular polypeptide therapies. additional examples of not-yet-approved materials that are steadily advancing towards clinical use are also featured. spherical assemblies such as virus-like particles (vlps), designed protein nanoparticles, and spherical peptide amphiphiles are highlighted, followed by fiber-forming systems such as fibrillizing peptides, fiber-forming peptide-amphiphiles, and filamentous bacteriophages. finding utilization in biomedical applications ranging between immunomodulation, drug delivery, tissue regeneration, defect repair, cell delivery, and combinations thereof. many self-assembling systems have been investigated in preclinical research, but among these, relatively few have been successfully translated into approved devices and therapeutics. those that are in current clinical use tend to possess the following features that have enabled their success: -chemical definition-highly specified control over material composition, assembly properties, and bioactivity, in contrast with biologically sourced materials -manufacturability-the extent to which the platform can be manufactured with relative ease at minimum cost and with maximum reproducibility -tunability-the ability to adjust the amount of multiple selected active components in a material with precision and reproducibility. synthetic supramolecular systems tend to feature this property well beyond naturally derived materials. selected platforms are discussed based on these features, because they have had a meaningful translation into clinical practice, or because they have had steady progress toward clinical translation. these include spherical assemblies such as virus-like particles, designed protein nanoparticles, and peptide amphiphiles (figure ) ; and elongated structures such as β-sheet nanofibers, fiber-forming peptide amphiphiles, and filamentous phage (figure ) . a timeline of their development is shown in figure . some of the first engineered supramolecular structures to be translated effectively have been virus-like particles (vlps), multiprotein constructs that self-assemble to mimic the organization, structure, and immunogenicity of native viruses but that lack infectious genetic materials ( figure a ). [ , ] their development has preceded other supramolecular materials discussed here (see timeline in figure ). vlps are potent immunogens, able to stimulate b cell/antibody responses, cd + t-cell responses, and cytotoxic t-cell responses. [ , ] owing to their lack of a viral genome, vlp-based vaccines circumvent some risks associated supramolecular materials composed of proteins and peptides have been receiving considerable attention toward a range of diseases and conditions from vaccines to drug delivery. owing to the relative newness of this class of materials, the bulk of work to date has been preclinical. however, examples of approved treatments particularly in vaccines, dentistry, and hemostasis demonstrate the translational potential of supramolecular polypeptides. critical milestones in the clinical development of this class of materials and currently approved supramolecular polypeptide therapies are described in this study. additional examples of not-yet-approved materials that are steadily advancing toward clinical use are also featured. spherical assemblies such as virus-like particles, designed protein nanoparticles, and spherical peptide amphiphiles are highlighted, followed by fiber-forming systems such as fibrillizing peptides, fiber-forming peptide-amphiphiles, and filamentous bacteriophages. over the past few decades, researchers have built an expansive toolbox of self-assembling peptides and proteins that offer unique advantages over traditional small molecules and polymers for a range of biomedical applications. the first examples of synthetic self-assemblies were bioinspired derivatives of native proteins, but an increasing familiarity with the design rules governing supramolecular assembly has more recently facilitated the de novo design of this class of materials. the ability to create predictable and engineerable supramolecular structures has led to the implementation of several of these materials within biomedical applications. in this review, we highlight self-assembling polypeptide materials that have been clinically translated. we will also discuss the advantageous properties and features that have enabled their clinical implementation. for recent reviews on preclinical work relating to this class of materials including self-assembling immunomodulating materials, [ , ] self-assembled materials for cell delivery, [ , ] and self-assembled biomaterials as a whole, [ , ] the reader is referred to the review articles indicated. properly designed polypeptides can self-assemble into a range of predictable structures including nanofibers, micelles, nanoparticles, and extended networks. these architectures are attenuated or inactivated live viruses, highlighting an advantage of supramolecular systems: they are more compositionally defined than the analogous biological structures, viruses. they also have advantages over subunit vaccines based on viral proteins or peptides conjugated to carrier proteins, which commonly require higher and more frequent dosing and adjuvants to be as effective as inactivated or attenuated viruses. [ ] as alternatives to these previous platforms, vlps were developed to display an array of epitopes that mimic the surface of native viruses more effectively than subunit or peptide vaccines, thus improving their immunogenic properties. [ ] vlps, like viruses, come in a range structures including those with a single capsid protein, multiple capsid proteins, or those without lipid envelopes. [ ] vlps consisting of multiple capsid proteins are expressed and assembled via subsequent processing or by co-expression of polycistronic genes within a cell. several additional aspects of vlps have contributed to their clinical translation. whereas the first translated vlp-based vaccines raised antibodies against the naturally occurring virus capsid proteins of the assembled structure, vlps can also be used as vehicles to display heterologous antigens associated with other infectious diseases. this modularity of the platform is described [ ] copyright , asbmb. (a, right) adapted with permission. [ ] copyright , elsevier. (b) adapted with permission. [ ] copyright , elsevier. (c) adapted with permission. [ ] become increasingly complex, they become increasingly more challenging to manufacture at a large scale. multi-subunit, chimeric, and other types of vlps must overcome these challenges. fortunately, a large assortment of expression systems (bacterial, yeast, insect, plant, mammalian, and cell-free) have been developed for the production of new vlp platform candidates. the idea of using noninfectious virus particles to develop prophylactic human vaccines first became attractive when noninfectious vlps composed of the surface antigen from the hepatitis b virus (hbsag, also known as the australia antigen), were discovered in human sera in . [ ] the first vlpbased human vaccine consisted of hbsag vlps derived from human plasma and was licensed in the united states in ( figure ). subsequently, after the advent of hiv/aids rendered plasma-derived products more challenging, the hbsag particles were produced recombinantly in yeast, and the first recombinant human vaccine, recombivax hb, was licensed by merck in . [ ] three years later in , engerix-b, a similar hbv vaccine, was licensed by glaxosmithkline. it took another years before the next recombinant vlp-based vaccine was licensed for human use. [ ] gardasil, licensed by merck in , is a vaccine against hpv that protects against hpv-related diseases such as cervical cancer. in , several laboratories concurrently observed that recombinantly expressed hpv l and l virus capsid proteins self-assembled into vlps resembling the native virion structure. [ ] in early , based on this observation, merck research laboratories (mrl) initiated an hpv vaccine program. at the time, a growing number of hpv genotypes were identified and associated with benign genital warts and or precursor lesions to cervical cancer. mrl devised a strategy to produce a quadrivalent hpv l vlp that would protect against cervical cancers (caused by hpv and ) as well as genital warts (caused by hpv and ). several concurrent studies in the s were additionally critical to the success of hpv vaccines. one important advancement was the development of an in vitro method of producing hpv virions. the hpv lifecycle is linked to human epithelial tissue differentiation which is difficult to achieve in vitro. kreider et al. overcame this challenge was overcome by developing a complex xenograft model in which human foreskin epithelial tissue was infected with hpv and grown under the renal capsule of athymic mice. this method allowed for the production of infectious hpv . [ ] in , animal challenge studies were published that continued to advance hpv vaccines. two studies demonstrated measurable serum antibody responses to vlp immunization and subsequent protection in later challenges. [ ] the third demonstrated the importance of maintaining the native vlp structure for optimal vaccine efficacy. [ ] another key contribution to the success of mrl's hpv vaccine came from work developing a manufacturing process for hbsag production in saccharomyces cerevisiae. it provided a foundation for the development of methods to design, express, and purify large amounts of hpv vlps. [ ] in , hpv l vlps were evaluated for the first time in humans as a monovalent vaccine during a phase i clinical trial to demonstrate safety and immunogenicity. the monovalent vaccine was chosen owing to the availability of robust models for the evaluation of immunogenicity, and because models for the other three hpv types had not yet been developed. the phase study outcomes were promising, with no reported significant adverse effects. a second study tested the ability of monovalent hpv l vlp efficacy in preventing hpv infection, providing the first demonstration that vaccination with hpv vlps could prevent disease. these favorable results supported the development of the multivalent vaccine and spurred the advancement of the program. in , after impressive efficacy data and an acceptable safety profile, the first hpv vlp vaccine, gardasil, was licensed (see timeline in figure ). [ , ] another hpv vlp human vaccine for the prevention of cervical cancer, cervarix, was developed by glaxosmithkline and licensed in the united states in . this vaccine includes hpv types and and has been produced using insect cells infected with recombinant baculovirus. [ ] cervarix demonstrated a . % efficacy against cervical intraepithelial neoplasia lesions containing hpv and and indicated cross-protection with the hpv types and , leading to the protection against % of cervical cancers. [ ] hecolin (xiamen innovax biotech), a recombinant vlp-based vaccine for prophylactic [ ] copyright , aaas. (a, right) adapted with permission. [ ] copyright , national academy of science. (b, left) adapted with permission. [ ] copyright , american chemical society. (b, right) adapted with permission. [ ] (c, left) adapted with permission. [ ] copyright , elsevier. (c, right) adapted with permission. [ ] use against hepatitis e virus infection, was licensed in china in . [ ] in the past decade, vlp vaccines have played a critical role in the improvement of human health and continue to be applied to new diseases. vlp-based vaccines are currently in clinical trials for the treatment of many infectious diseases: hbv, hiv, hpv, human parvovirus, influenza virus a, norwalk virus, ebola virus, and severe acute respiratory syndromerelated coronavirus. the inclusion of preclinical testing to this list broadens it even further, to diseases including chikungunya virus, west nile virus, and mumps virus. [ ] as mentioned in the introduction, one of the key strategic strengths of supramolecular systems is the modularity that they tend to possess. in vlps, this modularity is exemplified by chimeric vlps, in which epitopes of choice from various diseases are inserted into the particle-forming proteins. provided that the new epitope can be inserted in a way that does not disrupt self-assembly, and that presents the epitope on the surface of the particle so that it is both antigenic and immunogenic, the modularity of the system is maintained. [ ] by being able to insert epitopes of choice, the range of therapeutic applications becomes quite broad. for example, several clinical studies are currently evaluating chimeric vlp vaccines for the treatment of noninfectious diseases such as cancer (melanoma), [ ] neurodegenerative diseases (alzheimer's disease), [ ] autoimmune diseases (allergic rhinoconjunctivitis and asthmas), [ ] and other disorders. another approach for including chosen epitopes/ antigens is separately expressing the vlp and target protein and conjugating them together. this approach is often preferable and necessary due to differences in optimal expression conditions for each component. thus, postproduction methods have been developed to link the vlp and target antigen. this is achieved through genetic manipulation, coupling via supramolecular or covalent bonds, or by encapsulation of cargo by disassembling and reassembling purified vlps in the presence of the desired molecule. [ ] coupling chemistries range from the use of bifunctional crosslinkers, click chemistry, sortase-mediated attachment, polyhistidine/nta-ni + , or affinity-tag interactions to conjugate the vlp and target antigen [ ] notably, these postproduction-modified vlps have led to several platforms currently being tested in clinical trials. [ ] [ ] [ ] although a range of chemical cross-linking strategies have been developed for vlps, [ , ] conjugation of target antigens with multiple reactive sites can lead to heterogeneous coupling or unfavorable epitope display. [ ] the bachmann group addressed this challenge using the spytag-spycatcher system. [ ] spytag is a peptide that forms a spontaneous and irreversible isopeptide bond with spycatcher, its protein partner. [ ] the spycatcher-vlp platform is expressed in escherichia coli and mixed with spytag-antigen to form "plugand-display" decorated vlps, another highly modular, tunable approach that allows for incorporation of a wide variety of antigens to the vlp surface. the group reported that spycatcher-vlps decorated with the cidr or pfs antigens generated a robust antibody response are only a single immunization without requiring adjuvant. this platform shows promise in application such as drug delivery, enzyme scaffolds, biosensors, and cancer immunotherapy, [ ] and it mitigates the need for complex chimeric protein expression or the incorporation of unnatural amino acids, as would be necessary in coppercatalyzed azide-alkyne cycloaddition (click chemistry) or other chemoselective bioconjugation reactions. vlps, owing to their structural definition and flexibility in formulation, also make useful experimental tools for studying basic aspects of immunity. they usually range in diameter from - nm, an optimal size for drainage to lymph nodes and subsequent interactions with b cells. whereas the differentiation of naïve b cells into memory b cells has been extensively studied at the cellular and molecular level, the fate of memory b cells upon antigen re-encounter has been comparatively less well-studied. the bachmann group used a qβ vlp as a model system and were able to track vlp-specific b cells via flow cytometry and histology to follow naïve and memory b cell responses. [ ] they unexpectedly found that, during secondary b cell responses, secondary plasma cells are generated, whereas naïve b cells are recruited into a parallel primary b cell response. this phenomenon allows a plasticity of the memory b cell repertoire upon multiple antigenic exposures. shortcomings of vlps include their requirement for a continuous and well-regulated cold chain, which negatively impacts their distribution to the developing world. to address this challenge, the chackerian group has developed a vlp-based vaccine candidate that is compatible with spray drying, thus enhancing its stability over a broad range of temperatures. [ ] this platform targets a highly conserved, broadly neutralizing epitope from the hpv minor capsid protein, l . not only does this vaccine elicit high-titer and long-lasting antibody immune responses, [ ] but the spray dried vlps were highly immunogenic in a mouse model after being stored for months at either room temperature or °c. other constraints that vlps are subject to include the relatively small size of epitopes that can be accommodated within the particles. for example, large antigens such as hiv envelope and influenza hemagglutinin proteins are too large to be packaged within native vlps. the practicality of vlp platforms is also limited by manufacturing considerations. [ ] for example, vlps derived from e. coli, the most widely used and most efficient expression system in the biotechnology industry, have a high degree of heterogeneity in their physical properties, including the shape and diameter of the particles. such particles require post-purification disassembly and reassembly in optimized conditions. in addition, it may be possible to produce highly immunogenic vlp preparations, but the antigen might not be viable in the vlp context until stable formulations can be developed. formulations must be resistant to aggregation upon exposure to low salt and protein concentration, as well as protection against surface adsorption and aggregation as a result of heat stress and physical agitation in order to achieve the multiple-year stability required for a marketed vaccine. [ ] newer expression systems in conjunction with innovative purification strategies could determine the pace for the next generation of vlp-based vaccine candidates. for example, yeast, insect, and mammalian expression systems have been used to circumvent the limitations of bacterial expression such as sub-optimal ph and lipid compositions within the bacteria. additionally, the advancement of high-throughput biophysical and structural analyses of recombinant vlps may play a key role in the assessment of vlp candidates. electron microscopy, electrospray differential mobility analysis, atomic force microscopy, x-ray crystallography, and dynamic light scattering provide quantitative structural data for each vaccine candidate and play a valuable role in ensuring product robustness from the early clinical development stage and beyond. [ ] as an alternative to particles inspired by natural virus capsid proteins, fully designed protein nanoparticles have received considerable interest. although "bottom-up" approaches for designing nanomaterials were popularized over years ago, [ ] the development of supramolecular polypeptide materials into successful medical technologies was initially slow, hindered by the sheer diversity of possible self-assembled structures and the lack of design rules. in , the yeates group developed an approach based on molecular symmetry to fabricate protein assemblies having a range of predictable architectures. [ ] their strategy was a breakthrough in rational self-assembling protein design. in the past few years, the capacity to model and predict protein structures and energetics has increased along with computing power, leading to the computational design of de novo self-assembling protein nanoparticles. [ ] the baker group used naturally occurring oligomeric proteins as building blocks to design cage-like assemblies with accuracy. recently, they generated a hyperstable -subunit protein icosahedron via symmetric modelling coupled with computational protein-protein interface design. [ ] this structure is robust to genetic fusions, making it a notably modular platform that could be used in multivalent epitope display as well as drug delivery. taking inspiration from nature has also proven invaluable in the design of mechanically and chemically stable nanoparticles. in , raman et al. designed a self-assembling nanoparticle based on virus capsids via superposition of different protein oligomerization domains onto the symmetry axes of an icosahedron shown in figure b . the monomer building block consisted of a protein chain made of two coiled coils connected by a short linker region. the association between the coiled coils caused the assembly of monomers into a roughly spherical nanoparticle. [ ] the self-assembling protein nanoparticle's versatile and flexible design allow for optimization of biophysical and immunological properties making them a desirable vaccine platform. whereas synthetic peptides are usually not sufficiently immunogenic and require adjuvants, the burkhard group has developed a self-assembling protein nanoparticle platform that displays both b and t cell epitopes to produce a vaccine with self-adjuvanting qualities. [ ] this platform is currently under development to improve a malaria vaccine, "rts's" that is based on the circumsporozoite protein of plasmodium falciparum. [ ] the self-assembled protein nanoparticles are able to stimulate high titer, high avidity antibodies and present cd + t-cell eptiopes to stimulate il- and ifn-γ producing longterm memory t-cells in mouse models. the vaccine candidate fmp , based on this platform, is currently undergoing phase clinical trials. a principal advantage of designed protein nanoparticles is their chemical definition. the ability to use computational methods to design nanoparticles of different geometries and sizes opens the door to applications such as drug delivery, in which the shape and size of the delivery vehicle are crucial. however, the manufacturability of these platforms, similar to vlps, is a challenge due to their multi-subunit nature and the need to be recombinantly expressed. micellar nanocarriers composed of amphiphilic molecules have had particular success in the pharmaceutical realm as a tool to increase bioavailability, retention, and solubility of various drugs. [ ] however, in this review, we shall be focusing on peptide amphiphiles (pas) as they relate to our theme of the clinical translation of peptide-based materials. such structures self-assemble from molecules composed of a hydrophobic domain, usually an alkyl chain, and a hydrophilic peptide domain. [ , ] pa assembly is driven by hydrophobic-hydrophobic interactions in water, and bioactivity is programmed into the hydrophilic peptide head groups (see figure c ). for a comprehensive review on pas used in biomedical applications, the reader is directed to acar et al. [ ] targeting, diagnostic, and theranostic platforms have been derived from peptide amphiphile micelles (pams). to increase the size of hydrophobic head groups and push systems toward a micellar packing morphology, the micelles consist of a biologically active peptide attached to a hydrophobic alkyl tail via a bulky polyethylene glycol (peg) spacer. the peg spacer allows for enhanced blood circulation times while retaining the packing parameters necessary for micelle formation. [ ] these molecules are shown to circulate through the blood stream without causing blockage, and are cleared via the reticulo-endothelial system and renal system with % clearance and no toxicity after d. [ ] pams are currently in preclinical development for both cancer and atherosclerosis diagnostic applications. for cancer applications, fluorescently-labeled pas with the fibrin-binding peptide creka were used to target glioblastoma cells. upon intravenous administration to gl glioma-bearing mice, nontargeting micelles passively accumulated at the fibrin deposits characteristic of tumor vasculature. these micelles displayed enhanced tumor homing as early as h after administration without inducing cytotoxicity or tissue damage. [ ] toward a therapy for atherosclerosis, the creka targeting peptide, an antithrombin peptide called hirulog, and fluorescence molecules were assembled into theranostic micelles. [ ] this fibrin-targeting micelle could also be functionalized by the addition of the gadolinium chelator diethylenetriaminepentaacetic acid, allowing for plaque localization and visualization using t -weighted magnetic resonance imaging (mri) imaging. [ ] similar pams have been designed for the targeting of monocytes as a strategy to diagnose atherosclerosis via the areas of heightened immunological activity characterized by the disease. [ ] similarly to previously described vaccine nanoparticles, pa micelles are able to elicit either humoral or cell-mediated immunity without additional adjuvant. an antigen is simply conjugated to the tail domain of the molecule prior to selfassembly. black et al. was able to assemble cylindrical micelles from monomers consisting of a dialkyl tail conjugated to a peptide containing the known cytotoxic t-cell epitope from the model tumor antigen ovalbumin. [ ] these dic -ova micelles were able to stimulate ova-specific t-cells, offering in vivo protection from tumors without any additional adjuvant. this observation spurred additional immunological studies to expand upon the potential of peptide amphiphile micelle vaccine platforms. in , barrett et al. used a group a streptococcus (gas) b-cell antigen coupled to a dic tail which drove self-assembly of cylindrical micelles, to induce a micelle-mediated immune response (without adjuvant) that was stronger than seen with a conventional gold-standard vaccine formulation. [ ] although spherically assembled peptide amphiphiles have not yet reached regulatory approval for clinical applications, their versatility, modularity, and demonstrated success in preclinical work is encouraging. clinically translatable supramolecular materials are not limited to spherical morphologies. extended structures with high aspect ratios have also seen considerable development toward a variety of medical technologies (figure a-c) . prominent among these have been fibrillar assemblies of peptides and their derivatives. these structures have been investigated over the past years and have made progress toward therapies in hemostasis, dentistry, wound healing, and immunology. while studying structural biology in alexander rich's research group, s. zhang discovered a self-assembling β-sheet peptide based on the dna-binding protein, zuotin. [ ] the peptide formed amphiphilic tapes with two distinct surfaces, one hydrophobic and the other hydrophilic, and it also contained complementary charged residues that additionally favored this β-sheet folding (see figure b ). following this discovery, a mimic of the native peptide was designed by mutating the charged and hydrophobic residues, but leaving the pattern intact. [ ] this mimic demonstrated that self-assembly of peptides with these motifs was not a sequence-specific anomaly, but could be recreated in similar systems, forming the first steps toward design rules for fibrillizing peptides. the designer peptide was shown to also form macroscopic membranes and support the attachment of mammalian cells, demonstrating its utility as a biomaterial. [ ] subsequently, it was found that the original synthetic sequence rada -ii (raradada) and its modified form rada -i (rada) spontaneously formed hydrogels of entangled nanofibers in salt-containing solutions and cell culture media. [ ] mixtures of peptides bearing multiple bioactive groups could be incorporated into a single macroscopic gel by dosing in various amounts of monomeric peptides to the gel mixture. additionally, the incorporation of ligands or epitopes within the materials simply required extension of the peptide at either terminus with the desired sequence, thus lowering the barrier of synthetic difficulty for biological researchers. these gels have been subsequently developed toward neuronal regeneration, cytokine delivery, as biotinylated scaffolds for versatile protein delivery, and other applications. [ ] [ ] [ ] the release of proteins and small molecules from rada hydrogels largely depends on the size of the protein cargo regardless of charge and hydrophilic character, allowing a wide variety of biomolecules to be delivered in these gels. [ ] besides physical entrapment, a biotin-sandwich approach has been used to tether insulin-like growth factor (igf- ) to gels for long term localization of bioactive igf- in the scaffold. [ ] biotinylation provides the advantage of modularity within the hydrogel delivery system, as any biotinylated protein can be immobilized to biotinylated fibers via a streptavidin linkage. biotinylated fibers have been investigated for myocardial regeneration following infarction and showed success in rats when injected into infarcted hearts along with cardiac progenitor cells. [ ] although rada nanofibers have been investigated preclinically for a wide range of medical applications, they have had the most clinical success as a hemostatic agent. marketed under the trade name purastat by -d matrix ltd., [ ] the product is composed solely of peptide dissolved in sterile water, which forms fibers when in contact with biological fluids. this self-assembly provides a physical blockage in order to limit bleeding at the site of application. although this peptide's use as a hemostat is still being actively developed as a component of layer-by-layer wound dressings, [ ] the original rada -i peptide has been useful as a surgical hemostat for multiple surgeries because of the dense mesh it creates at neutral ph. the rada -i peptide which became purastat was first tested as a hemostatic agent in and demonstrated hemostasis in rats by stopping bleeding in under s when applied as a %- % aqueous solution to wounds in skin, brain, spinal cord, femoral artery, and liver. [ ] this rapid induction of hemostasis circumvented the need for components of the clotting cascade to activate the hemostat, it avoided the use of heat or pyrogenic substances, and it was effective in the presence of anti-coagulant therapies [ ] without causing pronounced tissue responses. [ ] although the peptide is slightly acidic, it did not cause significant inflammation in any animal or clinical studies, even when applied to the brain. [ ] also named tdm- , the rada -i peptide was first tested as a hemostat in human surgeries in japan during cardiovascular procedures, [ ] and later during endoscopic mucosal resection, [ ] with no treatment-related adverse events in either trial. purastat is limited to relatively low pressure hemorrhages in comparison to major arterial injuries, [ ] but has general utility as a versatile, biodegradable hemostat. clinical trials to monitor the post-market performance of purastat for vascular surgery (nct ), and to test the use of purastat during endoscopic submucosal dissection are scheduled to begin soon (nct ). purastat has already achieved ce marking in europe and has currently received medical device product registration approval in a number of countries including thailand, mexico, and indonesia, and australia. initial studies of the rada family of peptides inspired the development of general design principles for forming nanofibers, during which other peptides were shown to have similar self-assembling properties. notably, peptide p , developed by aggeli et al., would eventually lead to the development of the enamel regeneration product curodont. the first sequence designed by aggeli et al. in was a mimic of a β-sheet transmembrane protein. [ , ] this peptide was designed de novo based on patterns found in the transmembrane protein and was shown to form high aspect-ratio fibrils that tangled to form hydrogels independently of ph in a manner similar to the protein-inspired peptide on which it was based. [ ] currently, this peptide is on the market in the european union (eu) and switzerland in the form of a treatment for dental enamel caries, and it functions by creating a local environment that enhances enamel mineralization. [ ] upon injection, monomeric peptides assemble into nanotapes to create a d matrix that in some respects resembles the matrix environment necessary for enamel deposition. [ ] a major design improvement preceding the final curodont peptide sequence was the introduction of ph-sensitivity. in , aggeli et al. rationally modified the original peptide sequence so that it was negatively charged at ph > , thus creating electrostatic repulsion and allowing the peptides to remain in a monomeric, unassembled state. [ ] upon switching to more acidic ph, however, the acidic residues became protonated and monomers assembled in to β-sheet tapes. [ ] this ph-sensitivity allows for in situ assembly of peptides into nanofibers, which is hypothesized to improve the efficacy of curodont as the material is able to completely fill demineralized defects of varying shapes and sizes. this ph-sensitive assembly was specifically explored in the context of enamel remineralization in , where simulated intraoral conditions were employed to assess the performance of self-assembled scaffolds which were administered in a basic solution, allowed to fibriliize, and then incubated under cyclic ph. [ ] these studies were completed on enamel lesions formed on extracted human teeth and indicated that the p scaffold caused hydroxyapatite mineralization where the peptide solution was applied. [ ] thus, positive and significant results were obtained without the need for specialized application methods or a poorly translatable animal model, a significant advantage for the development of polypeptide biomaterials toward dental applications. after the enamel restorative properties of p were developed in preclinical models, credentis was founded in , curodont was launched as the company's first product, and its safety and efficacy were verified in a clinical trial to treat dental caries. [ ] in it received market approval (ce-label) for medical devices in the eu. in keeping with the original peptide design, the final formulation of curodont is composed of only a monomeric -amino acid peptide (p ) dissolved in water, with no additional bioactive agents. [ ] following a single application, the size and color of lesions are significantly improved, as observed one month after treatment. [ , ] peptide nanofibers have also increasingly received attention in immunological contexts, an area in which our group is active. however, because the focus here is on clinical development and these materials have not yet reached clinical trials, these applications will be only briefly described, and the reader is referred to other recent reviews for more expansive descriptions of this burgeoning field. [ , , , ] after the discovery that β-sheet fibrillized peptides can raise strong antibody responses without the requirement of supplemental adjuvant, [ ] these materials have been investigated preclinically toward a range of diseases and conditions including malaria, [ ] staphylococus aureus infections, [ ] influenza, [ ] west nile virus, cancer, [ ] and cocaine abuse. [ ] immunogenic peptide nanofibers are produced by co-assembling fibrillizing peptides extended with specific epitopes or haptens along with other fibrillizing peptides bearing t-cell epitopes. they stimulate antibody/b-cell responses, cd t-cell responses, and cd t-cell responses [ ] which can be raised in tunable magnitudes without causing inflammation. [ , , ] other recent extensions of fibrillizing peptide technologies have included assemblies containing both peptides and other therapeutically active compounds. in these drug-peptide formulations, hydrophobic, π-π, and electrostatic interactions induce the assembly of the molecules into fibrillar networks much in the same way as pure peptide systems. [ , ] a recently described example is a strategy in which the us food and drug administration (fda)-approved anticancer drug pemetrexed was conjugated to a four-amino acid peptide and used both as an mri contrast agent and to form drug depots near tumor sites. [ ] the dual function of the material (contrast and depot formation) was possible because the peptide-drug conjugate formed fibrillar hydrogels at high concentrations to form the drug depot, while lower concentrations in the circulation acted as the mri contrast agent. a range of other strategies have likewise employed fibrillizing peptides to alter the delivery or pharmacokinetics of various drugs. for example, hartgerink and co-workers showed that several different multivalent drug molecules such as clodronate, heparin, and suramin could be used to stabilize β-sheet fibrillar hydrogels by shielding the surface charges on the peptides that would otherwise inhibit gelation, thus forming drug depots. [ ] naphthalene-modified peptides have been explored to carry hydrophobic drugs such as curcumin, which require carrier transport due to their low solubility. [ ] these examples highlight considerable future potential for peptide nanofibers toward clinical applications. alongside the use of β-sheet peptides, peptide amphiphiles composed of a peptide head group and an alkyl tail are a related class of materials that have seen considerable interest for creating supramolecular nanofibers (see figure a ). although the self-assembly of peptide amphiphiles into spherical structures had been known for some time, [ ] the landmark paper by hartgerink, stupp, and colleagues catalyzed much interest in the ability of this class of molecule to form nanofibrous materials for specific biomedical applications. [ ] in this report, nanofibers spontaneously assembled from peptide amphiphiles into parallel bundles and promoted mineralization of hydroxyapatite. [ ] since then, fibrillar peptide amphiphile materials have been explored in a broad range of medical applications ranging between wound healing, [ ] bone healing, [ ] the delivery of proteins, [ ] nervous tissue repair, [ ] and others. additional chemistries have been developed to stabilize the materials. for example, toward applications where mechanical integrity is necessary, adhesive groups have been incorporated into the hydrophilic heads to render the fibrous gels self-healing after they are strained mechanically. [ ] a recent example of clinically directed peptide amphiphile nanofibers used them to deliver bioactive proteins such as bone morphogenic protein (bmp) to induce osteogenesis in an environment mimicking native bone growth. [ ] other examples have included nerve repair, which benefitted from the bioactivity and parallel alignment of fibrous scaffolds, [ ] and burn injuries, where heparin-mimetic gels induced the formation of vascularized, collagen-rich tissues. [ ] peptide amphiphiles have also been used to deliver bioactive cargo outside of the regenerative context, including the electrostatic complexation of antisense oligonucleotides to a cationic peptide head to form a depot for sustained release of oligonucleotide. [ ] because networks of peptide-based fibers are morphologically similar to the extracellular matrix, they have also been highly useful as in vitro culture materials [ ] and can be utilized as cell delivery vehicles, as demonstrated in the transplantation of islet cells [ ] and bone marrow-derived pro-angiogenic cells cultured prior to transplantation. [ ] although work with fibrous peptide amphiphiles has remained largely preclinical to date, is anticipated that the coming years will see many of these applications brought forward into approved therapeutics. filamentous bacteriophages (see figure c ) represent an additional step in the progression of nanofiber-forming biomaterials. although their production is considerably different than the chemical synthesis of pas and short peptides, their elongated morphology and polyamino acid composition are similar in some respects, and can be used to endow phage-based materials with similar properties to the other fiber-forming platforms discussed above. filamentous phages have highly engineerable coat proteins which allow the high density surface display of selected proteins. m phage are naturally filamentous, and they resemble peptide and peptide-amphiphile nanofibers in morphology as they are less than nm in diameter yet almost µm in length. because m bacteriophages are unable to infect mammalian cells, there is negligible risk of virulent infection when using these viruses in medical applications. for these reasons, they have been historically used as antimicrobial agents and are currently approved for use in food products, but have had seen limited use in clinical trials as therapeutics. a few examples exist where the tissue-targeting and tumor-homing abilities of full phage libraries were tested in humans. [ , ] bacteriophages were initially investigated as nanomaterials when they were observed to form liquid crystals, and they proved useful for the templating of inorganic structures by incorporating metal binding peptides on the viral coat proteins. [ , ] following the discovery of this functionality, their liquid crystalline behavior was utilized to form aligned matrices for neural cell culture by displaying arg-gly-asp (rgd) and ile-lys-val-ala-val (ikvav) peptides on the phage surface. [ ] this work included the use of standard phage display to select optimal -amino acid sequences for receptor binding, and the resultant filamentous phages were produced in e. coli, a relatively simple manufacturing process that is likely to be scalable. in another recent example of preclinical materials development using filamentous phages, their self-templating properties proved useful for controlling the direction of osteoblast growth using the orientation of the phage-based substrate. [ ] the use of both self-templated and fabricated directionality in phage substrates has allowed for the directional growth of human fibroblasts, [ ] proliferation and elongation of neural progenitor cells, [ ] and stimulated the differentiation of mesenchymal stem cells into osteoblasts when the phages displayed an osteogenic peptide on their surface. [ ] as in vivo injectable materials, phages have been used preclinically as carriers for magnetic nanoparticles for targeted imaging of cancerous tumors by displaying a high density of targeting ligands and metal binding peptides on the phage surface. [ ] a related study used a modified approach by conjugating a streptavidin-linked fluorophore to phages displaying tumortargeting and streptavidin-binding peptides on their surface for targeted cancer imaging without the use of metal particles. [ ] these studies demonstrated the importance of directional patterning in combination with the display of specific peptides on the phages. moreover, the incorporation of multiple bioactive phage populations into a single material only requires adjustment of the mixture of various deposited phages, so in this way these materials feature the modularity characteristic of supramolecular systems. similar to other nanofibrous materials, filamentous phage materials have received significant attention for inducing osteogenesis because they can form collagen-mimetic bundles for the mineralization of hydroxyapatite similarly to pa and β-sheet peptide nanofibers. display of anioinic peptides caused parallel assembly of individual phage in the presence of cations, followed by formation of oriented crystals when counterions were introduced owing to the local supersaturation of inorganic ions. [ ] this approach closely resembled the demonstration of pa mineralization presented by stupp and coworkers and illustrates conservation of biological processes across materials platforms. [ ] since the demonstration of phage assembly mineralization, osteogenesis studies have expanded to include presentation of a hydroxyapatite nucleating protein, dentin matrix protein- , as an alternative moiety for inducing crystal formation. [ ] tobacco mosiac virus (tmv), another rod-like virus was shown to cause differentiation of mesenchymal stem cells into bone cells as culture on the tmv coated substrate caused an upregulation of bmp- , osteocalcin, and calcium sequestration, which are all markers of bone development. [ ] recently, rgd-bearing phages were d-printed into a ceramic scaffold to induce osteogenesis and angiogenesis concurrently without the addition of exogenous vascular endothelial growth factor. [ ] because of the scale at which phage can be produced, they may have manufacturing advantages over other designer materials. over the past years, supramolecular assemblies of peptides and proteins have developed from single vaccines to a broad range of technologies that spans the breadth of biomaterials applications as drug delivery vehicles, scaffolds for tissue regeneration, and other therapeutics. [ , , , ] currently, several have achieved regulatory approval for clinical use ( table ) , primarily in the vaccine space. the advancement of these platforms can be attributed to many factors. peptides and proteins are more economical than ever to produce, and manufacturing efficiencies continue to be developed. the design rules for each subclass of materials has been significantly mapped in recent years. and strategies have been optimized for incorporating disease-specific ligands, epitopes, or other moieties within each platform. we expect the coming decades to witness the implementation of many new examples of supramolecular polypeptide therapies. the immunogenic features of peptide assemblies are advantageous for the development of synthetic vaccines and other engineered immunotherapies, a topic which has been recently reviewed by our group and others, [ , , , , ] but it is also important to note for other assemblies containing high densities of protein or peptide ligands/epitopes that such multivalent displays may induce unwanted immune responses. it remains to be seen whether such responses can be tolerated in specific applications, or if they could even be turned in the favor of the material's clinical performance. interestingly, neither curodont [ ] nor purastat [ ] contain specific ligands/epitopes, possibly avoiding immunogenicity that may be observed in trials of other nanofibrous materials containing additional protein or peptide functional components. additional future work may focus on investigating the biodistribution and pharmacokinetics of preclinical self-assembling materials. due to the dynamic nature of these materials, it is important to understand how the in vivo environment affects their long-term structural organization, retention, and clearance. in order to be clinically translated, these platforms must also be capable of large-scale production and stable storage. unlike traditional small molecules, these materials must generally be kept in monomeric or otherwise stable states of assembly prior to administration. each of these issues represents important considerations that have not yet been fully worked out for supramolecular materials. with several self-assembling platforms being discovered and developed over the past years, it is worth considering the cross-roads that lay ahead: should we focus on the development of the promising platforms discussed here, or should we continue searching for new, novel platforms? on the one hand, several of the aforementioned platforms have shown encouraging preclinical data and are being investigated in new disease models. on the other, the discovery of a new, more efficacious and versatile platforms could spur unforeseen therapies based on the self-assembling concept. either way, we predict that self-assembling peptide and protein technologies will continue making strides in the preclinical and clinical realms. proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci biomate rials k.m.h. and c.n.f. contributed equally to this work. research on supramolecular peptide biomaterials in our group has been supported by the national institutes of health under grant numbers r eb ; r ai ; r ca ; and r ar ). this progress report's contents are solely the responsibility of the authors and do not necessarily represent the official views of these agencies. key: cord- -ogu gmcb authors: da cunha, nicolau b.; cobacho, nicole b.; viana, juliane f.c.; lima, loiane a.; sampaio, kamila b.o.; dohms, stephan s.m.; ferreira, arthur c.r.; de la fuente-núñez, césar; costa, fabrício f.; franco, octávio l.; dias, simoni c. title: the next generation of antimicrobial peptides (amps) as molecular therapeutic tools for the treatment of diseases with social and economic impacts date: - - journal: drug discov today doi: . /j.drudis. . . sha: doc_id: cord_uid: ogu gmcb anti-infective drugs have had a key role in the contemporary world, contributing to dramatically decrease mortality rates caused by infectious diseases worldwide. antimicrobial peptides (amps) are multifunctional effectors of the innate immune system of mucosal surfaces and present antimicrobial activity against a range of pathogenic viruses, bacteria, and fungi. however, the discovery and development of new antibacterial drugs is a crucial step to overcome the great challenge posed by the emergence of antibiotic resistance. in this review, we outline recent advances in the development of novel amps with improved antimicrobial activities that were achieved through characteristic structural design. in addition, we describe recent progress made to overcome some of the major limitations that have hindered peptide biosynthesis. most drugs currently used therapeutically were obtained as naturally occurring molecules purified from microorganisms, plants, and animals [ ] . it is estimated that, from to , anti-infective agents based on natural product scaffolds accounted for up to % of all approved antibacterial new chemical entities (nces), thus attesting for the importance of natural products as the main source of therapeutics against pathogenic bacteria [ ] . in this context, microorganisms, notably members of the gram-positive phylum actinomycetes, have been the workhouse source of clinically approved antibacterial agents. the high diversity of candidate molecules extracted from microorganisms frequently leads to the discovery of new compounds with distinct mechanisms of action compared with drugs currently used for clinical purposes. however, in recent years, the traditional screening of drug candidates belonging to completely new antibacterial classes from microbial sources has suffered a considerable decline, mostly because of the presence of numerous, already well-characterized, molecules [ ] . this scenario is particularly worrisome because of the increasing emergence of bacterial resistance to antibiotics, notably in developing countries, where there is a widespread and indiscriminate use of antibiotics for clinical and veterinary purposes. the search for new therapeutic molecules for commercial applications is an ongoing in the pharmaceutical industry, which now is focusing on not only drug prospection, but also the modification of existing antibiotics in a timely fashion that meets the customer's needs [ ] . among the diverse naturally occurring anti-infective agents that have been discovered to date, amps are particularly important [ ] . these molecules are versatile, highly specific antimicrobial compounds that constitute promising candidates for commercial and clinical uses. amps are essential components of the innate immune system of vertebrates and the nonspecific host defense system of plants, fungi, and invertebrates that evolved over . billion years ago as potent anti-infective agents against different viruses, bacteria, fungi, and parasites. these small peptides usually comprise - amino acid residues and are - kda in size [ ] . commonly, they show a cationic structure rich in positively charged arginine and lysine residues, which favors the interaction between these peptides and microbial cytoplasmic membranes. however, although less common, there are also anionic amps, notably in plants. amps usually kill pathogens by interacting with membrane phospholipids, resulting in membrane permeabilization and subsequent disruption. these peptides also show different secondary structures, such as b-sheets stabilized by two or three disulfide bridges and often display a helical amphipathic structure [ ] [ ] [ ] . amps can target a variety of essential metabolic processes in the plasma membrane or at extra-and intracellular sites, and frequently exhibit immunomodulatory properties that stimulate cytokine production while repressing inflammation, can kill cancer cells, and promote wound healing [ ] . in eukaryotes, most natural amps are encoded by specific genes, which are constitutively expressed at basal levels and rapidly transcribed after induction by contact or exposition to invading pathogens. thereby, a variety of different amps can be found simultaneously in organisms, such as plants, in response to pathogen stimuli at different organs, such as roots, seeds, flowers, stems, and leaves [ ] . recent advances in in silico drug design and high-throughput screening of compound libraries based on protein-protein interactions have triggered a growing interest in the discovery of new antibacterial drugs from microbial, plant, and animal origins. such efforts have contributed to uncover a variety of molecules with novel structures and improvement of the currently available classes of antibiotic, such as b-lactams, ketolides, macrolides, glycopeptides, aminoglycosides, oxazolidinones, and other antiinfective agents [ ] . the use of viral vectors based on the tobacco mosaic virus (tmv) and potato virus x (pvx) for cloning and massively expressing amp genes in nicotiana benthamiana appears to be an interesting new approach to considerably enhance recombinant peptide production before structural and functional characterization. the rapid, high-throughput staggered biosynthesis of peptides using agroinfiltrated leaves of n. benthamiana constitutes the most promising strategy for delivering new antigenic peptides and vaccine candidates, addressing the interest of many private and state health institutes [ ] . finally, gene editing using the clustered regularly interspaced short palindromic repeats (crispr)-crispr-associated protein (cas) system is another molecular tool that could revolutionize the recombinant biosynthesis of amps [ ] . this approach specifically targets dna sequences by adding, removing, or replacing dna segments mediated by cellular dna repair mechanisms of site-specific double-stranded breaks. it allows the edition of specific genes within the host genome to integrate the fragments containing the coding sequence of a particular amp in a hotspot site to enable increased amp biosynthesis. working on a case-tocase scenario, this expression strategy could overcome major drawbacks in the production of amps, notably the low biosynthesis levels presented by plant and animal amps [ ] . the antimicrobial activity of a given amp is specifically related to its amino acid composition and physical chemical properties, such as positive net charge, flexibility, size, hydrophobicity, and amphipathicity [ , ] . marginal changes in peptide residue sequence are normally followed by major changes in antimicrobial activity [ ] . amps present different mechanisms of action, many of them well described in the literature. the molecular interactions of some amps and microorganisms rely on the variety of targets presented by microorganisms, particularly gram-positive and gram-negative bacteria. [ ] . generally, the mechanisms of action of amps can be classified into two basic groups: (i) the disruptive mechanisms, which are associated with membrane lysis; and (ii) the membrane undisruptive mechanisms, which focus on neutralizing intracellular targets [ ] . independently of the proposed group, the first step of any mechanism is the molecular interaction between the amp and the cytoplasmic membrane. the driving force of such interaction is the electrostatic force presented by the amp, which is normally cationic, and the polyanionic surface of bacteria [ , ] . differences in the cell wall composition among bacterial groups affect directly the mode of action of amps. gram-negative bacteria present three major layers in the cell envelope: (i) the outer membrane (om), comprising a lipid bilayer, mostly of lipopolysaccharide (lps) interlaced by teichoic acid, which has a major role in protection against the environment; (ii) the peptidoglycan cell wall, comprising repeated units of a disaccharide (n-acetyl glucosamine-nacetylmuramic acid) linked by pentapeptide side chains; and (iii) the cytoplasmic membrane-phospholipid bilayer. by contrast, in gram-positive bacteria, there is no om, but the peptidoglycan layer is thicker than in gram-negative bacteria [ ] . the disruptive mechanisms are presented in four classical models: (i) the toroidal model; (ii) the carpet model; (iii) the aggregate model; and (iv) the barrel model. recently, new disruptive models or models indirectly associated with membrane disruption were described: (i) the disordered toroidal model; (ii) the membrane thinning/thickening model; (iii) the charged lipid clustering model; (iv) the non-bilayer intermediate model; (v) the oxidized lipid targeting model; (vi) the anion carrier model; (vii) the nonlytic membrane depolarization mode; and (viii) the electroporation model. all the models are non-mutually exclusive, allowing that a single amp might present a multiple-hit strategy based on two or more simultaneous mechanisms. in the toroidal model, the amp binds the membrane and forms a 'flip-flop' translocation channel that opens the membrane vertically, with the amp remaining closely associated to the lipid head-groups throughout the process [ , ] . the carpet model provides the disruption of the membrane without the internalization of the amp. by contrast, the amp remains associated with the membrane until a crucial concentration of the peptide contributes to weaken the hydrophobic interactions of structural phospholipids and amps form a carpet structure that increase membrane disruption [ ] . in the aggregate model, the amps have a detergent-like role by interweaving the phospholipids and disaggregating them, similar to a true detergent. depending on the amp and the membrane composition, this mode of action does not provide the membrane rupture. instead, it just forms a thin channel for the crossing of amp and other molecules into the cell [ ] . the barrel model provides the formation of a regularly organized aggregate of amps that interacts and associate with the membrane. the amp oligomer leads to the formation of pores in the lipid bilayer by intimately interacting its hydrophobic side chains with hydrophobic parts of the membrane. the transmembrane pores allow the internalization of the hydrophilic part of the amp that faces the internal region of the membrane [ ] . lastly, there is the model that shares the most similarities with the four classic models: the disordered toroidal model. this mode of action provides a stochastic pore formation after inward lipid distortion that allows the aggregation of a maximum of two peptides in the center of the pore. however, in the external peripheral region of the pore, many peptides are set up before translocation [ , ] . the undisruptive mechanisms are based on the amp crossing the membrane because of the combined features of amp sequence and membrane composition, and the inhibition of some reactions of cellular metabolism, causing cell death. there are two different ways that an amp enters the cell. the first is an obscure spontaneous translocation across the membrane; the other is mainly the result of the presence of a secondary structure in the amp that causes membrane permeabilization. in this method, named the shai-matsuzaki-huang method, a a-helical amp binds parallel to the membrane. hydrophobic residues facing the membrane permit the internalization of part of the amps and the change of their organization to a transversal mode by forming toroidal pores [ , , ] . the other model suggests that the b-sheet amps are organized into a flat-aggregate form that allows the insertion of some aromatic residues in the membrane and the opening of thin translocational spaces in which the amps can cross the membrane [ ] . once across the membrane, the amps can target a variety of intracellular sites, such as gene promoters and coding sequences, mrna-binding sites, enzyme regulatory sites, and protein prefolding sites. such inhibitory interactions involve blocking both dna transcription and/or rna translation, or incorrect protein folding, triggering the failure of metabolic pathways and cell death [ ] . recently, a new undisruptive mechanism was discovered in amp-sensitive bacteria. species such as escherichia coli and salmonella spp. have evolved the ability to detect and prevent amp antibacterial activities by triggering pathways involved in sensing and amplifying resistance to cationic amps. resistance is provided by the phoq/phop system of e. coli, which comprises the activation of the amp detector kinase phoq by mg + and ca + , which phosphorylates and activates the transcription factor phop [ ] . the active phop activates pagp, which encodes a resistance enzyme against amps; mgta, encoding a transporter of mg + ; and hdea, encoding a chaperone responsive to acidic conditions. phosphorylated phop activates the transcription of the quee gene, encoding an enzyme that controls the biosynthesis of hypermodified guanosine found in rare trnas and, once overexpressed, inhibits cell division by downregulating the bacterial divisome [ , ] . sublethal concentrations of the c g amp, a highly cationic, amphiphilic peptide derived from the c-terminal sequence of human protein platelet factor , can trigger the phoq-dependent filamentation of wildtype e. coli by the overexpression of quee. the bacterial filamentation (tens to hundreds of microns in length) prevented fully bacterial growth and was detectable in genetically engineered strains with a phop-regulated promoter driving the transcription of a yellow fluorescent protein [ ] . another good example is buforin ii, a potent amp from chinese toad bufo gargarizans, with antimicrobial activity against a range of microorganisms. once inside the cell, buforin ii inhibits gene expression by associating with dna and mrna during transcription and translation. the same occurs with melittin, a bee venom peptide active against both gram-positive and gram-negative bacteria, as well as some pathogenic fungi, which is a typical example of nondisruptive amp, discovered during the late s, that supported the carpet model of microorganism inhibition detailed in [ , , ] . the development of antibiotics has had a major impact on modern medicine. however, the increasing emergence of antibiotic resistance and the limited development of novel classes of antibiotic over the past four decades has led to a scenario in which some infections are no longer treatable with available antibiotics [ ] . antimicrobial resistance (amr) is defined as the resistance of microorganisms to an antimicrobial against which they were once sensitive [ , ] . amr is an inevitable evolutionary outcome once all organisms develop genetic mutations that can improve fitness and lead to selection as a response to the selective pressure of the environment. in fact, more than % of pathogenic bacteria are resistant to at least one type of antibiotic [ ] . undesired selection of microbial cells with resistance-conferring mutations or other resistant elements represents the main drawback in long-term treatment efficiency of patients, leading to intensive medical research to discover new targets to overcome multidrug resistance (mdr) [ , , ] . the current classes of antibiotic face a constant threat represented by the diverse bacterial resistance mechanisms. the eskape pathogens (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species) represent the most dangerous pathogens to immunocompromised patients because they are commonly isolated as drug-resistant (dr) or mdr microorganisms [ ] . there are two major ways in which common bacteria evolve to become resistant: by intrinsic or acquired resistance against amps. intrinsic resistance occurs as a natural consequence of the presence of amps in the natural environment of bacteria, which develop mechanisms to resist antibiotic action [ ] . this can happen via passive or inducible mechanisms. passive resistance is always associated with less tight interactions between bacteria and amps because of the inherent accumulation of more positive charges in lipid a, and is more common in genera such as proteus, providencia, burkholderia, morganella, and serratia [ ] . inducible resistance is a consequence of perennial, reversible modifications at the molecular level in both gram-positive and gram-negative bacteria [ ] . acquired resistance is a product of high-fitness mutants that often contain more than one mutation that is unrelated to the amp selection. bacterial mutated genes provide altered genetic systems that allow bacterial growth in the presence of amps and can be identified by experimental procedures with and without amp in the culture media [ ] . the bacterial om and inner membrane architectures can be altered and sites for the ligation of amps can be protected in response to reduced levels of ca + , mg + , and specific proteins, and changes in lipid composition. this considerably reduces membrane fluidity and permeability to polymyxins, defensins, and cathelicidins [ , ] . the bacterial membrane phospholipid content, such as phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl), reflects a global state of protection against pore formation [ ] . the biosynthesis, turnover, and translocation of phospholipids to target sites in the membrane can be modulated by the expression of proteins closely related to resistance against amps [ , ] . profiles of cationic amp resistance can be shown by the % increase in the total content of cl in liposome model membranes [ ] . strains of s. aureus resistant to methicillin, for example, can increase lipid biosynthesis and translocation to the membrane by simply activating genes that encode multiple peptide resistance factor (mprf), cardiolipin synthase (cls), and phosphatidylglycerol synthase (pgsa) [ , ] . the affinity between the cell membrane and amps can be considerably reduced by minimizing the negative charge of the phospholipid bilayer (i.e. the lipid composition). in addition, twocomponent signal regulatory systems (tcs), such as the phoq/ phop system in p. aeruginosa and the apsr/apss in staphylococcus epidermidis, can act together with lipid modifications to enhance resistance against cationic amps [ ] . the cell wall is the outer barrier that acts as a secondary physical protection structure against pore formation in bacteria. it gives strength to the bacterial cell and influences the final cell format. amps often establish ionic and/or hydrophobic interactions with the cell wall. modifications of the polysaccharide bilayer of peptidoglycan and teichoic acids in the cell wall are particularly interesting in gram-positive bacteria. the amp-cell wall interactions can be avoided in s. aureus and staphylococcus xylosus, which present multiple copies of the dlt operon, a regulatory sequence that, when active, promotes d-alanylation of teichoic acids in the cell wall, reducing their anionic charges [ ] . in some gram-negative bacteria, the affinity of lipopolysaccharides (lps) in the om by cationic amps can be reduced by the increase of positively charged lipid a with substituents such as palmitate, phosphoethanolamine, and -amino- -deoxy-l-arabinose at -or -phosphate groups [ ] . these modifications in lipid a are also induced by the phoq/phop system and appear to be closely related to cationic amp resistance, mainly against polymyxin b, in gram-negative bacteria. glycosylation of lipid a was recently reported as another lipid modification that results in polymyxin resistance in ei tor vibrio cholerae [ ] . the addition of glycine to lipid a is regulated by the almefg operon, which controls the expression of several proteins (aime, almf, and almg) related to glycine activation and transfer to lipid a. glycine is activated by adenylation by alme, which transfers active glycine to the -phosphopantetheine group of almf. once active, almf donates the glycine to the hydroxylauryl chain of lipid a, a transfer reaction performed by almg [ ] . in bacteria, is commonly stated that the thicker the cell wall, the less efficient an antibiotic is, and, by extension, an amp will be. improving the cell wall thickness is a strategy adopted by s. aureus against erythromycin, vancomycin, acriflavine, and many amps. the cell wall peptidoglycan layer can increase in thickness by the upregulation of glutamine synthase in e. coli strains that are resistant to magainin ii, a characteristic absent in susceptible strains [ ] . some bacteria have an arsenal of molecules that prevent cellular metabolism from suffering stress caused by amps. several pathways can be up-or downregulated to increase the biosynthesis of proteases, modification of membrane sites recognizable by amps, overproduction of biofilms, and suppression of superficial elements related to pore formation [ ] . an inherent mechanism of amp resistance by bacteria is the partial or total proteolytic cleavage of amps. strains of s. aureus overproducing the metalloprotease aureolysin are resistant to cathelicidin. proteus mirabilis producing high amounts of the metalloproteases zapa and ll- can avoid the antimicrobial activity of b-defensin (hbd ) simply by breaking down the protein into six or nine innocuous peptides [ ] . other systems for the upregulation of the biosynthesis of bacteria proteases have been discussed elsewhere. the nisin resistance gene (nrs) of resistant strains of lactococcus lactis is a central element of nisin-controlled gene expression systems (nice). a protease encoded by the nrs gene cleaves the c terminus of nisin, conferring in vitro resistance for non-nisin-producing l. lactis [ ] . by analogy, the speb cysteine protease of streptococcus pyogenes catalyzes the proteolysis of ll- in vitro and in patients infected with resistant bacteria. under stress caused by amps, s. pyogenes synthesizes a g-related a -macroglobulin-binding (grab) protein that acts as a potent inhibitor of the protease inhibitor a -macroglobulin, forming a cluster named the 'grab-a -macroglobulin complex' that retains an active speb on the bacterial surface for posterior ll- cleavage, causing bacterial resistance [ ] . molecular traps that capture amps and biofilm production are also important resistance mechanisms evolved by different species of bacteria. for example, l. lactis expressing pilb, a pilus backbone protein in gram-positive bacteria, can trap cathelicidin along with the cell wall, avoiding contact with the cytoplasmic membrane. biofilm-producing bacteria can resist many different antibiotics. biofilm-mediated resistance results mainly from extracellular polymeric substance (eps), a liquid comprising mainly amyloid and adhesive fimbriae, extracellular dna, and exopolysaccharides that embeds multiple cells throughout the biofilm matrix [ ] . the eps extracellular dna of p. aeruginosa can induce resistance against polymyxin b and colistin in response to the chelating of environmental cations. the decrease in the concentration of biofilm cations modulates the upregulation of lps modification genes associated with resistance, a typical mechanism in resistant strains of p. aeruginosa [ ] . in addition to their natural antimicrobial activities, amps also have other potential applications in the therapy and treatment of disease. for example, some amps have antitumoral and immunomodulatory activities and these peptides are important drug candidates known as anticancer peptides (acp) and host defense peptides (hdp), respectively. several acps show improved absorption and higher specific cytotoxicity to tumor cells and fewer adverse effects compared with chemical agents. the high number of interactions between acps and tumor receptors could result from the presence of abundant anionic sites dispersed on the tumoral cell, resulting in rapid and selective binding and cell death [ ] . by contrast, hdps frequently show weak antimicrobial activity in mammals, but are potent triggers of the immune response through a variety of mechanisms that affect the innate immunity of hosts. these peptides have diverse structures and sequences because of constant interactions with different microbial cells presenting multiple infective strategies [ , ] . the innate immune systems of mammals show characteristics of nonspecific, quick antimicrobial therapies mediated by the activation of elements of resistance against pathogens [ ] . hdps act as triggers of immune responsive elements by several mechanisms, such as the upregulation of the expression of hundreds of genes in monocytes and epithelial cells, the induction of differentiation responses and chemokine synthesis, and promotion of angiogenesis, and inflammatory and wound-healing responses [ ] . some therapeutic peptides have already reached market status, with more than peptides currently available on the market in the usa. it is estimated that, in , approximately therapeutic peptides reached preclinical trials and were included clinical trials in the usa [ ] . despite the development of many potential pharmaceutical peptides, the road to market is restrictive because drug candidates must meet several requirements, such as similar or higher efficacy and tolerability compared with already existing analogous drugs, improved pharmacodynamics and pharmacokinetics, low toxicity, and safe use [ , ] . economic issues must also be satisfied, mainly relating to market competition, scalable production, and intellectual property. for these reasons, more than % of novel therapeutic candidates fail to achieve marketable status [ ] . regulations surrounding the use of such molecules are also determined based on the physicochemical properties and manufacturing of each peptide. the us food and drug administration (fda) usually ranks peptides as conventional drugs mostly because their chemical structures exceed residues, although exceptions are made mostly in the case of vaccines, which are ranked as biological products [ ] . in europe, the european medicines agency (ema) evaluates the source of the peptide [ ] . if the molecule was screened from a natural biological source, it is treated as a biological entity. by contrast, chemical entities are those that were chemically synthesized in vitro. in addition, manufacturing must guarantee the identity, purity, potency, and individual lot consistency [ , ] . examples of drug candidate failure are perhaps more common than might expected. the most famous case was the peptide magainin (pexiganan), a potent amp isolated from the african clawed frog xenopus laevis. after completion of phase clinical trials in , the fda did not approve its commercialization because the drug was not proved to be more effective compared with the antibiotics utilized during the trials [ ] . notable expansion in the peptide-based drug discovery field over the past years, encouraged the screening and testing of new drug candidates. the approval rate since for peptides is around %. this reflects an increasing number of peptides entering annual clinical trials, one in compared with in . most candidates that enter phase clinical trials are painkillers (> %), or anticancer and anticardiovascular disease agents. acps dominate phase ( %) and ( %) clinical trials, followed by painkillers, anti-infectious disease and antiallergen agents [ ] . historically, the search for new efficient amps was based on the high-throughput screening (hts) of biologically active molecules. this concept relies on the discovery of naturally occurring peptides using classic purification and in vitro and in vivo techniques for checking antimicrobial activity. many amps have been identified and tested against clinical and natural strains of pathogens using this approach [ ] . bioactive peptides obtained from natural sources have been under evolutionary pressure for millennia, and consistently show high stability and target affinity and/or specificity. however, naturally occurring amps are normally synthesized at low rates by their biological sources, many are susceptible to protease degradation, and have low bioavailability (i.e. the presence of bioactive molecules at usual low levels). despite the recent advances in hts techniques, this approach is laborious and it is difficult to produce high yields of peptides in a scalable fashion [ , ] . new amps with potent antimicrobial activities and lower propensity to select for drug resistance have been intensely investigated. in silico methodologies for the rational design of peptides aim to improve the biological activities and increase production efficiency, speeding up biosynthesis and decreasing production costs. these rational tailored peptides represent a new generation of designer drugs to simultaneously overcome pathogen resistance and enhance microbial killing [ ] [ ] [ ] . amp design is based primarily on structure-function relations of host-derived synthetic amps and computational analysis of surface interactions between the peptide and pathogen structures. there are three main ways to enhance peptide activities through computational design: (i) epitope and net charge engineering by de novo sequence optimization of amp motifs; (ii) changes in posttranslational patterns of glycosylated peptides by amino acid substitution in glycosylation sites; and (iii) engineering different peptides as chimeric molecules and/or biomaterial surfaces with amp properties [ , , ] . sequence optimization of motifs is mainly applied for the design of cationic a-helical amps with improved and specific antimicrobial activities and low toxicity to mammalian cells. diverse engineered cationic antimicrobial peptides (ecaps) have been synthetically produced in laboratories worldwide, and show a range of in vitro and in vivo antimicrobial activities. examination of structure-function relations is primarily performed for the prediction of protein interfaces to infer protein-protein and protein-lipid interaction networks [ , ] . interface prediction is based on the physicochemical properties of residues in interfaces of protein complexes and by overlapping interface and non-interface segments of protein motifs. the most prominent characteristics of amps for peptide design are the sequence conservation of interface residues, proportion of the types of amino acid residue, relative presence of secondary structures, solvent accessibility, and sidechain conformational entropy [ ] . some amps are synthesized as glycoconjugates, peptides carrying a variety of n-or o-linked glycans that are crucial for peptide recognition, binding, and protein-protein interactions. most surface glycans are post-translationally added in the golgi after the peptide has passed through the secretory pathway [ ] . plantderived amps are the main primary targets for glycosylation engineering that aims to improve biological activity against phytopathogens and to humanize glycosylation for clinical use in humans. plant n-glycans differ considerably from those in mammals. typically, mammalian a , fucose (n-acetylglucosamine of the core), b , n-acetylglucosamine (b-mannose of the core), and b , galactose combined with sialic acid and linked to the terminal n-acetylglucosamine are substituted in plants, by an a , fucose, a bisecting b , xylose, and a b , galactose and fucose a , -linked to the terminal n-acetylglucosamine, respectively [ , ] . another important issue concerning the humanization of amp glycosylation is avoiding the addition of allergenic glycoepitopes to the peptide surface, because humans are frequently allergic to plant a , fucose and b xylose. currently, the main efforts to minimize undesirable plant glycosylation of amps rely on avoiding the complete transit throughout the secretory pathway by confining peptides inside the endoplasmic reticulum, using n-or c-terminal retention signals, such as kdel. advances in the humanization of the glycosylation of proteins (i.e. the full substitution of non-mammalian host n-linked glycans for typical human glycans by knocking down plant xylosyl and fucosyltransferases and yeast mannosidases and expression of human glycosidases) is already a reality in the production of plant and yeast antibodies and interferons, but has not yet been fully applied to amps [ , ] . chimeric ecaps are another major group of engineered amps. they are synthesized chemically or recombinantly as fusion peptides or as antimicrobial surface-coating agents, based on the potential synergistic effect of multiple active epitopes that considerably enhances their antimicrobial activities [ ] . these features reveal an interesting aspect of chimeric ecaps, namely the potential to prevent bacterial colonization and biofilm formation, a promising approach to eliminate implant infections. after synthesis, the individual domains of chimeric ecaps must present solidbinding kinetics to nanoformulated surfaces or substrates without the loss of antimicrobial properties. these peptides remain one of the most promising engineered anti-infective agents to be popularized against opportunistic pathogens in postoperative care settings [ ] . naturally occurring amps are typically subject to proteolysis, because they comprise l-amino acids recognizable by proteases. to minimize peptide degradation, the rational design of sequences comprising analogous d-amino acids substituted for l-amino acids can consistently increase the peptide post-translational stability without altering biological function. given that the interactions between amps and the bacterial membrane are not strictly dependent on interactions mediated by specific receptors, the d-enantiomers of a peptide often retain the antimicrobial activity [ ] . another interesting modification of ecaps is the addition of polyalanine tails in the n or c terminus of the peptide. the amino acid alanine is moderately hydrophobic and, when polymerized as a repetitive peptide tail, can exceed the hydrophobic limit for insertion mechanisms in the cytoplasmic membrane. polyalanine peptides can not only be inserted into the membrane of microbes without causing significant phospholipid displacement, maintaining the bilayer integrity, but also be internalized within the cell for further modifications in metabolic pathways. therefore, polyalanine tails can induce undesirable peptide configurations that lead to the formation of peptide clusters that are unable to anchor lipid bilayers [ , ] . one of the most promising modifications of the ecap structure is peglyation: that is, the covalent addition of polyethylene glycol (peg) chains to peptides. peglyation provides improved structural stability and higher bioavailability of modified peptides and proteins. pegylated synthetic ecaps can retain their antimicrobial activity with higher target specificity, although superfluous covalently attached peg moieties can reduce the interactions between ecap and the target sites of the cytoplasmic membrane of bacteria [ , ] . when the peptide shows therapeutic functions, it must be injected into the bloodstream either in its pure form or nanoencapsulated. in such cases, the stability of the peptide must be preserved to maintain a high level of bioactivity. peptide cyclization is a major strategy to improve the serum stability of synthetic peptides. the joining of the n and c terminus backbone or the formation of internal cross disulfide bridges cyclizes the peptide, hiding proteolytic cleavage sites from specific cellular aminopeptidases [ , ] . the amidated c terminus of peptides appears to generally improve antimicrobial activity and stability. in general, amidated peptides exhibit higher antimicrobial activity. amidation of the c terminus affects the hydrophobic moment of synthetic peptides, corroborating to enhance interactions with the membrane. although this has been an important strategy to improve microbial death, it also appears to improve hemolytic activity, requiring case-by-case studies to evaluate the pros and cons of such modifications [ , ] . following the accurate prediction of structural changes, the engineered peptides can be chemically synthesized or routinely produced by many genetic engineering strategies (fig. ) . in this context, in silico interaction databases are the most valuable tools to predict the sites where peptides physically interact with the microbial cell and the optimization of motif architecture and net charge by amino acid replacements. many examples of rationally designed amps have already been described in the literature, most of which show true potential for future use in clinical settings. often, these ecaps present not only enhanced biological activities against mdr microorganisms, but also lower propensity to select for resistant bacteria in vitro compared with native analogs. table lists promising ecaps for therapeutic use against mdr microbes [ , ] . after prospection or improvements in silico, a selected amp must be produced on a large scale at a consistently high quality and under good manufacturing practice rules (gmp). the size and chemical properties of a given peptide will directly influence the choice of production strategy [ ] . there are at least four major strategies to achieve satisfactory yields of high-quality products: (i) solid-state chemical synthesis; (ii) recombinant microbe platforms; (iii) transgenic plants and animals; and (iv) cellfree expression systems. regardless of the method used, downstream processing is the crucial step before the commercialization of any peptide. this is the most expensive, time-consuming and testing phase of the production pipeline [ , ] . specially designed strains of bacteria or fungal accumulating induced mutations are typically utilized as reactors of naturally occurring amps. these strains can enhance protein synthesis and secretions, as well as upgrade peptide folding. under these circumstances, peptide yields can increase by three orders of magnitude, but in many cases, the production levels will still be below the standard production levels required [ ] . as an alternative to natural sources, the production technology can result in the chemical synthesis of partial or full peptide chains. there are three types of chemical synthesis: (i) the solution phase; (ii) the solid phase; and (iii) hybrid approaches [ ] . most of the commercially approved peptides, which are frequently small to medium in size, are synthesized by solution phase approaches [ , ] . this methodology provides standard protocols for the isolation, characterization, and purification of peptides. solidphase synthesis provides platforms for the production of large and structurally complex peptides on a large scale. the hybrid method combines characteristics of the two previous methodologies. although efficient for the production of active peptides, the three approaches are expensive because they enable prolonged to maximize peptide biosynthesis, genetically engineered bacteria and yeast cells are frequently explored as vehicles for the recombinant production of bioactive amps [ ] . many different amps have been synthesized in e. coli and pichia pastoris [ ] . despite the high therapeutic potential of recombinant amps, limited investment of companies and drawbacks in terms of poor yield, low quality, and unsatisfactory in vivo activity have restricted commercial development to only a few promising amps. regardless of these production limitations, some of these therapeutic peptides have reached advanced clinical trials before commercialization, as detailed in table . among the most important factors that limit the recombinant production of amps in microbial systems is the inner toxicity of the peptide toward host cells; however, this is not typically a limitation because many amps kill bacteria at very low, nontoxic concentrations. another concern is the low quality of the peptide product following post-translational modifications. under such circumstances, plants appear to be an interesting and promising alternative host system for the production of recombinant amps [ ] [ ] [ ] . although plants perform a range of post-translational modifications, low levels of recombinant biosynthesis of peptides are common, resulting in low quantities of purified products. however, a new transformative technology, called magnifection, has emerged as a platform for the fast production of large numbers of plant-derived recombinant proteins and peptides [ , , ] . developed by gleba and collaborators at the german biotech company icon genetics, magnifection is a transient expression platform that utilizes nicotinia tabacum or nicotinia benthamiana plants as efficient reactors for the production of massive yields of recombinant proteins, in a rapid, scalable fashion [ , ] . the process is based on the infiltration of whole plants with a suspension of transgenic agrobacterium tumefaciens cells carrying plasmids that encode viral rnas replicons (fig. ) . these gram-negative soil bacteria have key roles in infection and movement throughout plant tissues because they systemically spread through the plant to eventually reach most leaves and stems [ , ] . infiltrated plants contain viral vectors based on tobacco mosaic virus (tmv) or potato virus x (pvx) carrying amp-coding sequences, delivered by bacteria to be transiently expressed and amplified [ ] . these potent machines of transcript production use the viral machinery to enhance the production of viral proteins along with the selected amp, without stable transgene integration, resulting in massive yields of amps [ ] . speed is one of the main advantages of magnifection, because it provides expression kinetics that frequently reach the peak of peptide production - days after infiltration. such conditions allow the scale-up of plant infiltration by vacuum and peptide biosynthesis, combining elements of three biological systems (i.e. viral potent transcription, bacterial systemic spread, and plant accurate post-translational modifications) in a single expression strategy [ , ] . therefore, magnifection significantly reduces amp production costs because of a rapid and straightforward approach that results in the production of the first milligrams of amps in just weeks and up to kg in year, using a much-diluted a. tumefaciens suspension to infiltrate completely an entire green house. the high biomass of tobacco (> . kg ha À ) also contributes to the scaling up of peptide production, while reducing overall costs [ , ] . using magnifection, protein and peptide amounts frequently are - -fold higher than those observed for stable genetically transformed plants, yielding up to % of the total soluble protein (tsp). using this technology, amounts of up to g of peptides per kilogram of fresh agroinfiltrated leaves have been obtained routinely [ , , ] . the magnifection platform has experienced considerable success in the production of a variety of proteins and peptides, notably vaccines [ ] . the small size of antigen peptides and relatively simple chemical structures of some amps appear to fulfill the requirements of the platform. since , the magnifection system has been explored by the canadian biotech company medicago (http://www.medicago.com/) for the industrial production of a vaccine against h n flu in the usa. a us$ million budget financial deal was signed between medicago and the us department of defense agency defense advanced research projects agency (darpa) to develop million doses per month to avoid the risk of epidemic flu outbreaks [ , [ ] [ ] [ ] . the vaccine is currently in phase clinical trials, as is another vaccine against the variant virus h n , synthesized using the same platform [ ] . table details other vaccines already synthesized in tobacco using the magnifection system. although efficient for the transient biosynthesis of peptides, the addition of plant-specific post-translational modifications, notably the addition of n-glycans to peptides, is a potential limitation of the system and could lead to nonfunctional products or highly immunogenic vaccines. the threshold of economically accepted expression levels using magnifection is a restrictive factor similar to other previously explored recombinant platforms. although a good producer of full antibodies, the size of multimeric proteins constitutes another important challenge for the magnifection system, requiring the manipulation of two viral vectors to express two or more assembled polypeptides [ , ] . genome editing by crisprs and amp biosynthesis: advances, implications, and challenges screening and assembly is technically challenging. typically binds to short - -bp sequences. replacement of fragments longer than kb is difficult. off-target effects. target events in animals require screening. larger than zfns (coded usually by -kb dna). evidence that larger talens can lead to less specificity. off-target effects. crisprs are recently discovered bacterial adaptive immune systems that use a combination of short rnas and associated proteins to target specific sequences of dna for the generation of dsbs. crisprs revolutionized genome editing standards by presenting several technical advantages compared with previous systems, although they still have some disadvantages. easily adapted system to target and modify any genomic sequence. the cas protein, the main protein for dna recognition and cleavage, remains unchanged for any crisprs. easy to use to target numerous sites or across genomic libraries. use of multiplex-based guide rnas to simultaneously edit multiple sites. size of cas (cdna approximately . kb). expression levels and improve product quality. however, frequent gene silencing at the transcriptional level and instability of genes cloned in vectors for stable or transient expression remain major challenges limiting the efficient production of amps. another persistent issue is the poor quality of the peptides endogenously synthesized in bacteria, yeast, and plants, particularly resulting from undesirable post-translational modifications [ , ] . improved targeted genome engineering represents a sophisticated approach that could help minimize such limitations. over the past decade, alternative genome-editing tools, such as artificial engineered enzymes, zinc-finger nucleases (zfns), and transcription-activator-like effector nucleases (talens), have been successfully utilized to modify the genome of microbes, plants, and animals by adding, removing, or replacing segments of dna [ ] . a more efficient and less time-consuming technology for genome engineering was developed more recently, and appears to considerably expand the possible modifications of target sites in almost any sequenced genome: the clustered regularly interspaced short palindromic repeats system (crisprs) [ ] (box ). conventional genome-editing systems use synthetic nucleases to induce genomic double-stranded breaks (dsbs) at target sites. dsbs are targets of the imprecise cellular repair machineries either mediated by non-homologous end-joining (nhej) or homology directed repair (hdr), which require a donor dna template [ ] . crisprs constitute an incredibly versatile genome-editing platform derived from the s. pyogenes crispr-associated protein (cas ) [ , ] . there are three types of crisprs. type ii is the simplest, presented only by bacteria and comprises four proteins (i.e. cas , cas , cas , and cas ); it is the most widely used system for gene engineering. the most popular crispr-cas system is constituted by cas endonuclease proteins and crispr rnas [ ] . the nuclease cas can catalyze the precise cleavage of target sites assisted by two short helper rnas: crna and tracrna. the fusion of both rnas forms a hybrid single guide rna (sgrna) that binds to cas to form a supramolecular complex named rna-guided endonuclease, a sequence-specific recognizer complex that cleaves at specific sites in the genome, resulting in dsbs that are then repaired preferentially by hdr (in the case of bacterial genomes), thus altering the genome in a precise manner by site-specific modifications [ ] . the crispr-cas system has been used to introduce point mutations, modify gene function, generate gene knockouts, integrate foreign genes, repress and/or activate specific genes, deliver epigenetic modifications, and aid genomic loci accession by proteins. although limited studies report the utilization of the type ii crisprs-cas system to edit plant genomes, it has been exploited for editing other organisms [ ] [ ] [ ] . crispr/cas system ii technology description for amp biosynthesis mediated by genome edition. spacer acquisition: (a) formation of crisprs array by recognition and integration of foreign dna as spacer within the crispr locus, or fully synthesized by genetic engineering. the protospacers are non-coding region inserted in the bacterial dna with - bp. adjacent to each protospacer are found - bp short dna sequences termed protospacer adjacent motifs (pam) crrna processing: (b) the crispr array is transcribed as a long rna (pre-crrna) that is cleaved into crrnas with the help of cas proteins. an extra small rna (tracrrna) complementary to the repeat sequence is also synthesized. (c) the tracrrna pairs with the repeated region of crrna and helps in the processing of pre-crrna into crrna with the help of rnase iii for cleavage. interference stage: crrnas binds to ca proteins (d) to form a complex that recognizes foreign dna (e). a single multifunctional protein, cas , recruits crrna and tracrrna to cleave the recognized foreign dna using internal endonuclease domains (f). all the process is based in the recognition and pairing of the pam and the foreign dna. double strand breaks (dsbs) are generated (g). a donor dna containing the coding sequence of an amp of interest is integrated in the site of the dsbs by homologous recombination (i), and after gene expression, the amp is extracted and purified (h). in addition genome editing, the crispr-cas system can be utilized for other purposes within the cell. for instance, gene silencing by crispr interference (crispri) has proved to be a powerful tool to complement (and, in many cases, improve) previously described rnai, by the double formation of cas bound to sgrna and attached to the nontemplate strand of dna, blocking transcription [ ] . this approach can also be utilized to simultaneously silence multiple target genes (a process called multiplexing), thus overcoming one of the limitations of rnai in terms of its gene silencing potential. another interesting application of crispr-cas is to block transcription initiation in a specific and reversible manner [ ] . there is immense potential for the utilization of crisprs to improve the recombinant biosynthesis of amps in almost any organism. tailored genomes modified by crisprs can harbor amp coding sequences within the donor dna fragment inserted in a genomic expression hot spot, potentially boosting heterologous production of the peptide to unprecedented levels [ , ] (fig. ) . to reduce undesirable post-translational modifications of amps, it might be possible to knockout host glycosylase genes or substitute them with coding sequences of human glycosylases to humanize glycopeptides. this could contribute to enhance antimicrobial activity and therapeutic peptide quality (purity, lack of undesirable post-translational modifications, high levels, among others). in addition glycosylation, other post-translational modifications and peptide-processing pathways could be manipulated with crispr-cas. crispr-cas represents a promising technology to improve peptide engineering and biosynthesis, pushing the boundaries of the development of clinical drugs to new levels of sophistication [ , , ] . the major concern for the utilization of crisprs as a genomeediting tool is its potential secondary mutations or off-targets effects. this is a common scenario in human edited cells (however, on-target efficiency has been recently improved), but still rare in plants. to avoid off-target effects, it is imperative to properly design sgrnas that direct cas to an exact target in the genome, and efforts along these lines have been published recently. in addition, high levels of the nuclease cas relative to sgrna helps reduce the incidence of off-target effects [ ] . the cas nuclease can be further engineered into a nickase, which only cleaves a single strand of dna and the same strategy on the other strand enhances the specificity of site recognition and consistently minimizes off-target effects [ , [ ] [ ] [ ] [ ] . the prospect of novel, efficient amps is a crucial starting point to combat antibiotic-resistant microbial pathogens. the emergence of mdr bacteria is a tremendous global health problem that has been predicted to lead to the death of million humans per year by [ ] . microbes, plants, and animals are natural sources of therapeutic drugs and could also be a source of novel, biologically inspired, previously unknown antibiotics for human applications. in addition to their obvious importance, novel amps with high anti-infective properties are difficult to prospect and are naturally synthesized only at low levels in their respective host organisms. therefore, there is an urgent need to increase the efficiency and final yield of peptides during their production to allow their economic exploitation. new insights into the rational design of peptides are required to maximize amp variability and increase specificity and antimicrobial activity. currently, examples of synthetic and recombinant amps with tailored domains are now facing advanced clinical trials, with promising results. these rationally designed molecules present modified epitopes with improved net charge and enhanced antimicrobial activity, and represent a new generation of anti-infective agents with the potential to overcome mdr. the transient expression of amp genes in tobacco is a new technology with immense potential to considerably boost the heterologous production of new antibiotics and vaccines, in a fast, cheap, and efficient way. the use of potent viral vectors associated with the bacterial delivery of transcriptional units provides a scalable platform for the massive production of diversified amps, with potential desirable improvements in terms of processing and production costs. some drawbacks presented by plant expression systems are solved by the crispr system, a revolutionary genome-editing technology that presents myriad possibilities for genetic manipulation at the genomic level and provides unprecedented tools (crispri and crispra) to precisely control gene expression and the structural modification of amps. despite its current minor limitations (e.g. off-target effects), crispr could have a key role in the future development of clinical drugs as biotechnological antiinfective agents, including the rational biosynthesis of next-generation antimicrobials. overview: global and local impact of antibiotic resistance the emergence of peptides in the pharmaceutical business: from exploration to exploitation mechanisms and consequences of bacterial resistance to antimicrobial peptides antibiotic strategies in the era of multidrug resistance the expanding scope of antimicrobial peptide structures and their modes of action the antifungal plant defensin ahpdf . b is a beneficial factor involved in adaptive response to zinc overload when it is expressed in yeast cells engineered cationic antimicrobial peptides to overcome multidrug resistance by eskape pathogens magnifection: a new platform for expressing recombinant vaccines in plants crispr/cas for genome editing: progress, implications and challenges antimicrobial peptides targeting grampositive bacteria gram-positive bacterial cell envelopes: the impact on the activity of antimicrobial peptides antimicrobial peptides and their pore/ion channel properties in neutralization of pathogenic microbes understanding bacterial resistance to antimicrobial peptides: from the surface to deep inside induced bacterial cross-resistance toward host antimicrobial peptides: a worrying phenomenon new edge of antibiotic development: antimicrobial peptides and corresponding resistance correlation of cell membrane lipid profiles with daptomycin resistance in methicillin-resistant staphylococcus aureus cardiolipin prevents membrane translocation and permeabilization by daptomycin heterogeneity of mprf sequences in methicillin-resistant staphylococcus aureus clinical isolates: role in cross-resistance between daptomycin and host defense antimicrobial peptides daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content d-alanylation of teichoic acids promotes group a streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion lipopolysaccharide endotoxins antimicrobial peptide resistance of vibrio cholerae results from an lps modification pathway related to nonribosomal peptide synthetases a metalloproteinase karilysin present in the majority of tannerella forsythia isolates inhibits all pathways of the complement system a group b streptococcal pilus protein promotes phagocyte resistance and systemic virulence protein grab of streptococcus pyogenes regulates proteolysis at the bacterial surface by binding a -macroglobulin bacterial biofilm: structure, function, and antimicrobial resistance peptide therapeutics: current status and future directions antimicrobial and host-defense peptides as new anti-infective therapeutic strategies antimicrobial peptides (amps) as drug candidates: a patent review quality specifications for peptide drugs: a regulatorypharmaceutical approach antimicrobial peptides stage a comeback future directions for peptide therapeutics development rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? antimicrobial peptides in toroidal and cylindrical pores novel engineered peptides of a phage lysin as effective antimicrobials against multidrug resistant, acinetobacter baumannii antimicrobial peptides: versatile biological properties interaction-site prediction for protein complexes: a critical assessment biopharmaceutical production in plants: problems, solutions and opportunities n-glycosylation of plant recombinant pharmaceuticals production and glycosylation of plant-made pharmaceuticals: the antibodies as a challenge expression of rat b( , )-n-acetylglucosaminyltransferase iii in nicotiana tabacum remodels the plant-specific n-glycosylation end-tagging of ultra-short antimicrobial peptides by w/ f stretches to facilitate bacterial killing engineered chimeric peptides as antimicrobial surface coating agents toward infection-free implants d-enantiomeric peptides that eradicate wildtype and multi-drug resistant biofilms and protect against lethal pseudomonas aeruginosa infections membrane insertion and orientation of polyalanine peptides: a ( )n solid-state nmr spectroscopy investigation structural and functional characterization of a multifunctional alanine-rich peptide analogue from pleuronectes americanus chemistry for peptide and protein pegylation peg-peptide conjugates efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase global analysis of peptide cyclization efficiency the effects of the c-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems characterization of antimicrobial peptides toward the development of novel antibiotics antifungal plant defensins: mechanisms of action and production lanthipeptides: chemical synthesis versus in vivo biosynthesis as tools for pharmaceutical production chemical synthesis of proteins synthetic therapeutic peptides: science and market dengue fever virus and japanese encephalitis virus synthetic peptides, with motifs to fit hla class i haplotypes prevalent in human populations in endemic regions, can be used for application to skin langerhans cells to prime antiviral cd + cytotoxic t cells (ctls): a novel approach to the protection of humans expression systems for heterologous production of antimicrobial peptides current scenario of peptide-based drugs: the key roles of cationic antitumor and antiviral peptides a review of antimicrobial peptides and their therapeutic potential as anti-infective drugs plant antimicrobial peptides engineering viral expression vectors for plants: the 'full virus' and the 'deconstructed virus' strategies plant viral vectors for delivery by agrobacterium viral vectors for the expression of proteins in plants expanding the genetic editing tool kit: zfns, talens, and crispr-cas the crispr-cas system for plant genome editing: advances and opportunities the crispr/cas system for plant genome editing and beyond development and applications of crispr-cas for genome engineering double nicking by rna-guided crispr cas for enhanced genome editing specificity cas transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering antimicrobial resistance: tackling a crisis for the health and wealth of nations. hm government and the welcome trust the developing world urgently needs phages to combat pathogenic bacteria isolation and characterization of recombinant antibody fragments against cdc a from arabidopsis thaliana development of a recombinant vaccine against japanese encephalitis production of a modified peptide clavanin in pichia pastoris: cloning, expression, purification and in vitro activities cm-p : an antifungal hydrophilic peptide derived from the coastal mollusk cenchritis muricatus (gastropoda: littorinidae) design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt-resistance, and biocompatibility corn as a production system for human and animal vaccines a plant signal peptide-hepatitis b surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves biochemical safety evaluation of transgenic rice seeds expressing t cell epitopes of japanese cedar pollen allergens de novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity cathepsin-g interferes with clearance of pseudomonas aeruginosa from mouse lungs biological response on a titanium implant-grade surface functionalized with modular peptides modulation of proinflammatory activity by the engineered cationic antimicrobial peptide wlbu- . f res a phase iii, randomized, double-blind, placebo-controlled, study of iseganan for the reduction of stomatitis in patients receiving stomatotoxic chemotherapy topical antibacterial treatments for acne vulgaris: comparative review and guide to selection omiganan pentahydrochloride (mbi ), a topical -amino-acid cationic peptide: spectrum of antimicrobial activity and measurements of bactericidal activity histatins: antimicrobial peptides with therapeutic potential the big and small of drug discovery can innate immunity be enhanced to treat microbial infections? suspension-cultured by- tobacco cells produce and mature immunologically active house dust mite allergens plant-derived recombinant f , v, and f -v fusion antigens of yersinia pestis activate human cells of the innate and adaptive immune system immunogenicity of transgenic plant-derived hepatitis b surface antigen production of hiv- p protein in transgenic tobacco plants cloning of helicobacter pylori urease subunit b gene and its expression in tobacco (nicotiana tabacum l.) translational fusion of chloroplast-expressed human papillomavirus type l capsid protein enhances antigen accumulation in transplastomic tobacco plant-based vaccine: mice immunized with chloroplastderived anthrax protective antigen survive anthrax lethal toxin challenge severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine protection against tetanus toxin using a plant-based vaccine induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (gad) and il- expression of viral capsid protein antigen against epstein-barr virus in plastids of nicotiana tabacum cv. sr expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector key: cord- - e fchy authors: boisguérin, prisca; deshayes, sébastien; gait, michael j.; o'donovan, liz; godfrey, caroline; betts, corinne a.; wood, matthew j.a.; lebleu, bernard title: delivery of therapeutic oligonucleotides with cell penetrating peptides() date: - - journal: adv drug deliv rev doi: . /j.addr. . . sha: doc_id: cord_uid: e fchy oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. clinical translation has been often hampered by poor biodistribution, however. cell-penetrating peptides (cpps) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids. this review focuses on cpp-delivery of several classes of oligonucleotides (ons), namely antisense oligonucleotides, splice switching oligonucleotides (ssos) and sirnas. two main strategies have been used to transport ons with cpps: covalent conjugation (which is more appropriate for charge-neutral on analogues) and non-covalent complexation (which has been used for sirna delivery essentially). chemical synthesis, mechanisms of cellular internalization and various applications will be reviewed. a comprehensive coverage of the enormous amount of published data was not possible. instead, emphasis has been put on strategies that have proven to be effective in animal models of important human diseases and on examples taken from the authors' own expertise. in recent years there has been a dramatic re-evaluation of how genes are regulated and of gene expression modalities. two major sets of discoveries centre on the key roles of non-coding rnas, and in particular those involved in the rna interference (rnai) pathway, as well as the mosaic structure of eukaryotic genes. the antisense on strategy was proposed more than two decades ago for the artificial regulation of eukaryotic gene expression in cultured cells via the hybridization of short ons to mrna targets through the pioneering work of stephenson and zamecnik [ , ] . the immense potential of this strategy, which in principle only requires knowledge of the target mrna sequence, was quickly realized within both academic and industrial laboratories. interestingly, "mother nature" has also exploited this potential, as convincingly demonstrated in bacteria [ ] and later on in eukaryotes [ ] by their ability to capitalize on such potent biochemical and genetic tools. the best demonstration that the antisense concept is exploited came from the discovery that antisense genes are able to control transcription in bacteria (and with less experimental evidence in eukaryotic cells) and finely tune the expression of target genes. however, these natural antisense rnas turned out to be long and highly structured, and attempts to use this knowledge in the design of synthetic antisense genes proved disappointing. most studies so far in the antisense field have instead focussed on short singlestranded dna mimics, the hybridization of which allows recruitment of the cellular enzyme rnase h and as a consequence leads to the destruction of the rna target [ ] . somewhat unexpectedly, eukaryotic coding genes are transcribed as immature mrna precursors, the splicing of which by the complex nuclear machinery leads to intron removal. it became rapidly realized that a major outcome is the possibility of exon re-assortment and that this is the case for the majority of human genes. importantly in terms of potential clinical translation, several human diseases are associated with dysfunction of the splicing machinery (as in β-thalassemia) or with the preferential use of one splicing event rather than another. intervention with the process of exon selection using low molecular weight drugs has turned out to be difficult and the most promising strategy was proposed instead by ryszard kole and colleagues as detailed in section of this issue. in contrast to the "classical" antisense on strategy, splice switching oligonucleotides (ssos) are designed to prevent (or promote) the insertion of exons through high affinity binding at obligatory splicing sequences in the nuclear pre-mrna (for example donor or acceptor splice sites) and therefore rnase hincompetent on analogues must be used [ ] . the discovery of rna interference by fire et al. has also revolutionized our concepts of gene expression regulation [ ] . the current detailed knowledge of rnai processing and recognition by the rna-induced silencing complex (risc) has led to the possibility of rational design and synthesis of artificial sirnas able to recognize any mrna on the sole basis of their sequences. as for rnase h-competent antisense on, sequence-specific recognition of the rna target leads to its degradation by the risc-associated nuclease. further, several hundred human genes have now been identified to code for short stem-loop structures, known as micro rnas (mirnas), which are processed to allow targeting of the ′-utrs of mrnas and many such mrna targets have been identified [ ] [ ] [ ] . astonishingly, mirnas do not need to hybridize over their entire sequence to promote the down-regulation of an mrna target [ ] . as a consequence, a single mirna is able to regulate the expression of a complex set of mrnas, which are often related in terms of cellular function. although still incomplete, ongoing studies show that specific sets of mirnas control most cellular functions and that dysregulation of their expression is associated with many human diseases. regulation of the levels of certain mirnas or interference with their binding to mrna targets are both now heavily explored strategies. synthetic antisense ons, ssos, sirnas and mirnas have become routine and invaluable tools to dissect cellular functions and they can be efficiently transfected into most laboratory cell lines using commercially available reagents such as cationic lipid formulations. however, their systemic in vivo administration has been plagued by toxicity and by low efficiency in the presence of serum proteins. therapeutic developments centred on the use of naked ons have thus met with only limited successes. indeed, more than two decades of therapeutic developments have only led to three fda approved drugs, with two used for easier-to-manage topical ocular applications (fomivirsen as an antisense treatment for ocular cytomegalovirus infections in immunocompromised patients [ ] and ranibizumab, an aptamer for the treatment of macular degeneration [ ] ). of more promise is the recent approval in the us (but not yet in europe) of mipomersen [ ] , an antisense on for the treatment of homozygous familial hypercholesterolemia, which capitalizes on the accumulation of phosphorothioate (ps) ons in the liver after systemic administration. encouraging small scale clinical data have also been reported in the treatment of duchenne muscular dystrophy with ′-o-methyl phosphorothioate ( ′-omeps) ons (drisapersen [ ] [ ] [ ] ) and phosphorodiamidate morpholino oligonucleotides (pmo, eteplirsen [ ] [ ] [ ] ), as will be extensively discussed later in section of this chapter. despite these successes, it is generally agreed that degradation in biological fluids, passage across cellular barriers and intracellular trafficking are all limiting factors in nucleic acids-based therapies [ ] . extensive searches for on chemical modifications have improved their metabolic stabilities significantly as well as their affinities for rna targets, and have to some extent reduced off-target effects. no on chemical modification has significantly improved cellular uptake or tissue targeting, however. the design of efficient and non-toxic delivery vectors for on-based drugs has therefore become a major concern in both academic and industrial laboratories. among the many proposed tools for delivery, cell penetrating peptides (cpps) have appeared as an easy to implement strategy and have led to encouraging data at least in murine models of human diseases, as will be reviewed later in this chapter. this review does not pretend to be exhaustive and will be restricted ( ) to the cpp delivery of nucleic acids-based drugs and mainly to ssos and sirnas and ( ) to delivery strategies which have turned efficient in animal models of human diseases. the harnessing of cationic peptides to deliver drugs across biological membranes was first attempted by ryser and his colleagues [ ] . they demonstrated that an anticancer drug could be delivered as a poly-llysine (pll) conjugate in drug resistant cells in vitro and in vivo in mouse models [ ] . likewise, the chemical conjugation of antisense ons to pll led to the generation of a potent antiviral activity in several in vitro models of viral infections [ ] . unfortunately, conjugates were poorly characterized in view of the polydispersed character of commercial pll preparations and, more importantly, they led to acute cytotoxicity upon systemic administration in mice. it was discovered serendipitously by virologists that much shorter stretches of cationic peptides could promote the cellular uptake of macromolecules. for example, it was found that the full size purified hiv- tat protein trans-activates the hiv- ltr promoter when incubated with cells [ ] . more astonishingly, this same tat protein was able as a conjugate to promote the cellular internalization of the non-permeable protein β-galactosidase and this paved the way for the use of tat as a delivery vector for biomolecules. along the same lines, the purified antennapedia protein from drosophila was able to exert its transcriptional activity when incubated with nerve cells and this property was ascribed to a short peptide named penetratin [ ] . dissection of the tat protein did also show that cell penetration was due to a small, basic amino acids-rich peptide known as the tat peptide [ ] . initial studies of the cellular trafficking of both penetratin and tat suggested an unusual non-receptor dependent mechanism of direct translocation across the plasma membrane. although challenged later on as detailed in section , this mechanism and the possibility to use such so called cell-penetrating peptides (cpps) as non-viral delivery vectors for biomolecules, fostered a very large interest. many cpps were rapidly discovered and proposed as vectors for the transport of various drugs across biological membranes, starting from low molecular mass drugs to very large molecular entities such as nanoparticles. most cpps were designed for the transport of a chemically conjugated cargo (see table ). since the chemical conjugation and purification of negatively charged ons with the most popular cationic cpps has turned out to be difficult, most applications have concerned chargeneutral on analogues such as peptide nucleic acids (pnas) and pmo (see section ). the most advanced studies have involved the use of these conjugates for rna splicing regulation. early work described the use of penetratin for the delivery of conjugated ps ons [ ] and of transportan (another popular cpp) for pna transport [ ] . unexpectedly, in a well-characterized hela cell assay with a positive read-out ( fig. ) , splicing redirection using pna or pmo oligomers conjugated to various standard cpps (tat, penetratin or oligo-arginines) was not achieved in our research groups [ ] . hela pluc cells were stably transfected with a construction in which the coding sequence of the luciferase gene is interrupted by a mutated intron of the human β-globin gene. this mutation creates a ′splice site and activates a ′splice site. masking of the ′splice site by a rnase h-incompetent antisense on ( ) restores the production of functional luciferase mrna and protein. this assay was provided by kole and colleagues [ ] and allows a reliable and easy to implement comparative evaluation of on analogues and on-delivery vectors. intronic point mutations in a β-thalassemia globin gene activate cryptic splice sites leading to the aberrant splicing of this intron and as a consequence to a non-functional protein. masking of the mutated site with a steric-block on re-orients the splicing machinery toward complete removal of the intron and leads to the production of a correctly spliced mrna. this mutated intron has been introduced into the coding region of a reporter luciferase gene and the construction has been stably transfected in hela cells, which are available from atcc (atcc® ccl- ™). luciferase expression can be easily monitored enzymatically or by pcr. this assay is advantageous in providing a positive read-out with a low background and a large dynamic range. it has been adopted by many laboratories in the field thus allowing easy comparisons. extensive studies of their cellular trafficking revealed that these cpp-on conjugates were efficiently taken up by cells but remained stuck in endocytic vesicles. in keeping with this hypothesis, further incubation with chloroquine (an endosomolytic drug) or with saponin (a membrane-permeabilizing agent) allowed by-pass of this restriction [ ] . similar conclusions were reached using the same well-characterized assay by the nielsen group [ ] . understanding the mechanisms of cell uptake using cpps via comparison of literature data has proved extremely difficult, since the behaviours of cpps seem to be strongly influenced by experimental conditions. among relevant factors are cpp sequence, type of cargo, concentration (which seems to be crucial to foster endocytosis or direct translocation) and cell type [ ] . the first data showing strong activity in the hela splicing redirection assay were obtained with a derivative of oligo-arginines in which the spatial distribution of guanidinium side chains was optimized by the use of a non-natural aminohexanoic acid spacer [ ] . similarly, the addition of arginine residues on the n-terminus of penetratin also resulted in strong activity in this hela assay for pna conjugates [ ] . this led to the development of several arginine-rich peptides as pmo conjugates for use in muscle cells and in vivo mouse models of dmd as outlined in section . in addition to covalent conjugation, some cpps have been designed for use as complexes, particularly for sirna delivery (table ) . this is because, as previously alluded to, chemical conjugation and purification of ons and sirnas with cationic cpps has been difficult to achieve. for example, in the case of ons, the heitz and divita group described a new class of such cpps, with mpg as the lead compound, which can be complexed with negatively charged ons essentially through electrostatic interactions. this and other complexation peptides are detailed in section . since practically all cpps have a high cationic charge (due to lys and arg residues), it has proved extremely hard technically for covalent conjugation of most cpps to standard negatively charged (phosphodiester or phosphorothioate) ons because of aggregation or precipitation that can occur. it was possible for thiol linker-functionalised ons (for example mixmers of ′-o-methyl/lna steric blocking ons) to be conjugated with cysteine-functionalised classic cpps such as tat and penetratin to give disulphide linkages, if the conjugations were carried out in the presence of a denaturing agent such as formamide [ ] . in this way, it was found that fluorescent versions of such conjugates were taken up much better into endosomal compartments of model hela cells than unconjugated versions, but release into the nucleus to generate steric block rna targeting activity proved not to be achievable [ ] . a disulphide linkage is also not thought to be compatible with sufficient conjugate stability using systemic delivery, although lung delivery by disulphide-linked penetratin and tat conjugated sirna has been attempted [ ] . thus the few rare examples of peptide-conjugated negatively charged ons used in vivo have generally utilised a thiol-maleimide linkage between on and peptide (fig. ). in one case the on was ′functionalised during synthesis with an aminohexyl linker and the resulting amino group reacted with a bifunctional n-(gammamaleimidobutyrloxy)-succinimide (gmbs) reagent to give a maleimide derivative. the maleimide group was subsequently reacted with a cyscontaining peptide, a cationic cpp known as f , to furnish the desired conjugate ( fig. a ) [ ] . surprisingly, chain aggregation was not reported in this conjugation reaction. alternatively and more conveniently, -mer non-cationic peptides discovered by a phage display technique were functionalised at the nterminus by reaction with maleimidopropionic acid at the final stage of peptide synthesis and after purification were conjugated to a ′-o-me phosphorothioate on synthesized with a ′-thiohexyl linker ( fig. b ). such conjugates were designed for enhanced uptake in muscle and heart due to use of homing peptides selected by a phage display method [ ] . non-cationic homing peptides are likely to be explored further for delivery of ons to specific tissue types. indeed there was a recent report of a phage-selected space peptide conjugated covalently to sirna showing enhanced skin penetration [ ] . one might also expect increasing use of conjugations using the modern "click" chemistry, such as via the copper-catalysed reaction between an azido functionality and an alkyne group, where each of the peptide and on contains a clickable functionality [ ] or where the on contains a ′-o-propyargyl functionality [ ] . despite the large body of literature on the use of peptide-conjugated pnas in cell culture [ ] there are surprisingly few examples of their use in vivo. in the case of short peptide attachments to pna, these can be assembled directly on the n or c-terminus of the pna chain during solid-phase synthesis without needing any specific conjugation step. the amide couplings of protected amino acids are identical to that of protected pna monomers as long as a compatible protecting group scheme is used. in the standard syntheses of pna, it has been common to add a few lys residues in any case, particularly to enhance the aqueous solubility of the pna. thus the first report of pna activity involved merely use of (lys) n-terminally functionalised pna (the lys residues effectively acting as a cpp) in an in vivo splicing assay using a green fluorescent protein reporter that was up-regulated through redirection of splicing by the pna [ ] . further, in vivo results were obtained using (lys) derivatives of pna, synthesized in the same continuous way on the n-terminus of the pna [ ] . the results showed that an (lys) -pna conjugate was rapidly cleared from the circulation but distributed relatively broadly in a mouse with highest concentrations reached in liver, kidney, and spleen. only very low amounts were detected in lungs, heart, skeletal muscle and testes, however. in vivo analysis was extended to an amphipathic lys and leu-containing d-peptide and another rich in arg and homo-arg as pna conjugates, again synthesized continuously on solid phase [ ] . neither showed splicing redirection in liver and only the lys/leu peptide-pna conjugate showed activity in kidney, but both showed significant activity in adipose tissue. however, the lack of potency compared to rnase-h activating ons and their perceived toxicity profile at high doses led to abandonment of peptide-pna conjugates as a drug modality for isis pharmaceuticals. a continuously synthesized nuclear localization signal (nls) peptide (pkkkrkv) attached to a pna was also reported to be active in a severe combined immunodeficiency (scid) mouse model in intronic targeting of c-myc in burkitt's lymphoma-aimed therapy [ ] . further, a penetratin peptide was synthesized continuously at the c-terminus of a triplex-forming pna to target chromosomal dna for genetic modification of haematopoietic progenitor cells in mice [ ] . promising microrna knockdown results were obtained also when an antisense lys-pna-(lys) derivative was tested in mouse spleen for targeting microrna- [ ] . very recently an anti-microrna pna disulphide-conjugated (fig. a) to a tumour-targeting phlip peptide has been shown to have potent activity in a mouse model of lymphoma [ ] . the only other example of a peptide-pna active in vivo that was not synthesized by continuous solid-phase assembly was an arg-rich cpp pip a (and related pip b) conjugated through a thioether bridge by reaction of a c-terminal cys residue on the peptide to an nterminal bromoacetyl group on the pna (fig. b ) [ ] . the conjugates were active by intramuscular injection into the mdx mouse, a model of duchenne muscular dystrophy (dmd), but later higher activity was found in vivo when using pmo rather than pna as the on material [ ] . despite results of mixed fortunes in vivo for cpp conjugates of pna, these materials deserve further investigation especially in the case of tissues that are harder to reach with other types of on. a new method of parallel synthesis of arrays of cpp-pna conjugates (known as selpepcon) could prove useful for pre-screening of suitable drug candidates in cell assays or for example by intramuscular delivery by far the majority of successfully in vivo used cpp conjugates have utilised pmo as on cargo. the application of cpp-pmo to muscular dystrophies is outlined in section . regarding peptide-pmo (p-pmo) synthesis, suffice it to say that it has not been possible to date to use continuous synthesis methods for p-pmo conjugates, since synthesis has been restricted to commercial pmo suppliers. methods of conjugation are therefore limited to what is available commercially regarding functionalised pmo or instead must employ un-functionalised pmo. practically all cpps used with pmo to date have been arg-rich starting from the initial observations of moulton and colleagues [ ] . early work involved use of a bi-functional cross-linker to couple a ′piperazino pmo through a maleimide linkage to the peptide (fig. a ). later the ′-piperazine was directly conjugated through an amide linkage using c-terminally activated peptides (fig. b ) [ ] . ′-conjugation can also be carried out with commercially available amino-link functionalised pmo by direct coupling to the c-terminal carboxylic acid of the peptide through an amide link [ , ] . more generally now, the secondary amine of the ′-morpholino group is coupled directly to the c-terminal carboxylic acid of the pmo via an amide link ( fig. c) [ , ] . for direct amide conjugation, it is not necessary for arg-containing peptides to be chemically protected. however, lyscontaining cpps require a protecting group on the epsilon amino group removable after conjugation. direct ′-amide conjugation of un-functionalised pmo also allows for peptides to be conjugated to the pmo containing for example alkyne functionalities to allow for azide click coupling of a fluorescent label to the p-pmo [ ] . in addition click chemistry conjugation features in an adaptation for p-pmo of the selpepcon method of parallel conjugate synthesis (fig. d ) [ ] . combinations of compatible conjugation techniques such as use of click chemistries are likely to dominate future p-pmo synthesis methods. the term muscular dystrophy describes a large group of hereditary diseases characterized by progressive weakness and degeneration of skeletal muscle [ ] . a variety of gene therapies are being developed for this clinically and genetically heterogeneous group of disorders, including antisense on mediated splice modulation (sso). recent advances have placed duchenne muscular dystrophy (dmd) at the forefront of developments in sso therapy. in order to overcome the remaining obstacles to the success of this approach, peptide-conjugated ssos have been extensively investigated in animal models of dmd. dmd is the most common subtype of muscular dystrophy in the uk [online mendelian inheritance in man (omim [ ] ) database reference: ] affecting one in new born boys. this severe, x-linked recessive disease results from mutations in the dmd gene [ ] . the disorder is characterized by progressive muscle degeneration and wasting, along with the emergence of respiratory and cardiac complications, ultimately leading to premature death [ ] . the majority of mutations underlying dmd are genomic deletions, encompassing multiple exons thereby producing a premature truncation of the open reading frame and resulting in the absence of the dystrophin protein. dystrophin is an integral component of the dystrophin associated protein complex (dapc), forming a crucial connection between the intracellular actin-cytoskeleton and the extracellular matrix. exon skipping therapy utilises ssos to target specific regions of the dmd transcript, inducing the exclusion of individual exons, leading to the restoration of aberrant reading frames and resulting in the production of an internally deleted, yet largely functional, dystrophin protein [ ] . following development in animal models, two ao chemistries have undergone proof-of-concept studies and repeat systemic doseescalation studies, which have now been completed in the clinical trial setting [ , [ ] [ ] [ ] ] . systemic administration of both pmo and ′ omeps ssos have yielded specific exon exclusion and partial restoration of dystrophin protein in peripheral muscle of dmd boys. despite the undoubted potential of exon skipping on therapy for dmd, the successful application of this approach is currently limited by the relatively inefficient targeting of skeletal muscle, as well as by the inadequate targeting of ssos to other affected tissues such as the heart [ ] . as such, cationic cpps, which may be co-administered by conjugation with ssos, have shown dramatic improvement in delivery and induce high levels of dystrophin correction in muscle at comparatively low doses. the majority of work has been performed in the mdx dmd mouse model, which is the most widely utilised model in the dmd field. the dystrophic murine phenotype arises as a result of a spontaneous mutation in exon which encodes for a premature termination site, thus preventing the production of dystrophin protein [ , ] . the targeted exclusion of exon in this mouse models the therapeutic strategy in patients by restoring the production of dystrophin protein. the majority of cpps utilised in the dmd field (tables and ) may be covalently linked to pmo [ , ] . however, another class of noncovalent peptides from the transportan family has been engineered to deliver anionic ssos such as ′omeps, as detailed in section [ ] . a number of modifications of stearylated tp [ ] gave rise to cpps capable of inducing high splice correction in vitro in murine h k mdx myotubes (pepfect ; [ ] and pepfect ; [ ] ) ( table ) . it should be noted that the in vivo efficacies of these cpps have yet to be assessed. the covalent class of cpps comprises subtypes, specifically oligoarginine derivatives, phage peptides, and penetratin derivatives. oligoarginines spaced by -aminohexanoic acid and/or β-alanine exhibit high splicing efficiency and serum stability in vitro [ ] [ ] [ ] . the (rxr) peptide was the first cpp conjugate to be administered in the mdx mouse at a range of doses, time-intervals and via different delivery routes. generally, a single intravenous administration induced high dystrophin exon skipping in skeletal muscle, diaphragm and, for the first time, in heart [ ] . another arginine-rich peptide, (rxrrbr) peptide (b-peptide), identified from a screen using the egfp- splicing reporter mouse model [ ] , also gave rise to impressive exon skipping notably in the heart, when delivered using higher doses and via the retro-orbital route [ ] . improvements in cardiac function such as resistance to dobutamine stress testing and improvements in end systolic volume and end diastolic volume were also observed. the identification of phage motifs from phage display-libraries allows the specific homing of a target tissue. as such, peptides with preferential binding to muscle and cardiac tissue were identified specifically for the treatment of dmd. these include muscle specific peptide (msp) which exhibits enhanced in vivo muscle binding capacity [ ] , a -mer peptide (m ) which revealed better splicing efficiency over msp [ ] , and a -mer peptide (p ) which exhibited a moderate improvement in splicing activity over naked sso [ ] . the beneficial tissue targeting attributes of phage peptides may be further enhanced when combined with the delivery efficiency of cpps, to develop a chimeric peptide. this was demonstrated when msp was coupled to the bpeptide to determine the combined efficacy in mdx mice [ , ] . the specific orientation of these peptides was crucial to dystrophin splicing activity, and when coupled in the configuration 'b-msp-pmo' revealed a - fold improvement in skeletal muscle restoration compared to b-pmo [ ] . however, no improvement in dystrophin restoration was observed in cardiac muscle. more recently, the pna/pmo internalization peptide (pip) series was derived from the parent penetratin peptide [ , ] . subsequent modifications including the addition of arginine residues to the nterminus (r -penetratin) [ ] , the addition of a c-terminal cysteine residue, and the utilisation of disulphide conjugation methods [ ] fashioned this group of peptides into the established 'pip' sequence conformation, consisting of a central hydrophobic core flanked on either side by arginine-rich sequences. additional in vivo screening of pip-pmo compounds was carried out to identify optimal cpps for dystrophin splicing and correction in mdx mice. in the pip -series (pip e-o), the core sequence, ilfqy, was retained and there was alteration in the composition and length of the flanking regions [ ] . the number of arginine residues ranged between and , and the number and placement of -aminohexanoic acid (x) and ß-alanine (b) spacer residues also varied in the flanking regions (table ). pip e-, pip j-, pip l-, and pip n-pmo resulted in the greatest number of dystrophin positive fibres following intramuscular administration (tibialis anterior muscle). of these highly efficient cpps, pip e-pmo induced the highest levels of exon skipping and dystrophin restoration body wide including in the heart, following a single mg/kg intravenous administration. when directly compared to b-pmo, pip e-pmo was shown to restore considerably greater dystrophin protein levels in the heart (intravenous administration comparison). as b-pmo and pip e-pmo comprised similar arginine sequence and content, it was deduced that the core region of pip e-(ilfqy) was responsible for the splicing activity in heart. therefore, the pip -series was designed with identical flanking regions to pip e and the core sequence was altered (table ) . pip a-, pip b-and pip f-pmo, which maintained a amino acid core, exhibited the greatest dystrophin splicing activity in heart over the previous lead candidate, pip e-pmo [ ] (fig. ) . peptides such as pip c-and pip d-pmo, with a shortened core, resulted in a substantial reduction in efficacy. as cardiac and respiratory complications are the leading causes of death amongst dmd patients, the ability of pip -pmo to restore cardiac function is vital. these compounds demonstrated their restorative ability in a long term, low dose administration study ( mg/kg over month time-course) in which % dystrophin protein was restored in heart and the onset of cardiomyopathy was prevented in an exercised mdx mouse model (unpublished data). liver and kidney toxicity has been assessed weeks after the administration of pip-pmo conjugates in mdx mice with no sign of toxicity [ , ] . further toxicology studies are currently being pursued however have not been published yet. of course, continuous optimisations to dose, pharmacokinetics and toxicity are underway which will facilitate the progression of this class of cpps to clinical trial. the development of cpp-on conjugates for the treatment of dmd has served as a foundation for the treatment of other diseases facing similar antisense on delivery challenges. antisense ons have been used to modulate rna processing in the triplet repeat disorder myotonic dystrophy [omim: , ]. this degenerative disease arises fig. . immunohistochemical staining of dystrophin following pip a-pmo treatment. dystrophin staining in the tibialis anterior and heart of c bl/ , untreated mdx and pip a-pmo treated mdx mice. the treated cohort received a single, intravenous . mg/kg dose, and tissues were harvested weeks later. from the expansion of pathogenic microsatellite repeats within noncoding regions of either the dystrophia myotonica-protein kinase (dmpk) or cchc-type zinc finger, nucleic acid binding protein (cnbp) gene loci [ , ] . a variety of antisense on strategies have been used to combat the effect of these toxic rna expansions, although currently the most promising is the use of steric block ons [ ] . using this approach, ons were designed to bind to the dmpk repeat regions, therefore preventing the detrimental sequestering of rna-binding proteins to within the expansion. due to the multisystemic nature of myotonic dystrophy, a cpp conjugated antisense on strategy has recently been tested in a mouse model to induce body wide distribution [ ] . spinal muscular atrophy [omim: , , , ] is a neuromuscular disorder that results from the loss of motor neurons and skeletal muscle atrophy. this autosomal recessive disease is caused by mutations in the survival of motor neuron (smn ) gene. the related smn gene undergoes alternative splicing due to a single nucleotide polymorphism, and therefore lacks exon in the majority of transcripts, resulting in low protein expression. in order to functionally compensate for the loss of smn protein in patients, ssos have been used to promote the inclusion of exon in smn transcripts, and therefore increase the production of smn protein [ ] . substantial headway has been made with naked ssos in animal models of sma [ ] and clinical trials are ongoing. however, the effective delivery of antisense ons to motor neurons in the cns constitutes a major challenge, which may be combatted by cpp conjugation. as described, multiple diseases may benefit from antisense on therapy and indeed some have reached clinical trial and demonstrated significant therapeutic potential. these successes may be further amassed with the utility of cpps, which is a rapidly evolving field of research. whilst studies pertaining to toxicity and bio-distribution are underway, it is anticipated that cpp-on therapies will reach clinical trial in the near future. amongst antiviral applications, cpp-pnas were designed several years ago for targeting the hiv- trans-activation responsive element tar [ , ] . reassuringly, a tat-pna was found to be non-toxic in mice at high ( mg/kg) doses [ ] . however, no further in vivo antiviral studies of cpp-pna have been published since. by contrast, cpp-pmo antiviral applications have been plentiful for a range of viruses, such as dengue, cocksackie, ebola and marburg viruses [ ] . only a few in vivo studies of cpp-directed antiviral delivery have been published, all of these involving arg-rich cpps, notably (rxr) xb or r f , as conjugates of pmo (table ) . for example, (rxr) xb-pmo blocked viral replication of human respiratory syncytial virus (rsv) expression in balb/c mice, where the pmo sequence was targeted to the start of the coding region [ ] . there was also a reduction in lung viral titres seen when mice were treated h after infection, but not h after infection. this suggested that the pmo must be present in the cell soon after infection if it is to have an impact on virus production. promising results were obtained in pre-and post-infection (rxr) xb-pmo treatment of -week old piglets infected with porcine reproductive and respiratory syndrome virus (prrsv) both in reducing viremia and interstitial pneumonia [ ] . an (rxr) xb-pmo conjugate, where the pmo was targeted to the ′-terminus of the genomic rna strand, was found to be substantially more active than naked pmo in reducing viral titers in livers of mice infected with murine hepatitis virus (mhv), a coronavirus, and protected mice from tissue-associated liver damage [ ] . cpp-pmo treatment also prolonged survival in two lethal challenge models. however in the case of high dose viral challenge and delayed treatment, the cpp-pmo was not protective and there was some evidence of toxicity in the diseased mice. ag mice treated by intra-peritoneal injection with (rxr) xb-pmo targeted to the ′-terminus or the ′-cyclization sequence (cs) regions of the dengue virus (denv- ) pre-infection (but not post-infection) were able to increase the average survival time to up to days, but viral rebound was seen eventually [ ] . similarly, dosing of (rxr) xb-pmo targeted either to the ′-terminus or the ′-cs region of west nile virus (wnv) rna partially protected mice from viral challenge but higher doses caused toxicity [ ] . unconjugated pmo had no efficacy. although the cpp-pmo approach is very promising in principle for targeting viral rna, an alternative antiviral approach involves use of pmo analogues with cationic piperazine groups within the pmo. such "second generation" pmos, known as pmoplus™, have now generally supplanted cpp-pmo in antiviral applications and these have been reviewed [ , ] . cpp-pna was suggested more than years ago for targeting specific essential bacterial genes [ ] and subsequently both cpp-pna and cpp-pmo have been studied extensively in bacterial cell culture [ ] , but surprisingly few experiments showing in vivo efficacy have been published. the first proof of principle in vivo was shown in where a single intravenous injection of a (kff) k-pna targeted to acpp (an essential gene) rna administered into escherichia coli k- infected balb/c mice min after bacterial challenge reduced bacterial blood titres and enhanced survival of the infected mice [ ] . similar results were obtained for the arg-rich (rff) rxb-pmo conjugate targeted to the same acpp gene when dosed in mice h after infection with e. coli w [ ] . the effect on potency of varying the peptide sequence was investigated in this model and (rxr) xb was found to be the most potent of various arg-rich cpps studied in mice [ ] . more recently, cpp-pna targeting rpod, a gene that encodes an rna polymerase primary σ subunit essential for bacterial growth, showed broad inhibition in multidrug-resistant escherichia coli, salmonella enterica, klebsiella pneumoniae, and shigella flexneri both in cells and in vivo, with (rxr) xb cpp as a pna conjugate having higher activity in general than (kff) k cpp [ ] . (rff) r-pmo targeted to acpp or gyra genes was found to be highly effective to increase mouse survival time when mice were challenged with the deadly ames strain of bacillus anthracis (the causative agent of anthrax) [ ] , suggesting that such treatment of anthrax-infected humans might one day become possible. however, just like in the case of pathogenic viruses, cationic backbone-containing pmo (pmoplus™) although not achieving the same efficiency as cpp-pmo [ ] , may prove to have a more favourable toxicity profile for therapeutic development. as briefly mentioned above, the non-covalent strategy is based on short peptides which are able to form complexes with cargoes without requiring any cross-linking or chemical modifications [ , ] . most of the cpps used in the non-covalent approach have an amphipathic character which enables a combination of electrostatic and hydrophobic interactions with the cargo. the amphipathicity may arise from either the primary structure or the secondary structure. primary amphipathic peptides can be defined as the sequential assembly of hydrophobic residues with hydrophilic residues, whereas secondary amphipathic peptides are generated by the conformational state that allows distribution of hydrophobic and hydrophilic residues on opposite sides of the molecule [ ] . though originally based on amphipathic peptides, the non-covalent approach has been extended to peptides and peptidic analogues that are able to self-assemble with ons to form stable cpp:on complexes [ ] and several of them have been reported to improve on delivery into mammalian cells [ , ] . the non-covalent approach was originally developed for the delivery of large molecules and several peptides able to bind and condense dna such as gala, kala, jts have been used to improve gene delivery [ , ] . in , the divita and heitz group designed the mpg peptide for the delivery of single and double stranded short ons [ ] and the strategy was then extended to proteins and peptides by the development of pep- [ ] . since mpg, numerous cpp:on complexes have been developed for the delivery of different types of ons including ao, sso and sirna. the main advantage of the non-covalent strategy over covalent conjugation lies in its simplicity and the protection that cpp:on complexes confer to the on from digestion by nucleases [ ] . compared to a chemical cpp-on conjugate, the non-covalent complexes usually involve a one-step process consisting of a simple mixing of both partners, cpp and on [ ] (fig. ) . cpp:on complexes do not require any chemical cleavage, prevent steric hindrance between peptide and on, favour a better release of the on inside the targeted cells and facilitate modifications to increase specificity for the on and/or the target [ ] . with regard to the mechanism of internalization, there is no consensus point of view for the cell entry of non-covalent complexes (see section ). however, as these complexes rely on electrostatic and hydrophobic interactions between positively charged cpps and negatively charged ons, their cellular uptake mechanism could be also controlled by structural polymorphism of peptides, particle stability and the nature of complexes/membrane interactions [ ] . interestingly, some cpp:on non-covalent complexes were shown to enter cells through a direct translocation process [ ] . most non-covalent cpps enable a wide range of chemical modifications and combinations and the modular nature of both peptides and resulting cpp:ons particles also allows new developments. the first developments of mpg were based on peptide/on interactions and investigations of cpp:on affinity by fluorescence spectrometry to measure the dissociation constant (kd) as well as the optimal molar ratio and/or charge ratio to obtain stable complexes [ , ] . a molar ratio (mr) of peptides per on and a charged-corrected (n/p) ratio of positively charged amino acids (n) for a phosphate group (p) were estimated [ ] . similar approaches were used for other non-covalent cpps such as pep- for hypna-ppna [ ] , cady, c and mpeg-pcl-ch r h c for sirna condensation [ ] [ ] [ ] [ ] . fluorescence assays were also combined to circular dichroism (cd) analyses to monitor conformational changes that might occur in the presence of the on and during cpp:on complex formation [ , [ ] [ ] [ ] . although both fluorescence and cd investigations suggest interactions and formation of cpp:on complexes, the use of gel shift assays has been generalized to demonstrate the formation of complexes [ , ] . however, none gives a clear characterization of cpp:on particles in terms of colloidal properties (size, charge and shape). studies on colloidal properties of cpp:on complexes have therefore been witnessed over recent years. methods used for other nanomedicines were applied in order to characterize the size, surface charge and morphology of these cpp:on complexes. in , law et al. were the first to report the hydrodynamic diameter and the zeta potential of r :sirna non-covalent complexes [ ] . after uv-visible absorbance and cd investigations suggesting r /sirna interactions, size and zeta potential were measured for several charge ratios (+/−) and revealed that r forms particles with sirna, with a hydrodynamic diameter of~ μm at sirna saturation [ ] . zeta potential increased with the hydrodynamic diameter of particles, supporting the positive contribution of the guanidinium group of r in the surface charge. size and zeta potential have to be estimated in parallel since, according to dvlo theory for colloidal systems, low zeta potential suggest low electrostatic repulsion which induce aggregation of particles whereas high absolute value of zeta potential are indicative of stable particles suspension [ ] . in this light, investigations of size and charge were generalized to other cpp:on complexes, for different types of ons (sirna, sso, and as-on). however, these parameters might be very different according to the cpp, the nature of the on or the formulation protocol [ ] . for example, kim et al. identified small r :sirna nanoparticles with a nm diameter and a + mv zeta potential for a charge-corrected n/p ratio of in pbs [ ] whilst cady:sirna nanoparticles have a size of nm and surface charge of + mv whatever the mr (mr to mr ) in mm nacl [ ] . cady:sirna nanoparticles have a polydispersity index (pdi) of . , suggesting homogeneous and unimodal nanoparticles [ ] . for other cpp:on complexes, the size and the biological activity clearly vary with the mr [ ] . size and charge can also depend on the environment which is important since nanoparticles stability and homogeneity might vary according to the nature of the buffer, as described for c m :sirna particles [ ] . in addition physiological conditions such as serum addition can influence colloidal properties. pepfect :sirna complexes form homogenous unimodal nanoparticles of - nm (pdi~ . - . ), which remained stable in water, whilst the presence of serum proteins induces larger particles of - nm with a wider distribution [ ] . cpp:on complexes can also be stabilized by specific excipients, such as lactose [ ] , % mannitol [ ] or albumin [ ] . since the shape of nanoparticles also influences the bloodstream circulation half-life [ ] , the morphology of pbn was studied by electronic microscopy and atomic force microscopy (afm) and most studies pointed to globular or spherical nano-objects [ , , ] . in fine colloidal characterization suggested a redefinition of the cpp:on complexes, and with regard to chemical modifications that were included in the design of non-covalent cpp as well as the discrepancy in the formulation of complexes, the more appropriate term of "peptide-based-nanoparticles" (pbns) was proposed [ , ] . in this light, formulation of pbn clearly became the key point to consider for further developments, especially with regard to in vivo administration. as pbns aim for in vivo perspectives, they have to be improved for membrane permeation and cell targeting abilities, whilst being biocompatible and biodegradable, with minimal cytotoxicity and inflammatory response (see section . ). few pbn have been successfully used for the in vivo delivery of antisense ons, ssos or sirna [ ] [ ] [ ] , , ] . with regard to sirna, the first reports on pbn-mediated delivery of sirna in vivo involved mpg-Δ nls peptide and a cholesterol oligo-d-arginine (chol-r ) [ , ] . mpg-Δ nls was used for the in vivo delivery of sirna targeting oct- into mouse blastocytes. oct- / (pou f ) is one of the earliest transcription factors expressed in the embryo and both the pluripotency and the fate of es cells depend upon a tight control of oct- / expression. mpg-Δ nls -based nanoparticles induced a sirna-mediated inhibition of up regulation of oct- / in es cells which prevents their specification toward the mesoderm and their differentiation into cardiomyocytes [ ] . similarly, injection of pbn in the inner cell mass of blastocysts impairs cardiogenesis in early embryos [ ] . chol-r was applied for the delivery of sirna targeting vegf (sivgef) in ct- cells xenografted tumour. intratumoural administration of chol-r :sivgef complexes induced a significant inhibition of tumour growth associated with a pronounced decrease in vegf level in the tumour, suggesting a synergistic effect between the d-amino acids of the cpp and the cholesterol moiety [ ] . in a similar approach, mpg-Δ nls was modified in a shortened (mpg- ) and cholesterol-functionalized (mpg- -chol) peptide analogue. mpg- -mediated delivery of sirna targeting cyclin b induces a significant down regulation of both protein and mrna levels and a systemic administration of mpg- -based nanoparticles targeting cyclin b prevented tumour growth in a xenografted tumour mouse model [ ] . intravenous injections of . mg/kg reduced tumour growth for % by day , and administration of . mg/kg pbn entirely abolished tumour growth [ ] . in addition formulation of a mix of mpg- and mpg- -chol ( %) in pbn revealed that cholesterol increases the biodistribution of sirna in the tumour by maintaining sirna in the plasma [ ] . cytokine levels in the plasma were quantified in order to assess the ability of mpg- :sirna and mpg- /mpg- -chol:sirna formulations to induce innate immune response in vivo. no increase in cytokine level was observed h after injection of any formulation, suggesting the lack of immune response induction [ ] . in a similar approach, pepfect was tested for in vivo rnai silencing. the hypoxanthine phosphoribosyltransferase (hprt ) gene was targeted due to the long cellular half-life of the protein (~ h) and the minimal impact on the viability of the transfected cell. systemic administration of pepfect :sirna nanoparticles ( mg/kg) induced a significant down regulation (n %) of the hprt mrna level in liver, kidney, and lung, without induction of immune response [ ] . in addition, pepfect -based nanoparticles were also used for rnaimediated silencing of luciferase (luc-sirna) in mice expressing luciferase in the liver and intravenous injection of pepfect :luc-sirna ( mg/kg) displayed decrease of bioluminescence for weeks, reaching % gene silencing whilst naked luc-sirna was unable to induce a rnai response [ ] . likewise, chol-r , r :sirna nanoparticles were formulated for sirna-mediated knockdown of human epidermal growth factor receptor (her- ) in human ovary adenocarcinoma cells (sk-ov- ). a reduction of her- expression was associated with the inhibition of sk-ov- xenograft tumour growth [ ] . intratumoural injection of r :sirna nanoparticles, every days at a dose of μg sirna per mouse, significantly reduced tumour growth without significant toxicity [ ] . furthermore, different strategies were investigated to stabilize the nanoparticles and to increase blood circulation such as exemplified by albumin-or polyethylene glycol (peg)-coated nanoparticles. for example, hou and co-workers have shown that albumin-coated formulation of p rhh exhibits remarkable transfection efficiency attributable to ph triggered nanoparticle disassembly [ ] . furthermore, tanaka and co-workers have demonstrated that intravenous injection of mpeg-pcl-ch r h c/sirna complexes had a significantly higher anti-tumour effect in sarcoma-bearing mice [ ] . most non-covalent cpps tested in vivo have been formulated for sirna delivery. however the non-covalent strategy is also intended to deliver other types of ons, such as-ons or ssos, in vivo. in this light, pep- was formulated with the antisense cyclin b hypna-ppna and the resulting pbn were evaluated on pc xenografted mice [ ] . pbn were administrated through intratumoural or intravenous injection every days, and the biological effect of the antisense hypna-ppna was evaluated by monitoring tumour growth during weeks after injection. intratumoural administration of pbn induced a % inhibition of tumour growth at a mg on dose and more than % at a mg dose, whereas intravenous injection only reduced tumour growth by % at a mg dose. consequently in order to improve the in vivo stability of pbn, small amounts of pegylated-pep- were included in formulation [ ] . intravenous administration of mg of on with pbn containing % of pegylated-pep- significantly improved pbn stability and inhibited tumour growth by more than %, which was - -fold more efficient than un-pegylated-pep- [ ] . these data emphasize the potential of non-covalent cpp for in vivo as-on delivery and the importance of pegylation on nanoparticle stability. in addition to the physicochemical properties of pbn, some specific characteristics of formulations have to be addressed. parameters such as affinity, molar or charge ratios, excipients, size and colloidal properties have a strong influence on biological interactions and minor changes can have a major impact on drug delivery efficacy [ ] . thus, as in the fda draft guidance for liposomal products, the physicochemical properties and specifications should include: morphology, net charge, particle size of pbn, spectroscopic data, light scattering index, in vitro release, content of peptide-engaged in pbn versus free peptide, biodegradation of the pbn. one of the most challenging questions concerning cpps is the mechanism by which these peptides enter cells. early studies using fluorescent dyes linked to cpps concluded that internalization was energy-and temperature-independent. however, this was later found to be an experimental artefact due to strong cpp adherence to the cell membrane leading to fluorescence overestimation and/or to fixation protocols using methanol allowing the cpp-dye complex to enter cells [ ] . recent studies have suggested that cpp uptake takes place by both endocytosis and energy-independent translocation, with the balance between these two pathways influenced by factors such as cpp sequence [ ] , temperature and cpp concentration [ ] [ ] [ ] [ ] . it is now admitted that both biophysical methods, cell biology assays and biological end-points to assess activity need to be used to dissect the cell import mechanism. molecules approaching a cell first encounter a layer of oligosaccharidesthe endothelial glycocalyx which is a network of membranebound proteoglycans and glycoproteins, covering the endothelium luminally [ ] . proteoglycans in particular carry large o-linked oligosaccharides consisting of highly negatively charged repeating disaccharide units, the glycosaminoglycans (gags), such as heparan sulphates (hs). gags are involved in the cellular binding and uptake of several viruses [ , ] as well as cationic cpps and polycationic nanoparticles [ , ] . oligo-arginines bind to hs with affinities in the upper nanomolar range suggesting that gags may act as cpp receptors (reviewed in [ ] ). for the interaction of cationic cpps with gags, the number of positive charges (number of arginines) was shown critical [ ] . as examples, tp -pna and penetratin-pna conjugates are mainly internalized via macropinocytosis after initial hs interaction on the cell surface [ , ] . beside the important role of the positive charges, sagan and co-workers also show a strong positive correlation between the number of tryptophan residues, gag binding and cell uptake [ ] . likewise, cady:sirna complexes interact with hspgs, which probably allow the binding and accumulation of the particles at the cell surface as demonstrated by electromobility gel shift assay and nanoparticle dissociation [ ] . several models to explain a direct membrane translocation of cpps have been proposed, such as the formation of inverted micelles, pore formation (toroidal or barrel slave), the carpet model and the sinkingraft model. these models have been initially proposed for the uptake of membrane-active peptides (reviewed in [ ] [ ] [ ] ) and then adapted for cpps, such as oligo-arginines [ , ] or transportan [ ] . likewise, the amphipathic mpg and cady peptides are internalized via direct membrane translocation. on the basis of physico-chemical investigations (e.g., circular dichroism, fourier transform infrared and electrophysiological measurements on model membranes), two very similar models have been proposed for mpg:sirna and cady:sirna pbns based on the formation of transient pore-like structures [ , ] . a partial conformational change takes place upon mpg complexation with nucleic acids, and an increase in β-sheet content upon association with the cell membrane [ ] . cady:sirna pbns were characterized in more details (reviewed in [ ] ). cady-mediated cellular uptake of sirna is extremely rapid [ ] . moreover, internalization and biological activity of cady:sirna occurred even at °c and under inhibition of mitochondrial oxidative phosphorylation revealing an energy-independent mechanism [ ] . fluorescence microscopy revealed that neither transferrin, nor rab co-localized with cady:sirna nanoparticles whereas co-localization with lysotracker was observed to some extent [ ] . these data further support an uptake mechanism largely independent of classical endocytosis with a degradation of the nanoparticles via the lysosomal pathway. apart for the few cases described in section . , the present consensus is that cell uptake occurs mainly by endocytosis for arginine-rich cpps at least at low concentrations [ ] . whether clathrin-coated pit endocytosis, macropinocytosis, or another endocytic route is used is still a matter of debate and the route might differ with cell type, nature of the payload, or concentration [ , ] . the internalization mechanism was mainly characterized using the hela splicing redirection assay (fig. ) by correlating cellular uptake and biological efficiency of cpp-pna or cpp-pmo conjugates. several conjugates have been analysed in detail such as (lys) -pna-lys [ ] , (rxr) -pmo [ ] or pip b-pna [ ] . cell uptake is energy-dependent and leads to sequestration of conjugates in cytoplasmic vesicles after an endocytic mechanism of internalization. the limitations provided by the endosome sequestration of the payload are well illustrated by the two following sets of data. pepfect , a stearylated version of transportan [ ] , is able to complex ssos and these pepfect :sso pbns are rather efficient in the hela splicing-redirection assay. however, a significant part of the complexes still remains entrapped in endosomes and can be released upon chloroquine treatment [ ] . based on these results, endosomolytic trifluoromethylquinoline (qn) moieties were grafted on the pepfect cpp leading to pepfect with largely improved cytoplasmic delivery of the transfected on and an increased splicing redirection activity. likewise, pepfect was efficient to transfect sirnas and to promote gene silencing at low concentrations at variance to the pepfect formulation [ ] . along the same line and more recently, we could relate the high efficiency of pip a-pmo in h k mdx skeletal muscles with an efficient release from endocytic vesicles after internalization via caveolaedependent endocytosis. at variance, this same conjugate is internalized by clathrin-dependent endocytosis in primary mdx cardiomyocytes, is less efficiently released from endocytic vesicles and has a lower exon skipping activity [ ] . several strategies can be used to study the cellular internalization of cpp-on conjugates or cpp:on complexes [ , ] . . biophysical characterization of cpp/on using circular dichroism, infra-red spectroscopy, dynamic light scattering or by insertion studies into phospholipid layers. . inhibition of a specific endocytic pathway and assessment of its effect on cpp/on internalization and biological activity. . co-localization microscopy studies of the association of cpp/on with fluorescently labelled endocytic markers the association of cpps with membranes induces the modification of several physical properties, such as the surface pressure of monolayer (langmuir blodgett) and the secondary structure of the peptide (ft-ir, cd, etc.). deformation of the lipid bilayer due to hydrophobic and hydrophilic interactions between the components as well as the peptide localization in the membrane can be also assessed using nmr, x-ray diffraction, coupled plasmon waveguide resonance, epr or fret [ ] . assessing endosomal escape directly has turned difficult and can only be monitored using artificial models such as liposomal leakage assays. different protocols were developed using large unilamellar vesicles (luvs) with entrapped dyes such as calcein [ ] , carboxyfluorescein [ ] or ants (fluorescent dye)/dpx (quencher) [ , ] . in cellulo, clathrin-mediated endocytosis can be predominantly characterized by the use of transferrin. its uptake in cells can indeed be inhibited by a clathrin-specific sirna or via cell transfection cells with a mutant form of dynamin [ ] . however, dynamin is now known to regulate other endocytic and membrane trafficking pathways. alternatively pharmacological inhibitors, such as chlorpromazine or dynasore (a dynamin inhibitor), can be used to inhibit this pathway even if these reagents cause significant cell toxicity and have to be used over short incubation times [ ] [ ] [ ] . potassium depletion or hypertonic medium incubation is also commonly used to investigate clathrin-mediated endocytosis [ , ] . caveolae are invaginations of the plasma membrane that are localized in lipid rafts. a number of ligands, such as cholera toxin b, sv virus and albumin have been shown to be internalized more or less specifically via caveolae. the glycosphingolipid analogue lactosylceramide (laccer), a fluorescent probe, was also shown to be internalized via this route and may represent a more selective marker for this pathway [ ] . a range of pharmacological inhibitors of this pathway have been described and, in the main, they are agents that deplete cholesterol synthesis (lovastatin), agents that rapidly extract cholesterol from lipid rafts (methyl-ß-cyclodextrin), and other cholesterol-interacting molecules such as the antibiotics nystatin and filipin. dowdy, futaki and their colleagues have shown that tat and other arginine-rich cpps induce a ubiquitous form of fluid-phase endocytosis termed micropinocytosis [ , ] . for example, the uptake of octaarginine (r ) peptide by hela cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (eipa) and the f-actin polymerization inhibitor cytochalasin d, suggesting a role for macropinocytosis in the uptake of the peptide. as mentioned above, cpps are taken up primarily by endocytic pathways, and in order to promote endosomal escape and increase the transfection efficiency of cpps, many different strategies have been used. we have mentioned the insertion of endosomolytic moieties in section . and additional strategies are described below. since endosomes have a low ph, researchers have incorporated histidine residues or appended oligo-histidine tails in the cpp sequence, aiming at capitalizing on a "proton sponge" effect (see review [ ] ). due to its protonation at ph . , the imidazole ring of histidine (which is a weak base) counterbalances the accumulation of protons generated by a specific atpase inside acidic vesicles, neutralizes the lumen of endocytic vesicles and increases their osmolarity. as a consequence, the endosomal vesicles swell and their content is delivered into the cytosol. this has been exploited with success for the tat-cpp [ ] , for microsphere coated with ornithine and histidine repeats (o h ) and for self-assembling nano-constructs of amphiphilic copolymers (see review [ ] ). mason and co-workers have focused on amphipathic α-helical peptides incorporating ph sensitive residues [ ] . the histidine residues in the lah peptide are uncharged at neutral ph but when the ph of the endosomal lumen drops, the side chains become protonated, large numbers of peptides are released from the complex and adopt a conformation and alignment in the membrane that induces membrane disorder [ , ] . other groups have focused on the addition of cholesterol or on fatty acid modifications. for example, chol-r improves sirna delivery in a mouse model bearing a subcutaneous tumour [ ] . tat-pna-mediated splice correction is increased by up to two orders of magnitude when conjugated with decanoic acid [ ] . stearylation of cpps represents another strategy to increase endosome escape and as a consequence to increase the transfection efficiency of sirna [ ] and phosphorothioate ′-ome rna [ ] as already detailed in section . for in vivo applications, the most frequently modification involves the grafting of a polyethylene glycol (peg) moiety to prevent rapid clearance. as reported for pep- -mediated delivery of antisense-cyclin b -charged-pna which blocks tumour growth in vivo upon intratumoural and intravenous injection, pegylation of pep- significantly improves complex stability and efficiency [ ] . in the context of clinical translation, it will be crucial to improve the tissue specificity of cpps through their functionalization. the screening by in vivo phage display has enabled the identification of numerous peptides that home specifically to various organs under normal or pathological conditions [ ] . for example the five residues homing peptides creka, which was identified by in vivo screening of phagedisplayed peptide libraries in tumour-bearing mmtv-pymt transgenic breast cancer mice [ ] , has been combined to the pvec peptide to yield a cpp with tumour homing specificity [ ] . despite their huge potential, the clinical use of nucleic acids-based drugs has been limited by their poor biodistribution. cell penetrating peptides have therefore been considered as a possible strategy to improve passage across biological barriers and intracellular delivery as reviewed extensively in this chapter. both covalent conjugates with neutral antisense on mimics and noncovalent complexes with either charged antisense ons or sirnas have been engineered. several such constructs have undergone extensive evaluation in animal models (mainly in mice) of both acquired (e.g., viral infections or cancers, in particular) and genetic (e.g., duchenne muscular dystrophy) human diseases. cpp-delivery has been convincingly shown to increase significantly the efficiency of these nucleic acids cargoes. clinical trials have however not yet been started to our knowledge. extensive studies of biodistribution and of possible toxic effects have in particular to be completed. despite many studies, mechanisms responsible for the extravasation and for the cellular trafficking of these cpp-drug conjugates or complexes are still poorly understood. direct translocation across cell membranes appears to be operational in some instances. in most cases however, cell internalization occurs through endocytosis and escape from endocytic vesicles limits biological efficiency. likewise, cpp delivery does not occur with a similar efficiency in all tissues after systemic administration. understanding better these limitations will obviously help in engineering of new cpp generations with a superior potential to target various tissues (and pathological conditions) and to deliver their payload within the appropriate cellular compartment. efforts in these directions have already been started as described in this chapter but much remains to be done. inhibition of rous sarcoma viral rna translation by a specific oligodeoxyribonucleotide inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide regulation by small rnas in bacteria: expanding frontiers the antisense transcriptomes of human cells antisense technology: a selective tool for gene expression regulation and gene targeting therapeutic potential of splice-switching oligonucleotides potent and specific genetic interference by double-stranded rna in caenorhabditis elegans targeting rna to treat neuromuscular disease use of 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current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers an endosomolytic tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for dna transfection multifunctional polymeric micelles for delivery of drugs and sirna cationic amphipathic histidine-rich peptides for gene delivery effective endogenous gene silencing mediated by ph responsive peptides proceeds via multiple pathways design and evaluation of histidine-rich amphipathic peptides for sirna delivery improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (catlip) domain octaarginine-modified multifunctional envelope-type nano device for sirna delivery of nucleic acids with a stearylated (rxr) peptide using a non-covalent co-incubation strategy homing peptides as targeted delivery vehicles biomimetic amplification of nanoparticle homing to tumors design of a tumor homing cell-penetrating peptide for drug delivery acknowledgements lo'd was supported by a grant from the french muscular dystrophy association afm (programme number ). the work in the laboratory of mjg was supported by the medical research council (mrc programme number u ). bl and mjaw were supported by the afm grant "advances in oligonucleotide-mediated exon skipping for dmd and related disorders". work in the laboratory of pb and sd was partly funded by the feder (fonds européen de développement regional)/la région languedoc-roussillon, by the agence nationale de la recherche (anr) and by the centre national de la recherche scientifique (cnrs). key: cord- - t s authors: choy, wai-yan; lin, shu-guang; chan, paul kay-sheung; tam, john siu-lun; lo, y m dennis; chu, ida miu-ting; tsai, sau-na; zhong, ming-qi; fung, kwok-pui; waye, mary miu-yee; tsui, stephen kwok-wing; ng, kai-on; shan, zhi-xin; yang, min; wu, yi-long; lin, zhan-yi; ngai, sai-ming title: synthetic peptide studies on the severe acute respiratory syndrome (sars) coronavirus spike glycoprotein: perspective for sars vaccine development date: - - journal: clin chem doi: . /clinchem. . sha: doc_id: cord_uid: t s background: the s (spike) protein of the etiologic coronavirus (cov) agent of severe acute respiratory syndrome (sars) plays a central role in mediating viral infection via receptor binding and membrane fusion between the virion and the host cell. we focused on using synthetic peptides for developing antibodies against sars-cov, which aimed to block viral invasion by eliciting an immune response specific to the native sars-cov s protein. methods: six peptide sequences corresponding to the surface regions of sars-cov s protein were designed and investigated by use of combined bioinformatics and structural analysis. these synthetic peptides were used to immunize both rabbits and monkeys. antisera collected week after the second immunization were analyzed by elisa and tested for antibody specificity against sars-cov by immunofluorescent confocal microscopy. results: four of our six synthetic peptides (s , s , s , and s ) elicited sars-cov-specific antibodies, of which s (residues – ) and s (residues – ) exhibited immunogenic responses similar to those found in a parallel investigation using truncated recombinant protein analogs of the sars-cov s protein. this suggested that our s and s peptides may represent two minimum biologically active sequences of the immunogenic regions of the sars-cov s protein. conclusions: synthetic peptides can elicit specific antibodies to sars-cov. the study provides insights for the future development of sars vaccine via the synthetic-peptide-based approach. severe acute respiratory syndrome (sars) is a lifethreatening infectious respiratory disease with symptoms including fever, dry cough, headache, dyspnea, and hypoxemia ( ) . a novel type of human coronavirus (sars-cov) was identified to be the etiologic agent of sars ( ) ( ) ( ) . the coronaviruses belong to a diverse family of large, enveloped, single-stranded positive sense rna viruses that replicate in the cytoplasm of animal host cells ( ) . there are three groups of coronaviruses; mammalian viruses are found in groups and , and avian viruses are found only in group ( ) . in the past, human coronaviruses (hcovs) found in both group (hcov- e) and group (hcov-oc ) were associated only with mild upper respiratory tract diseases ( ) , whereas the novel sars-cov appears to be the first human coronavirus responsible for severe disease in humans. phylogenetic analysis indicates that sars-cov does not belong to any of the above three groups, which contain all other known coronaviruses, including the human coronaviruses; this suggests that sars-cov did not arise by recombination or mutation of the known coronaviruses ( , ) . sars-cov contains a rna genome of ϳ kb. its overall genome organization is typical of other coronaviruses, with five major open reading frames (orfs) encoding the replicase polyprotein, which comprises two-thirds of the genome, and the four major structural proteins. the nucleocapsid (n) protein and the membrane (m) protein interact to form a spherical core, whereas the spike (s) glycoprotein, and the envelope (e) protein constitute the viral envelope ( - ) . in addition to these proteins, the genome of sars-cov also encodes for other uncharacterized structural and nonstructural viral proteins. the distinct crown appearance on the surface of sars-cov is attributed to the s protein. on coronavirus infection, the s protein recognizes and binds to species-specific host cell receptors, and the conformational changes induced in the s protein would then facilitate the fusion between the viral envelope and host cell membranes ( - ) . the potential receptor-binding site of sars-cov should lie within the surface region of s protein, and it would be a good target for the development of synthetic peptide-based vaccines against sars-cov. to date, there is no specific treatment for sars, and the control of sars-cov infection by vaccination is not yet available. it is possible for sars to recur in a seasonal pattern similar to other similar respiratory diseases; thus the development of an effective vaccine is necessary for sustained control of sars. although both attenuated and inactivated coronaviruses could be used for vaccine development, these types of sars vaccines bear a potential risk of unpredictable sars outbreak. in this study, we focused on the use of biologically active synthetic peptides for viral protein neutralization, which aimed to block the viral invasion by eliciting an immune response that could specifically recognize and neutralize the s protein of sars-cov. our study could also provide insights into future vaccine development against sars-cov by the synthetic peptide-based approach and help to achieve better understanding of sars to allow better preparation for possible recurrences of sars. the genome sequence of sars-cov used for peptide design in this study was based on the sars-cov cuhk-su complete genome sequence downloaded from gen-bank (http://www.ncbi.nlm.nih.gov/entrez; accession number: ay ). orf finder (http://www.ncbi.nlm. nih.gov/gorf/gorf.html) was used to determine the orf of the sars-cov s glycoprotein, netnglyc . server (http://www.cbs.dtu.dk/services/netnglyc/) was used to predict potential n-glycosylation sites, and netphos . server (http://www.cbs.dtu.dk/services/netphos/) was used to predict potential phosphorylation sites. the secondary structure and hydrophobicity analyses on the sequence of the s protein were performed with dnasis max (ver. . ) software (miraibio), and peptide sequences that correspond to the potential surface regions of the s proteins were designed based on the combined data from the above protein analyses. the three-dimensional structures of these peptides in the solvated state were simulated on an insight ii molecular modeling platform (accelrys) running on silicon graphics workstations. the six designed peptides were synthesized by a solidphase technique with an applied biosystems a peptide synthesizer on amide resins using standard fmoc synthesis with hbtu/hobt coupling. the nh termini of peptides were acetylated on the resin with g/l acetic anhydride in dimethyl formamide. the acetylated peptides were cleaved from the resins, and side-chainprotecting groups were removed by cleavage solution containing g/l ethanedithiol and g/l thioanisole in trifluoroacetic acid. the peptides were then precipitated with cold diethyl ether, washed, and lyophilized. the cleaved peptides were purified with agilent technologies series analytical and preparative hplc systems using linear gradients formed from the solvent systems a ( ml/l trifluoroacetic acid in h o) and b ( . ml/l trifluoroacetic acid in acetonitrile) ( ) . the identities of purified peptides were confirmed by mass spectrometry on an ettan tm matrix-assisted laser desorption/ionization time-of-flight pro mass spectrometer (amersham biosciences). keyhole limpet hemocyanin (klh) was dissolved in phosphate-buffered saline (pbs) at ph . to a final concentration of g/l. approximately mg of synthetic peptide was added to ml of klh solution (i.e., ϳ - mol peptides/mol of klh). the mixture was sonicated in a water bath for min; mg of n-hydroxysuccinimide and mg of -ethyl- -( -dimethylaminopropyl)carbodiimide hydrochloride were then added, and the mixture was stirred at room temperature for min. the mixture was loaded on a hitrap tm desalting column (amersham biosciences), and the eluate was collected as the klh-conjugated peptide solution. before the immunization of rabbits and monkeys, preimmune sera were collected and confirmed to be sars-cov negative by real-time quantitative reverse transcription-pcr ( ) . in the primary injection, . ml of complete freund's adjuvant was added to . ml of purified antigen with a peptide concentration of g/l (either conjugate-free peptide or klh-conjugated peptide) and emulsified. each rabbit (male new zealand white; weight, - kg) and monkey [male macaca fascicularis; weight, - kg; the monkey strain m. fascicularis was chosen in this study because it has been demonstrated previously as a potent host of sars-cov that developed sars symptoms after sars-cov infection ( ) ] was immunized with ml of emulsion by subcutaneous injection at five different sites on day . the rabbits and clinical chemistry , no. , monkeys were then boosted by subcutaneous injection of an emulsion containing . ml of incomplete freund's adjuvant and . ml of purified antigen on days and . the first batch of rabbit and monkey antisera was collected week after the second immunization. titrations of both the rabbit and monkey antisera against the corresponding peptide or peptide conjugate were determined in duplicate by elisa as described previously ( ) , and the results were averaged. the purified antigens of the six synthetic peptides (either conjugatefree peptide or klh-conjugated peptide) were diluted to ϳ mg/l in pbs; l of the diluted antigen solution was added to the corresponding well of a -well microplate and incubated at °c overnight. the wells were washed three times with . ml/l tween in pbs; l of pbs containing ml/l skim milk and . ml/l tween was then added to each well and incubated at °c for h for blocking. the rabbit and monkey antisera against the synthetic peptides were diluted in a twofold series to : , : , : , : , : , : , : , and : in pbs containing ml/l skimmed milk and . ml/l tween , and l of diluted antiserum was added to each well. the plate was incubated at °c for h. after plates were washed three times with pbs containing . ml/l tween , bound antibody was measured by use of either alkaline phosphatase-conjugated goat anti-rabbit igg or alkaline phosphatase-conjugated goat anti-monkey iggy ( l/well of : dilution of igg in pbs containing ml/l skimmed milk and . ml/l tween ). after incubation at °c for h and three washes with pbs containing . ml/l tween , l of developing solution ( mg of p-nitrophenyl phosphate added to ml of substrate buffer containing ml of diethanolamine, mg of mgcl ⅐ h o, and . mg of nan ; adjusted to ph . with naoh and brought to ml with milliq h o) was added to each well. absorbance was monitored by a microplate reader at nm. both the rabbit and monkey preimmune sera were used as negative controls in this assay. the rabbit and monkey antisera against the synthetic peptides were diluted to : -fold with pbs, and l of each diluted serum was added to a well of the slide that was coated with sars-cov-infected african green monkey kidney vero cells and incubated at °c for h. the slide was washed three times with pbs and air dried. in each well of the slide, l of the diluted conjugate ( : ) and evan blue ( : ) were added, and the slide was incubated at °c for h. the slide was washed three times with pbs, air dried, and mounted. the mounted slide was observed with a confocal microscope. noninfected african green monkey kidney vero cells and the preimmune sera of both the rabbits and monkeys were used as negative controls in this assay. the orf encoding for the s glycoprotein of sars-cov is nucleotides in length, comprising . % of the total genome. the s protein is the largest structural viral protein in sars-cov and is composed of amino acids with an estimated molecular mass of . kda; its isoelectric point (pi) is . . there are potential sites of n-linked glycosylation and potential sites of phosphorylation, including serine, threonine, and tyrosine residues. the s protein of sars-cov demonstrates Ͻ % identity with the previous identified s proteins of other known coronaviruses. the secondary structure and the hydrophobicity of the s protein were predicted by use of dnasis max (ver. . ) software. because the crystal structure of the sars-cov s protein is not yet available, we designed six peptide sequences (s , s , s , s , s and s ) ϳ - amino acid residues in length (table ) , which corresponded to the surface regions of s protein based on the combined data from protein analyses. most of them were expected to adopt a helix-loop motif and were hydrophilic; they were relatively widespread throughout the sequence of the s protein. these six synthetic peptides were used as either conjugate-free or klh-conjugated peptides for immunization of both the rabbits and monkeys. the three-dimensional structures of these six synthetic peptides in solvated states were simulated on an insight ii molecular modeling platform, and they were expected to adopt helix-loop motifs at their relatively stable energy states. the first batch of the rabbit and monkey antisera against the six synthetic peptides was collected week after the second immunization and was tested for antibody specificity against the corresponding antigen (either conjugatefree or klh-conjugated peptide) by elisa analysis. as shown in figs. and , all of these antisera were specific to their corresponding peptide antigens. both the rabbit and monkey preimmune sera were used as negative controls in this assay. these rabbit and monkey antisera were then tested for antibody specificity against sars-cov by immunofluorescent confocal microscopy. the antisera were incubated with african green monkey kidney vero cells infected by sars-cov obtained from a sars patient. the results of immunofluorescent confocal microscopy are shown in fig. and summarized in table . for rabbit antisera, positive results were observed in samples r_s _klh, r_s r_s _klh, and r_s _klh, which were elicited by their corresponding antigens: s -klh conjugate, s peptide, s -klh conjugate, and s -klh conjugate, respec-tively. for monkey antisera, positive results were observed in samples m_s , m_s , m_s _klh, m_mix, and m_mix_klh, which were elicited by their corresponding antigens: s peptide, s peptide, s -klh conjugate, mix peptide, and mix-klh conjugate, respectively. noninfected african green monkey kidney vero cells and the preimmune sera of both the rabbits and monkeys were used as negative controls in this assay. the positive results indicated that most of the sars-cov was localized abundantly in the cytoplasm of the infected cells, which further confirmed the replication of sars-cov in the cytoplasm of animal host cells. in this study, we designed a total of six peptide sequences, s -s (table ) , that corresponded to the surface regions of sars-cov s protein based on the combined bioinformatics data from protein analyses. these six synthetic peptides were used to immunize both rabbits and monkeys. the results of immunofluorescent confocal microscopy indicated that four of the six synthetic peptides, s , s , s , and s , raised antibodies that could specifically recognize sars-cov, which suggested the efficacy of using synthetic peptides as antigens to elicit specific immune responses. in a parallel study carried out at tsinghua university using recombinant protein approaches, similar results were obtained. pang's team ( ) at tsinghua university has generated a series of truncated recombinant protein analogs (sg , analog of residues - ; s , analog of residues - ; and s , analog of residues - ) of the sars-cov s protein to characterize the structural and functional properties of the s protein. their immunoassays indicated that the sg , s , and s analogs of the s protein were recognized by the igg in sera from recovered sars patients. their sg analog is suggested to be the most specific immunogenic region on the s protein. our work clearly demonstrated that the immunogenic region of their sg analog (residues - ) could be further delineated to residues - , which corresponds to our s peptide (table ). in addition, our s peptide (residues - ; table ) could be another minimum and significant immunogenic region of their s and s analogs of s protein. in addition, free s peptide is capable of inducing specific antibodies in monkeys. there are several factors contributing to the ability of the four synthetic peptides, i.e., s , s , s , and s , to elicit the production of sars-cov-specific antibodies. the design of the peptide sequences was deliberately targeted to the surface region of the s protein. by use of sequence homology analysis, secondary structure, and hydrophobicity predictions, we could predict the potential surface region of the s protein. most of the designed peptides were expected to adopt a helix motif to give a rigid structure to enhance antigenicity. to further enhance the antigenicity of these peptides, later work may include introducing several constraints to these peptides to increase structural rigidity, such as by adding linkers to cyclize the linear peptides or disulfide bonds for peptide stabilization. in addition, from the in silico energy minimization studies of our peptides, the predicted loop size carried on the peptide may reflect its antigenicity; thus, the loop structures of these peptides could be further adjusted and strengthened to optimize the corresponding antigenicity. in addition, our initial attempts at antibody production using the six synthetic peptides had deliberately avoided the regions rich in posttranslation modifi-cations, such as glycosylation and phosphorylation, to simplify the situation for preliminary analysis. on the basis of our studies, which demonstrated that four of the synthetic peptides could elicit the production of sars-cov-specific antibodies, future experiments will be carried out to address the protective nature of these antibody responses in monkeys. this can be done by vaccinating noninfected monkeys with these synthetic peptides. after exposure of these vaccinated monkeys to live sars-cov, their health status could be monitored to determine the efficacy of these synthetic peptides as potential sars vaccines. the monkey strain m. fascicularis would be a good choice for this animal test because this strain has previously been demonstrated as a potent host of sars-cov that developed sars symptoms after sars-cov infection ( ) . because of the high infectiousness of sars-cov, a biosafety p /p level laboratory is required whenever performing any experiment using live sars-cov to prevent unpredictable outbreaks of sars. it is possible for sars to recur in a seasonal pattern, as do other, similar respiratory diseases; thus the development of an effective vaccine is necessary for the sustained control of sars. to date, vaccination against sars is not available. a synthetic peptide-based approach could be a rapid initial step for providing useful information for sars-related research. the results of synthetic peptide studies enable preliminary information about the possible antigenic sites on the native viral protein to be generated, which provides insights for generating a recombinant protein analog that could better mimic the conformation of the epitope regions. moreover, the biophysical properties and binding kinetics of these synthetic peptide candidates could be systematically characterized to allow elucidation of the structural and functional relationships of different sars-cov proteins. databases designed to contain future "novel" sars-related rna and protein sequences are being constructed, and updating of these databases will be an ongoing task to monitor "polymorphisms" in the viral rna and protein sequences. the quality (biologically active sequences) and quantities of our peptide stocks can be easily adjusted to accommodate the ongoing investigation of polymorphisms in the sars-cov genome that produce changes in the primary sequences of the viral protein. eventually, we will establish peptide databases that cover the motifs corresponding to the biologically active functional and structural regions of the sars-cov proteins. our experimental data are informative and could advance the understanding of sars, which is necessary if we are to be prepared for possible recurrences of this disease. Ϫ m_mix_klh ϩ a mix peptide and mix-klh conjugate were prepared by mixing the six synthetic peptides (s , s , s , s , s , and s ) in equal amounts. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome the biology of coronaviruses viruses and bacteria in the etiology of the common cold characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus mechanisms and enzymes involved in sars coronavirus genome expression the coronavirus surface glycoprotein coronavirus spike proteins in viral entry and pathogenesis the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex high performance liquid chromatography of peptides and proteins: separation, analysis and conformation quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus the elisa guidebook initial postgenomic studies of sars coronavirus. presented at advances in protein sciences, the croucher foundation advanced study institute this project is supported by the sars special rgc grant cuhk / m. we are also grateful for the generous special sars funding from greaterchina technology group ltd. key: cord- -arivkkj authors: chu, ling-hon matthew; choy, wai-yan; tsai, sau-na; rao, zihe; ngai, sai-ming title: rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (sars) coronavirus c-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry date: - - journal: protein science doi: . /ps. sha: doc_id: cord_uid: arivkkj severe acute respiratory syndrome coronavirus (sars-cov) c-like protease ( cl(pro)) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-sars drug development. here we present a rapid and high-throughput screening method to study the substrate specificity of sars-cov cl(pro). six target amino acid positions flanking the sars-cov cl(pro) cleavage site were investigated. each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the “cartridge replacement” approach and was subjected to enzymatic cleavage by recombinant sars-cov cl(pro). susceptibility of each peptide substrate to sars-cov cl(pro) cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms). the hydrophobic pocket in the p position at the protease cleavage site is crucial to sars-cov cl(pro)-specific binding, which is limited to substitution by hydrophobic residue. the binding interface of sars-cov cl(pro) that is facing the p ′ position is suggested to be occupied by acidic amino acids, thus the p ′ position is intolerant to acidic residue substitution, owing to electrostatic repulsion. steric hindrance caused by some bulky or β-branching amino acids in p and p ′ positions may also hinder the binding of sars-cov cl(pro). this study generates a comprehensive overview of sars-cov cl(pro) substrate specificity, which serves as the design basis of synthetic peptide-based sars-cov cl(pro) inhibitors. our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis. ; peiris et al. ) . coronaviruses are large, enveloped, single-stranded positive-sense rna viruses that replicate in the cytoplasm of the infected host cell (siddell et al. ) . phylogenetic studies reveal sars-cov does not belong to any of the three known groups of the coronaviridae family; thus, it is suggested that sars-cov defines a distinct fourth group of coronaviruses (marra et al. ; rota et al. ) . the rna genome of sars-cov is , kb in length, with five major open reading frames (orfs) encoding the replicase polyprotein, the spike (s) protein, the envelope (e) protein, the membrane (m) protein, and the nucleocapsid (n) protein (marra et al. ; rota et al. ) . investigations of various sars-cov proteins can facilitate the development of vaccines and drugs against the sars disease. the pivotal role of sars-cov surface s protein in mediating viral entry sheds light on the generation of s protein-specific neutralizing antibodies (gallagher and buchmeier ; choy et al. ) , and some of the experimental vaccines have been tested in the murine viral replication model recently (bisht et al. ; yang et al. ) . meanwhile, the . -kda sars-cov main protease, also known as the sars-cov c-like protease (sars-cov cl pro ) is being considered extensively as an attractive and promising target for anti-sars drug design, owing to its important biological role in the viral life cycle (anand et al. ) . sars-cov cl pro mediates extensive proteolytic processing of two overlapping replicase polyproteins, pp a ( kda) and pp ab ( kda), to yield the corresponding functional polypeptides that are essential for sars-cov replication and transcription processes (herold et al. ; ziebuhr et al. ; rota et al. ) . there are at least conserved sars-cov cl pro cleavage sites on the sars-cov polyprotein pp ab, in which the substrate specificity involves preferentially the leu-glnfl sequence in the p and p positions at the cleavage site, respectively (ziebuhr et al. ; heygi and ziebuhr ; anand et al. ) . sars-cov cl pro comprises three domains, including the catalytic domains i and ii that form the nterminal chymotrypsin fold, and the unique extra helical domain iii in c-terminal that is important for regulating the activity and specificity of sars-cov cl pro (shi et al. ). the reported x-ray crystal structures of the sars-cov cl pro allows the investigation of possible interactions of sars-cov cl pro with its substrates, which contributes to the development of specific protease inhibitors as potential anti-sars drugs (anand et al. ; yang et al. ) . a better understanding of the specific enzyme substrate-binding mechanism during the sars-cov cl pro proteolytic cleavage process is required for the rational design of an effective protease inhibitor with strong binding affinity to sars-cov cl pro . to screen the substrate specificity of sars-cov cl pro in a rapid and highthroughput manner in contrast to the traditional tedious procedures, we applied the matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (maldi-tof ms) analysis in combination with the novel "cartridge replacement" solid-phase peptide synthesis approach to investigate the biological significance of amino acid residues in the p , p , p , p ¢, p ¢, and p ¢ positions that are flanking the conserved gln residue in the p position at the sars-cov cl pro cleavage site (schechter and berger ; fan et al. fan et al. , . owing to the sensitivity of maldi-tof ms, the protease cleavage progress of a mixture of different peptide substrates can be monitored simultaneously to generate comprehensive data on the substrate specificity of sars-cov cl pro . therefore, all of the possibilities of amino acid substitutions in a particular position of the peptide substrate can be easily investigated in the same reaction. our study can provide insights into the molecular mechanism of the sars-cov cl pro cleavage process and reveal the feasibility of developing synthetic peptide-based protease inhibitors as potential drugs against sars-cov and other coronavirus infections. furthermore, the rapid approach we described in this study could be widely applicable for monitoring the cleavage activities of other proteases, such as allowing the rapid screening of potential protease inhibitors of the human immunodeficiency virus (hiv) as the basis of drug development. in addition, automations are highly compatible and feasible with our experimental procedures to cope with the increasing demand of high-throughput analysis in proteomic research. substrate specificity preferences of sars-cov cl pro positive control of the sars-cov cl pro cleavage assay the ps peptide was amidated and acetylated at the c and n termini, respectively. the corresponding amino acid sequence of ps was based on one of the sars-cov cl pro cleavage sites on the sars-cov bj polyprotein pp ab (residues - ) and was used as the positive control in the enzymatic cleavage assay to assess the activity of the recombinant sars-cov cl pro . the original ps peptide peak with m/z value of . before the enzymatic assay was absent from the mass spectrum after the cleavage reaction ( fig. ) as it was being cleaved into two peptide fragments with their corresponding m/z values (data not shown), which validated the enzymatic activity of the recombinant sars-cov cl pro that we prepared. in this study, we used maldi-tof ms analysis in combination with the solid-phase peptide synthesis approach to examine the biological significance of amino acid residues in a total of six target positions at the sars-cov cl pro cleavage sites, including the p , p , and p positions at the amino side of the p position; and the p ¢, p ¢, and p ¢ positions at the carboxyl side of the p position (table ) . based on the flexibility in changing the contents of each amino acid cartridge, we designed a novel peptide synthesis strategy that we named as the "cartridge replacement" solid phase peptide synthesis. our "cartridge replacement" strategy consists of two screening procedures: ( ) primary screening and ( ) secondary screening. . primary screening using the "cartridge replacement" strategy by using the "cartridge replacement" strategy, the corresponding amino acid cartridge at the target position under investigation was deliberately replaced by another peptide peak (*) was resolved before cleavage assay (a- ) and was absent from the mass spectrum after the reaction (a- ). (b) p position. for ps peptide substrates, all peptide peaks were resolved on the mass spectrum before cleavage assay (b- ), and only the peptide peaks corresponding to leu (*) and phe (**) substitutions in the p position were absent, whereas all other peaks remained after the cleavage reaction (b- ). (c) p position. for ps peptide substrates, all peptide peaks were resolved on the mass spectrum before cleavage assay (c- ), and only the peptide peak with pro (*) substitution in the p position remained after the reaction (c- ). (d) p position. for ps peptide substrates, all peptide peaks were resolved on the mass spectrum before cleavage assay (d- ), which were all absent from the mass spectrum after the reaction (d- ). (e) p ¢ position. for ps peptide substrates, all peptide peaks were resolved on the mass spectrum before cleavage assay (e- ), and the peptide peaks with pro (*), asp (**), and glu (***) in the p ¢ position remained after the reaction (e- ). (f) p ¢ position. for ps peptide substrates, all peptide peaks were resolved on the mass spectrum before cleavage assay (f- ), and the peptide peaks with pro (*) and ile/leu (**) in the p ¢ position remained after the reaction (f- ). (g) p ¢ position. for ps peptide substrates, all peptide peaks were resolved on the mass spectrum before cleavage assay (g- ), which were all absent from the mass spectrum after the reaction (g- ). cartridge containing a mixture of standard amino acids in equal molar ratios during the solid-phase peptide synthesizing process so as to generate a mixed pool of different peptide substrates with single amino acid residue alteration at the target position. each batch of the mixed peptide substrates was subjected to in vitro enzymatic cleavage assay by recombinant sars-cov cl pro in a single reaction and then analyzed by maldi-tof ms to detect the susceptibility of each of the peptides to cleavage by sars-cov cl pro . the results from the primary screening of the in vitro enzymatic cleavage assay of the six batches of synthetic peptide substrates (namely ps , ps , ps , ps , ps , and ps ) with amino acid substitutions in their corresponding target positions are shown in figure and summarized in table . before the enzymatic assay, the corresponding peaks of the peptides in each substrate pool were resolved on the mass spectrum with their exact m/z values, which demonstrated the validity of solid-phase peptide synthesis using the "cartridge replacement" approach to generate a pool of peptides with single amino acid difference at the target position. the results also demonstrated the capability of maldi-tof ms to discriminate most of the peptide species with single amino acid difference. p , p , and p positions at the amino side of the sars-cov cl pro cleavage site for ps peptide substrates with amino acid substitutions in the p position, only the original peptide (m/z value of . ) and the peptide with phe substitution for leu in the p position (m/z value of . ) were cleaved, with their corresponding peptide peaks absent from the mass spectrum after the enzymatic assay, the amino acids flanking the sars-cov cl pro cleavage sites are labeled from the amino terminus to the carboxyl terminus as follows: -p -p -p fl p ¢-p ¢-p ¢, as described previously (schechter and berger ) . b the control peptide substrate is based on the amino acid sequence of one of the sars-cov cl pro cleavage sites on the sars-cov bj polyprotein pp ab (residues - ). c the corresponding amino acid at this position is substituted by a mixture of standard amino acids in equal molar ratios. the positions flanking the sars-cov cl pro cleavage sites are labeled from the n terminus to the c terminus as follows: -p -p -p fl p ¢-p ¢-p ¢, as described previously (schechter and berger ) . b the original amino acid residue in the position under investigation before the substitution with standard amino acids. c the identity of the amino acid in the particular peptide that substituted the residue at the position under investigation. d all peptides other than those in the opposite column were not cleaved by sars-cov cl pro . e all peptides other than those in the opposite column were cleaved by sars-cov cl pro . f all peptides were cleaved by sars-cov cl pro . whereas all other peptides were not cleaved, with their corresponding peaks remaining. for ps peptide substrates with amino acid substitutions in the p position, only the peptide with pro substitution for val in the p position (m/z value of . ) was not cleaved, with its corresponding peptide peak remaining in the mass spectrum after the enzymatic assay, whereas all other peptides were cleaved successfully. for ps peptide substrates with amino acid substitutions in the p position, all of the peptides were cleaved completely, with their corresponding peaks absent from the mass spectrum after the enzymatic assay. p ¢, p ¢, and p ¢ positions at the carboxyl side of the sars-cov cl pro cleavage site for ps peptide substrates, only three peptides with pro substitution (m/z value of . ), asp substitution (m/z value of . ), and glu substitution (m/z value of . ) for ser in the p ¢ position, respectively, were not cleaved. for ps peptide substrates, only two peptides with pro substitution (m/z value of . ) and ile/leu substitution (m/z value of . ) for gly in the p ¢ position, respectively, were not cleaved. for ps peptide substrates with amino acid substitutions in the p ¢ position, all of the peptides were cleaved completely, with their corresponding peaks absent from the mass spectrum after the enzymatic assay. . secondary screening using the "cartridge replacement" strategy after the primary screening procedure, we generated the comprehensive cleavage profiles of the corresponding peptide substrates of each target amino acid position simultaneously, based on the "cartridge replacement" strategy. however, there were peptides (carrying ambiguous residues like ile/leu and gln/lys that carry the identical or close masses) in which true cleavage might not be identified with absolute confidence. a secondary screening procedure was necessary, and we proceeded to the synthesis of target peptide substrates into defined batches, particularly for those peptides with identical or close molecular masses, which resulted in ambiguous peaks for detection on the mass spectra in the primary screening procedure. the results of the in vitro enzymatic cleavage assay of five peptide batches (namely, ps - , ps - , ps - , ps -i, and ps -l) are shown in figure and summarized in table . for ps - peptide batches, only the original peptide with leu in the p position (m/z value of . ) was cleaved, with its corresponding peptide peaks absent from the mass spectrum after the enzymatic assay, whereas the other three peptides with pro, cys, and glu substitutions for leu in the p position were not cleaved, with their corresponding peaks remaining. for ps - and ps - peptide batches, not all of the peptides were cleaved, with their corresponding peaks remaining. for ps -i and ps -l peptide batches, both the peptides with ile substitution (m/z value of . ) and leu substitution (m/z value of . ) for gly in the p ¢ position, respectively, were not cleaved, with their corresponding peaks remaining. based on the mass spectrometry results from the in vitro enzymatic cleavage assay (table ) , four target positions including p , p , p ¢, and p ¢ positions located at the sars-cov cl pro cleavage sites were further analyzed by the insight ii molecular modeling platform, and the molecular docking results are shown in figures and . the control peptide, ps , was docked to the predefined active site of sars-cov cl pro , with the conserved gln residue located at close proximity to cys -his residues at the catalytic dyad of sars-cov cl pro (fig. ) , in which this total energy data ( . kcal/mol) generated from energy minimization was used as the reference for comparing the docking results using other peptide substrates. this slightly positive total energy value is due to the relatively high repulsive van der waals energy attributed by the narrow binding pocket on sars-cov cl pro . for the ps peptide with phe substitution in the p position, the total energy of . kcal/mol was similar to that of the ps control peptide ( . kcal/mol), which further confirmed the tolerance of phe substitution at the p position at the cleavage site (fig. ) . on the other hand, the total docking energy for ps peptide with pro substitution in the p position ( . kcal/mol); ps peptides with pro substitution ( . kcal/mol), asp substitution ( . kcal/mol) and glu substitution ( . kcal/mol) in the p ¢ position; and ps peptides with pro substitution ( . kcal/mol), ile substitution ( . kcal/mol), and leu substitution ( . kcal/mol) in the p ¢ position were at least fivefold higher than the total energy of the ps control peptide ( . kcal/mol). the relatively high total docking energy revealed the unfavorable cleavage reactions of these peptides by sars-cov cl pro (fig. ). we have introduced a rapid and high-throughput screening method to generate a comprehensive overview of the substrate specificity preferences of sars-cov cl pro via a combinatory approach that uses maldi-tof ms and the novel "cartridge replacement" solidphase peptide synthesis strategy. research on the substrate specificity preferences of sars-cov cl pro is gaining increasing attention, owing to the biological significance of sars-cov cl pro in the viral life cycle and the feasibility to design specific sars-cov cl pro inhibitors as potential anti-sars drugs. it has been explicitly demonstrated that sars-cov cl pro cleaves the coronavirus replicase polyproteins at no less than conserved sites, preferentially involving the leu-glnfl sequence in the p and p positions at the cleavage site, respectively (ziebuhr et al. ; heygi and ziebuhr ; anand et al. ) . to further extend our knowledge of the substrate specificity of sars-cov cl pro , we aimed to comprehensively study the biological importance of amino acid residues in six selected positions (namely p , p , p , p ¢, p ¢, and p ¢) flanking the conserved p position at the sars-cov cl pro cleavage site by a rapid and high-throughput approach (table ) . from the primary screening procedure, the results of the ps , ps , ps , and ps peptide substrates can be interpreted precisely. from the results of ps and , and gln (q; m/z= . ) substitutions were resolved on the mass spectrum before cleavage assay (c- ), which all were remaining after the reaction (c- ). the . monoisotopic peak with high intensity (*) belongs to the mother peak of the peptide with asp (d) substitution. (d) ps -i peptide batch. the peak of peptide with ile (i; m/z= . ) substitution was resolved on the mass spectrum before cleavage assay (d- ), which remained after the reaction (d- ). (e) ps -l peptide batch. the peak of peptide with leu (l; m/z= . ) substitution was resolved on the mass spectrum before cleavage assay (e- ); which remained after the reaction (e- ). ps peptide substrates (fig. ) , all peptide peaks were absent from the mass spectra after the sars-cov cl pro cleavage reaction: the sars-cov cl pro can cleave all of the peptides regardless of the types of amino acid substitutions at the p ¢or p positions. for ps and ps peptide substrates, the corresponding peptide peaks (m/z value of . for ps and m/z values of . , . , and . for ps ) retained on the mass spectra could be clearly resolved after the cleavage reaction (fig. ) ; the sars-cov cl pro cannot cleave those peptides for which the nonoverlapping m/z values were shown, and hence, those uncleaved peptides can be easily identified. for ps peptide substrates, there were only two peptide peaks that vanished after the cleavage reaction. although these two peaks can be identified as corresponding to their exact m/z values, several remaining peptide peaks with close masses introduced ambiguity in the mass spectra. in addition, for ps peptide substrates, there were only two peptide peaks found on the mass spectra after the cleavage reaction. one of the peptide peaks (m/z value of . ) may contain two other peptide entities with exact m/z values, i.e., the ile/ leu containing peptide. in fact, this result was also observed in one of the cleaved peaks of ps peptide substrates (m/z value of . ). therefore, a secondary screening procedure using the defined batch synthesis approach (table ) was used to solve the above problem. in addition to the well-known conserved gln residue in the p position at the sars-cov cl pro cleavage site, the p position, which is exclusively occupied by leu residue, also serves as another important determinant of substrate specificity. our peptide cleavage results indicated that only the peptide substrate with phe replacement in the p position was also favorable for sars-cov cl pro cleavage ( fig. ; table ), with similar total energy level ( . kcal/mol) in molecular docking when compared with the total energy of the positive control peptide ( . kcal/mol) with leu in the p position. also, the results of the three peptide batches (ps - , ps - , and ps - ) in the secondary screening procedure further confirmed the results obtained from the primary screening procedure. the ps - peptide batch showed that ile was intolerant in the p position for sars-cov cl pro . our results demonstrated consistency with other findings (fan et al. (fan et al. , , suggesting that the p position serves as an important hydrophobic pocket for sars-cov cl pro -specific binding; thus, the peptide substrate with replacement of the original leu with the hydrophobic phe in the p position was still susceptible to sars-cov cl pro cleavage (fig. ) . from the results of a previous time course cleavage experiment (data not shown), we observed that the figure . interaction between sars-cov cl pro and ps control peptide as predicted by molecular docking. overview (left) of the interaction between the sars-cov cl pro (purple) and peptide substrate ps (red) and the zoom-in view (right) of the conserved gln residue (gray) in the p position at the cleavage site of the ps control peptide being docked into the catalytic dyad of sars-cov cl pro , which is composed of the cys (orange) and his (yellow) residues. sars-cov cl pro proteolytic cleavage rate of the positive control peptide was higher than that of the peptide with phe substitution in the p position, which accounted for the dominant occurrence of leu in the p position. other studies also demonstrated that sars-cov cl pro favorably tolerates phe and val substitutions, and to a lesser extent, for met and ile substitutions in the p position (fan et al. (fan et al. , , hence revealing the significance of the p hydrophobic pocket in the determination of sars-cov cl pro substrate specificity. furthermore, the p ¢ position, which is frequently occupied by ser residue, also contributes to the substrate specificity of sars-cov cl pro considerably. our peptide cleavage results revealed that the p ¢ position is highly unfavorable to the substitution by pro, asp, and glu residues ( fig. ; table ), which is consistent with molecular docking results showing their total energy levels being higher than that of the positive control peptide by more than fivefold. our findings on the intolerance of asp or glu substitutions in the p ¢ position suggest that the acidic residues at the binding interface of the sars-cov cl pro active site are probably in close proximity and sterically complementary to the p ¢ position of the substrate, leading to electrostatic repulsions between the negatively charged acidic residues that hinder the substrate binding to sars-cov cl pro . our molecular docking results also demonstrated that the p ¢ position is proximal to the glu and asp residues at the sars-cov cl pro active site (fig. ) , which further confirmed our interpretations. also, the unfavorable substitution by pro in the p ¢ position could be accounted for by the steric hindrance arising from its cyclic side chain (fig. ) , which obstructs the substrate binding to sars-cov cl pro . previous studies also demonstrated that small aliphatic residues such as ser, ala, and gly are favored in the p ¢ position (fan et al. ) . moreover, our results demonstrated that the substrate specificity of sars-cov cl pro are less dependent on the p ¢ and p positions at the cleavage site. the substitutions of pro, ile, or leu in the p ¢ position and the substitution of pro in the p position in the peptide substrates are unfavorable for sars-cov cl pro cleavage ( fig. ; table ), in which these bulky pro residue and bbranching ile and leu residues may cause steric hindrance to binding of sars-cov cl pro with its substrates (fig. ) . the results of ps -i and ps -l peptide batches further confirmed the results of the primary screening: neither ile nor leu is tolerant in the p ¢ position for sars-cov cl pro . on the other hand, the peptide cleavage results showed that the p ¢ and p positions have no effect on determining the substrate specificity preferences of sars-cov cl pro ( fig. ; table ). in contrast to providing quantitative measurements on the kinetic data of the interaction between sars-cov cl pro and the substrates, the scope of our studies mainly focuses on the description of a useful tool for rapid and comprehensive screening of substrate specificity for sars-cov cl pro , which is also applicable to the studies of other proteases. by combining the maldi-tof ms and synthetic peptide-based approaches, peptide-cleavage studies on the defined equal molar mixture of a batch of peptide substrates before and after the protease cleavage reaction could be performed simultaneously on a single maldi-tof ms analysis. the maldi-tof mass spectrometer is figure . comparison between the molecular models of different selected peptide substrates docked to the active site of sars-cov cl pro . the cys (orange) and his (yellow) residues in the catalytic dyad of sars-cov cl pro (purple) and the conserved gln residue (gray) in the p position at the cleavage site of the peptide substrate (red) were shown. (a) ps peptide with phe (green) in the p position was in close proximity to the active site of sars-cov cl pro . peptide substrates that were unfavorable for sars-cov cl pro cleavage were deviated away from the active site of sars-cov cl pro , which include ps peptide with pro (blue) in the p position (b); ps peptide with pro (blue) in the p ¢ position (c); ps peptide with asp (blue) in the p ¢ position that was in close proximity to the acidic residues (brown) at the active site (d); ps peptide with glu (blue) in the p ¢ position that was in close proximity to the acidic residues (brown) at the active site (e); ps peptide with pro (blue) in the p ¢ position (f); ps peptide with ile (blue) in the p ¢ position (g); ps peptide with leu (blue) in the p ¢ position (h). a delicate and sensitive instrument that enables clear and precise discrimination of different peptides, even with single amino acid difference. therefore, each of the peptide substrates with equal molar ratios in the same mixture can be monitored simultaneously on a single maldi-tof ms analysis before and after the protease cleavage reaction. our approach is advantageous over the traditional methods that are restricted to testing single peptides with defined condition one at a time and the setup of a total of identical experimental conditions for every analysis. after generating the comprehensive cleavage profile on the peptides simultaneously based on the primary screening procedure of the "cartridge replacement" strategy, we can easily identify some of the biologically important residues taking part in the recognition process. nevertheless, we employed the secondary screening procedure using the defined batch-synthesis approach to synthesize target peptides in different batches for exact identification purpose (ambiguous residues like ile/leu and gln/lys that carry identical or close masses). our approach provides an option in which the resolution of the maldi-tof mass spectrometer instrument is not sensitive enough to distinguish between species with -da mass difference. also, maldi-tof ms analysis is tolerant to impurities; therefore, the reaction mixture can be analyzed directly after a simple desalting procedure, which reduces the workload of subsequent downstream purifications of cleavage products prior to analysis. for the synthesis of peptide substrates, we deliberately applied the novel "cartridge replacement" strategy, which was based on the flexibility on modifying the solid-phase peptide synthesis process, in which each amino acid cartridge can be filled with different contents of amino acids in defined ratios, to generate the desired combinations of mixed peptide substrates according to experimental needs. by the incorporation of maldi-tof ms analysis and "cartridge replacement" peptide synthesis approach, we presented a useful tool that is widely applicable to studies that involve the monitoring of protease cleavage activity, which includes the rapid screening of potential protease inhibitors of other coronaviruses and the human immunodeficiency virus (hiv). on the basis of our studies, future experiments will be carried out to address the effectiveness of various synthetic peptidebased inhibitors in blocking the protease activity, with the ultimate aim of anti-sars drug development. in this study, we have combined the sensitivity, the high resolution of maldi-tof ms, and the flexibility of the "cartridge replacement" solid-phase peptide synthesis approach to generate sars-cov cl pro proteolytic cleavage data rapidly in a comprehensive manner. our studies definitely provide insights into the full automation of such experimental procedures to meet the increasing demand of high-throughput and automated proteomic studies. the coding sequence of sars-cov cl pro used for cloning was based on the sars-cov bj strain complete genome sequence downloaded from genbank (accession no. ay ; http://www.ncbi.nlm.nih.gov/entrez). the construction of the pgex- cl pro plasmid has been described previously by dr. rao's team at tsinghua university (yang et al. ) . the pgex- cl pro plasmid was transformed into escherichia coli strain bl (de )-competent cells and plated on luria-bertani (lb) agar containing ampicillin ( mg/ml)/ chloramphenicol ( mg/ml) and grown overnight at °c. a single colony was inoculated into ml of lb-broth containing ampicillin ( mg/ml) and grown overnight at °c with shaking. this overnight culture was then transferred into l of lb-broth containing ampicillin ( mg/ml) for large-scale protein expression at °c with shaking. when the culture was grown to an od nm of . , it was induced with . mm isopropyl- -thio-b-d-galactopyranoside (iptg) and grown for an additional h at °c. cells were harvested by -min centrifugation at g at °c, and the bacterial cell pellet was resuspended in lysis buffer containing mm tris-hcl (ph . ), mm nacl, mm dithiothreitol (dtt), . mm edta, and . mm phenylmethylsulfonyl fluoride (pmsf), and homogenized by sonication. the lysate was centrifuged at , g for min at °c, and the supernatant was loaded onto a glutathione sepharose b affinity column (amersham biosciences) equilibrated with the lysis buffer. the column was then washed with · column volume of lysis buffer. to cleave the gst-tag from the gst- cl pro fusion protein, prescission protease (amersham biosciences) was added into the column, and the mixture was incubated overnight at °c. the sars-cov cl pro recombinant protein was eluted with the lysis buffer, concentrated to mg/ml by microcon centrifugal filter devices (millipore), and then stored at - °c. "cartridge replacement" solid-phase peptide synthesis the sequence of peptide substrate used in this study was based on one of the sars-cov cl pro cleavage sites on the sars-cov bj polyprotein pp ab (residues - ) ( table ) . the biological significance of amino acid residues in the p , p , p , p ¢, p ¢, and p ¢ positions that are flanking the conserved gln residue in the p position at the cleavage site was investigated one at a time using the novel "cartridge replacement" solid-phase peptide synthesis strategy. in a standard procedure of solid-phase peptide synthesis, each amino acid cartridge that contains a particular type of amino acid is placed side-by-side in the peptide synthesizer according to the target peptide sequence, and the peptide synthesis begins with the cartridge containing the first amino acid at the carboxyl terminus of the sequence. in this study, we utilized flexibility in changing the contents of each amino acid cartridge and designed a novel peptide synthesis strategy that we named as the "cartridge replacement" solid-phase peptide synthesis. prior to peptide synthesis, the corresponding amino acid cartridge at the target position under investigation was replaced by another cartridge containing a mixture of standard amino acids in equal molar ratio, such that a mixed pool of synthetic peptides differing by a single amino acid residue was generated as the end product after complete synthesis of the substrate sequence. six sets of such peptide substrate pools together with the original peptide substrate were synthesized with this approach (table ) using a standard solid-phase peptide synthesis protocol as described previously (choy et al. ). the ps and ps peptide substrates require further analysis by a secondary screening. for the ps peptide substrates, the peptides with close masses were separated into three batches: ps - , ps - , and ps - . for the peptide synthesis of the ps - batch, the corresponding leu cartridge at the p position was replaced by a cartridge containing a mixture of cys, glu, leu, and pro in an equal molar ratio. for the peptide synthesis of the ps - batch, the corresponding leu cartridge at the p position was replaced by a cartridge containing a mixture of asn, lys, and val in an equal molar ratio. for the peptide synthesis of the ps - batch, the corresponding leu cartridge at the p position was replaced by a cartridge containing a mixture of asp, gln, ile, and thr in an equal molar ratio. for the ps peptide substrates, only the ile and leu substitutions were further investigated and separated into two batches: ps -i and ps -l. for the peptide synthesis of ps -i and ps -l batches, the corresponding gly cartridge at the p ¢ position was replaced by a cartridge containing ile or leu, respectively. all of the peptide substrates were synthesized by the solidphase technique with an in-house applied biosystems a peptide synthesizer on preloaded resins using standard -fluorenylmethoxycarbonyl (fmoc) synthesis chemistry with piperidine deprotection and -( h-benzotriazole- -yl)- , , , tetramethyluronium hexafluorophosphate (hbtu)/n-hydroxybenzotriazole (hobt) activation. the amino (nh ) termini of peptides were acetylated on the resin with acetic anhydride capping solution containing . m acetic anhydride, . m n,n-diispopropylethylamine (diea), . m n-hydroxybenzotriazole (hobt) in n-methylpyrrolidone (nmp). the acetylated peptides were cleaved from the resins, and side-chain protecting groups were removed by cleavage solution containing g/l ethanedithiol (edt) and g/l thioanisole in trifluoroacetic acid (tfa). the cleaved peptides were then precipitated with cold diethyl ether, washed, and lyophilized. the identities of the synthetic peptides were confirmed by mass spectrometry on an ettan maldi-tof pro mass spectrometer (amersham biosciences). the in vitro peptide cleavage assay was performed in a total reaction volume of ml (ph . ), containing . mm recombinant sars-cov cl pro , . mm sum total concentration of the peptide substrate batch, mm na hpo , mm nacl, mm edta, and mm dtt, with overnight incubation at °c. cleavage products were desalted by ziptip u-c (millipore), and the corresponding peptide peaks before and after the cleavage reactions were resolved by mass spectrometry on an ettan maldi-tof pro mass spectrometer (amersham biosciences) or an abi maldi tof/tof analyzer (applied biosystems). for acquisition of mass spectra, . -ml aliquots of samples were dispensed onto the maldiplate, followed by a . -ml matrix solution ( g/l a-cyano- -hydroxycinnamic acid in % acetonitrile/ . % trifluoroacetic acid). all spectra were acquired in a reflectron-positive ion mode and accumulated from laser shots with acceleration of kv and pulsed extraction. angiotensin iii (m/z value of . ) and adrenocorticotropic hormone (m/z value of . ) were used as standards for internal calibration of maldi-tof ms spectra. all three-dimensional molecular modeling studies were performed on an insight ii molecular modeling platform (accelrys software inc.) run on silicon graphics octane workstations (silicon graphics inc.). the consistent valence force field (cvff) was selected in all simulations. the x-ray crystal structure of the sars-cov cl pro ( uk ) was downloaded from the protein data bank (pdb; http://www.rcsb.org/pdb) and used as a starting model. the catalytic active site of chain a in the sars-cov cl pro structure without the bound cmk peptide was used in the study. hydrogen atoms were added for the structure of sars-cov cl pro chain a using the builder module. then, the force field potentials and partial charges were assigned. the resulting structure was subsequently subjected to energy minimization using the dis-cover module and was used as the initial structures for molecular docking. since the proteolytic activity sars-cov cl pro mainly relies on the active nucleophile cys and the acid-base catalyst his , the region containing all residues ( . Å radius) around this cys-his dyad was selected as the target site and was used to set up as a grid with the docking module. the nonbond energies of this region were precalculated on the grid. different peptide substrates were then docked into the target site by manual docking using the docking module. the best docking position was based on the docking energy of the peptide/sars-cov cl pro complex. thousands of orientations of peptides were searched by maneuvering in the cys-his dyad region of sars-cov cl pro manually until reaching the energy minimum. the resultant complex was subjected to energy minimization and molecular dynamics simulation using the discover module. a dielectric constant of . was used throughout the energy minimization and molecular dynamics simulation. the system was equilibrated at k for psec, during which time the potential energy of the system reached a stable value. a time step of fsec was used to integrate the equation of motion. the final conformation of the structure was obtained through iterations of steepest descent energy minimization. ultimately, the total docking energy between the peptide substrate and sars-cov cl pro in the energy-minimized complex was calculated using the docking module. the docking energy included the van der waals and electrostatic energies, which were the components of the intermolecular energy. coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice synthetic peptide studies on the severe acute respiratory syndrome (sars) coronavirus spike glycoprotein: perspective for sars vaccine development identification of a novel coronavirus in patients with severe acute respiratory syndrome biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus c-like proteinase the substrate specificity of sars coronavirus c-like proteinase coronavirus spike proteins in viral entry and pathogenesis conservation of substrate specificities among coronavirus main proteases nucleotide sequence of the human coronavirus e rna polymerase locus a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong the genome sequence of the sars-associated coronavirus coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome on the size of the active site in proteases dissection study on the severe acute respiratory syndrome c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors the biology of coronaviruses the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor a dna vaccine induces sars coronavirus neutralization and protective immunity in mice characterization of a human coronavirus (strain e) c-like proteinase activity virus-encoded proteinases and proteolytic processing in the nidovirales we thank dr. zihe rao's research team at tsinghua university, in particular haitao yang, xiaodong zhou, and xiaoyu xue, for their generosity in providing us with the pgex- cl pro construct. this project is supported by the sars special rgc grant and special sars funding from greaterchina technology group ltd. key: cord- -de lmzl authors: hu, han; guo, nan; chen, shuhua; guo, xiaozhen; liu, xiaoli; ye, shiyi; chai, qingqing; wang, yang; liu, binlei; he, qigai title: antiviral activity of piscidin against pseudorabies virus both in vitro and in vivo date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: de lmzl background: swine-origin virus infection spreading widely could cause significant economic loss to porcine industry. novel antiviral agents need to be developed to control this situation. methods: in this study, we evaluated the activities of five broad-spectrum antimicrobial peptides (amps) against several important swine-origin pathogenic viruses by tcid( ) assay. plaque reduction assay and cell apoptosis assay were also used to test the activity of the peptides. protection effect of piscidin against pseudorabies virus (prv) was also examined in mouse model. results: piscidin (piscidin ), caerin (caerin . ) and maculatin (maculatin . ) could inhibit prv by direct interaction with the virus particles in a dose-dependent manner and they could also protect the cells from prv-induced apoptosis. among the peptides tested, piscidin showed the strongest activity against prv. moreover, in vivo assay showed that piscidin can reduce the mortality of mice infected with prv. conclusion: in vitro and in vivo experiments indicate that piscidin has antiviral activity against prv. swine-origin virus infection is one bottleneck for the development of the porcine industry worldwide. the pathogenic viruses isolated from pigs including pseudorabies virus (prv), porcine reproductive and respiratory syndrome virus (prrsv), porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and rotavirus (rv) are commonly observed in china. the viral infection is also responsible for the secondary infection by bacteria which has greatly increased the application of antibiotics [ , ] . in the past decades, the efforts to alleviate pig viral diseases have focused on the development of vaccines to enhance the adaptive immunity of the hosts [ , ] . however, some of the evolving viruses could escape from the host immunity through different kinds of strategies. several viral diseases have broken out in recent years among pig herds, such as the reemergence of swine-origin influenza in [ ] , ped in late [ ] and pseudorabies in [ ] . thus, there is an urgent need to develop novel potential agents to kill these viruses or block their infection. amps are common host defense molecules in nearly all forms of life. ever since their discovery, amps have gained worldwide attention as important alternatives in the field of disease prevention and immune modulation [ , ] . previous studies mostly focused on the protection effect of amps against bacterial and fungal infection [ ] . later on, amps were also reported to be effective against viral infection [ ] . it has been reported that some alpha-helical peptides can act as virucidal agents. for example, caerins and maculatins isolated from amphibian skin could completely inhibit human immunodeficiency virus type (hiv- ) infection after virus is exposed to peptides within minutes [ ] . piscidins, discovered in the mast cells of fish, could reduce the infectivity of several important fish-origin viruses [ ] . indolicidin, a natural -amino acid antimicrobial peptide isolated from bovine neutrophils, could directly kill hiv- [ ] . bovine lactoferricin derived from bovine lactoferrin has been reported to exert antiviral activity to fight against human cytomegalovirus (hcmv), herpes simplex virus type (hsv- ), hsv- , and adenovirus [ ] . other antimicrobial agents, such as human defensins which is a group of beta-sheet peptides, can inhibit enveloped viruses such as hsv- and hsv- , hiv- , vesicular stomatitis virus (vsv), influenza virus, and hcmv by directly inactivating viruses [ ] . therefore, amps might be promising agents against viral infections. prv, a large enveloped dna virus, is a swine neurotropic herpesvirus [ ] . although the pigs are the natural reservoir for the virus, most mammals and some avian species are also susceptible to prv. prv infected animals may die from central nervous system disorders [ ] . prv infection poses a severe threat to pig industry and either attenuated live or inactivated vaccines are usually used to control the disease [ ] . although vaccination can suppress development of the disease, vaccines cannot eliminate virus infection. mutant isolates emerged and caused pseudorabies outbreak in [ ] . thus, novel antiviral agents should be developed as a complementary to vaccination. in one of our recently published papers, we described the antiviral activity of caerin against porcine epidemic diarrhea virus (pedv) in vitro [ ] . in this study, we investigated the activity of five amps including piscidin, caerin, maculatin, lactoferricin b, and indolicidin against several porcine-origin viruses. more inhibitory activity of piscidin, caerin, and maculatin against prv was also explored. the in vivo protection effect of piscidin against prv infection was further investigated. chen et al found that piscidin has the anti-inflammatory and anti-nociceptive properties in inflammatory animal models [ ] . kumar et al reported the obvious anti-endotoxin and anti-bacteria properties of piscidin- analogues both in vitro and in vivo [ ] . the peptides (table ) used in this study were synthesized on an automated solid-phase peptide synthesizer by neweast biosiences inc. (wuhan, china) [ ] . the crude peptides were purified via a reverse-phase highpressure liquid chromatography (rp-hplc) using a c column (waters xbridge). the elution was conducted using a water-acetonitrile linear gradient ( - % of acetonitrile) containing . % (v/v) trifluoroacetic aicd (tfa). finally, the purity and accurate masses of the product peptides were determined using hplc and mass spectrometry, respectively. the prv strains of ea, hnxx, , and tgev wh- were propagated in pk- cells based on the method reported in previous studies [ ] . rv tm-a was propagated in rhesus monkey kidney cell line (ma ) [ ] . prrsv ya was cultured in marc- cells [ ] . pedv strain ch/ynkm- / was cultured in vero cells as described before [ ] . the virus suspension was stored at − °c until used. in our initial topical screening assays, the viruses were pre-incubated with peptides ( μg/ml) for h at °c before they were added to the target cells. peptide-free controls (virus plus cell culture medium only) were incubated in parallel. after incubation, the tcid of peptidetreated viruses and peptide-free viruses was measured. residual virus was calculated according to the following formula, residual infectivity = b/a × % ("a" represent tcid of residual virus non-treated by the amps (control), "b" represent tcid of residual virus after treated by amps). a -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) cell proliferation assay was used to assess cell viability as described previously [ ] . briefly, pk- cells were added to -well tissue-culture plates (coster, usa) and incubated overnight at °c. serial two fold dilutions of amps ranging from to . μg/ml were added into the plate. the cells were incubated with amps at °c for h, and the morphology of the cells was evaluated under light microscope. then, the cells were further incubated with mtt solution for h and the cell pellets were collected for measurement of absorbance at nm by an elisa reader. the antiviral activity was evaluated by plaque reduction assay [ ] at different infection stages: pre-infection and post-infection, as well as direct inactivation of the viruses. briefly, the direct effect of peptides on virus per se was analyzed by the following procedures. monolayer of to analyze the post-infection effect of the peptides on virus growth, cells were infected with the same amount of virus mentioned above at °c for h. subsequently, the cells were washed times with dmem and treated by the peptides at different concentrations for h at °c. the peptides were removed from the cell culture and replaced with the overlaid medium. the resulting pfu titer was determined as described above. to understand the pre-infection effect of the peptide on virus, the cells in -well tissue culture plates were treated with peptides at serial concentrations at °c for h. then, cells were washed with dmem for times, followed by infection with viruses. the cells were covered with the overlaid medium for plaque assays. cell apoptosis was analyzed with annexin v-fitc kit (nanjing keygen biotech. co., ltd). briefly, fitc-conjugated annexin v ( μl/well) and propidium iodide (pi, μl/well) were added to the cells infected by amptreated viruses. then, the obtained mixture was incubated at room temperature for min in the dark prior to fluorescence observation. cell apoptosis was determined on basis of the observation soon after initiating apoptosis. cells translocated the membrane phosphatidylserine (ps) from the inner surface of the plasma membrane to the cell surface. thereafter, ps can be easily detected by staining with a fluorescent conjugate of annexin v (green), a protein that has a high affinity to ps. cellular late apoptosis was determined by cell staining with pi (red). the results were analyzed by fluorescence microscope (nikon, japan) at hpi. in vivo prv challenge assay the to week-old specific-pathogen-free balb/c mice were purchased from the experimental animal center of zhongnan hospital of wuhan university (china) and randomly divided into six groups consisting of mice each. individuals in each group of mice were anesthetized with - % isoflurane gas and challenged by the intra-footpad injection with μl of dmem containing × tcid of prv-ea in the presence or absence of , , , . μg/ml piscidin. after days, the surviving mice were challenged with × tcid of prv again. mice viability and behaviors were monitored on a daily basis. in the first days after the challenge, the mice commonly did not exhibit severe symptoms. however, mice gradually showed neurological symptoms in the subsequent days during the monitoring period and the post-challenged mice were monitored since day post prv infection once every - h for days to obtain the survival information of the mice. the level of prv infection symptoms was scored for every mouse by the following system: = posture normal, absence of neurological symptoms, = mild neurological symptoms: excitation, unrest, occasional itching, and foot swollen; = severe neurological symptoms: ataxia, severe pruritus, and self-mutilation, biting and bleeding of the footpad. mice were euthanized in the chamber with co gradually filled when the score reached . on day , three mice from control group and noncontrol groups were euthanized and brain tissues were collected and transferred to % paraformaldehyde. the tissues were dehydrated in ascending grades of ethyl alcohol i.e. , and % ethanol. afterwards, the tissues were cleared with xylene. slides were stained with haematoxylin and eosin (h&e) stain and analyzed through a light microscope. all of the animal experimental protocols were approved by the ethics committee of huazhong agricultural university according to hubei province laboratory animal management regulations (hzaumo - ). during the experiments, mice were offered ad libitum access to water and food in a controlled environment of a h light/dark cycle. all efforts have been made to reduce suffering of animals. the antiviral effects of the peptides (maculatin, caerin, piscidin, lactoferricin b, indolicidin) were investigated in vitro against several viral pathogens that severely threaten the porcine industry. four enveloped viruses were used in this assay including the prv ea strain, pedv ynkm strain, tgev wh- strain, and prrsv ya isolate, as well as one non-enveloped rv tm-a strain. the peptides exhibited different inhibitory activities against these viruses (fig. ) . the relative potency of the peptides against prv was: piscidin≈caerin>ma-cualtin>indolicidin> lactoferricin b. piscidin and caerin showed strong virucidal activity with the residual infectivity being around . % while indolicidin and lactoferricin b exhibited mild antiviral activity. in descending order of potency, the peptides against pedv ranked as follows: caerin>piscidin> maculatin>lactoferricin b > indolicidin. caerin had the most potent activity with the residual infectivity being . %. the assay results indicated, that the order of potency versus prrsv was: caerin>lactoferricin b > piscidin>maculatin>indolicidin. caerin exhibited the best inhibitory activity with the residual infectivity being . %, separately. the assay results of antivirus activity against tgev indicated the activity order as follows: piscidin>lactoferricin b > indolicidin>maculatin>caerin. piscidin was the most potent peptide versus tgev with the residual infectivity being . %. the assay result also indicated that prrsv and tgev were less sensitive to the tested peptides, compared with other viruses (fig. ) . when it comes to rv, the relative potency order was: piscidin>caerin>maculatin>lactoferricin b > indolicidin. piscidin displayed the most potent activity against rv with residual infectivity being . %. piscidin and caerin exhibited activity against almost all the viruses with the residual infectivity being lower than % with exception of piscidin versus prrsv and caerin versus tgev. caerin showed the greatest inhibitory activity against pedv with the residual infectivity being . %, which was the lowest value among all the viruses tested. maculatin exhibited strong activity against prv ( . %) and mild activity against rv ( . %). however, it showed limited activity against pedv, prrsv, and tgev. lactoferricin b and indolicidin were not as potent as other peptides to fight against viruses. lactoferricin b showed stronger inhibitory activity against tgev than against other tested viruses with residual infectivity being . %, and residual infectivity of indolicidin versus prv was . %. we examined the effects of amps on cell viability after incubating pk- cells with different concentrations of each peptide. cytotoxicity was evaluated using mtt method (fig. ) . the non-linear regression analysis result indicated that the % toxic concentration (tc ) of caerin, maculatin, piscidin, and indolicidin were . , . , . , and . μg/ml, respectively. indolicidin was fig. spectrum of antiviral activity of the peptides (tcid assay). peptides ( μg/ml) and viruses were incubated at °c for h before they were added to the target cell monolayers. after incubation for - h, tcid of the virus was recorded. the bars represent ± se. three separate assays were conducted (n = ). the dashed line means % residual infectivity fig. cytotoxic properties of the peptides. pk- cell monolayers were incubated with peptides. the cytotoxicity was measured by mtt assay (n = ). the cell survival rates at different peptide concentrations were plotted and the dashed line means % cell survival less cytotoxic than piscidin, caerin and maculatin. lactoferricin b exhibited the weakest cytotoxic activity and it did not exhibit obvious cytotoxic activity even at maximum concentration of μg/ml. to determine whether the inhibitory activity against prv by maculatin, caerin, piscidin was strain-specific or not, we tested the activity of these peptides against different prv isolates including prv-hnxx (a newly prv isolate in china in ) and prv- (modified bartha strain). we found that all the three peptides displayed inhibitory activities against all the tested strains (prv hnxx, prv , and prv-ea) (fig. a) . this finding indicated that the activity of the peptides versus prv was not strain-specific. when the concentration of the peptides increased from to μg/ml, the residual infectivity of prv decreased (fig. b) . this trend was the most obvious for maculatin with the residual infectivity decreasing from . to . %, while the groups treated with piscidin and caerin exhibited a decrease by more than folds in the residual infectivity. these data indicated that these peptides inhibited the virus in a dose-dependent manner. in order to better understand the inhibitory effect of amps on the propagation of prv-ea, we examined a b fig. prv inhibitory activity displayed by maculatin, piscidin and caerin (tcid assay). a prv of ea, hnxx strain and isolate were treated with the peptides ( μg/ml) for h before they were added to the cell monolayers. the bars represent means ± standard errors of the means of three separate experiments (n = ). *, statistically significant difference by one-way anova with tukey post hoc test (p < . ), ns means not significant. b prv ea was treated with peptides ( μg/ml) for h before added to the cell monolayers. the effects of the peptides at μg/ml and at μg/ml against prv ea were compared whether amps directly damaged virus particles or indirectly interacted with the host cells pre-or post-infection. plaque-reduction assay was also performed to confirm the peptides' antiviral activity. the viruses were incubated with the peptides prior to infection. the result showed that all the three peptides inhibited the virus infection in a dose-dependent manner (fig. ) . at the concentration of μg/ml, pisicidin, caerin and maculatin were found to inhibit most of the prv particles. pisicidin exhibited the strongest activity and obvious inhibitory ability at concentrations above μg/ml. the % effective concentration (ec ) values of the peptides were calculated using probit analysis by ibm spss (version , new york, usa). the analysis revealed that ec values of caerin and maculatin were . μg/ml and . μg/ml, respectively. ec of piscidin was the lowest ( . μg/ml) among the peptides tested, which indicated that piscidin was the most potent antiviral peptide against prv in this study. subsequently, the experiments were conducted to further determine whether the peptides could function at post-(curative) or pre-infection (prophylactic) stage. the cell monolayers were treated with piscidin, caerin, and maculatin before (prophylactic) or after (curative) the virus binding stage, separately. no inhibitory effect of the three peptides on prv could be detected (the obtained data not shown). this implied that these peptides probably inhibited prv infection by directly interacting with the virus particles. the cell apoptosis assay showed that prv could induce both early and late apoptosis of pk- cells (fig. ) . the late apoptosis rate of cells infected by the peptides-treated viruses decreased as the concentration of the peptide increased (fig. b) . the inhibition of late cellular apoptosis was obvious at the concentration of μg/ml (fig. a) . in the control group of the in vivo assay, mice (n = ) were injected with μl of piscidin ( μg/ml) by the intra-footpad route. all mice survived and behaved normally. prv was injected into mice with or without piscidin at various dosages ( . , . , , μg/ml). and the surviving mice were re-challenged with prv on day (fig. ) . the mice survival rate was recorded for days. protected against prv infection (fig. ) . however, one mouse from the . μg/ml peptide-treated group and nine mice from the . μg/ml group died within days. to further characterize the prevention effect of piscidin against brain damage induced by prv, the brain sections of mice from the control group, prv-infected, and co-injection group were collected respectively and subjected to pathological examination. the blood stasis was observed in the brain of mice from prv-infected group (fig. a) . the brain of mice from co-injection group showed no specific symptom. after days, the surviving mice were re-challenged with prv at × tcid . the groups co-injected with piscidin and prv failed to provide protection with the survival rates dropping to % at the end of the experiment and the mice in the μg/ml peptide treated group all died. we previously reported the proof-of-concept usage of amps as the bactericidal agents against pathogenic bacteria isolated from pig herds [ ] . the five antimicrobial peptides used in this study have been reported to inhibit the growth of various kinds of viruses including hiv, hsv- , hsv- , etc. [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however there has been limited information about the inhibitory activity of these peptides against the swine-origin viruses. considering this, this study evaluated the effect of these peptides against several porcine viral pathogens. three peptides (caerin, piscidin, maculatin) exhibited inhibitory activity against prv, pedv, tgev, prrsv, and rotavirus. plaque reduction assay showed that the prv infection could be inhibited in a dose-dependent manner by direct treatment of the peptides. a previous study indicated that caerin and maculatin could inhibit hiv infection without affecting t-cell viability and that the activity of caerin was more potent than that of maculatin [ ] . in our research, we further tested the activity of caerin and maculatin against prv, pedv and prrsv. compared with maculatin, caerin showed better activity against most of the tested viruses except tgev (fig. ) . the fact that caerin has a longer α-helix than maculatin, which enables caerin to disintegrate the membrane more potently, might explain the better activity of caerin. usually, the bacterial membrane and viral envelope were reported to be the main targets of the amps. for example, hiv envelope integrity was directly disrupted by caerin and maculatin [ ] . however in this study, tgev and prv which both acquired their envelopes from pk- , exhibited different levels of sensitivity to the tested peptides. in addtion to its envelope, physical characteristics of the virus such as viral size, surface area, and morphology might affect its sensitivity. hnp- also demonstrated a -fold difference in activity against different enveloped viruses [ ] . it should also be noted that the antiviral activity of the amps tested in this study is only observed at concentrations that are already somewhat cytotoxic at h and could be even more so at h or longer. to our surprise, piscidin, caerin, and maculatin a b fig. piscidin protects mice from prv-induced death. a. processed brain sections from control, co-treated, and prv infected groups were subjected to haematoxylin and eosin (h&e). the representative h&e results were shown. b. the mice injected with piscidin or prv, co-treated with piscidin and prv were divided into different groups. surviving mice were challenged with prv on day again. survival status of control and experiment groups were monitored on a daily basis for days (n = ). in the group of piscidin ( μg/ml), mice were heavily infected and died on day and . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( μg/ml) + prv, mice were heavily infected and died on day . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( μg/ml) + prv, mouse was heavily infected and died on day . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( μg/ml) + prv, mice were heavily infected and died on day and . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( . μg/ml) + prv, mice were heavily infected and died on day . and another mice with severe neurological symptoms were euthanized respectively for animal welfare reasons; in the group of piscidin ( . μg/ml) + prv, mice were heavily infected and died on day and . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; a total of mice from all groups survived and were euthanized by the end of the experiment could consistently inhibit the growth of rv, a non-enveloped rna virus. among these three peptides, piscidin showed the strongest inhibitory activity. it has been reported that piscidin possessed significant antiviral activity to fight against both frog virus and channel catfish virus [ ] . the reduction of fv infectivity resulting from the application of piscidin is due to its capacity to interact with the essential lipid membrane of fv [ ] . based on these results, it could be speculated that there might be other targets for these peptides in addition to the viral envelope, which need further exploration. lactoferricin b and indolicidin showed weak or no activity against the five tested viruses compared with caerin, maculatin, and piscidin. robinson et al reported that the ic of indolicidin against hiv- ranged from μg/ml to μg/ml [ ] . the weak activity of indolicidin against the tested viruses in this study was probably due to the low concentration we used ( μg/ml). it has been reported that amps could inhibit both extracellular and intracellular viruses [ ] [ ] [ ] . our plaque reduction assay result indicated that piscidin, caerin, and maculatin could inhibit prv by directly interacting with the virus. addition of the peptides pre-or post-infection could not help inhibit the prv infection. vancompernolle et al reported that caerin inhibited hiv infection by disrupting the virion envelope [ ] . chinchar et al also reported that amphibian-origin esculentin- p (e p) and ranatuerin- p (r p) could inactivate the channel catfish virus (ccv) by directly interacting with the virions [ ] . the re-emergence of pseudorabies in china since has caused a huge economic loss to the pig farms. three prv strains, i.e. ea, and hnxx, were chosen for further evaluation of peptide's antiviral activity. the peptides inhibited replication of the three prv strains. this is of great importance in fighting against the mutant viruses resulting from natural selection or antibodyinduced events. on the other hand, these peptides had some cytotoxicity on pk- cells (fig. ) . piscidin has been reported to have antiviral and antibacterial properties in vitro [ , ] . however, this had not been tested in vivo. in this study, the data showed that coinjection of prv with piscidin at the concentration of above μg/ml could offer protection against prv infection. even when the concentration of piscidin decreased to . μg/ml, the survival rate of mice could reach %. huang et al previously reported that th - , as an amp isolated from tilapia, could offer % protection against jev infection at μg/ml [ ] . compared with th - , piscidin was more effective even at low concentration. as huang et al noted, th - treated mice surviving from the first jev infection remained alive after second challenge with jev at day [ ] . however, our in vivo studies exhibited a different result. the possible reason needs further exploration. this study indicated that piscidin, maculatin and caerin could inhibit the infection of prv, prrsv, pedv, tgev and rotavirus. among the peptides examined in this study, piscidin showed the strongest antiviral activity against prv both in vitro and in vivo. and it could block the prv-induced cell apoptosis as well. abbreviations amp: antimicrobial peptide; prv: pseudorabies virus; tcid : tissue culture infective dose assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction s domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen pathogenesis and transmission of swine-origin a(h n ) influenza virus in ferrets new variants of porcine epidemic diarrhea virus, china antimicrobial peptides: do they have a future as therapeutics? in: antimicrobial peptides defensins: antimicrobial peptides of innate immunity antimicrobial peptides: promising alternatives in the post feeding antibiotic era the immunology of host defence peptides: beyond antimicrobial activity inhibition of hiv infection by caerin antimicrobial peptides the chemistry and biological activities of peptides from amphibian skin secretions generation of a dual-target, safe, inexpensive microbicide that protects against hiv- and hsv- disease a review of the design and modification of lactoferricins and their derivatives defensins at the mucosal surface: latest insights into defensin-virus interactions generation and characterization of ul null pseudorabies virus variant in vitro and in vivo role of the pseudorabies virus gi cytoplasmic domain in neuroinvasion, virulence, and posttranslational n-linked glycosylation vaccines against pseudorabies virus (prv) caerin . suppresses the growth of porcine epidemic diarrhea virus in vitro via direct binding to the virus the use of the antimicrobial peptide piscidin (pcd)- as a novel anti-nociceptive agent single amino acid substitutions at specific positions of the heptad repeat sequence of piscidin- yielded novel analogs that show low cytotoxicity as well as in vitro and in vivo anti-endotoxin activity broad activity against porcine bacterial pathogens displayed by two insect antimicrobial peptides moricin and cecropin b a crispr/cas and cre/lox system-based express vaccine development strategy against re-emerging pseudorabies virus establishment and clinical application of a multiplex reverse transcription pcr for detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine group a rotavirus porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonbeta production by interfering with the rig-i signaling pathway porcine epidemic diarrhea virus orf gene prolongs s-phase, facilitates formation of vesicles and promotes the proliferation of attenuated pedv the ubiquitin-proteasome system is required for the early stages of porcine circovirus type replication antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus inactivation of viruses infecting ectothermic animals by amphibian and piscine antimicrobial peptides anti-hiv- activity of indolicidin, an antimicrobial peptide from neutrophils antiviral peptides targeting the west nile virus envelope protein novel influenza virus ns antagonists block replication and restore innate immune function fusion core structure of the severe acute respiratory syndrome coronavirus (sars-cov): in search of potent sars-cov entry inhibitors inactivation of frog virus and channel catfish virus by esculentin- p and ranatuerin- p, two antimicrobial peptides isolated from frog skin piscidin: antimicrobial peptide of rock bream modulation of the immune-related gene responses to protect mice against japanese encephalitis virus using the antimicrobial peptide, tilapia hepcidin - publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. availability of data and materials data and materials are available upon request by the corresponding author.ethics approval and consent to participate all of the animal experimental protocols were reviewed and approved by the ethics committee of huazhong agricultural university. not applicable. the authors declare that they have no competing interests. key: cord- -giakcaki authors: xu, wan-xiang; wang, jian; tang, hai-ping; chen, ling-han; lian, wen-bo; zhan, jian-min; gupta, satish k.; ji, chao-neng; gu, shao-hua; xie, yi title: a simpler and more cost-effective peptide biosynthetic method using the truncated gst as carrier for epitope mapping date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: giakcaki there is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. here, we describe improved biosynthetic peptide (bsp) method using a newly developed plasmid pxxgst- as vector, which has a viral e gene in the cloning sites of pxxgst- . it is crucial to employ pxxgst- instead of pxxgst- , since it makes use of the bsp method simpler and easier to perform, and more cost-effective for epitope mapping. these merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of bp inserted between bamh i and sal i sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing / mer peptides by running polyacrylamide gel electrophoresis. the protocol involves the following core steps: (i) design of plus and minus strands of dna fragments encoding overlapping / mer peptides; (ii) chemical synthesis of the designed dna fragments; (iii) development of r-clones using pxxgst- vector expressing each / mer peptide fused with truncated gst protein; (iv) screening r-clones by running the cell pellets from each induced clone on sds-page gel followed by sequencing of inserted dna fragments for each verified r-clone; and (v) western blotting with either monoclonal antibodies or polyclonal antibodies. this improved gst -bsp method provides a powerful alternative tool for epitope mapping. for rational vaccine design or development of diagnostics, it is imperative to map all functionally relevant, specific and/or conserved linear (continuous) b cell epitopes (bce) of target proteins. several methods have been described to define linear bces, which can be broadly classified into four categories: i) chemically synthetic peptide (csp) and its chip method [ ] [ ] [ ] [ ] [ ] ii) biosynthetic peptide (bsp) by gene fragment expression, phage display random-peptide or antigen-fragment libraries and expression-pcr technique, etc [ ] [ ] [ ] [ ] [ ] [ ] ; iii) epitope prediction based on computational tools relating to several physical or chemical parameters of a given protein; and iv) sequencing of proteolytic fragments applying enzymatic and chemical reagents. however, performance of these methods, including their numerous improved or derivative methods, is far from ideal because of the various limitations. in particular, for classical csp method commonly used, it is not easy to identify antibody-recognizing bce minimal motifs even when using monoclonal antibodies (mabs) in most cases, or impossible to reveal igg-epitome on a target antigen using polyclonal antibodies (pabs) that is the basis of finding all desired functional bces. recently, many research groups tried to use bsp method similar to csp method for antibody identification or bce mapping of antigenic segments of interest, on which several genes encoding core streptavidin (stv), glutathione s-transferase (gst), and β-galactosidase (gal) were used as carriers of bsp [ ] [ ] [ ] [ ] [ ] [ ] . these explorations, including our construction of minimum short fragment encoding amino acids (aa) fused with stv gene [ ] and finding of weak antigenic range (~ to kda) in bacterial proteins (fig a and b) , resulted finally in the development of original bsp method capable of arbitrarily expressing overlapping / mer peptides. it employed a truncated gst gene encoding residues (gst ) as carrier in pxxgst- ( fig c) [ ] , and the subsequent improvement of applying the enhanced chemiluminescence (ecl) reagents in western blotting for bce mapping [ ] . although the method has been used to reveal igg-epitomes of three major proteins from human papillomavirus type (hpv ) and in other related studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it still had potential faults that needed to be overcome for convenience of users, which were: i) it is unable to purify the double enzyme-digested pxxgst- product with base pairs (bp), because it is only bp shorter than the bp product generated by inadequate digestion leading to lower efficiency of constructing recombinant (r-) clones; and ii) setting gst control was required to screen the r-clone of expressing gst (gst + mer peptide) protein due to the~ kda of migration rate difference between both proteins, otherwise it is inconvenient to distinguish the~ . kda difference between gst and gst (gst + aa) proteins expressed by r-and self-ligation clones. it also has same trouble for determining self-ligation clone by running sds- % polyacrylamide gel electrophoresis (page), due to the~ . kda difference between the gst and control gst proteins that were expressed by pxxgst- and pxxgst- ( fig d) . it is likely that these clones expressing gst protein could be mistakenly regarded as r-clones and subsequently followed-up for sequencing leading to wasteful expenditure. in our newly improved method (named gst -bsp), these shortcomings have been well addressed through using pxxgst- ( fig e) , which can make it more convenient and efficient for cloning and screening r-clones of overlapping / mer peptides, because of the strategy to insert a longer dna fragment between both target cloning sites of pxxgst- . obviously, the bp in length of selected e gene is sufficient to distinguish two different enzyme-cut products of bp and bp, and the~ . kda of e protein composed of aa can make the gst -e fusion protein out of the to kda weak antigenic area, and thus no longer require any protein control for screening r-clones of / mer peptide due to non-existence of any nonspecific band like gst protein within the area of cell proteins from a self-ligation clone of pxxgst- . the gst -bsp method presented here not only keeps characteristics and virtues of original bsp approach but also is simpler and easier to perform, and more costeffective. here, we describe the method and its standardized protocol for general laboratory application. our data suggest that the gst -bsp method reported herein is an excellent alternative tool for bce/epitome mapping and identification of mab-recognizing minimal motif. the preparation of rabbit antisera to r-huzp c - segment was conducted in accordance with the guidelines for the care and use of laboratory animals, and was approved by the ethics committee of shanghai institute of planned parenthood research (sippr). the procedures were in accordance with guidelines established by the ethics committee of sippr. ethical approval for the unpublished research project no. bai b was granted by the ethical committee of the sippr ( - ). three male new zealand white rabbits purchased from sippr-bk lab animal co., ltd. (shanghai, china) were caged at controlled temperature of ˚c. all the performed experiments were in full compliance with standard laboratory animal care protocols approved by the institutional animal care committee of sippr. these rabbits were immunized intramuscularly with mg of purified e. coli-expressed recombinant human zona pellucida glycoprotein- corresponding to amino acid residues from to (r-huzp c - ) emulsified in . ml of complete freund's adjuvant and . ml of pbs at multiple sites on rabbit's back. three booster immunizations of . mg r-huzp c - protein in incomplete freund's adjuvant were administered at -week intervals. sera were collected from each rabbit seven days after each vaccination and stored at - ˚c. the thermo-inducible plasmids of pxxgst- and pxxgst- were constructed as described earlier [ ] . the plasmid pxxgst- was constructed by our group, which had a hpv -e gene (genbank no: x ) inserted between bamh i and sal i sites in pxxgst- , but failed to express gst -e fusion protein in the various prokaryotic expression systems used by us. the rabbit antisera raised against r-huzp b - [ ] and r-huzp c - segments were prepared previously, the mab c p [ ] (american research products inc., usa), and goat anti-rabbit/mouse igg conjugated to horseradish peroxidase (hrp) (proteintech group, usa) were purchased from shanghai sangon co., china. all gene fragments encoding short peptides of interest were synthesized by the sbs genetech co., ltd, shanghai. dna sequencing of inserts in each r-clone was performed by shanghai generay biotechnology co., ltd, china. the molecular cloning of synthesized dna fragments was done as described previously [ ] , which has been briefly described in the 'protocol' as described later in this section. step one: designing series of overlapping short peptides. design all / mer peptides with an overlap of aa residues covering the full-length of the target protein or a longer segment sequence for the first round of antigenic peptide mapping, as well as design a set of mer peptides with overlapping aa residues and covering each reactive / mer peptide sequence for the second round of precise bce motif identification. note: it is recommended to design overlapping mer peptides, as the cost for synthesizing oligonucleotide of less than nt in length is lower than that of oligonucleotide of more than nt. also, when using a mab or cross-reactive mab to map bce, it is desirable to express two or several longer segments of the target protein so as to determine its shorter antibody reactive segment followed by designing overlapping / mer peptides. doing so can save the cost, time and workload in following steps - . step two: designing oligonucleotides of plus and minus strands. design all plus and minus strands of dna fragments encoding each designed / mer peptides of the target protein, having cohesive ends for bamh i and sal i restriction enzymes at their 'and ' ends, so that they could be directly used to insert into the bamh i and sal i sites downstream of the gst gene in plasmid pxxgst- after their annealing in pairs. also, a taa termination codon is designed between each / mer peptide and sal i site, so as to avoid possible false blotted bands of / mer peptides for bce and minimal motif mapping, which will be caused by four gsvd residues encoded by bp present in cloning site region of pxxgst- ( fig e) . in short, the designed plus and minus strands of encoding fragment should include sequences of '-gatcc and taag- ', as well as '-tcgactta and g- ' at their both ends (fig a and b ). as mentioned in step one, it is just close to nt that is oligonucleotide corresponding to nt encoding mer peptide together with nt at their both ends. note: it is emphasized that design of the target gene fragments at this step is very convenient, that is, the consideration of the optimal codon usage for expression of each short peptide fused with gst in e. coli is not essential as more than of the constructed / mer peptide fusion proteins have been successfully expressed by using their dna fragments from known prokaryotic and eukaryotic genes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, it should be ascertained that whether there is another potential bamh i or sal i site within the oligonucleotide sequence, if any, it needs to be modified because it will disturb annealing of the paired oligonucleotides and its subsequent cloning. step three: chemical synthesis of the designed dna fragments. once the plus and minus stands of encoding gene fragments have been designed, they can be synthesized and page-purified by the sbs genetech co., ltd, shanghai. all page-purified dna fragments should be stored at - ˚c prior to use. step four: conduct annealing reactions of page-purified dna fragments in pairs. . to make μm stock solution of each synthesized dna fragment by adding appropriate volume of double distilled h o (ddh o) into tubes based on the report provided by the biotechnical service company, respectively. . take respectively μl of the stock solution from two tubes in pairs into a . ml of new tube, and add μl of ddh o to make μm final concentration of each dna fragment. . heat tubes up to ˚c in an electric heating block, anneal for min, and then allowed them to cool gradually to room temperature (rt). step five: development of r-clones for expressing each / mer peptide. . perform the ligation reactions using above each annealed dna fragment and constructed vector of pxxgst- digested with bamh i and sal i enzymes, and then purified using qiaquick gel extraction kit after running . % agarose gel electrophoresis at v for min. the ligation reaction was carried out at ˚c overnight using the following μl of reaction mixture in . ml tube (table ) , in which all components were mixed by flicking and spin briefly (refrigerated mikro centrifuge). . employ the above ligation mixture in each tube to transform the e. coli bl (de ) competent cells according to the standard procedures [ ] . grow overnight each clone on solid luria broth (lb)-ampicillin (amp) plates at ˚c, respectively. it should be observed that there are about to clones on the lb plates next morning. note: if there was no growth of clones on a lb-amp plate after repeating twice, it suggests that they were incorrect or wrongly paired dna fragments and need to be resynthesized. . grow overnight each clone from lb-amp plates in ml of liquid lb-amp medium at ˚c, subsequently dilute : in fresh lb-amp medium and were grown at ˚c at rpm to an od of . - . . the clones were induced at ˚c for - h. harvest each cell pellets by centrifugation at x g for min at ˚c. cell pellets obtained from ml culture of expressed / mer peptide fusion proteins were boiled in μl of x sample loading buffer ( mm tris-hcl, ph . , % sds, . % bromophenol blue, % glycerol and mm β-mercaptoethanol) for min. peptide biosynthetic method for epitope/epitome mapping step six: screening of r-clones by running sds-page. take~ μl of each cell suspension from prepared individual thermo-induced bacterial cultures to run sds- % polyacrylamide gel for screening r-clones, and cell proteins resolved by conducting electrophoreses at v for h. the gels were stained with coomassie brilliant blue g for analyzing the bands of fusion proteins. note: as shown in fig , all r-clones can be easily determined by observing difference between gst fusion proteins expressed in lanes - and control gst protein expressed by pxxgst- in lane . approximately kda difference can be observed even when expressing overlapping mer peptide fusion proteins as compared to gst protein. however, as shown in lane , there was no specific / mer-peptide fusion protein band appeared as compared to lanes - when self-ligation of pxxgst- happened due to inadequate enzyme digestion, suggesting that it no longer needs any additional control (lane or ) on the gels for the / mer peptides expressed specifically in r-clones when using pxxgst- vector in the improved gst -bsp method. step seven: sequencing of insert for each determined r-clone. conduct sequencing of all synthesized dna fragments inserted into plasmid pxxgst- from r-clones verified by above sds-page analysis through the shanghai biosune biotechnology co., ltd, china. it is recommended to use the primer '-ggccatcatacgttatatag- '. note: for ensuring that all amino acid sequences of expressed short peptides are accurate, it is important to check the correctness of each synthesized dna fragments by sequencing after determining r-clones and before conducting bce and minimal motif mapping (fig c) , because this step can avoid possible error in bce mapping due to possible bp missense mutation or tubes numbering errors happened when synthesizing a large number of dna fragments. therefore, elimination of deleterious errors becomes necessary in many cases. of course, for saving cost and time if needed, it might be carried out after finishing two rounds of bce and fine motif mapping to check those dna fragments encoding reactive / mer peptides. step eight: determination of bce recognized by mabs/pabs in western blot. proteins on sds-page gel were electrotransferred onto . μm nitrocellulose membrane, on which complete transfer was ensured by staining membrane with . % ponceau s. non-specific antibody-binding sites on the membrane were blocked with blocking phosphate-buffered saline (pbs) containing . % tween and % skim milk powder or preimmune rabbit serum sample overnight at ˚c, and probed with antisera raised against r-huzp c that was pre-adsorbed with host cell lysates or mab c p ( : or : dilution in pbs) for h at rt. following washing four times, specific bands on the membrane were visualized by using goat anti-rabbit igg or goat anti-mouse igg conjugated with hrp at : dilution. the blot was developed by using the ecl plus western blotting detection reagents. peptide biosynthetic method for epitope/epitome mapping note: to produce clear blotted bands in western blotting, it is suggested that before use, the antiserum against r-protein may be absorbed on host cell lysates as described earlier [ ] , which facilitate in removal of cross-reactive antibodies to e. coli proteins. in order to know how many bces are there among three neighboring reactive mer-peptides of p - (fig a and b) , which were blotted in first round of antigenic peptide mapping in our study on epitome decoding of huzp protein, we first expressed a set of mer-peptides (p - ) covering the full-length sequence of reactive p according to procedures of steps to in the protocol, and then they were subjected to sds-page, and transferred onto . μm nitrocellulose membrane. the western blotting was performed by standard method with : -diluted rabbit antisera to r-huzp c. specific antigen-antibody reactions on the membrane were visualized by applying goat anti-rabbit igg conjugated to hrp and ecl plus western blotting detection kit with higher sensitivity according to the manufacturer's instructions. as shown in fig c and d , a precise bce motif (pltlelq) was defined in reactive p according to the common sequence highlighted in yellow color within reactive p - peptides. the mapped motif was in good agreement with the pltlel motif of huzp that was recognized by rabbit anti-native porcine zp igg in another previous study [ ] , which only had a residue q at its c-terminus more than that of the latter, suggesting that rabbit immune system could recognize the common antigenic site shared among homologous proteins of pig and human, no matter whether they were against native protein or not. obviously, it is impossible to identify a bce among these reactive peptides (p - ) using rabbit antisera to r-huzp c, if not applying the gst -bsp method. by the way, other two bce motifs in reactive p and p have also been identified using a set of overlapping mer peptide of p in completed study on epitome decoding of huzp glycoprotein, which one (dknygsy - ) was present in the aa overlapping region of p -p and another (yygvgdyp - ) at the c-terminus of p (fig ) . there is a need to identify the fine bce motif recognized by a non-conformational mab, and the present gst -bsp method is a powerful and economic technique for such purpose. to save cost and workload, express two or more bigger segments of a large protein at first so as to determine a reactive segment, and then map bce motif using / mer peptides for a specific mab. for a known cross-reactive mab; however, another strategy may be adopted to find one or several potential segments of % conservation or with a residue difference among homologous proteins through sequence alignment. the following is a practical example for the latter case. the fine bce e - motif (ygd/xtl) of hpv -e was found to be highly conserved among most known high risk-hpvs (hr-hpvs) including hpv and hpv in our previous study [ ] , and the commercially available mab c p of hpv -e can cross-react with hpv -e protein [ ] . therefore, in order to know whether the mab also could recognize the e - epitope, the western blotting tests were performed using our previously expressed p octapeptide containing ygdtl motif and r-e protein of hpv . the mab did not react with p , but recognized r-protein of hpv -e . thus, another highly conserved site (el/ yrhy) with only a residue difference among them was found via sequence alignment of e proteins from hpv , and (fig a) . based on bce minimum motif of three aa [ , ] , thus, a panel of dna inserts encoding mer to mer peptides (the addition of three aaa residues at their n-terminus is only for the convenience of constructing r-clones) were designed, synthesized, annealed, and cloned into bamh i and sal i sites within pxxgst- , resulting in five r-vectors. after constructing and expressing each r-clone, cell pellets from induced r-clones were used to perform western blotting with mab c p . finally, the bce fine motif (elrhy) of hpv -e and a cross-reactive pentapeptide (eyrhy) among several homologous proteins were characterized, which the consensus pentamer sequence el/yrhy was based on blotted results of p and p (fig b and c ). meanwhile, it was yet found from the mapping result that besides hpv -e , the cross-reactive mab c p can recognize hpv -e as well as oncogenic hpv -and -e proteins that were according to subsequent eyrhy alignment of homologous e proteins (fig a) , suggesting the importance of identifying fine bce motif for a cross-reactive nonconformational mab. in shanghai, people's republic of china, the cost of oligonucleotide synthesis is currently about usd . /nt (rmb yuan . /nt) for oligonucleotides of length less than nt ( nt for encoding mer peptide that included nt of cohesive sequence at their both ends), and usd . (rmb yuan . ) per base for length more than nt ( nt for encoding mer peptide). enzymes (dna ligase, bamh i and sal i), molecular weight markers and other reagents until got a sequence-confirmed r-clone, which is far below present market price (usd~ . to . or rmb yuan~ . to . per residue in shanghai sangon co., china and the biomatik co., usa) of / mer csp with > % purity used in bce mapping. the cost-effectiveness of the gst -bsp method is obvious when conducting bce fine motif or epitome mapping to a macromolecular protein. most importantly, it can obtain more definitive and trustworthy results of bce mapping by applying such bsps than csps [ ] , and these r-plasmids expressing / mer bsps can also be stored at - ˚c for future possible use. most of the methods pertaining to mapping of bces have employed csp strategy [ ] , but it failed to reveal epitome of a target antigen using this strategy due to the limitation of being unable to use pabs to map a bce fine motif and/or more within several contiguous reactive peptides as shown in fig that resulted in a limited number of mapped bces [ ] [ ] [ ] . for example, not including a bce present in the c-terminus transmembrane-like domain, only three bces on mature huzp were identified by rabbit pabs in elisa assay when using a panel of overlapping mer csps covering huzp sequence [ ] , whereas it was shown by our group that there are at least seven bces present in the protein by western blotting with rabbit pabs and sets of mer bsps [ ] . for non-conformational mabs, there are several reports on using sets of overlapping / mer csps or mer peptide library to identify bce minimal motifs [ ] [ ] [ ] . due to limited availability of / mer peptides synthesized on polypropylene pins and the high cost to purchase csps of high-purity, this method has limited applications. further, most of mapped mer antigenic peptides were inadvertently referred as "epitopes" [ ] [ ] [ ] [ ] , even being mer peptide [ ] , although it is well recognized that smaller number of the aa residues are needed for binding to the antibody [ ] . it is likely that within a mapped / mer peptide there may be two and more bces [ , , [ ] [ ] [ ] . moreover, the mab-recognizing fine motif has not yet been stipulated as a needful criterion or parameter for manufacturing a mab(s) as drug, although all know it is one of the major features to distinguish one mab from another, and could broaden application range of a mab. for instance, the commercially available mab c p raised against hpv -e protein can be used to identify hpv / / -e as well, besides known hpv -e according to the mapping results in fig . in early explorations of bsp, the stv (expression plasmid ptsa ), gst (pgex- p) and gal (pwr ) proteins were used as carriers to express / mer peptides for bce mapping or antibody identification [ ] [ ] [ ] [ ] [ ] [ ] . these endeavors suggested the feasibility to develop bsp approach in addition to csp strategy. however, these efforts failed to show the merits of bsp approach leading to a standard protocol suitable for general laboratory application. major drawbacks of applied bsps in above studies were: i) expressed mer peptide fusion proteins need to be purified before western blotting even when using chicken sera to sars-cov and sars convalescent sera [ ] [ ] [ ] , with this operation step it obviously increases the research cost and work load, and takes much longer time; ii) it is inconvenient to select each r-clone of mer bsps before sequencing of insert, which needs to conduct the double enzyme restriction to identify insert, and iii) the position of expressed short peptides fused with gst or stv was not in the widest weak antigenic range of bacterial proteins, and thus was unsuitable for bce mapping with pabs, etc. the present gst -bsp method as described has fully embodied practical approach. the outstanding advantages of gst -bsp method are as follows: i) using the truncated gst as carrier, it makes the expressed / mer peptides fusion proteins in the weak antigenic area of bacterial proteins, and thus permits directly using them to map each bce fine motif and epitome of target protein; ii) employing pxxgst- enables users to extract double enzymecut pxxgst- by running agarose gel electrophoresis, and thus greatly reduce the self-ligation of pxxgst- via removing products produced by inadequate digestion that may happened sometimes; iii) screening r-clones on the sds-page gels no longer needs any control, because as shown in lane of fig , there are no band similar to gst protein in the to kda area of cell proteins, which will interfere with screening r-clones of mer peptide in the ~ kda area for self-ligation of pxxgst- ; iv) employing thermo-inducible expression system instead of isopropyl-β-d -thiogalactoside (iptg) induction reduces cost; v) it is less expensive than csp method due to cheaper cost of oligonuleotide synthesis than peptides; vi) it is clearer and more credible for the blotted bands in western blotting due to use of the ecl luminescence reagents than others such as , '-diaminobenzidine (dab) or alkaline phosphatase (ap) kit; vii) it is adaptable for most laboratories due to its relative simplicity. in conclusion, based on the improved gst -bsp method using the pxxgst- vector, we have constructed more than one hundred of r-clones to express / mer and minimum mer peptides (via adding three alanine residues to its n-terminus, fig c) fusion proteins in our ongoing studies of epitome mapping. the epitopes recognized by pabs to r-huzp c and mab cip against hpv -e have been mapped. hence, we believe that the gst -bsp method offers a much simpler option which is more cost-effective, reliable and adaptable for general laboratories. it will facilitate studies on epitope/epitome mapping of numerous target proteins and bce motif identification of non-conformational mabs. use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid general method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids strategies for epitope analysis using peptide synthesis epitope mapping using synthetic biotin-labeled peptides applications of peptide arrays prepared by the spot-technology efficient mapping of protein antigenic determinants vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice mapping of viral epitopes with prokaryotic expression products universal promoter for gene expression without cloning: expression-pcr expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen. application to the human thyrotropin receptor high-resolution mapping of gens and b f epitopes on myelin-associated glycoprotein by expression pcr expression and purification of three fusion proteins containing a single b-cell epitope (β , β or β ) of human chorionic gonadotropin beta subunit immunogenic comparison for two different recombinant chimeric peptides (cp and cp ) containing one or two copies of three linear b cell epitopes from β-hcg subunit identification of two antigenic epitopes on sars-cov spike protein expression and antigenic epitopes mapping of receptior binding domain on the spike protein of severe acute respiratory syndrome coronavirus identification and antigenic epitope mapping of immunodominant region amino residues to on the spike protein of the severe acute respiratory syndrome coronavirus a peptide of foot-and-mouth disease virus serotype asia generating a neutralizing antibody response, and an immunostimulatory peptide minimal motif mapping of a known epitope on human zona pellucida protein- using a peptide biosynthesis strategy mapping of minimal motifs of b-cell epitopes on human zona pellucida glycopotein- epitomics: igg-epitome decoding of e , e and l proteins from oncogenic human papillomavirus type mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein- using monoclonal antibodies fine epitope mapping of the central immunodominant region of nucleoprotein from crimean-congo hemorrhagic fever virus (cchfv) fine mapping and conservation analysis of linear b-cell epitopes of peste des petits ruminants virus nucleoprotein antisera preparation and epitope mapping of a recombinant protein comprising three peptide fragments of the cystic fibrosis transmembrane conductance regulator vaccination with an epitope peptide of izumo to induce contraception in female mice immunogenicity of recombinant human zona pellucida- peptides expressed in e. coli and efficacy of their antisera to inhibit in vitro human sperm-egg binding identification of human papillomavirus type e polypeptide in cells derived from human cervical carcinomas molecular cloning: a laboratory manual surface conformational and linear epitopes on hpv- and hpv- l virus-like particles as defined by monoclonal antibodies evaluation of the contraceptive potential of recombinant human zp and human zp peptides in a primate model: their safety and efficacy mapping of dominant b-cell epitopes of a human zona pellucida protein (zp ) mapping of b cell epitopes on the zona pellucida protein of a marsupial, the brushtail possum (trichosurus vulpecula) identification of epitopes of monoclonal antibodies to porcine zona pellucida β glycoprotein, a homologue of the mouse/human sperm receptor identification of b-epitopes in the human papillomavirus e open reading frame protein human papillomavirus type e and e antibodies in human sera: increased anti-e prevalence in cervical cancer patients epitope mapping of antibodies using a cell array-based polypeptide library epitopes on protein antigens: misconceptions and realities papillomavirus-like particle (vlp) vaccine displaying hpv l epitopes induces cross-neutralizing antibodies to hpv a pan-hpv vaccine based on bacteriophage pp vlps displaying broadly cross-neutralizing epitopes from the hpv minor capsid protein, l identification of broad-genotype hpv l neutralization site for pan-hpv vaccine development by a cross-neutralizing antibody writing -original draft: wan-xiang xu, satish k. gupta. wan-xiang xu, satish k. gupta. key: cord- - d mn ok authors: gouveia, duarte; miotello, guylaine; gallais, fabrice; gaillard, jean-charles; debroas, stéphanie; bellanger, laurent; lavigne, jean-philippe; sotto, albert; grenga, lucia; pible, olivier; armengaud, jean title: proteotyping sars-cov- virus from nasopharyngeal swabs: a proof-of-concept focused on a min mass spectrometry window date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: d mn ok rapid but yet sensitive, specific and high-throughput detection of the severe acute respiratory syndrome coronavirus (sars-cov- ) in clinical samples is key to diagnose infected people and to better control the spread of the virus. alternative methodologies to pcr and immunodiagnostic that would not require specific reagents are worth to investigate not only for fighting the covid- pandemic, but also to detect other emergent pathogenic threats. here, we propose the use of tandem mass spectrometry to detect sars-cov- marker peptides in nasopharyngeal swabs. we documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. simili nasopharyngeal swabs spiked with different quantities of purified sars-cov- viral material were used to develop a nanolc-ms/ms acquisition method, which was then successfully applied on covid- clinical samples. we argue that peptides adetqalpqr and gfyaqgsr from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a min window in the tested conditions. these results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry. the new severe acute respiratory syndrome-related coronavirus (sars-cov- ) is the causative agent of covid- , the coronavirus disease that was first reported in december in the city of wuhan, china ( ) . due to its easy inter-human transmission, sars-cov- has since quickly spread worldwide, causing more than million covid- diagnosed infections and more than thousand deaths officially reported as off mid june (https://covid .who.int/). the rapid, sensitive and specific detection of the sars-cov- virus in large cohorts of clinical samples is of utmost importance to identify infected people and control the propagation of the virus by specific containment measures. at the same time, being able to catch the numerous sars-cov- variants represents an opportunity to identify attenuated forms of the virus ( ) . however, the occurrence of specific mutations, especially deletions, may challenge current molecular detection methodologies. the research community has been placing great efforts in the development of quick and accurate detection tests ( , ) . the gold standard in diagnostics relies on the amplification and measurement of the viral rna by reverse transcription polymerase chain reaction (rt-pcr). rt-pcr is highly specific and achieves a good compromise between speed ( - min) and sensitivity. however, due to the great demand for pcr-based testing, shortage of rna extraction kits and pcr reagents may have limited the testing capacity in some countries at the early stage of the pandemic ( ) . besides, rt-pcr testing of clinical samples may be in some case less efficient due to nucleic acid variations in the targeted regions -primers or their close vicinity -that could affect the amplification rate ( , ) . for these reasons, alternative detection strategies that address these concerns should be developed to complement conventional tools. immunoassays, whole-genome sequencing ( ) and mass spectrometry (ms) ( ) technologies are commonly suggested alternatives to pcr-based assays. among these, new generation ms offers a highly sensitive technology that allows the rapid identification of thousands of proteins present in a single sample. the typing of organisms by tandem mass spectrometry (ms/ms), commonly referred to as "proteotyping", is based on the identification of specific peptide sequences that allow the unambiguous identification of organisms ( ) ( ) ( ) . the uniqueness of the mass to charge ratios and fragmentation patterns measured in ms/ms allows identifying peptides that differentiate organisms at the subspecies level. although classical ms-based identification of pathogens in the clinical setting is based on whole-cell maldi-tof technology ( ) , the field has thrived with the increases in speed, sensitivity, and accuracy of new ms instrumentation in the last decade. the coupling of new generation instruments with the separation power of liquid chromatography makes lc-ms/ms a valuable technology to implement in the routine of clinical laboratories. despite their high potential, the application of lc-ms/ms approaches for virus proteotyping is still scarce. among the few examples available in the literature, lc-ms/ms was shown to be able to detect purified influenza virus ( ) and human metapneumovirus in clinical samples ( ) . because of the considerable damages of the covid pandemic, the mass spectrometry community quickly proposed to mobilize its efforts at helping to understand the molecular mechanisms of infection ( ) ( ) ( ) and at improving detection methods ( ) . several research groups started investigating ms-based quantification of peptides for the detection of sars-cov- in clinical samples, but these results are not yet published ( ) ( ) ( ) ( ) . these preliminary results indicate that targeted ms, in which the mass spectrometer is programmed to precisely detect and quantify a limited number of peptides of interest, can be successfully applied to virus detection. targeted ms is considered as the gold standard for peptide quantification due to its higher sensitivity when compared to shotgun proteomics approaches. nevertheless, this approach has a much lower throughput and is commonly used to test hypotheses on a subset of proteins of interest, in contrast to discovery shotgun proteomics. by being more flexible, the latter provides a more comprehensive picture of the viral peptidome, including the detection of variant sequences because of the possibility of detecting peptides without any previous knowledge of their sequences. here we established the proof of concept of the use of ms/ms for the rapid proteotyping of sars-cov- from clinical samples. we recently published a dataset from a shotgun lc-ms/ms experiment performed with sars-cov- infected cells and proposed a list of specific viral peptides that could be used for the development of targeted approaches ( ) . interestingly, we observed that some sars-cov- specific peptides eluted from the lc column at narrow windows of retention time. here, we used lc-ms/ms with an orbitrap instrument (q exactive hf) for analyzing the peptidome from nasal swabs spiked with different quantities of viral material. by using a short lc gradient focusing on the region of interest identified in our previous study, we tested the detection of the virus in samples containing different quantities of viral peptides, as well as covid- clinical samples, paving the way for the development of time-efficient viral diagnostic tests based on an alternative platform. nasopharyngeal swab collection and processing for reference matrices. two nasopharyngeal swabs were collected using a sterile polyester swab with semi-flexible polystyrene handle (puritan) from two healthy volunteers (swabs r and r ). each swab was soaked into a tube containing µl of sterile water, incubated for min at room temperature, and then rinsed with µl of sterile water. the biological material from the µl of solution was precipitated with the addition of µl of trichloroacetic acid at % (w/v) and centrifugation at , g for min. the supernatant was discarded. the hardly visible pellet was dissolved into µl of lds x containing % beta-mercaptoethanol, heated for min at °c, and centrifuged briefly. for each swab sample, a volume of µl of lds x sample was deposited on a sds-page gel and run for min. after migration, the gel was rinsed with water, stained with simply blue safestain (invitrogen), and destained overnight in water. the two polyacrylamide gel bands corresponding to the whole proteome of each matrix were excised, processed as described ( ) , and then subjected to trypsin gold proteolysis (promega) using . % proteasemax surfactant (promega). the nasal matrix peptide fractions were µl for each swab. peptides from the nasal swab matrices were analysed with a q-exactive hf mass spectrometer (thermo) coupled with an ultimate lc system (dionex-lc) and operated in datadependent mode as previously described ( ) . a volume of µl of peptides was injected, desalted onto an acclaim pepmap c pre-column ( µm, Å, µm id x mm), and then resolved onto a nanoscale acclaim pepmap c column ( µm, Å, µm id x cm) with a -min gradient at a flow rate of . µl/min. the gradient was developed from to % of ch cn, . % formic acid in min, and then from to % in min, washed, and re-equilibrated. peptides were analysed with scan cycles initiated by a full scan of peptide ions in the orbitrap analyser, followed by high-energy collisional dissociation and ms/ms scans on the most abundant precursor ions (top method). full scan mass spectra were acquired from m/z to at a resolution of , with internal calibration activated on the m/z . signal. ion selection for ms/ms fragmentation and measurement was performed applying a dynamic exclusion window of sec and an intensity threshold of x . only ions with positive charges + and + were considered. vero e (atcc, clr- ) cells were cultured at °c in % co in dulbecco's modified eagle's medium (dmem, gibco, themofisher) supplemented with % fetal calf serum (fcs) and . % penicillin-streptomycin. the sars-cov- strains -ncov/italy-inmi (genbank mt ) was provided by the lazzaro spallanzani national institute of infectious diseases (rome, italy) via the evag network (european virus archive goes global). sars-cov- stocks used in the experiments had undergone two passages on vero e cells and were stored at - °c. virus titer was . x plaque forming units (pfu)/ml, as determined by standard plaque assay (three dilutions in duplicates). all experiments entailing live sars-cov- were performed in our biosafety level facility and strictly followed its approved standard operating procedures. vero e cells ( x ) seeded into cm flasks were grown to cell confluence in ml dmem supplemented with % fcs and . % penicillin-streptomycin for one night at °c under % co . they were infected at multiplicity of infection (moi) of . . cells were harvested at days post infection (dpi) and viral suspension was recovered after centrifugation at , rpm for min to remove cell debris. ml of the viral suspension were laid on ml of % (w/v) sucrose cushion prepared in nacl . m, edta m m, mm tris hcl buffer (ph . ) (tne buffer) in ultra-clear ml tubes (beckman coulter). samples were centrifuged at , rpm for h at °c. pellets were solubilised in l of cold tne buffer and a volume of . ml was laid on a five step - % (w/v) sucrose gradient prepared in ultra-clear ml tubes (beckman coulter). the tubes were centrifuged at , rpm for h at °c in a beckman sw rotor. after recovery of the virus band, the viral suspension was inactivated by incubation with betapropiolactone at a final concentration of . % for h at °c. plaque assay titration was used to quantify the purified virus and validate the viral inactivation. the inactivated purified virus sample (equivalent to . x pfu/ml) was quantified in terms of protein concentration ( . mg/ml) by uv spectrophotometry. a volume of µl was mixed with µl of lds x to obtain a protein fraction of . mg/ml. after denaturation at °c for min, a volume of µl ( . µg of proteins) was deposited on a nupage - % gel (invitrogen) and subjected to min electrophoretic migration. the whole proteome was excised as a single polyacrylamide gel band and subjected to trypsin proteolysis as previously described ( ) . an aliquot of µl of peptides was extracted. ms/ms analysis was performed to confirm the high content of viral proteins in this sample (data not shown). sars-cov- viral peptides ( µl) were diluted in µl of h o, . % tfa. after mixing, µl of this tube was removed and diluted with µl of h o, . % tfa. this was repeated several times to obtain a one third dilution cascade of viral peptides. two series of simili swabs were prepared in parallel. the two peptide fractions obtained from nasopharyngeal swabs ( µl) were diluted with µl of h , . % tfa. a volume of µl of this diluted matrix was added to each simili swab samples, giving a final volume of µl per sample. thus, each simili swab contained the equivalent of . % of the proteins harvested by a nasal swab. two biological replicates were prepared using each nasal swab matrix. a volume of µl per sample was injected in the q-exactive hf tandem mass spectrometer. they were analysed in the same conditions as above except that the gradient was developed from to . % of ch cn, . % formic acid for min at a flow rate of . µl/min. the min ms/ms acquisition started min after injection. nasopharyngeal swabs were collected from covid- diagnosed adult patients as routine medical controls and tested by rt-pcr assay for detecting sars-cov- in a nasopharyngeal sample (swabs t -t ). this study was approved by the institutional review boards of the university hospital of nîmes, france ( - - ). patients have been previously be informed that part of these samples could be used for research purpose and agreed. each swab was soaked into a tube containing ml of phosphate buffered saline (ph . ) sterile solution and transferred in the biosafety level facility. the biological material was precipitated with the addition of . ml of trichloroacetic acid at % (w/v). after centrifugation, the supernatant was discarded and the pellet was dissolved into µl of lds x containing % betamercaptoethanol, heated for min at °c, and deposited on a nupage - % gel (invitrogen). the proteins were subjected to min electrophoresis and treated as described here above to obtain tryptic peptides. ms/ms acquisition was done as for the simili swabs. the min ms/ms acquisition started min after injection with an inclusion list comprising m/z values corresponding to viral peptides. ms/ms spectra from the nasopharyngeal swabs were searched against the generalist ncbinr database ( , , sequences totalling , , , amino acids) with the mascot daemon . . search engine (matrix science). the search parameters were as follows: fulltrypsin specificity, maximum of two missed cleavages, mass tolerances of ppm on the parent ion and . da on the ms/ms, carbamidomethylated cysteine (+ . ) as a fixed modification, and oxidized methionine (+ . ) and deamidation of asparagine and glutamine (+ . ) as variable modifications. psms with an fdr< % were selected for peptide inference. peptides were assigned to taxa using the unipept . web interface ( ) with default parameters (equate i/l, filter duplicate peptides). ms/ms spectra from the simili sars-cov- contaminated swabs and from the covid- nasopharyngeal swabs were assigned with the mascot daemon . . search engine (matrix science) as follows: the spectra were first queried against the crap_contaminants_ - - .fasta file and then against the swissprot_human_isl_ _ - - database ( , sequences totalling , , amino acids) in follow-up mode and with the decoy option activated. this last database is the merge of the sars-cov- viral proteins and the swissprot human proteome. the mascot search was performed with the same parameters as above. all peptide matches presenting a mascot peptide score with a fdr lower than % were assigned to protein sequences. ms peak areas were evaluated with skyline ( ) . briefly, we created spectral libraries based on the dat files from each mascot search (cut-of . ) and uploaded the ms full scan information contained in the raw files. the protein database previously used for the mascot search was used as background proteome. only the viral proteins were added to the target panel. peptide settings were matched to those used in the mascot search. peak peaking was manually checked for all peptides. the mass spectrometry and proteomics data acquired on simili swabs have been deposited to the proteomexchange consortium via the pride partner repository ( ) with the dataset identifiers pxd and . /pxd . to assess the performance of shotgun ms-based proteomics in detecting sars-cov- peptides in a background matrix consisting of nasopharyngeal swab protein material, we experimentally created tryptic peptidomes from i) a purified virus solution obtained from vero e cells infected with a sars-cov- reference strain, and ii) nasopharyngeal swabs obtained from two healthy volunteers (figure ) . we first characterized the nasal peptidomes and searched for the presence of detectable microorganisms by metaproteomic data analysis. then, the virus peptidome was serially diluted into nasopharyngeal swab peptidomes to obtain two sets of seven tubes containing from ng (equivalent to infectious particles) to . ng (equivalent to infectious particle) of viral protein material. the fourteen samples were subsequently analyzed by lc-ms/ms. a window of min of acquisition within a min lc gradient was adjusted to target the region of elution of five previously identified virusspecific peptides ( ) . the rationale for focussing the mass spectrometry measurements on these peptides was their remarkable sequence conservation amongst the numerous sars-cov- strains sequenced to date or/and their specificity to the novel coronavirus ( ) . these peptides were the following: eitvatsr, gfyaegsr, htpinlvr, iaghhlgr, and adetqalpqr. while known variants exist for the latter, the other four peptides are conserved along the several sars-cov- sequenced genomes. the ms/ms spectra acquired over min on the two nasopharyngeal swabs were analyzed to infer the main microbial components present in these samples as such presence should be taken into account for creating an ad hoc database for ms/ms interpretation. swabs r and r yielded , and , ms/ms spectra, respectively, from which , and , were attributed to , and , peptide sequences from organisms present in the ncbinr database (fdr < %). these peptide sequences were analyzed with the unipept tool ( ) to assess the biodiversity present in each sample through their taxon-specificity characteristics based on the lowest common ancestor approach. only a small proportion of the peptide sequences mapped by unipept belonged to microorganisms (table s ) . a rather low number ( and ) of peptides from the r and r swabs, respectively, were attributed to bacteria, archaea or fungi. these corresponded to . % and . % of the mapped peptide sequences, respectively. to exclude false positive identifications, we applied a threshold of at least threetaxon specific peptides for organism validation at the species level, corresponding to . % of the total number of species-specific peptides ( and in each sample), as suggested by ( ) . thus, one low-abundant corynebacterium was identified in sample r , namely corynebacterium accolens, with specific peptides. in swab r , corynebacterium propinguum, corynebacterium pseudodiphtheriticum, and dolosigranulum pigrum could be identified at the species taxonomical rank with , and specific peptide sequences, respectively. simili swabs containing specific quantities of sars-cov- virus and the equivalent of . % of the nasal matrix protein material collected during sampling were analysed by ms/ms with a short gradient. we first confirmed on the most diluted fraction that the bacterial signal was negligible for both fractions, thus not to consider at the ms/ms attribution search stage. for this, the two datasets were searched against the generalist database ncbinr to check for the presence of non-human peptides in the swab peptidomes. the unipept analysis of the detected peptide sequences showed that only and peptides, from replicate and , respectively, were attributed to bacteria and no bacterial species could be confidently identified ( table s ) . the results from the short gradient ms analysis on the simili swabs against the specific human/virus database yielded , ms/ms spectra recorded in the fourteen samples. from these, , were attributed to , peptide sequences with a fdr below % (table s ) . this data allowed for the identification of , protein groups (table s ) . a small fraction of peptide-to-spectrum matches (psms), corresponding to . % of the total psms, allowed identifying different viral peptide sequences, including the five peptides of interest. the peptides report for structural proteins from the virus: peptides from the nucleocapsid protein (n), peptides from the spike protein (s), and peptides from the membrane glycoprotein (m). at least one viral peptide was identified in all samples independently of the concentration of the viral material, from ng ( pfu) to ng ( pfu). however, no peptide from the virus was identified in the sample containing viral peptides corresponding to . ng ( pfu). the heatmap in figure displays the ms peak areas, the number of psms attributed to each peptide in each sample, and the number of viral peptides identified in each sample. the five peptides with the highest ms peak areas across samples were the following: eitvatsr, gfyaegsr, lnqlesk, adetqalpqr, and kadetqalpqr. among them, eitvatsr, gfyaegsr, and adetqalpqr are between the five peptides of interest. peptide htpinlvr was the seventh most abundant. inversely, peptide iaghhlgr was amongst the peptides with the lowest ms peak areas, along with peptides msecvlgqsk, lddkdpnfk, and eidrlnevak. as expected, the number of identified peptides decreased with the decreasing viral load in the sample. while all peptides were identified in the initial dilution containing ng of viral proteins ( pfu), in highly-diluted samples containing ng of viral proteins ( pfu) and ng ( pfu), the virus was proteotyped with only and peptides, respectively. in these samples only peptides from protein n were detected (adetqalpqr, kadetqalpqr, and gfyaegsr). generally, the peptides from protein n were the most consistently detected across samples. despite being among the peptides with higher peak areas in the chromatograms, peptides of interest htpinlvr and eitvatsr were only detected in the simili swabs containing an estimated ng of viral proteins ( pfu). on the other hand, the two other peptides of interest from protein n, gfyaegsr and adetqalpqr, allowed virus proteotyping in the sample containing ng of viral proteins ( pfu). of note, the peptide identified in the condition with ng of viral proteins ( pfu) is a miss-cleaved version of the adetqalpqr peptide: kadetqalpqr. peptide asanlaatk, that had not been previously selected among the "best" candidates for sars-cov- proteotyping ( ), was detected in the dilution with ng of viral proteins ( pfu) and was the most sensitive peptide from protein s. with the lc gradient used in this experiment and the delay for starting the acquisition, the retention times of peptides are minus - minutes compared to those described in our previous paper. peptides are generally well distributed along the gradient, with some exceptions of peptide pairs that co-elute: kadetqalpqr/rvdfcgk, qlqqsmssadstqa/cygvsptk, adetqalpqr/gfyaqgsr, or htpinlvr/eidrlnevak. six peptides elute in the first ten minutes of the gradient: iaghhlgr, kkadetqalpqr, asanlaatk, lnqlesk, kadetqalpqr, and rvdfcgk. of the utmost interest, three of the most conserved and well-detected peptides, eitvatsr, adetqalpqr, and gfyaqgsr, elute in a -min window between and min of the gradient. nasopharyngeal swabs were sampled from nine covid- diagnosed patients with different clinical manifestations (moderate symptoms and asymptomatic) and at different postdiagnostic stages ( table ) . due to the complexity of the samples, an inclusion list of m/z signals corresponding to the five peptides of interest as well as other sars-cov- peptides detectable in this gradient region ( ) was added to the acquisition method to increase the likelihood of their detection. table s reports this inclusion list which contained m/z values for different precursors from different viral peptide sequences. the short gradient ms analysis on these clinical samples yielded between and , ms/ms spectra recorded per sample. sixty-five spectra were attributed to viral peptide sequences with a fdr below % (table s ) . this data allowed for the detection of six peptides reporting for two viral proteins (table s ) : lddkdpnfk, kadetqaipqr, kkadetqaipqr, adetqaipqr, gfyaegsr from protein n, and eitvatsr from protein m. the heatmap in figure displays the ms peak areas, the number of psms attributed to each peptide in each sample, the number of viral peptides identified in each sample, and the result from the pcr testing performed on the same sample. the virus was confidently proteotyped in clinical swabs t and t , with four and five peptides respectively. peptide eitvatsr was identified in swab t with two spectral counts, but virus detection in this sample cannot be validated since this peptide is not specific to sars-cov- ( ). as shown in table , swabs t and t correspond to patients that were diagnosed as sars-cov- positive by rt-pcr with relatively clear viral loads (ct values of and , respectively) and were sampled and days after their diagnostic and confinement. ms/ms samples were negative for swabs that yielded a relatively low pcr signal (ct of and for swabs t and t ), with undetectable pcr signal (swabs t , t , t , and t ) or days after their diagnostic (swab t ). the negative ms/ms signals for swabs t , t , and t patients are explained by the very low viral load probably present in these samples. to foster the development of alternative detection methods for sars-cov- , we performed a proof-of-concept study to assess the potential of ms/ms for proteotyping sars-cov- : i) in simulated nasal swabs containing different quantities of viral peptides; and ii) in nasopharyngeal swabs from covid- diagnosed patients. the two nasal peptidomes collected from healthy donors for the first experiment were first analyzed with a gradient of min to check for the presence of detectable microorganisms from the natural microbiota. a search against a generalist database such as ncbinr detected only trace levels of very low abundant bacteria commonly found in the nasal tract ( ) , thus confirming the absence of a measurable microbiome in the swab samples. based on this metaproteomic analysis, we used a human-only database as representative of the nasopharyngeal matrices for the subsequent analysis. the simili sars-cov- contaminated swabs contained a fixed amount of swab peptidome, plus a precise amount of viral peptidome corresponding to the expected quantities extracted from ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), and . ng ( pfu) of sars-cov- . it is important to note that the virus produced in vero e cells and purified on sucrose gradient is only partially infectious, and thus the data are also presented in quantities of viral proteins. the real number of viral particles could be much higher in these samples and could be roughly estimated as the molecular weights of each viral protein are known and if the numbers of molecules per virus particle were documented for sars-cov- . here, we refer to the infectious dose as this is the most important parameter in terms of health concern, but the ratio of infectious particles in the nasopharyngeal swabs of patients may drastically differ from the purified virus fraction used here, and could even fluctuate during the course of the pathology. the strategy proposed for the analysis of these simili swabs consisted in a shotgun ms analysis based on a short acquisition of min with a short lc gradient. for the clinical samples, we added an inclusion list of viral peptides in the ms method. the inclusion list allowed forcing the fragmentation of candidate viral peptide ions contained in the background matrix, even when they were not included in the top from the data dependent acquisition method. the shotgun strategy resulted in the detection of viral peptides in six out of the seven conditions tested for the simili swab experiment. from the five peptides of interest, gfyaqgsr and adetqalpqr proved to be the most detectable and most sensitive in this background matrix, allowing proteotyping the virus up to the condition of ng of viral material ( pfu). one of the most interesting result was the omnipresence of peptide adetqalpqr and its two miss-cleaved versions kadetqalpqr and kkadetqalpqr. these peptides were consistently detected in out of identifications in the six most abundant conditions from the simili swab experiment. peptide kadetqalpqr was identified in all simili swabs from figure . these results clearly show that adetqalpqr, despite being prone to missedcleavages, is one the most abundant and ionisable peptides and should be the main target for proteotyping sars-cov- . this result was confirmed from the analysis of the clinical swab samples, since peptides adetqalpqr, kadetqalpqr, and kkadetqalpqr were undoubtedly the most abundant in samples from covid- patients (figure and table ). in our previous work, we showed that this peptide sequence is also specific to sars-cov- , but presented several variants among the available sars-cov- genomes ( ) . therefore, when targeting this peptide for viral detection with ms/ms we can also take into account both its missed-cleaved versions, and its different variants. surprisingly, the high intensity peptide eitvatsr was only identified in simili swabs with high concentration of viral proteic material (figure ) . by analyzing the ms and ms/ms spectra from these samples, we confirmed that this peptide co-eluted with another intense precursor from the background matrix that was fragmented simultaneously. the low mascot ion score attributed to these spectra hindered the confident identification of this peptide. this coelution effect is most likely due to the use of the short chromatographic gradient, and one way to tackle it would be to use smaller isolation windows for fragmentation. this parameter was tested for the analysis of clinical swabs, but little or no improvement was observed. no ms/ms spectra were validated at fdr % for this peptide in swabs t and t , even with the presence of a ms peak corresponding to this peptide in swab t . this peptide is therefore problematic in this type of matrix and probably not suited for tracking sars-cov- in nasal swab samples with our specific experimental setup. the distribution of the peptides along with the chromatogram from figure shows that the two most detectable peptides gfyaqgsr and adetqalpqr eluted in a narrow window of retention time between - min in simili swab samples. for the clinical swab samples, we observed that the retention time for these two peptides was . ± . min for peptide adetqalpqr, and . ± . minutes for peptide gfyaqgsr as established with skyline (table s ). in the light of these new results, we argue that targeting peptides adetqalpqr and gfyaqgsr with an extra short lc gradient of min coupled to the enrichment of these hydrophilic peptides prior the lc injection could be one way to develop quick and robust assays for detection of the virus in clinical samples and gain in signal/noise ratio. besides their high intensity, these peptides provide the needed specificity for a confident assay: peptide gfyaegsr is highly conserved among different sars-cov- genomes, and peptide adetqalpqr is specific to sars-cov- . the simultaneous detection of these two peptides could provide therefore unequivocal evidence for the presence of the virus. interestingly, a recent not yet published study showed the high potential of the same two peptides by using a targeted proteomics assay ( ) . the authors report limits of detection in the mid-attomole range corresponding to theoretically , sars-cov- particles in their specific experimental set-up. besides shortening the lc gradient to less than three min, sample preparation can also be optimized to develop more rapid peptidome preparation assays and remove too hydrophilic and too hydrophobic peptides that could saturate the chromatography column. here, we performed a sds-page gel and in-gel proteolysis with trypsin to denature proteins and to remove any mass spectrometry-chromatography deleterious compounds that could be present in the nasal swab. this procedure is known to not be optimal as only % of the peptide material deposited on the gel is recovered. the literature is becoming rich in alternative sample preparation protocols for ms-based proteomics. for example, we recently proposed a proteotyping assay based on sp magnetic beads for protein purification and digestion in roughly min ( ) . being easily adapted to -well plates and robotization, sp based digestion is the method of choice for quick, high-throughput, and highly reproducible proteome digestions, as recently demonstrated ( , ) . such sample preparation may further significantly increase the sensitivity of the tandem mass spectrometry proteotyping proposed in the present work. furthermore, more sensitive instrument and ms acquisition modes could be tested to gain further sensitivity. in conclusion, we tested in this study the potential of lc-ms/ms based methods for proteotyping sars-cov- in nasopharyngeal swabs. with a min ms-acquisition window, we were able to identify and quantify several virus-specific peptides that allowed proteotyping the virus in simulated swabs and clinical swabs from covid- patients. we argue that peptides adetqalpqr (and its variant forms) and gfyaqgsr from the nucleocapsid protein are of utmost interest to develop quick and robust targeted assays for proteotyping the virus in nasopharyngeal swab samples. further research must be done to validate their usefulness and their limits of detection in clinical samples, and develop the shortest possible pipeline. ja conceived the study with help from dg, gm, lg, and op. gm and jcg performed the mass spectrometry experimental work. jpl and as contributed the medical covid- swab samples. fg, sd, and lb contributed the sars-cov- biological material. dg, lg, gm, op and ja analysed the data. dg and ja wrote the manuscript with help from lg. table s . list of m/z values of the inclusion list; table s . unipept taxonomical analysis of swab r , swab r , and simili swab r - pfu samples; table s . list of peptide-to-spectrum matches (psms) assigned from the simili sars-cov- swabs (fdr< %); table s . list of proteins from the simili sars-cov- swabs and their spectral counts; table s . list of peptideto-spectrum matches (psms) assigned in the clinical samples (fdr< %); table s . list of proteins identified in the clinical samples and their spectral counts; table s . skyline report with experimental data on precursor ions from the nine swabs from covid- patients. patients were numbered from "swab t " to "swab t ". a pneumonia outbreak associated with a new coronavirus of probable bat origin the importance of naturally attenuated sars-cov- in the fight against covid- development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis detection of sars-cov- in different types of clinical specimens overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for covid- detection 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proteomics of sars-cov- -infected host cells reveals therapy targets the covid- ms coalition-accelerating diagnostics, prognostics, and treatment peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data taking the shortcut for high-throughput shotgun proteomic analysis of bacteria rna-binding proteins are a major target of silica nanoparticles in cell extracts the unipept metaproteomics analysis pipeline skyline: an open source document editor for creating and analyzing targeted proteomics experiments the pride database and related tools and resources in : improving support for quantification data evaluating the impact of different sequence databases on metaproteome analysis: insights from a lab-assembled microbial mixture the human nasal microbiota and staphylococcus aureus carriage evaluation of sample preparation methods for fast proteotyping of microorganisms by tandem mass spectrometry automated sample preparation with sp for low-input clinical proteomics the authors are indebted to dr silvia meschi (national institute for infectious diseases "lazzaro spallanzani" irccs, via portuense , rome, italia) for making the human -ncov strain -ncov/italy-inmi ( n- ) available. this publication was supported by the european virus archive goes global (evag) project that has received funding from the european union's horizon research and innovation programme under grant agreement n° . the authors are also grateful to the french alternative energies and atomic energy commission (cea), and the anr program "phylopeptidomics" (anr- -ce - - ) that supported part of this study. the authors have declared no conflict of interest. key: cord- -w spqqt authors: huan, yuchen; kong, qing; mou, haijin; yi, huaxi title: antimicrobial peptides: classification, design, application and research progress in multiple fields date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: w spqqt antimicrobial peptides (amps) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. amps have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. the emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of amps, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. this review introduces the progress of research on amps comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. the research progress on antivirus peptides, especially anti-coronavirus (covid- ) peptides, has been introduced given the covid- pandemic worldwide in . alexander fleming discovered lysozyme in , and this discovery marked the birth of modern innate immunity. since then, antibiotics and antimicrobial peptides (amps) have been discovered. a total of , amps have been reported in the antimicrobial peptide database (apd ) updated on august , . different types of amps have the following commonalities: their number of amino acid residues is between and (average: . ), and almost all amps are cationic (average net charge: . ). however, several anionic amps also exist, and they have several acidic amino acids like aspartic acid and glutamic acid (malkoski et al., ; schittek et al., ; lai et al., ) . the anti-microbial resistance of microorganisms is becoming increasingly serious with the abuse of antibiotics in medicine, agriculture and animal husbandry, especially in developing countries. research from kenya has detected substantial amounts of antibiotic residues in edible meat (ayukekbong et al., ) . the prevalence of vancomycin-resistant enterococcus (vre) and methicillin-resistant staphylococcus aureus (mrsa) is increasing in clinical medicine, so the countermeasures are urgently needed to address these bacterial infections. however, from the http://aps.unmc.edu/ap/ perspective of pharmaceutical companies, the development of new antibiotic drugs results in low profit. thus, replacing antibiotics has become a consideration in the pharmaceutical, agricultural, animal husbandry, and food industries. research on amps is continuously developing and considerable amounts of data on amps have been stored in amp databases. however, the mechanism of amps remains incompletely understood, and further work needs to be performed to determine the relationship between different physicochemical properties to obtain low-cost and highly safe amps with remarkable antimicrobial effects and the specificity and a high capacity for synergies of amps should also be further developed (lazzaro et al., ) . the diversity of natural amps causes difficulty in their classification. amps are classified based on ( ) source, ( ) activity, ( ) structural characteristics, and ( ) amino acid-rich species (figure ). the sources of amps can be divided into mammals (human host defense peptides account for a large proportion), amphibians, microorganisms, and insects according to statistical data in apd . the amps found in oceans have also attracted widespread attention. mammalian antimicrobial peptides are found in human, sheep, cattle, and other vertebrates. cathelicidins and defensins are the main families of amps. defensins can be divided into α-, β-, and θ-defensins depending on the position of disulfide bonds (reddy et al., ) . human host defense peptides (hdps) can protect human from microbial infections but show different expressions in every stage of human growth. for example, cathelicidin ll- , a famous amp derived from the human body, is usually detected in the skin of newborn infants, whereas human betadefensin (hbd- ) is often expressed in the elderly instead of the young (gschwandtner et al., ) . hdps can be identified in many parts of the body such as skin, eyes, ears, mouth, respiratory tract, lung, intestine, and urethra. besides, amps in human breast milk also play an important role in breastfeeding because it can decrease the morbidity and mortality of breastfeeding infants (field, ) . what's interesting is that casein (peptide derived from β-casein - aa), identified in colostrum, shows different levels in preterm human colostrum and term human colostrums . dairy is an important source of amps, which are generated through milk enzymatic hydrolysis. several amps have been identified from α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions, and the most famous peptide obtained is lactoferricin b (lfcinb) (sibel akalın, ) . furthermore, whether the amps derived from dairy products can be used for dairy preservation is also an interesting subject to develop. in addition to antimicrobial activity, hdps, such as cathelicidins and defensins, also affect immune regulation, apoptosis, and wound healing (wang, ) . antimicrobial peptides from amphibians play an important role in the protection of amphibians from the pathogens that have induced the global amphibian population decline (rollins-smith, ). frogs are the main source of amphibian amps and the most famous amp from frogs is magainin; the skin secretions of frogs from genera xenopus, silurana, hymenochirus, and pseudhymenochirus under the pipidae family are rich in amps (conlon and mechkarska, ) . furthermore, cancrin, which has an amino acid sequence of gsaqpykqlhkvvnwdpyg, has been reported as the first amp from the sea amphibian rana cancrivora (lu et al., ) . this marks a broader source of amps of amphibians. figure | classification of antimicrobial peptides. (semreen et al., ) . myticusin-beta is an immune-related amp of mytilus coruscus and a promising alternative to antibiotics (oh et al., ) . moreover, ge , known as pardaxin, is a marine amp and the ge -based vaccine has shown the ability to enhance antitumor immunity in mice (huang et al., ) . the activity of amps can be divided into categories according to the statistics of the adp database. these categories can be summarized as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (hiv), and anti-tumor peptides (figure ). antibacterial peptides account for a large part of amps and have a broad inhibitory effect on common pathogenic bacteria, such as vre, acinetobacter baumannii, and mrsa in clinical medicine and s. aureus, listeria monocytogenes, e. coli in food and salmonella, vibrio parahaemolyticus in aquatic products. many natural and synthetic amps like nisin, cecropins and defensins have shown good inhibition activity to gram-positive bacteria and gram-negative bacteria. in recent research, amps p (yirkirrffkklkkilkk-nh ) and p (syerkinrhfktlkknlkkk-nh ), which are designed based on aristicluthys nobilia interferon-i, inhibit mrsa and show a low cytotoxicity . antifungal peptides are a subclass of amps that address fungal infections with enhanced drug resistance. many afps have shown excellent anti-fungal activities against common pathogenic fungi, such as aspergillus and candida albicans in clinical medicine, yeast, filamentous fungi (e.g., aspergillus flavus), mold in food and agriculture. except for brevinin, ranatuerin, cecropins, many synthetic peptides also show good antifungal activity. for example, aurh , derived from aurein . , can effectively treat c. albicans infection, which has a lethal rate up to % (madanchi et al., ) . aflatoxin, which is a carcinogen produced by a. flavus, is harmful to the human body. many afps can inhibit the growth of a. flavus. for example, an afp with a sequence of fpshtgmsvppp can inhibit the growth of a. flavus md . a total of antifungal peptides isolated from lactobacillus plantarum te and their mixture can reduce a. flavus spore formation in fresh maize seeds (muhialdin et al., ) . moreover, two chemically synthesized radish amps show a good inhibitory effect against different yeast species, such as zygosaccharomyces bailii and zygosaccharomyces rouxii (shwaiki et al., ) . viruses cause serious harm to human life and huge economic losses to the animal husbandry. the covid- , which is the recent outbreak, has caused great loss of lives and properties. furthermore, foot-and-mouth disease virus, avian influenza virus (aiv), and hiv are long-term threats to human life. so, it is extremely urgent to solve these problems, and antiviral peptides provide new ways. antiviral peptides show a strong killing effect on viruses mainly by ( ) inhibiting virus attachment and virus cell membrane fusion, ( ) destroying the virus envelope, or ( ) inhibiting virus replication (jung et al., ) (shown in figure ). a recent report has shown that amp epi- mediates the inactivation of virus particles and has good inhibitory activity against foot-and-mouth disease virus . moreover, infectious bronchitis virus (ibv) is the pathogen of infectious bronchitis and the inoculation of swine intestinal amp (siamp)-ibv mixed solution remarkably reduced the mortality of chicken embryos compared with the ibv infection group, showing the good inhibitory activity of siamp on ibv (sun et al., ) . anti-hiv peptides are a subclass of anti-viral peptides. the most important examples of these peptides include defensins (including αand β-defensins, which have different mechanisms), ll- , gramicidin d, caerin , maximin , magainin , dermaseptin-s , dermaseptin-s , siamycin-i, siamycin-ii, and rp (madanchi et al., ) and antiviral peptide fuzeon tm (enfuvirtide) has been commercialized as an anti-hiv drug (ashkenazi et al., ) . due to the global spread of the covid- (figure a ), the antiviral peptides against the coronavirus will be discussed in more detail. coronaviruses (covs) belong to the family coronaviridae; they are enveloped viruses with a positive-sense single-stranded rna genome and have a helical symmetry (franks and galvin, ) . covs, including severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (mustafa et al., ) , and the recent outbreak of covid- have caused serious threats to human life and property. covs can cause lifethreatening respiratory diseases and the viral particle is formed by spike glycoprotein (s), the envelope (e), the membrane (m), and the nucleocapsid (n) (vilas boas et al., ) . it should be noted that their infectivity requires viral spike (s) protein. fusion inhibitor peptides combine with the s protein to interfere with its folding and prevent infection. besides, the s domain of the sars-cov s protein contains heptad repeat hr and hr sequences. peptide hr (hr : sltqinttlldltyemlslqqvvkalnesyidlkel) and its lipid-binding peptide is highly similar or even identical to the near-membrane portion of s protein ferredoxin, which interferes with refolding into post-fusion fusion-catalyzing domains (fds) (du et al., ; park and gallagher, ) . according to recent research, the lipopeptide ek c , derived from ek (sldqinvtfldleyemkkleeaikkleesyidlkel), is the most effective fusion inhibitor against covid- s proteinmediated membrane fusion . homology modeling and protein-peptide docking showed that temporin has potential therapeutic applications against mers-cov (marimuthu et al., ) . two amps from the non-structural protein nsp of sars-cov, k , and k , can inhibit sars-cov replication (ke et al., ) . furthermore, rhesus thetadefensin (rtd- ) treated animals have a marked reduction in mortality in the presence of sars-cov while the peptide alone shows airway inflammation and the one possible mechanism of action for rtd- is immunomodulatory (wohlford-lenane et al., ) . in general, amps against coronavirus can be roughly classified as i) peptides derived from hr , hr and rbd subunits of the spike protein, ii) peptides derived from other amps, iii) peptides derived from non-structural protein (mustafa et al., ) . furthermore, molecular docking analysis indicated that peptides were employed to disrupt the interaction between covid- and ace (angiotensin-converting enzyme ) to inhibit covid- entrance in cells ( figure b ) (souza et al., ) . finally, it should be noted that this therapy lacks clinical trials and the main method of animal experiments is an intranasal administration. this reminds us that nasal drug delivery (ndd) is a potential therapy for amps as anti-coronavirus drugs. besides, the antiviral database avpdb includes numerous antiviral peptides. parasitic protozoa can cause diseases in human and animals through a variety of routes, including animal-to-person or person-to-person contact, water, soil, and food (chalmers et al., ) . and with the increase in parasite drug resistance, the need for new treatments has increased. antiparasitic peptides show their killing effect on parasites which cause diseases such as malaria and leishmaniasis (mangoni et al., ; rhaiem and houimel, ) and amps like cathelicidin, temporins-shd show high inhibition activity against parasites (abbassi et al., ) . in recent research, epi- , a marine synthetic amp, can remarkably inhibit trichomonas vaginalis by destroying its membrane (neshani et al., ) . the peptide jellein derived from bee royal jelly which has introduced above and -amino acid amp kdel (lysine, aspartic acid, glutamic acid, and leucine) has shown a significant effect on the leishmania parasite (cao et al., ; zahedifard et al., ) . however, it should be noted that their mechanisms are not the same. cyanobacterial peptides differ from higher-eukaryote amps because their antiparasitic action depends on specific protein targets. thus, these target parasites can be distinguished accurately even though they belong to the same family or genus (rivas and rojas, ) . the acps show anticancer mechanisms by ( ) recruiting immune cells (such as dendritic cells) to kill tumor cells, ( ) inducing the necrosis or apoptosis of cancer cells, ( ) inhibiting angiogenesis to eliminate tumor nutrition and prevent metastasis, and ( ) activating certain regulatory functional proteins to interfere with the gene transcription and translation of tumor cells (wu d. et al., ; ma et al., ) . tritrpticin and its analogs induce considerable toxicity toward jurkat cells in vitro, whereas indolicidin and puroindoline a can also act as acps (arias et al., ) . it should be noted that both net charge and hydrophobicity play important roles in optimizing the anticancer activity of acps and they can constrain and influence each other. thus, achieving a balance between net charge and hydrophobicity is important for better anticancer activity. besides the peptide mentioned above, anti-inflammatory, anti-diabetic peptides, spermicidal peptides etc. have been noticed, but they are not the same as antimicrobial peptides. simply put, anti-inflammatory peptides decrease the release of inflammatory mediators and inflammatory cytokines (nitric oxide, interleukin- , and interleukin- β) and some of them also inhibit inflammatory signals like nf-κb, mapk, and jak-stat pathways (meram and wu, ; gao et al., ) . anti-diabetic peptides play their function by modulating the g proteincoupled receptor kinase (grk / ) or activating glucagonlike peptide- (glp- ), glucagon receptors (marya et al., ; graham et al., ) . however, it is not accurate to classify these types of peptides as amps and bioactive peptides may be more convincing. proline is a typical non-polar amino acid. pramps behave differently from other amps, that is, they enter bacterial cytoplasm by the inner membrane transporter sbma instead of killing bacteria through membrane destruction (mattiuzzo et al., ) . once in the cytoplasm, pramps target ribosomes and block the binding of aminoacyl-trna to peptidyltransferase center or trap decoding release factors on the ribosome during the termination of translation to interfere with protein synthesis (seefeldt et al., ) . for instance, tur a, which is an orthologous amp of bovine pramp bac discovered from tursiops truncatus, interferes with the transition from the initial phase to the extension phase of protein synthesis by binding to ribosomes. in addition, different pramps lack a high sequence similarity but have short motifs containing repeating proline and arginine (arg) residues (e.g., -ppxr-in bac and -prpxin bac ) (mardirossian et al., (mardirossian et al., , . although pramps mainly kill gram-positive bacteria, ppr-amp , a proline-rich amp identified from crab (scylla paramamosain), exhibits antimicrobial activity against gram-positive and gram-negative bacteria (imjongjirak et al., ) . besides, pieces of research have shown that pramps have immunostimulation activity (li w. et al., ) . tryptophan (trp), as a non-polar amino acid, has a remarkable effect on the interface region of the lipid bilayer, whereas arg, as a basic amino acid, confers peptide charge and hydrogen bond interactions, which are essential properties to combine with the bacterial membrane's abundant anionic component. and it seems that trp residues play the role of natural aromatic activators of arg-rich amps by ion-pair-π interactions (walrant et al., ) , thereby promoting enhanced peptide-membrane interactions (chan et al., ) . in addition to indolicidin and triptrpticin which both are famous amps that rich in arg and trp residues. octa (rrwwrwwr) is also a typical trp-and arg-rich amp that inhibits gram-negative e. coli and pseudomonas aeruginosa and gram-positive s. aureus. and short trp-and arg-rich amps designed based on bovine and murine lactoferricin have also shown strong inhibitory action against bacteria (strøm et al., ; bacalum et al., ) . histidine is a common basic amino acid, and histidine-rich amps show good membrane permeation activity. hv is a histidinerich amp designed based on rr(xh) xdpgx(yh) rr-nh (where x represents i, w, v, and f). this peptide increases the permeability of bacterial cell membranes to cause cell membrane rupture and death. in addition, hv inhibits bacterial movement in a concentration-dependent manner and shows a strong anti-inflammatory effect by inhibiting the production of tumor necrosis factor α (tnf-α) ). an amp designed based on octa has shown good therapeutic potential by replacing its arg residues with histidine (bacalum et al., ) . furthermore, l h , which is designed based on the linear cationic amphiphilic peptide magainin, also shows good antibacterial activity and cell penetration properties by inserting four histidine sequences in leucine and alanine (lointier et al., ) . the r group of glycine is generally classified as a non-polar amino acid in biology. glycine-rich amps, such as attacins and diptericins, widely exist in nature (lee et al., ; kwon et al., ) . these peptides contain % to % glycine residues, which have an important effect on the tertiary structure of the peptide chain. a glycine-rich amp derived from salmonid cathelicidins activates phagocyte-mediated microbicidal mechanisms, which differ from the mechanism of conventional amps (d'este et al., ) . furthermore, the glycine-rich central-symmetrical gg is an ideal commercial drug candidate against clinical gramnegative bacteria . antimicrobial peptides can be divided into four categories based on their structures including linear α-helical peptides, β-sheet peptides, linear extension structure, and both α-helix and β-sheet peptides ( figure ) (lei et al., ) . moreover, progressively cyclic peptides and amps with more complex topologies (including lasso peptides and thioether bridged structures) are reported (koehbach and craik, ) . the membrane-targeting mechanisms of amps can be described through models, including the pole and carpet models and the pole model can be further divided into the toroidal pore and barrel-stave models (figure ). the toroidal pore model is also known as the wormhole model. in this model, amps vertically embedded in the cell membrane accumulate and then bend to form a ring hole with a diameter of - nm (matsuzaki et al., (matsuzaki et al., , . the typical examples of this model are magainin , lacticin q, and arenicin. furthermore, cationic peptides, including tc , tc , and bp , compromise the membrane barrier by creating fluid domains (omardien et al., ) . antimicrobial peptides aggregate with each other, penetrate the bilayer of the cell membrane in the form of multimers, and form channels that result in the cytoplasmic outflow. in severe cases, amps can induce cell membrane collapse and lead to cell death (lohner and prossnigg, ). for instance, alamethicin performs its pore-forming activity by using this model. besides, hairpin amp protegrin- can form stable octameric β-barrels and tetrameric arcs (half barrels) in implicit and explicit membranes by simulations (lipkin and lazaridis, ) . antimicrobial peptides are arranged parallel to the cell membrane. their hydrophilic end faces the solution, and their hydrophobic end faces the phospholipid bilayer. amps will cover the membrane surface that similar to a carpet and destroy the cell membrane in a 'detergent'-like manner (oren and shai, ) . however, this pore-forming mechanism requires a certain concentration threshold and the required concentration of amps is high. human cathelicidin ll- exhibits its activity through this mechanism, and amps with β-sheet structure also play a role in this model (shenkarev et al., ; corrêa et al., ) . polarized light-attenuated total reflection fourier transform infrared spectroscopy (atr-ftir) was used to study the effect of amp cecropin p on the bacterial cell membrane and found that it was an applied flat on the surface of the pathogen's cell membrane to destabilize and eventually destroy the cell membrane . membrane targeting mechanisms (the cell membrane composition differences of bacteria and fungi shown in figure ) can be further refined to address the large differences in the lipid composition of the cell membranes of bacteria, fungi, and mammals. the main lipids in cell membranes include glycerophospholipids (gpls), lysolipids, sphingolipids, and sterols. phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl) are the most common anionic lipids in bacteria, whereas phosphatidylcholine (pc), phosphatidylinositol (pi), pe, and phosphatidic acid (pa) are the main gpls in fungal cell membranes (ejsing et al., ; singh and prasad, ; li et al., ) . furthermore, fungal cell membranes are more anionic than mammalian cell membranes and have higher pc content. meanwhile, ergosterol is the sterol found in the plasma membrane of lower eukaryotes, such as fungi, whereas that of animals contains cholesterol (faruck et al., ) . many amps take advantage of differences in membrane components to exert their effects. antimicrobial peptides are promising to be anti-biofilm agents but it should be noticed that they are different from the cell penetrating peptides (cpps) which typically comprise - amino acids and can translocate across the cell membrane. cpps could be categorized according to physicochemical properties into three classes: cationic, amphipathic, and hydrophobic, but anti-biofilm peptides have stricter requirements for these physicochemical properties. anti-biofilm peptides target the biofilms by different mechanisms including ( ) degradation of signals within biofilms; ( ) permeabilize within cytoplasmic membrane/eps; ( ) modulating eps production etc. and then can address chronic multi-resistant bacterial infections (pletzer et al., ; ribeiro et al., ; guidotti et al., ; derakhshankhah and jafari, ; rajput and kumar, ) . for instance, saap- , synthesized based on ll- , showed activity to prevent biofilm formation by s. aureus and a. baumannii (crunkhorn, ) . the way of amps entering cells is direct penetration or endocytosis. after entering the cytoplasm, amps will identify and act on the target. depending on the target, amps can be divided into the following categories. antimicrobial peptides affect transcription, translation, and assembly into functional peptides through molecular chaperone folding by interfering with related enzymes and effector molecules. for example, bac - targets ribosomes to inhibit protein translation (mardirossian et al., ) , whereas tur a inhibits protein synthesis in e. coli and thermus thermophilus by inhibiting the transition from the initial phase to the extension phase. however, the differences between tur a and bac also lead to various ways of binding to ribosomes and interacting with the ribosomal peptide exit tunnel (mardirossian et al., ) . but some amps' have different targets. for instance, genome-wide transcription shows that the amp dm can affect many important intracellular pathways of protein biosynthesis . chaperones are key proteins for correctly folding and assembling newly synthesized proteins and make them have stereoisomerism, which makes amps have cell selectivity and can prevent cytotoxicity. according to a previous review: both pyrhocoricin and drosocin can prevent dnak from refolding misfolded proteins by inducing a permanent closure of the dnak peptide-binding cavity (kragol et al., ; le et al., ; wrońska and boguś, ) . antimicrobial peptides can affect key enzymes or induce the degradation of nucleic acid molecules to inhibit nucleic acid biosynthesis. indolicidin, a c-terminal-amidated cationic trprich amp with amino acids, specifically targets the abasic site of dna to crosslink single-or double-stranded dna and it can also inhibit dna topoisomerase i (subbalakshmi and sitaram, ) . tfp (tissue factor pathway inhibitor) - tc , which is an amp from tongues, enters the cytoplasm of target cells after the rupture of cell membrane and then degrades dna and rna (he et al., ) . many amps can inhibit various metabolic activities by inhibiting protease activity. for example, histatin has a strong inhibitory effect on the proteases secreted by the host and bacteria. amps enap- and indolicidin inhibit microbial serine proteases, elastase, and chymotrypsin (le et al., ) . cathelicidin-bf is a peptide isolated from the venom of bungarus fasciatus, it can effectively inhibit thrombin-induced platelet aggregation and further block protease-activated receptor (shu et al., ) . antimicrobial peptides inhibit cell division by inhibiting dna replication and dna damage response (sos response), blocking the cell cycle or causing the failure of chromosome separation (lutkenhaus, ) . for instance, app (glaraltrllrqltrqltra), which is an amp with amino acid residues, can efficiently kill c. albicans because of its cell-penetrating efficiency, strong dna-binding affinity, and ability to induce s-phase arrest in intracellular environment . mciz, which has amino acid residues, is an effective inhibitor of bacterial cell division, z-ring formation, and localization (cruz et al., ) . moreover, it has been reported that several afps have damaging effects on the organelles of fungi. for example, histintin can interact with mitochondria, causing the production of ros, and inducing cell death (helmerhorst et al., ) . in addition to intracellular targets, differences in cell wall composition, such as lipopolysaccharide (lps), lipid a and mannoproteins, are potential targets for amps. specifically, gram-positive and gram-negative bacteria are classified based on their bacterial cell wall structure. gram-positive bacteria have a layer of cross-linked peptidoglycan, whereas gram-negative bacteria have an additional outer membrane with an inner leaflet containing only phosphatidic acid and an outer leaflet made of lps. lps has numerous negatively charged phosphate groups, which combine with a salt bridge with a divalent cation (e.g., ca + and mg + ) to form an electrostatic network (nikaido, ) . this electrostatic zone is the main barrier against hydrophobic antibiotics and causes the low permeability of gram-negative bacteria. the main components of the fungal cell wall are mannoprotein, β-glucans and chitin (polymers of , -β-n-acetylglucosamine) and the mutations in the relevant genes of the lps pathway and phospholipid trafficking provide resistance to the amps (cabib, ; spohn et al., ) . mannoproteins in fungal cell walls include a variety of proteins, including structural proteins, cell adhesion proteins (floccrin and lectin) and enzymes involved in cell wall synthesis and remodeling (hydrolytic enzymes and transglycosylase). these proteins differ from human cell membrane proteins and are potential targets of afps (rautenbach et al., ) . furthermore, teichoic acid and lipoteichoic acid in the cell wall are also potential targets of amps and these theories could support the design of amps with low cytotoxicity. antimicrobial peptides have good application prospects. however, amps have the following problems. ( ) amps damage the cell membrane of eukaryotes and cause hemolytic side effects; ( ) rising production costs and technical problems limit their manufacture; ( ) their stability is limited at certain ph; ( ) amps have reduced activity under the presence of iron and certain serum; ( ) amps are easily hydrolyzed by proteases. therefore, the ideal amp should meet the following characteristics: (i) high antimicrobial activity; (ii) low toxicity to mammalian membranes; (iii) high protease and environment stability; (iv) low serum binding capacity and (v) ease of access and low cost production . therefore, designing amps to achieve the desired effect has attracted increasing attention. the rational design of antibacterial peptides should focus on the following five aspects: chain length, secondary structure, net charge, hydrophobicity, and amphiphilicity and these have been mentioned in many studies and this review will focus more on several specific methods of antimicrobial peptide design. site-directed mutation refers to the redesign of natural antimicrobial peptides by adding, deleting or replacing one, or several amino acid residues (torres et al., ) . the de novo design of peptides attaches importance to the design of amphiphilic amps (guha et al., ) . for example, gala is a well-known de novo-designed amp. amphipathic α-helical peptide gala is created by placing protonatable glutamic acid residues in most positions with the spacing of i to i + (goormaghtigh et al., ) . the repeated sequence (xxyy)n, where x and x are hydrophobic amino acids, y and y are cationic amino acids, and n is the number of repeat units, is designed based on the hydrophobicity cycle that mimics natural α-helical amps and successfully designs broadspectrum α-helical amps. sequences (lkkl) and (wkkw) . have the highest selectivity (khara et al., ) . moreover, l l k m w model peptides are also de novo-designed peptides. amphipathic helical properties were conferred by using leucines and lysines, and two tryptophan residues were positioned at the amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. among the model peptides, l k w has good anti-mrsa activity (lee et al., ) . sequence templates can be obtained by comparing a large number of structurally homologous fragments of natural amps (such as hdps) and extracting conservative patterns based on the type of residue (such as charged, polar, hydrophobic, etc.) (zelezetsky and tossi, ) . based on the modification, the parameters, such as helix formation tendency, cationic, amphiphilicity and overall hydrophobicity, can be systematically changed. for instance, cecropin, magainin, protegrin, and lactoferrin have all been used as amp templates (fjell et al., ) . peptides can form nanostructures, such as micelles, vesicles, nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and can increase or impart antibacterial activity to amps during the self-assembly of peptides. for example, kld- (kld) is a self-assembling peptide with amino acid residues that can adopt nanostructures and are known for their tissue engineering properties. the addition of arg residues in kld shows no remarkable change in its β-sheet secondary structure and the self-assembly characteristics of the forming nanostructures (tripathi et al., ) . dimer structure can also be used to enhance the antimicrobial activity of amps and reduce toxicity, but membrane-destabilizing effects are reduced after dimer formation (malekkhaiat häffner and malmsten, ). various chemical modifications of amps, including residue phosphorylation, the addition of d-amino acids or unnatural amino acids (homoarginine), cyclization, halogenation, acetylation, and peptidomimetics, have been used to improve the stability of peptides against proteases. given that the enzyme is stereospecific, the incorporation of unnatural d-amino acids into the amp sequence can reverse the stereochemistry and prevent protease degradation (zhong et al., ) . the so-called peptidomimetics, whose main elements mimic the structure of peptides, are usually produced by modifications, such as chain extension or heteroatom incorporation of existing peptides (patch and barron, ) . ornine, which is an unnatural residue with a positive charge and has a high resistance to protease activity, is also used in non-chemical modification. replacing trp residues with family residues, such as β-dihydrophenylalanine, can stabilize secondary structures and improve antibacterial properties (maurya et al., ) . halogenation is highly related to the activity, specificity, and stability of amps. in the latest report, halogen is introduced into jelleine-i which is a short peptide isolated from the royal jelly of honeybees (apis mellifera) by replacing phenylalanine with a halogenated phenylalanine analog, increasing the antibacterial activity in vitro and anti-biofilm activity. in addition, the proteolytic stability of jelleine- is increased by - times by halogenation (jia et al., ) . the halogenated peptidomimetic α,α-disubstituted β-amino amides are also promising bacteriostatic drugs that have inhibitory effects on more than multi-resistant clinical isolates of gram-positive and gram-negative bacteria (paulsen et al., ) . halogenation is also related to the specificity of amps. the o-fluorine substitution in phenylalanine residues maintains the activity of temporin l on e. coli but leads to the loss of activity on s. aureus and p. aeruginosa (setty et al., ) . three modes of cyclisation, including cyclisation via disulfide bonds, head-to-tail cyclisation and internal bonding between side chains, have been found in natural amps. the synthesis of disulfide bonds often complicates the development of synthetic peptides. the circularisation of the main chain of arenicin- molecule resulted in increased activity against drug-resistant clinical isolates but caused no substantial effect on cytotoxicity (orlov et al., ) . the hdps tachyplesins i, ii, and iii and their cyclic analogs cti, ctii, and ctiii, respectively, have similar structures and activities and can resist bacterial and cancer cells. the cyclisation of the backbone reduces the hemolytic activity and improves the stability of the peptides whilst maintaining effective anticancer and antibacterial activities (vernen et al., ) . capping refers to the addition of specific motifs or modifications, such as amidation at the c-terminus and acetylation at the n-terminus, rendering amps with more natural peptide characteristics. post-translational modifications play an important role in the function of amps and are the most commonly used in peptide design. the c-terminal rana box (consisting of a c-terminal cyclic heptapeptide with a conservative disulfide bond) and amide group are important c-terminal capping methods. for example, the c-terminal amide group of maximin h can enhance antibacterial efficacy without increasing lytic ability (dennison et al., ) . the n-terminal lipidated analog c vg krkp shows enhanced antibacterial activity against various gram-negative bacteria. the functions of n-terminal lipidation include (i) increasing lps neutralization, (ii) increasing stability to proteases and peptidases, and (iii) reducing cytotoxicity (datta et al., ) . furthermore, hydrophobic end labeling is a commonly used method to increase the activity of antimicrobial peptides. acyl lipid peptides have a linear or cyclic structure in which one or more hydrocarbon tails are connected to the n-terminus of a short oligopeptide (chu-kung et al., ) . lipopeptides have covalently attached hydrophobic moieties, such as sterols or fatty acids. aromatic amino acid terminal labeling is also the main hydrophobic terminal labeling method. tryptophan (w) and phenylalanine (f) are the commonly used aromatic amino acids. their large and polarisable residues have an affinity for the interface, and the w/f tag is also sensitive to the differences between ergosterol and cholesterol and can prevent self-assembly. this condition results in low aggregation numbers and high critical aggregation concentrations (schmidtchen et al., ) . peptide conjugation has been the goal of most research in recent years to produce active and stable amps with high selectivity. different side chains or amp fragments can be used aside from the repetition of the same amino acid motifs. for example, conjugating fatty acids with a length of - carbon atoms to the th or th side chain of the d-amino acids of ano-d , improves antibacterial selectivity and anti-biofilm activity. in addition, the new peptide exhibits high stability against trypsin, serum, salt, and different ph environments (zhong et al., ) . the conjugation of different amps can also be performed. for example, the hybrid peptide (pa -gnu ) constructed by the addition of pa to gnu has a high activity and specificity to p. aeruginosa . smamps include a broad family of molecular entities based on the structure and function of amps. however, their backbones are not entirely based on α-amino acids, including β-amino acid oligomers, arylamide oligomers, and phenylene ethynylenes (michael henderson and lee, ) . for instance, smamp , which is a potential drug for intravenous treatment, causes no drug resistance and has a strong inhibitory effect on mrsa and vancomycin-resistant enterococcus faecium (tew et al., ) . peptoids are peptide isomers, in which the side chain is bonded to the main chain nitrogen instead of α-carbon or poly-nsubstituted glycine in which the side chain is connected to amide nitrogen instead of the α-carbon on the main chain (andreev et al., ) . for example, the cationic peptide sa (iowagolfolfo-nh ) and its poly-n-substituted glycine homolog spo (ninonwnangnonlnfnonlnfno-nh ) inhibit the planktonic and biofilm formation of a. baumannii strains, which are susceptible to multi-drug resistance (sharma et al., ) . motifs with specific functions have been reported increasingly. these motifs can be repeated units for combining into new antimicrobial peptides, or specific amino acid combination units appearing at the end (such as capping) of or even in the peptide chain. this motif includes two tripeptide structures, including gly-gly-his or val-ile-his, which are added at the end of the peptide chain. atcun-containing amps in the presence of hydrogen peroxide and ascorbic acid combine with cu + to induce the valence of copper ions between + and + oxidation states and form an atcun-cu (ii) complex, generating ros by fenton-like reactions. extracellular polymeric substances (eps) are important for biofilms and can enhance the resistance of cells to antibacterial agents (flemming, ) . atcun-amps have been used to degrade environmental dna, which is one of the major components of eps. several related practical applications have been reported. for example, the biological activity against carbapenem-resistant enterobacteriaceae is increased by adding this motif to the n-terminus of an alpha-helical amp (such as cm ). besides, the cu-atcun derivative of ov- containing a c-terminal ggc sequence showed high levels of membrane permeation and lipid peroxidation. the concept of catalytic metal drugs has attracted widespread attention although the concept is still in its infancy because of the role of metal ions (alexander et al., ; agbale et al., ) . rana box: rana box is a heptapeptide motif (cglxglc) from the nigrocin family. rana box consists of two cysteine residues that are separated by four or five other residues on the side and can form a cyclic disulfide bond. rana box peptide has shown structural analogies with polymyxin (colistin), and the primary structure of the rana box motif is important in determining bacteriostatic activity (kozić et al., ) . the deletion of the 'rana box' motif will cause the amp antibacterial effect to disappear, but replacing the natural 'rana box' sequence of amps with amidated phenylalanine can expand its efficacy against antibiotic-resistant microorganisms, including mrsa and p. aeruginosa, and reduce cytotoxicity. this phenomenon also shows that the effect of the motif on amps needs to be determined based on the specific situation and is not completely beneficial (bao et al., ) . the lps binding motif (g-wkrkrf-g) can produce a broad spectrum of antibacterial activity when introduced into the c-terminus of temporin- ta and temporin- tb (close isoforms of temporin) (mohanram and bhattacharjya, ) . antifungal peptides have a conserved gxc(x − ) c γ-core motif (residues - , gkcykkdnic; d-isomer) at its n-terminus, which is a cation part of the ring. this conserved motif interferes with the integrity of the plasma membrane of the cell (yount and yeaman, ) . conserved γ-core motifs are directly involved in protein-membrane interactions and strongly contribute to membrane binding (utesch et al., ) . if replace d-phe -pro sequence in peptide chain with d-phe- -abz turn motif ( -abz is an abbreviation of -aminobenzoic acid d-amino acid) in amp tyrc a, and nuclear magnetic resonance shows that this change retains the β-hairpin structure. unlike the traditional β-turn motif, the d-phe- -abz motif can be used as a tool for β-hairpin libraries. the hydrophobic peptide can be formed into the nucleated β-hairpin formation by adding the d-phe- -abz motif. moreover, the inclusion of this part in two designed cationic amphiphilic peptides can produce broadspectrum antibacterial activity and low hemolysis rate (cameron et al., ; cameron et al., ) . the ngr motif is composed of asn-gly-arg, and amps with this structure have strong cytotoxicity ( table ). the data indicate that the new amps containing ngr may bind to cd + or αvβ + tumor cells by binding to cd or αvβ , respectively, to exert anti-tumor activity, especially on cd + tumor cells . the central gxxxg motif can induce strong self-assembly and have been already used in the design of amps (brosig and langosch, ; krauson et al., ) . bovine lactoferrin b is an amp composed of amino acid residues and has antibacterial, antifungal, and antiparasitic activities. the multivalent molecules lfcinb ( - ) and lfcinb ( - ) contain the lfcinb ( - ) motif (rrwqwr) and show inhibition activity against e. coli, p. aeruginosa, and s. aureus. chimeric peptide chimera containing two motifs, namely, the rrwqwr of lfcinb ( - ) and the rllr of bfii computer design includes simple statistical modeling, structureactivity relationships study (abdel monaim et al., ) , neural networks (müller et al., ) , deep learning (veltri et al., ) , word embedding (hamid and friedberg, ) and machine learning. for example, a machine learning method by matlab is proposed based on the concept of scoring the contribution of each amino acid's antibacterial activity (wu x. et al., ) . the genetic algorithm was used to design the amphiphilic α-helical peptide guavalin , which has an uncommon amino acid composition (three tyrosine and three glutamine residues) and interestingly causes membrane hyperpolarization, which is a different mechanism from those of other amps (porto et al., ) . two research methods have been developed based on the research background of quantitative structure-activity relationships: prediction method based on amp therapeutic index and the identification of novel potential amps from the expressed sequence tag database based on the principles of the highly conserved signal peptide subclasses related to amps (juretić et al., ) . in this way, a variety of amp variants can be obtained. if combined with high-throughput screening, it can effectively obtain the desired amp. for instance, some new amps are designed by the combinatorial peptide library of melittin and show higher activity and lower cytotoxicity (krauson et al., ) . cations, such as na + and mg + , may affect amp activity . however, the different valences of metal ions have varied effects on amps. for example, divalent cations show stronger antagonism to bacteria than monovalent cations with thanatin and s-thanatin, which are insect amps (wu et al., ) . in the presence of nacl, the signal response during the association phase remarkably decreased in single-cycle and multi-cycle kinetic experiments, resulting in a decreased association rate. this occurrence may be caused by the shielding effect of nacl between the cationic peptide and the zwitterionic membrane. another possible reason is that na + can bind to the phospholipid bilayer, where the ions interact with the phosphate and the carbonyl oxygen of lipid head groups (sabapathy et al., ) . the reduced activity of synthetic peptide [rllr] under high salt concentration is possibly caused by the destruction of its α-helix structure. table shows that several amps, including histatin, myxinidin, and hepcidin, contain atcun motifs (amino terminal copper and nickel with xxh sequence). iron is the most abundant metal ion in human saliva, but the combination with this metal ion results in the loss of the α-helix of histatin and greatly reduces its antifungal activity (puri et al., ) . however, the coordination of copper (ii) and nickel (ii) ions can induce the formation of ros, which is essential for bactericidal activity (jeżowska-bojczuk and stokowa-sołtys, ). anionic amps have a large number of negatively charged aspartic and glutamic acid residues (lakshmaiah narayana and chen, ) . they require zinc as a functional cofactor and the zinc complex shows stronger antibacterial activity (jiang et al., ) . several of these amps use metal ions to form cationic salt bridges with the negatively charged components of the microbial membrane to penetrate the membrane. anionic amps may attach to ribosomes or inhibit ribonuclease activity when in the cytoplasm (jeżowska-bojczuk and stokowa-sołtys, ). metal ions also affect the self-assembly of peptides. these ions can recognize specific amino acids, such as lysine and glutamic acid, and may form salt bridges between peptide molecules to induce peptide self-assembly. for example, zn + can stabilize the aggregation of peptides on the cell membrane, which results in the enhanced antibacterial effect of dcd- l in the presence of zn + (tian et al., ) . ph many amps are stable and retain their antimicrobial activity in a wide ph range. amps have enhanced activity at low ph because of their basic properties. this condition is related to the protonation of histidine at acidic ph, which promotes electrostatic interactions with anionic surfaces, including lps and the anions of phospholipids, and subsequently enhances antibacterial properties. the effect of ph on the antibacterial activity of amps varies. for example, thanatin's activity at neutral ph is slightly higher than that under acidic conditions. by contrast, the activity of xylan on e. coli, listeria, and c. albicans is remarkably higher at ph . than at ph . (holdbrook et al., ) . the inactivation of the histidinecontaining amp c g-his under low ph conditions involves ph-dependent changes in the state of the aggregates in the solution, because the aggregates, which are sensitive to ph and lipid composition, may be affected by binding and conformation. peptides can also enhance bacterial membrane permeability at low ph (hitchner et al., ) . thrombin-derived c-terminal peptides (tcps) will also change the mode of cd (a protein that is abundant in human plasma) from anti-inflammatory mode to bacterial elimination mode from ph . to ph . (holdbrook et al., ) . a dimer (e.g., p- ) can be created to provide amps with resistance to a higher ph range. the sensitivity of this ph-sensitive amp can be used to achieve a certain targeting effect in practical applications. in addition, charge interaction is one of the most important factors in peptide self-assembly. ph affects the charge state of amino acid and substituent functional groups. therefore, adjusting the ph is the most common method for controlling peptide assembly and disassembly (tian et al., ) . proteases have a strong destructive effect on amps. for instance, ll- , which has the strongest inhibitory effect on chlamydial infection, is inhibited by the protease chlamydial protease-like activity factor (cpaf) secreted by chlamydia (tang et al., ) . studies have been focused on the design of amp carriers to solve this problem (lewies et al., ; nordström et al., ) . the presence of chitosan-silica solid support of kr- peptide can protect it be hydrolyzed by α-trypsin, and the degree of protection is increased by % compared with the free kr- (diosa et al., ) . however, several enzymes, such as protease , esterase and phosphatase , cut the blocking group of the peptide and trigger the self-assembly of the peptide, which positively affects amps (tian et al., ) . antimicrobial peptides can regulate pro-inflammatory reactions, recruit cells, stimulate the proliferation of cells, promote wound healing, modify gene expression and kill cancer cells to participate in the immune regulation of human skin, respiratory infections, and inflammatory diseases (de la fuente-núñez et al., ) . for example, α-defensins hnp- , hnp- , and hnp- showed effective antibacterial activity against adenovirus, human papilloma virus, herpes virus, influenza virus and cytomegalovirus. pulmonary diseases, such as idiopathic pulmonary fibrosis, alveolar proteinosis, and acute respiratory distress syndrome, show elevated levels of amps (guaní-guerra et al., ) . likewise, amps secreted by the paneth cells in the mammalian gut are important to shape the gut microbiota (bevins and salzman, ) . the application of amps in medicine, such as dental, surgical infection, wound healing and ophthalmology is developing now. but there are only three amps that have been approved by fda including gramicidin, daptomycin, and colistin. dental caries, endodontic infections, candidiasis, and periodontal disease are common diseases in the human oral cavity. dental caries is a prevalent oral disease and some acidogenic bacteria like streptococcus sp. are the main cariesassociated pathogens (izadi et al., ) . several amps have good application potential. for instance, peptide zxr- (fkiggfikklwrslla) has shown potent activities against pathogenic bacteria of dental caries, streptococcus mutans, streptococcus sobrinus, and porphyromonas gingivalis and peptide pac- (clinical trial identifier: nct ) that has been sold over the counter in taiwan for treating oral candidiasis (chen l. et al., ) . in surgical infection and wound healing: surgical infection occurs after surgery, burns, accidental injury, skin disease, and chronic wound infections have a serious hazard to human life (thapa et al., ) . several amps have shown the therapeutic potential of these diseases. for example, amp pxl shows pronounced efficacy as an anti-infective agent in burn wounds in mice and amp d a has been in the third phase of clinical trials for treating burn wound infections (björn et al., ) . in ophthalmology: human eyes are prone to be infected by several organisms including bacteria and fungi in which s. aureus, streptococcus pneumoniae, p. aeruginosa, aspergillus spp., and c. albicans are the most relevant pathogens (silva et al., ) . although amps such as lactoferricin b, protegrin- exhibited antimicrobial activity against these pathogenic bacteria, their application in the field of ophthalmology is only at the theoretical stage. with the popularity of contact lenses and the increase in cases of related eye infections, antimicrobial peptides have shown good application prospects in ophthalmology (khan and lee, ) . additional methods need to be performed for the application of amps as drugs in medicine. the main strategies include ( ) constructing precursors to reduce cytotoxicity and improve protease stability, ( ) using amps in combination with existing antibacterial agents, ( ) inducing the correct expression of amps with appropriate drugs and using engineering probiotics as vectors to express amps. for example, in the field of wound repair, different formulation strategies, such as loading amps in nanoparticles, hydrogels, creams, gels, ointments, or glutinous rice paper capsules, have been developed to effectively deliver amps to the wound (borro et al., ; thapa et al., ) . in recent research, the sponges developed from modified starch and hs-peg-sh are covalently immobilized with amp showed effective antibacterial activity (yang et al., ) . more technical means, including pheromone-labeled amps, local environment-triggered amps (enzyme precursor drug release system, ph-activated amps, etc.), have been developed to improve the targeting mechanism of amps. furthermore, nanotubes, quantum dots, graphene, and metal nanoparticles have been proposed to be a potential method to enhance drug delivery of amps (magana et al., ) . hybrid peptides have also been used to build targeting peptides. for example, pa , which is a p. aeruginosa-targeting peptide, was combined with gnu (a broad-spectrum amp) to construct a hybrid peptide (pa -gnu ) that targets oprf protein and has good bactericidal activity and specificity . furthermore, some antibiotics, for instance, daptomycin (a lipopeptide), lugdunin which is a -membered cyclic peptide consists of amino acid residues plus a thiazolidine moiety and telavancin (a glycopeptide) have been widely used for the clinic (durand et al., ; lampejo, ) . although they are antibiotics, they have provided broader ideas for the design of amps. food preservatives have potential harm to the human body. therefore, natural preservatives are being advocated by more people. amps have a good inhibitory effect on common bacteria and fungi in food, and many amps are resistant to acids, alkalis, and high temperatures are easily hydrolyzed by proteases in the human body. thus, amps are a promising alternative to preservatives. nisin is a bacteriocin produced by l. lactis subspecies. lactic acid bacteria have been widely used as food preservatives. nisin is categorized as generally recognized as safe (gras) by the us food and drug administration (fda) and is used as a food preservative in other countries (khan and oh, ) . however, only nisin and polylysine are currently approved by the fda as food additives (santos et al., ) . pedocin pa- , a bacteriocin consisting of amino acids produced by a diplococcus, is also used as a food preservative and is sold on the market under the trade name alta . pedocin pa- is used as a food additive to inhibit the growth of l. monocytogenes, which can cause meat deterioration (settanni and corsetti, ) . enterocin as- is an amp used to preserve cider, fruit and vegetable juices, and enterocin ccm is used to preserve soy milk (rai et al., ; santos et al., ) . encapsulating bacteriocins into liposomes is a new method used to overcome the problems of amps in food applications (such as proteolytic degradation or interaction with food ingredients) (da silva malheiros et al., ) . moreover, active packaging by adding amps is a novel packaging method that has great potential in the food industry. for instance, ε-poly-l-lysine is used in conjunction with starch biofilms to show good inhibitory effects on aspergillus parasiticus (aflatoxin producer) and penicillium expansum and nisin have the potential to be dairy preservative because it is a highly surface-active molecule (luz et al., ) . the european union banned the use of animal growth promoters in animal feed in . thus, a new antibacterial strategy is needed. many amps are the potential to be used in poultry, swine, and ruminants breeding and aquaculture because they can improve production performance (liu et al., ; bao et al., ) , immunity and promote intestinal health and some of them have a stronger inhibitory effect on bacterial inflammation if used with antibiotics cote et al., ) . for example, siamp has a good effect on the treatment of ibv in chicken (sun et al., ) . by adding swine gut intestinal antimicrobial peptides (sgamp), broilers showed higher average daily gain and feed efficiency under chronic heat stress conditions (hu et al., ) . frog caerin . , european sea bass dicentracin and nk-lysine peptides (nklps) have good inhibitory effects on nodavirus, septicaemia haemorrhagic virus, infectious pancreatic necrosis virus and spring viremia carp virus, which are devastating to fish farming (león et al., ) . the amp in soybean meal fermented by b. subtilis e effectively inhibits v. parahaemolyticus and vibrio alginolyticus and enhances the resistance level of litopenaeus vannamei against v. parahaemolyticus when added to feeds (cheng et al., ) . for agriculture, the plant pathogenic infection of bacteria and fungi causes the loss of economy, for instance, aspergillus flavus infection of corn and peanuts, citrus green mold caused by penicillium digitatum, gray mold disease caused by botrytis cinerea on strawberries and geotrichum citriaurantii infection of citrus fruit all cause great harm to the growth and post-harvest of agricultural products (liu et al., ; liu et al., ) . several afps have shown prospect to control these problems. however, the practical application of antimicrobial peptides in the transportation and preservation of agricultural products is still lacking, because the use of antimicrobial peptides will greatly increase the cost in the transportation of fruits and vegetables (application examples of amps in these four fields are shown in table ). antimicrobial peptides constitute a global research hotspot, but many key issues in design and application need to be solved urgently. several restrictive factors hinder the application of amps. the interaction of multidisciplinary subjects, such as biology, materials science, chemistry, bioinformatics, molecular informatics and pharmacy can further develop prospective amps. computer molecular dynamics simulation, cell membrane simulation, and more methods are being applied to study the mechanism of amps. how to further understand the correlation between amps and various targets instead of conducting one-sided experimental research might improve experimental designs to obtain stronger systemic and scientific demonstrations. on this basis, further animal experiments are required instead of simple cell-level experiments to test the effect of amps under complex physiological conditions. several complicated methods, such as the chemical method of peptidomimetics and non-natural amino acid modifications, have been applied in designing amps to solve the problem of protease hydrolysis. most methods use chemical substrates, but the cost of these methods cannot be ignored in practice. in addition, chemical synthesis and the use of engineered bacteria are currently the mainstream for such procedures. finding a better biological preparation method, reducing the cost and increasing the yield is important problems in practical application. furthermore, studying the amp expression of the organism itself and finding a better expression vector are necessary for mass production in the future as more amps in nature are discovered. further research is needed on the reported amps to solve the problem on structure-function relationship. as a branch of peptide drugs, amps need to progress with the advancement of medical science against the background of the current low success rate of the clinical application of amps. more attention can be 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harvested strawberries biological activity and structural aspects of pgla interaction with membrane mimetic systems membrane pore-formation correlates with the hydrophilic angle of histidine-rich amphipathic peptides with multiple biological activities the first antimicrobial peptide from sea amphibian regulation of cell division in e. coli antimicrobial packaging based on ε-polylysine bioactive film for the control of mycotoxigenic fungi in vitro and in bread nucleation and growth of pores in , -dimyristoyl-sn-glycero- -phosphocholine (dmpc) / cholesterol bilayer by antimicrobial peptides melittin, its mutants and cecropin p in vitro and md simulation study to explore physicochemical parameters for antibacterial peptide to become potent anticancer peptide antimicrobial peptides of the vaginal innate immunity and their role in the fight against sexually transmitted diseases the value of antimicrobial peptides in the age of resistance influence of self-assembly on the performance of antimicrobial peptides kappacin, a novel antibacterial peptide from bovine milk temporins, small antimicrobial peptides with leishmanicidal activity the host antimicrobial peptide bac - binds to bacterial ribosomal proteins and inhibits protein synthesis the dolphin proline-rich antimicrobial peptide tur a inhibits protein synthesis by targeting the bacterial ribosome proline-rich peptides with improved antimicrobial activity against e. coli, k. pneumoniae, and a. baumannii insilico alpha-helical structural recognition of temporin antimicrobial peptides and its interactions with middle east respiratory syndromecoronavirus structural characterization of lacticin , a two-peptide lantibiotic with synergistic activity anti-diabetic potential of peptides: future prospects as therapeutic agents translocation of a channel-forming antimicrobial peptide, magainin , across lipid bilayers by forming a pore an antimicrobial peptide, magainin , induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation role of the escherichia coli sbma in the antimicrobial activity of proline-rich peptides mechanism of action of novel synthetic dodecapeptides against candida albicans anti-inflammatory effects of egg yolk livetins (α, β, and γ-livetin) fraction and its enzymatic hydrolysates in lipopolysaccharideinduced raw . macrophages promising antimicrobial agents designed from natural peptide templates lollipop'-shaped helical structure of a hybrid antimicrobial peptide of temporin b-lipopolysaccharide binding motif and mapping cationic residues in antibacterial activity antifungal activity determination for the peptides generated by lactobacillus plantarum te against aspergillus flavus in maize seeds recurrent neural network model for constructive peptide design current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review epinecidin- , a highly potent marine antimicrobial peptide with anticancer and immunomodulatory activities molecular basis of bacterial outer membrane permeability revisited. microbiol degradable dendritic nanogels as carriers for antimicrobial peptides myticusin-beta, antimicrobial peptide from the marine bivalve bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer mode of action of linear amphipathic α-helical antimicrobial peptides ponericins, new antibacterial and insecticidal peptides from the venom of the ant pachycondyla goeldii redesigning arenicin- , an antimicrobial peptide from the marine polychaeta arenicola marina, by strand rearrangement or branching, substitution of specific residues, and backbone linearization or cyclization expression systems for heterologous production of antimicrobial peptides lipidation increases antiviral activities of coronavirus fusion-inhibiting peptides mimicry of bioactive peptides via nonnatural, sequence-specific peptidomimetic oligomers antimicrobial activity of amphipathic α,α-disubstituted β-amino amide derivatives against esbl -carba producing multi-resistant bacteria; effect of halogenation, lipophilicity and cationic character use of click chemistry for obtaining an antimicrobial chimeric peptide containing the lfcinb and buforin ii minimal antimicrobial motifs anti-biofilm peptides as a new weapon in antimicrobial warfare in silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design iron binding modulates candidacidal properties of salivary histatin antimicrobial peptides as natural bio-preservative to enhance the shelf-life of food anti-biofilm peptides: a new class of quorum quenchers and their prospective therapeutic applications antifungal peptides: to be or not to be membrane active antimicrobial peptides: premises and promises targeting leishmania major parasite with peptides derived from a combinatorial 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temporin l through introduction of fluorinated phenylalanine nkl- : a novel antimicrobial peptide derived from zebrafish nk-lysin that inhibits bacterial growth and enhances resistance against vibrio parahaemolyticus infection in yesso scallop cationic antimicrobial peptide and its poly-n-substituted glycine congener: antibacterial and antibiofilm potential against a. baumannii the unique antimicrobial peptide repertoire of stick insects molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers antimicrobial peptide cathelicidin-bf inhibits platelet aggregation by blocking protease-activated receptor . intern anti-yeast activity and characterisation of synthetic radish peptides rs-afp and rs-afp against food spoilage yeast dairy-derived antimicrobial peptides: action mechanisms, pharmaceutical uses and production proposals the importance of antimicrobial peptides and their potential for therapeutic use in ophthalmology comparative lipidomics of azole sensitive and resistant clinical isolates of candida albicans reveals unexpected diversity in molecular lipid imprints a molecular docking study revealed that synthetic peptides induced conformational changes in the structure of sars-cov- spike glycoprotein, disrupting the interaction with human ace receptor integrated evolutionary analysis reveals antimicrobial peptides with limited resistance antimicrobial activity of short arginine-and tryptophan-rich peptides mechanism of antimicrobial action of indolicidin swine intestine antimicrobial peptides inhibit infectious bronchitis virus infectivity in chick embryos chlamydiasecreted protease cpaf degrades host antimicrobial peptides antimicrobial peptides from different plant sources: isolation, characterisation, and purification antimicrobial activity of an abiotic host defense peptide mimic topical antimicrobial peptide formulations for wound healing: current developments and future prospects role of peptide selfassembly in antimicrobial peptides peptide design principles for antimicrobial applications variants of self-assembling peptide, kld- that show both rapid fracture healing and antimicrobial properties a computational modeling approach predicts interaction of the antifungal protein afp from aspergillus giganteus with fungal membranes via its γ-core motif synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the rwqwr motif against gram-positive and gram-negative strains deep learning improves antimicrobial peptide recognition characterization of tachyplesin peptides and their cyclized analogues to improve antimicrobial and anticancer properties antiviral peptides as promising therapeutic drugs evolutionary plasticity of insect immunity ionpair-π interactions favor cell penetration of arginine/tryptophanrich cell-penetrating peptides human antimicrobial peptides and proteins high specific selectivity and membrane-active mechanism of the synthetic centrosymmetric α-helical peptides with gly-gly pairs antimicrobial peptides: promising alternatives in the post feeding antibiotic era control of green and blue mold and sour rot in citrus fruits by the cationic antimicrobial peptide paf rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease heat shock proteins (hsp , , , and ) in galleria mellonella (lepidoptera) hemolymph are affected by infection with conidiobolus coronatus (entomophthorales) peptide-based cancer therapy: opportunity and challenge effects of cations and ph on antimicrobial activity of thanatin and s-thanatin against escherichia coli atcc and b. subtilis atcc in vitro and in vivo activities of antimicrobial peptides developed using an amino acidbased activity prediction method inhibition of sars-cov- (previously -ncov) infection by a highly potent pancoronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion fabricating antimicrobial peptide-immobilized starch sponges for hemorrhage control and antibacterial treatment multidimensional signatures in antimicrobial peptides secretory production of antimicrobial peptides in escherichia coli using the catalytic domain of a cellulase as fusion partner comparative study of different forms of jellein antimicrobial peptide on leishmania parasite identification and biochemical characterization of a new antibacterial and antifungal peptide derived from the insect sphodromantis viridis alpha-helical antimicrobial peptides-using a sequence template to guide structure-activity relationship studies antimicrobial activity and mechanism of the human milk-sourced peptide casein an antimicrobial peptide containing ngr motif has potent antitumor activity against cd + and cd -tumor cells antimicrobial peptides conjugated with fatty acids on the side chain of d-amino acid promises antimicrobial potency against multidrug-resistant bacteria isolation and structure of corticostatin peptides from rabbit fetal and adult lung characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity we thank the national key r&d program of china ( yfd ). key: cord- - y kkmby authors: pessi, antonello title: cholesterol‐conjugated peptide antivirals: a path to a rapid response to emerging viral diseases date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: y kkmby while it is now possible to identify and genetically fingerprint the causative agents of emerging viral diseases, often with extraordinary speed, suitable therapies cannot be developed with equivalent speed, because drug discovery requires information that goes beyond knowledge of the viral genome. peptides, however, may represent a special opportunity. for all enveloped viruses, fusion between the viral and the target cell membrane is an obligatory step of the life cycle. class i fusion proteins harbor regions with a repeating pattern of amino acids, the heptad repeats (hrs), that play a key role in fusion, and hr‐derived peptides such as enfuvirtide, in clinical use for hiv, can block the process. because of their characteristic sequence pattern, hrs are easily identified in the genome by means of computer programs, providing the sequence of candidate peptide inhibitors directly from genomic information. moreover, a simple chemical modification, the attachment of a cholesterol group, can dramatically increase the antiviral potency of hr‐derived inhibitors and simultaneously improve their pharmacokinetics. further enhancement can be provided by dimerization of the cholesterol‐conjugated peptide. the examples reported so far include inhibitors of retroviruses, paramyxoviruses, orthomyxoviruses, henipaviruses, coronaviruses, and filoviruses. for some of these viruses, in vivo efficacy has been demonstrated in suitable animal models. the combination of bioinformatic lead identification and potency/pharmacokinetics improvement provided by cholesterol conjugation may form the basis for a rapid response strategy, where development of an emergency cholesterol‐conjugated therapeutic would immediately follow the availability of the genetic information of a new enveloped virus. copyright © european peptide society and john wiley & sons, ltd. the spectrum of infectious diseases that threaten human health is rapidly broadening: the number of previously unknown conditions that have emerged since exceeds , with a new disease discovered on average more than once a year [ , ] . according to a world health organization (who) report, between the years and , a record of worldwide epidemic events occurred [ ] . a comprehensive examination of episodes of emerging infectious disease outbreaks between and , after controlling for reporting bias, came to the conclusion that the incidence of the events has increased significantly over time [ ] . for viral diseases in particular, in addition to the constant fear of a new wave of pandemic influenza, the list of emerging or reemerging viruses includes hiv [ ] . at the time when this manuscript was being finalized, the largest and longest outbreak of ebola virus was still spreading [ ] . the latter virus, together with nipah virus, hantavirus, lassa virus, and marburg virus, is on the centers for disease control and prevention list of bioterrorism agents. for most of these viruses, the therapeutic options are limited if not completely absent. the best description of the features of an outbreak of a novel virus comes from the sars pandemic of / the first major emerging disease threat of the st century. in the short period between the who global alert (march ) and the who announcement that the pandemic was over ( july ) , sars affected people, caused deaths, disrupted international travel, and cost huge business losses. sars presented many of the features of the most feared pandemic diseases: a respiratory disease that is transmitted from person to person, with a long asymptomatic incubation period, and symptoms very similar to other commonplace illnesses. at the time of its first appearance, its causative agent (sars-associated coronavirus) was unknown, and there was neither a diagnostic test nor a specific treatment. the international response to the new threat was extremely efficacious, leading to the isolation of sars-cov [ ] and sequencing of its genome in the record time of just weeks [ , ] . the measures that proved efficacious in containing the disease were isolation, quarantine, travel surveillance, increased personal hygiene, and personal protection equipment (masks, gloves, and eyeglasses). as to pharmacological treatments, however, a who expert panel review concluded that it was not possible to determine whether any of the treatments used for sars-infected patients did some benefit and suggested that some treatments might have actually been harmful [ ] . as the sars case vividly illustrates, our ability to quickly isolate and genetically fingerprint the causative agent of new viral diseases is not matched by an ability to develop suitable treatments. this is not surprising, because drug discovery typically requires additional information to that simply deriving from knowledge of the viral genome. against this background, peptides may represent a special case, because for enveloped viruses, it is generally possible to identify the sequence of candidate peptide inhibitors directly from genomic information. for all enveloped viruses, fusion between the viral and the target cell membrane represents an obligatory step of the life cycle. interfering with this process is a well-established therapeutic strategy [ ] that has led to the development of the peptide fusion inhibitor enfuvirtide (fuzeon ® , also known as t , roche, basel, switzerland), which is in clinical use for hiv [ ] . viral fusion is mediated by specialized proteins, which harbor a structural motif that consists of a repeating pattern of seven amino acids: the heptad repeat (hr). the most prevalent class (class i) of fusion proteins typically has two hr regions: the first one close to the n-terminus (hrn), adjacent to the fusion peptide, and the second one close to the c-terminus (hrc), immediately preceding the transmembrane domain. it is generally accepted that in the key intermediate of viral fusion, the so-called prehairpin intermediate, bridging the viral and cell membranes, the hrn and hrc regions are separated, and the hrn forms a trimeric coiled coil ( figure ). folding of the hrc onto the hrn trimer leads to the formation of a six-helical bundle ( hb), and in this process, the two membranes are brought in close apposition, which leads to their fusion [ ] (figure ). peptides corresponding to the sequence of the hr regions can bind to the prehairpin intermediate, prevent its transition to the hb, and block fusion (figure (a) ). this is the mechanism of inhibition of enfuvirtide [ ] , which applies to several other peptides derived from the hr regions of many viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the characteristic sequence pattern of the hr provides an important advantage for the development of peptide-based antivirals, because hr can be easily identified through computer programs (e.g. learncoil [ ] or multicoil [ , ] ). this was the case for sars-cov, where hr regions were immediately identified [ ] and led to the development of peptide inhibitors [ , ] , in the absence of any structural information on the fusion protein. this came later and largely confirmed the predictions [ ] [ ] [ ] [ ] . this pathway to drug development offers the opportunity to develop a specific antiviral in a very short timeframe. however, not all hr-derived peptides display the same antiviral potency as the hiv fusion inhibitor enfuvirtide. a major factor at play is the difference in fusion kinetics: viruses that unlike hiv transition through the hairpin intermediate very rapidly offer a shorter window of opportunity for peptide inhibitors that, accordingly, show reduced potency [ ] [ ] [ ] . if the peptide lead derived from genomic information has insufficient potency, it is necessary to modify it through peptide engineering strategies that, although often successful [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , may be time consuming. an alternative approach, which does not require any change in the native hr sequence, exploits the role of cholesterol in viral fusion. cholesterol plays a key role in the fusion and budding of enveloped viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , with hiv as the most studied case. it has been shown that cholesterol depletion from virus-infected cells suppresses virus production [ ] and that depletion of cholesterol from either virions or target cells inhibits virus-cell fusion [ ] [ ] [ ] [ ] . hiv is able to influence the cholesterol metabolism of the host cell [ ] [ ] [ ] [ ] , and its lipid membrane is highly enriched in cholesterol [ ] . moreover, a link has been established between low levels of cholesterol in antigen-presenting cells and the status of long-term nonprogressor, a rare infected individual who controls disease progression in the absence of antiretroviral therapy [ , ] . the role of cholesterol in viral fusion is related to its role in membrane fluidity and in particular in the formation of lipid rafts [ , [ ] [ ] [ ] [ ] [ ] . accordingly, the composition of the lipid membrane of hiv is highly enriched in cholesterol and sphingomyelin [ , , ] . moreover, cd , the primary receptor for hiv, is enriched in lipid rafts [ ] . in , ingallinella et al. proposed that addition of a cholesterol group to an hr-derived peptide would augment its affinity for membranes and in particular for the cholesterol-rich lipid rafts. the resulting increase in local concentration at the site of action would translate into enhanced antiviral potency (figure (b) ). application of this concept to the hr-derived peptide c yielded c -chol, where the cholesterol group was attached, via a thioether bond, to the side chain of a cys residue added to the c-terminus, with an intervening gly-ser-gly spacer. cholesterol conjugation brought a dramatic improvement of antiviral potency: depending on the viral strain tested, c -chol was -to -fold more potent than unconjugated c and -to -fold more potent than enfuvirtide [ ] . no such improvement was apparent when cholesterol was substituted with palmitic acid [ ] , a lipid that is both a poorer membrane anchor and is not enriched in lipid rafts [ ] . numerous examples have since accumulated that cholesterol conjugation can improve the antiviral potency of hr-derived peptides from many enveloped virusesthese include in vitro and in vivo studies (table ) . cholesterol conjugation has also been successfully applied to a covalent inhibitor derived from c , where an isothiocyanate group on the side chain of asp targets lys in hrn [ ] . interestingly, in line with the hypothesized mechanism of action of cholesterol conjugation, another lipid that is enriched in the hiv lipidome, dihydrosphingosine [ ] , also augments potency when conjugated to an hr peptide; in this case, one derived from the hrn instead of the hrc [ ] . cholesterol-conjugated peptides show improved pharmacokinetics and are active in vivo cholesterol conjugation also solves an outstanding problem of peptide therapeutics, the short in vivo half-life because of enzyme degradation and rapid clearance [ ] . an effective way to improve peptide pharmacokinetics (pk) is conjugation to lipids [ ] , which drive binding to serum proteins. the most typical derivatization is with long chain fatty acids, but derivatization with cholesterol has also been explored [ ] [ ] [ ] . in a comparative subcutaneous pk study in mice at the concentration of . mg/kg, c was undetectable in plasma after h, while nm of c -chol was still detectable h after the injection: a concentration ≈ -fold higher than the ic measured against multiple hiv- strains [ ] . in another example, a single dose of mg/kg of a cholesterolconjugated inhibitor of nipah virus, administered intraperitoneally to golden hamsters, was sufficient to achieve a plasma concentration > -fold higher than the in vitro ic for h [ ] . a single intramuscular injection of . mg/kg of a cholesterolconjugated inhibitor of newcastle disease virus (ndv) administered to chickens yielded a plasma concentration of nm at h and ≈ nm after h, to be compared with the in vitro ic = . nm and ic = nm [ ] . overall, the pk of a cholesterol-conjugated inhibitor is expected to be at least comparable withand likely better thanthe pk of enfuvirtide, which is administered twice daily by subcutaneous injection [ ] . one further possibility to potentiate the activity of hr-derived peptide inhibitors is to exploit multimerization, another modification that can be implemented on the native sequence of the peptide. multimerization brings an avidity effect [ ] , which effectively reduces the k off of the inhibitor fusion protein complex and increases the potency of fusion [ , ] and entry [ ] inhibitors. the effect of cholesterol conjugation, which increases the k on by facilitating the encounter of the peptide with the viral target protein, is complementary: the two modifications can therefore work in concert. a reagent that can simultaneously dimerize the peptide and install a cholesterol group onto it is described in figure [ ] . trimers or higher-order multimers could be prepared by the same strategy, but the maximum benefit of multimerization is achieved at the level of a dimer [ , ] . the reagent is stable upon prolonged storage at À °c. notably, the same cysteine-containing precursor peptide used to produce the monomeric cholesterol-conjugated peptide can be reacted with the cholesterol dimer-forming moiety: this enables parallel synthesis of the cholesterol-conjugated monomer and dimer (figure ) , which can then also be tested in parallel to establish the optimal inhibitor. using the reagent in figure , dimeric cholesterol-conjugated inhibitors were prepared for hiv [ ] , nipah [ ] , and measles [ ] virus (mv), which were more potent and effective than the corresponding cholesterol-conjugated monomers. potentiation of a trimeric hiv peptide inhibitor [ ] by cholesterol conjugation [ ] has independently been reported by another laboratory. since its discovery in , the scope of cholesterol conjugation has considerably increased, and it now includes examples for retroviruses, paramyxoviruses, coronaviruses, orthomyxoviruses, henipaviruses, and filoviruses (table ). for some of these viruses, in vivo efficacy has been demonstrated in a suitable animal model [ , , ] . for example, porotto et al. showed that once-daily intraperitoneal administration of a cholesterol-conjugated inhibitor of nipah virus to golden hamsters, an established model of infection, effectively prevented what would otherwise be fatal nipah virus encephalitis. prophylactic treatment was % efficacious when initiated days before viral challenge and % efficacious when initiated concurrently with the infection. notably, in light of the highly lethal nature of this virus, treatment days after infection led to survival of % of the animals [ ] . the same laboratory used intranasal infection of mv in suckling mice expressing the human slam transgene as a model to evaluate the efficacy of cholesterol-conjugated hr-derived peptides. infection with mv causes a lethal acute neurological syndrome, which was completely prevented, in % of the animals, by prophylactic once-daily administration of a cholesterol-conjugated dimer derived from the sequence of the mv hrc domain [ ] . li et al. used cholesterol conjugation to improve the potency of their previously described inhibitor of ndv [ ] . intramuscular injection of the cholesterol-conjugated peptide day before virus infection, and then every days, protected % of the chickens from different serotypes of ndv [ ] . moreover, treatment of the animals days after infection resulted in % protection [ ] . two interesting features of these studies deserve further comment: first, in the aforementioned three studies, the peptide was detectable in the brain h after administration [ , , ] , indicating that cholesterol conjugation may enable penetration of the blood-brain barrier, a difficult feat for drugs in general and for biologics in particular [ ] . second, the modest but observable degree of protection observed at days postexposure ( % in the porotto et al. [ ] and % in the li et al. [ ] study) is comparable with the results reported for monoclonal antibodies against ebola virus in macaques ( % efficacy when dosed days after exposure) [ , ] , including the zmapp antibody cocktail [ ] , which is being used as an emergency therapeutic in the latest ebola outbreak [ ] . it must be noted though that all the examples reported so far come from viruses featuring class i fusion proteins. while being the most prevalent class, it does not include major pathogens such as hepatitis c virus and dengue virus, both belonging to class ii, and herpes simplex virus belonging to class iii. class i proteins are mainly α-helical and form hbs as the postfusion structure, as exemplified by hiv (figure ). class ii fusion proteins are characterized by trimers of hairpins composed of β-structures [ ] , with a fusion loop located at the tip of an elongated β-sheet, in place of a fusion peptide upstream of an α-helix. class iii proteins form trimers of hairpins by combining a central α-helical trimeric core, similar to class i proteins, with fusion loops structurally similar to class ii proteins [ ] . despite these structural differences, all fusion proteins adopt a hairpin structure [ ] that is susceptible to inhibition. for example, although the entry of herpes viruses (featuring a class iii fusion protein) is a complex process, not yet fully clarified, in which multiple glycoproteins are involved, several laboratories have reported that peptides derived from the hr regions of gb and gh of herpes simplex virus type , bovine herpes virus type , and human cytomegalovirus are able to inhibit infection [ ] . for dengue virus, helical domains have been identified in the stem region of e protein (a class ii protein) that are critical for virus assembly and entry [ ] . peptide inhibitors derived from this membrane-binding region have been described [ ] [ ] [ ] , one of which inhibits all four virus serotypes [ ] . moreover, de novo computational design of peptide inhibitors has been successful [ , ] . the work by xu et al. in particular [ ] indicates how the same computational approach may yield candidate peptide inhibitors for fusion proteins of all classes. given the ubiquitous importance of cholesterol-rich lipid rafts in viral fusion, it is expected that cholesterol conjugation may enhance the antiviral potency also for peptides derived from class ii and iii fusion proteins. however, no data in this regard have yet been reported. the combination of bioinformatic lead identification (lead id) and potency/pk improvement provided by cholesterol conjugation may form the basis for a rapid response strategy to emerging viral diseases (figure ) . the project would begin immediately after the genome of the new pathogen is made available. the lead id phase would consist in the bioinformatic analysis (e.g. via learncoil [ ] or multicoil [ , ] ) of the newly sequenced genome to identify the hr regions; it could last a few days. as discussed in the previous section, the computational identification of inhibitory peptides for viruses utilizing class ii or iii fusion proteins would be less straightforward, and the number of candidate peptides to be tested likely larger. however, given the high throughput of parallel solid-phase figure . rapid response strategy based on cholesterol-conjugated fusion inhibitors. from the sequenced genome of the emerging virus, the hr regions are identified bioinformatically. this corresponds to the lead identification (lead id) phase. in the lead optimization (lead opt) phase, candidate hr peptides are synthesized in the form of suitable precursors for cholesterol conjugation and reacted with the reagents shown in figure . the cholesterol-conjugated monomer and dimer are tested for antiviral activity. the lead opt cycle continues until a peptide is found potent enough to be moved to large-scale production. peptide synthesis and the modularity of the cholesterol conjugation reaction, the initial number of target sequences should not represent a key limiting factor in the lead optimization (lead opt) phase. in lead opt, candidate peptides would be synthesized in the form of suitable precursors for cholesterol conjugation and reacted with the reagents shown in figure or similar ones. the cholesterol-conjugated monomers and dimers would be tested for antiviral activity. in the logic of off-the-shelf reagents for immediate use, variations of the reagents of figure to optimize the length of the linker joining the monomers might provide considerable increase in potency [ ] , as shown by kay et al. [ ] . haloacetyl or maleimide-functionalized cores with polyethylene glycol spacers of different length could all be reacted with the same precursor to rapidly select the most potent inhibitor. the lead opt cycle would continue until a peptide was found, potent enough to be moved to large-scale production. with a relatively small number of candidate peptides, and parallel synthesis and conjugation, it is expected that the lead opt phase would be rapid, optimally a few weeks. large-scale production of the candidate peptide therapeutic could begin immediately, because the protocol for lab-scale and large-scale solid-phase peptide synthesis are essentially the same. preclinical evaluation (formulation, animal pk to predict the human dose) could proceed in parallel with the good manufacturing practices batch production. abbreviated preclinical toxicology, in connection with the 'animal rule' [ ] , would be justified in an emergency situation. the strategy outlined in the previous section could provide in a very short timeframe a cholesterol-conjugated therapeutic specific for the emerging virus. although not necessarily the optimal drug for the new disease, it would represent a key complement to preventive measures and could enable better control of the initial outbreak. further research efforts may then lead to a substitute therapy with increased efficacy and/or better ease of administration. one could even envisage setting up a core laboratory that routinely runs the lead id and lead opt stages on newly identified viruses of potential concern, in advance of an actual outbreak of disease. the limited resources necessary could be part of the emergency and preparedness response set out by the centers for disease control and prevention (http://emergency.cdc.gov/) or the global alert and response by who (http://www.who.int/csr/en/) for preparing for and responding to public health emergencies. emerging and reemerging diseases: a historical perspective emerging viral 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and polymorphic interactions of two heptad-repeat regions of the sars virus s protein structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core sensitivity of hiv- to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics a quantitative and kinetic fusion protein-triggering assay can discern distinct steps in the nipah virus membrane fusion cascade kinetic dependence of paramyxovirus entry inhibition broad antiviral activity and crystal structure of hiv- fusion inhibitor sifuvirtide short-peptide fusion inhibitors with high potency against wild-type and enfuvirtide-resistant hiv- design of helical, oligomeric hiv- fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus design of a novel hiv- fusion inhibitor that displays a minimal interface for binding affinity remodeling of gp -c peptide leads to highly effective inhibitors of the fusion of hiv- with target cells inhibition of hiv type infectivity by constrained alpha-helical peptides: implications for the viral fusion mechanism short constrained peptides that inhibit hiv- entry hydrocarbon doublestapling remedies the proteolytic instability of a lengthy peptide therapeutic design and evaluation of sifuvirtide, a novel hiv- fusion inhibitor lipid rafts are involved in sars-cov entry into vero e cells cholesterol enhances mouse hepatitis virus-mediated cell fusion a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry the major cellular sterol regulatory pathway is required for andes virus infection lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles ebola virus entry requires the cholesterol transporter niemann-pick c cholesterol dependence of varicella-zoster virion entry into target cells influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion plasma membrane rafts play a critical role in hiv- assembly and release membrane raft microdomains mediate lateral assemblies required for hiv- infection role for human immunodeficiency virus type membrane cholesterol in viral internalization role of cholesterol in human immunodeficiency virus type envelope protein-mediated fusion with host cells nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type -infected t cells human immunodeficiency virus impairs reverse cholesterol transport from macrophages nef increases the synthesis of and transports cholesterol to lipid rafts and hiv- progeny virions nef mobilizes lipid rafts in macrophages through a pathway that competes with abca -dependent cholesterol efflux the hiv lipidome: a raft with an unusual composition alterations in cholesterol metabolism restrict hiv- trans infection in nonprogressors new clues to understanding hiv nonprogressors: low cholesterol blocks hiv trans infection evidence for budding of human immunodeficiency virus type selectively from glycolipid-enriched membrane lipid rafts galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions lipid composition and fluidity of the human immunodeficiency virus lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes role of lipid rafts in virus replication the pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation addition of a cholesterol group to an hiv- peptide fusion inhibitor dramatically increases its antiviral potency membrane targeting of lipid modified signal transduction proteins a multi-functional peptide as an hiv- entry inhibitor based on self-concentration, recognition, and covalent attachment sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant hiv- converting peptides into drug leads by lipidation structure-activity and protraction relationship of longacting glucagon-like peptide- derivatives: importance of fatty acid length, polarity, and bulkiness glucagon-like peptide /glucagon receptor dual agonism reverses obesity in mice dpp-iv-resistant, long-acting oxyntomodulin derivatives inhibition of nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry a cholesterol tag at the n terminus of the relatively broad-spectrum fusion inhibitory peptide targets an earlier stage of fusion glycoprotein activation and increases the peptide's antiviral potency in vivo enfuvirtide: a fusion inhibitor for the treatment of hiv infection dependence of avidity on linker length for a bivalent ligand-bivalent receptor model system potent d-peptide inhibitors of hiv- entry design of a potent d-peptide hiv- entry inhibitor with a strong barrier to resistance designed oligomers of cyanovirin-n show enhanced hiv neutralization a general strategy to endow natural fusion-protein-derived peptides with potent antiviral activity fatal measles virus infection prevented by brain-penetrant fusion inhibitors design of a modular tetrameric scaffold for the synthesis of membrane-localized d-peptide inhibitors of hiv- entry characterisation and evaluation of antiviral recombinant peptides based on the heptad repeat regions of ndv and ibv fusion glycoproteins drug transport across the blood-brain barrier successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies therapeutic intervention of ebola virus infection in rhesus macaques with the mb- monoclonal antibody cocktail experimental ebola drugs enter the limelight virus membrane fusion virus membrane-fusion proteins: more than one way to make a hairpin peptide inhibitors against herpes simplex virus infections the helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry peptide inhibitors of dengue-virus entry target a late-stage fusion intermediate peptide inhibitors of flavivirus entry derived from the e protein stem release of dengue virus genome induced by a peptide inhibitor structural optimization and de novo design of dengue virus entry inhibitory peptides computational identification of self-inhibitory peptides from envelope proteins new drug and biological drug products; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. final rule viral entry inhibitors targeted to the membrane site of action capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide, a new antiviral strategy for influenza virus c-peptide inhibitors of ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking key: cord- -hkvll xh authors: yang, zheng rong title: peptide bioinformatics- peptide classification using peptide machines date: journal: artificial neural networks doi: . / - - - - _ sha: doc_id: cord_uid: hkvll xh peptides scanned from whole protein sequences are the core information for many peptide bioinformatics research subjects, such as functional site prediction, protein structure identification, and protein function recognition. in these applications, we normally need to assign a peptide to one of the given categories using a computer model. they are therefore referred to as peptide classification applications. among various machine learning approaches, including neural networks, peptide machines have demonstrated excellent performance compared with various conventional machine learning approaches in many applications. this chapter discusses the basic concepts of peptide classification, commonly used feature extraction methods, three peptide machines, and some important issues in peptide classification. proteins are the major components for various cell activities, including gene transcription, cell proliferation, and cell differentiation. to implement these activities, proteins function only if they interact with each other. enzyme catalytic activity, acetylation, methylation, glycosylation, and phosphorylation are typical protein functions through binding. studying how to recognize functional sites is then a fundamental topic in bioinformatics research. as proteins function only when they are bound together, the binding sites (functional sites) and their surrounding residues in substrates are the basic components for functional recognition. studying consecutive residues around a functional site within a short region of a protein sequence for functional recognition is then a task of peptide classification that aims to find a proper model that maps these residues to functional status. a peptide classification model can be built using a properly selected learning algorithm with similar principles to those used in pattern classification systems. the earlier work was to investigate a set of experimentally determined (synthesized) functional peptides to find some conserved amino acids, referred in protease cleavage site prediction, we commonly use peptides with a fixed length. in some cases, we may deal with peptides with variable lengths. for instance, we may have peptides whose lengths vary from one residue to a couple of hundreds residues long when we try to identify disorder regions (segments) in proteins [ ] . figure . shows two cases, where the shaded regions indicate some disorder segments. the curves represent the probability of disorder for each residue. the dashed lines show % of the probability and indicate the boundary between order and disorder. it can be seen that the disorder segments have variable lengths. the importance of accurately identifying disorder segments is related to accurate protein structure analysis. if a sequence contains any disorder segments, x-ray crystallization may fail to give a structure for the sequence. it is then critically important to remove such disordered segments in a sequence before the sequence is submitted for x-ray crystallization experiments. the study of protein secondary structures also falls into the same category as disorder segment identification, where the length of peptides that are constructs for secondary structures varies [ ] . because the basic components in peptides are amino acids, which are nonnumerical, we need a proper feature extraction method to convert peptides to numerical vectors. the second section of this chapter discusses some commonly used feature extraction methods. the third section introduces three peptide machines for peptide classification. the fourth section discusses some practical issues for peptide classification. the final section concludes peptide classification and gives some future research directions. currently, three types of feature extraction methods are commonly used for converting amino acids to numerical vectors: orthogonal vector, frequency estimation, and bio-basis function methods. the orthogonal vector method encodes each amino acid using a -bit long binary vector with bit assigned a unity and the rest zeros [ ] . denoted by s a peptide, a numerical vector generated using the orthogonal vector method is x Î b ×| s | , where b = { , } and | s | is the length of s . the introduction of this method greatly eased the difficulty of peptide classification in the early days of applying various machine learning algorithms like neural networks to various peptide classification tasks. however, the method significantly expands the input variables, as each amino acid is represented by inputs [ , ] . for an application with peptides ten residues long, input variables are needed. figure . shows such an application using the orthogonal vector method to convert amino acids to numerical inputs. it can be seen that data significance, expressed as a ratio of the number of data points over the number of model parameters, can be very low. meanwhile, the method may not be able to properly code biological content in peptides. the distance (dissimilarity) between any two binary vectors encoded from two different amino acids is always a constant (it is if using the hamming distance or the square root of if using the euclidean distance), while the similarity (mutation or substitution probability) between any two amino acids varies [ - ] . the other limitation with this method is that it is unable to handle peptides of variable lengths. for instance, it is hard to imagine that this method could be used to implement a model for predicting disorder segments in proteins. an example of using the orthogonal vector for converting a peptide to numerical vector for using feedforward neural networks, where each residue is expanded to inputs. adapted from [ ] the frequency estimation is another commonly used method in peptide classification for feature extraction. when using single amino acid frequency values as features, we have the conversion as s ‫ۋ‬ x Î ᑬ , meaning that a converted vector always has dimensions. however, it has been widely accepted that neighboring residues interact. in this case, di-peptide frequency values as features may be used, leading to the conversion as s ‫ۋ‬ x Î ᑬ . if tri-peptide frequency values are used, we then have the conversion as s ‫ۋ‬ x Î ᑬ . this method has been used in dealing with peptides with variable lengths for peptide classification, for instance, secondary structure prediction [ ] and disorder segment prediction [ ] . one very important issue of this method is the difficulty of handling the large dimensionality. for any application, dealing with a data set with , features is no easy task. the third method, called the bio-basis function , was developed in [ , ] . the introduction of the bio-basis function was based on the understanding that nongapped pairwise homology alignment scores using a mutation matrix are able to quantify peptide similarity statistically . figure . shows two contours where one functional peptide (sknypivq) and one nonfunctional peptide (sdgngmna) are selected as indicator peptides. we use the term indicator peptides , because the use of some indicator peptides transform a training peptide space to an indicator or feature space for modeling. we then calculate the nongapped pairwise homology alignment scores using the dayhoff mutation matrix [ ] to calculate the similarities among all the functional/positive and these two indicator peptides as well as the similarities among all the nonfunctional/negative peptides and these two indicator peptides. it can be seen that positive peptides show larger similarities with the positive indicator peptide (sknypivq) from the left panel, while negative peptides show larger similarities with the negative indicator peptide (sdgngmna) from the right panel. we denote by s a query peptide and by r i the i th indicator peptide and each has d residue, the bio-basis function is defined as k(s, r ) = exp (s, r ) (r , r ) note that s j and r ij are the j th residues in the query and indicator peptides, respectively. the value of m ( s j , r ij ) can be found from a mutation matrix. figure . shows the dayhoff mutation matrix [ ] . from eq. , we can see that k ( s , the equality occurs if and only if s = r i . the basic principle of the bio-basis function is normalizing nongapped pairwise homology alignment scores. as shown in fig. . , a query peptide ( iprs ) is aligned with two indicator peptides ( kprt and ykae ) to produce two nongapped pairwise homology alignment scores a ( å = + + + = ) and b ( å = + + + = ) respectively. because a > b , it is believed that the query peptide is more likely to have the same functional status (functional or nonfunctional) as the first indicator peptide. after the conversion using the bio-basis function, each peptide s is represented by a numerical vector x Î ᑬ m or a point in an m -dimensional feature space, where m is the number of indicator peptides. note that m = in fig. . . the bio-basis function method has been successfully applied to various peptide classification tasks, for instance, the prediction of trypsin cleavage sites [ ] , the prediction of hiv cleavage sites [ ] , the prediction of hepatitis c virus protease cleavage sites [ ] , the prediction of the disorder segments in proteins [ , ] , the prediction of protein phosphorylation sites [ , ] , the prediction of the o-linkage sites in glycoproteins [ ] , the prediction of signal peptides [ ] , the prediction of factor xa protease cleavage sites [ ] , the analysis of mutation patterns of hiv- fig. . dayhoff matrix. each entry is a mutation probability from one amino acid (in rows) to another amino acid (in columns). reproduced from [ ] with permission drug resistance [ ] , the prediction of caspase cleavage sites [ ] , the prediction of sars-cov protease cleavage sites, [ ] and t-cell epitope prediction [ ] . as one class of machine learning approaches, various vector machines are playing important roles in machine learning research and applications. three vector machines-the support vector machine [ , ] , relevance vector machine [ ] , and orthogonal vector machine [ ] -have already drawn the attention for peptide bioinformatics. each studies pattern classification with a specific focus. the support vector machine (svm) searches for data points located on or near the boundaries for maximizing the margin between two classes of data points. these found data points are referred to as the support vectors . the relevance vector machine (rvm), on the other hand, searches for data points as the representative or prototypic data points referred to as the relevance vectors . the orthogonal vector machine (ovm) searches for the orthogonal bases on which the data points become mutually independent from each other. the found data points are referred to as the orthogonal vectors . all these machines need numerical inputs. we then see how to embed the bio-basis function into these vector machines to derive peptide machines. the support peptide machine (spm) aims to find a mapping function between an input peptide s and the class membership (functional status). the model is defined as the bio-basis function. as iprs is more similar to kprt than ykae , its similarity with kprt is larger that that with ykae , see the right figure. reproduced from [ ] with permission where w is the parameter vector, y = f ( s , w ) the mapping function, and y the output corresponding to the desired class membership t Î {- , }. note that - and represent nonfunctional and functional status, respectively. with other classification algorithms like neural networks, the distance (error) between y and t is minimized to optimize w . this can lead to a biased hyperplane for discrimination. in fig. . , there are two classes of peptides, a and b . the four open circles of class a and four filled circles of the class b are distributed in balance. with this set of peptides, the true hyperplane separating two classes of peptides, represented as circles, can be found as in fig. . (a) . suppose a shaded large circle belonging to class b as a noise peptide is included, as seen in fig. . (b) ; the hyperplane (the broken thick line) is biased because the error (distance) between the nine circles and the hyperplane has to be minimized. suppose a shaded circle belonging to class a as a noise peptide is included as seen in fig. . (c) ; the hyperplane (the broken thick line) also is biased. with theses biased hyperplanes, the novel peptides denoted by the triangles could be misclassified. in searching for the best hyperplane, the spm finds the set of peptides that are the most difficult training data points to classify. these peptides are referred to as support peptides . in constructing a spm classifier, the support peptides are closest to the hyperplane within the slat formed by two boundaries ( fig. . d ) and are located on the boundaries of the margin between two classes of peptides. the advantage of using an spm is that the hyperplane is searched through maximizing this margin. because of this, the spm classifier is robust; hence, it has better generalization performance than neural networks. in fig. . (d) , two open circles on the upper boundary and two filled circles on the lower boundary are selected as support peptides. the use of these four circles can form the boundaries of the maximum margin between two classes of peptides. the trained spm classifier is a linear fig. . a hyperplane formed using conventional classification algorithms for peptides with a balanced distribution. b and c hyperplanes formed using conventional classification algorithms for peptides without balanced distribution. d hyperplane formed using spm for peptides without balanced distribution. the open circles represent class a , the filled circles class b , and the shaded circle class a or b . the thick lines represent the correct hyperplane for discrimination and the broken thick lines the biased hyperplanes. the thin lines represent the margin boundaries. the γ indicates the distance between the hyperplane and the boundary formed by support peptides. the margin is γ. reproduced from [ ] with permission combination of the similarity between an input peptide and the found support peptides. the similarity between an input peptide and the support peptides is quantified by the bio-basis function. the decision is made using the following equation, y = sign {Σ w i t i k ( s , r i )}, where t i is the class label of the i th support peptide and w i the positive parameter as a weight for the i th support peptide. the weights are determined by a spm algorithm [ ] . the relevance peptide machine (rpm) was proposed based on automatic relevance determination (ard) theory [ ] . in spm, the found support peptides are normally located near the boundary for discrimination between two classes of peptides. however, the relevance peptides found by rpm are prototypic peptides. this is a unique property of rpm, as the prototypic peptides found by a learning process are useful for exploring the biological inside. suppose the model output is defined as where t is a similarity vector and w = ( w , w , . . ., w n ) t . the model likelihood function is defined as follows: using ard prior to computation can prevent overfitting the coefficients by assuming each weight follows a gaussian where α = (α , α , . . . , α n ) t . the bayesian learning method gives the following posterior for the coefficients: where the parameters (covariance matrix Σ , the mean vector u , and the hyperparameters for weights s ), which can be approximated: and the marginal likelihood can be obtained through integrating out the coefficients: in learning, α can be estimated in an iterative way: a formal learning process of the rpm is then conducted. the following condition is checked during learning: where θ is a threshold. if the condition is satisfied, the corresponding expansion coefficient is zeroed. the learning continues until either the change of the expansion coefficients is small or the maximum learning cycle is approached [ ] . figure . shows how the rpm selects prototypic peptides as the relevance peptides, compared with the spm, which selects boundary peptides as the support peptides. first, we define a linear model using the bio-basis function as y = kw ( ) where k is defined in eq. . the orthogonal least square algorithm is used as a forward selection procedure by the orthogonal peptide machine (opm). at each step, the incremental information content of a system is maximized. we can rewrite the design matrix k as a collection of column vectors ( k , k , . . . , k m ), where k i represents a vector of the similarities between all the training peptides and the i th the opm involves the transformation of the indicator peptides ( r i ) to the orthogonal peptides ( o i ) to reduce possible information redundancy. the feature matrix k is decomposed to an orthogonal matrix and an upper triangular matrix as follows: where the triangular matrix u has ones on the diagonal line: the orthogonal matrix satisfies where h is diagonal with the elements h ii as the space spanned by the set of orthogonal peptides is the same space spanned by the set of indicator peptides, and eq. can be rewritten as we can define an error model as follows: suppose e ~ n ( , ), the pseudoinverse method can be used to estimate a as follows: because h is diagonal, the element in a is then the quantities a and w satisfy the triangular system the gram-schmidt or the modified gram-schmidt methods are commonly used to find the orthogonal peptides then to estimate w [ , ] . hiv/aids is one of the most lethal and transmittable diseases, with a high mortality rate worldwide. the most effective prevention of hiv infection is to use a vaccine to block virus infection [ ] . however, hiv vaccine is difficult to develop because of the expense and complexity in advancing new candidate vaccines. although some efficient models and integrated hiv vaccine research enterprises have been proposed [ ] , there is little hope that an hiv vaccine would be developed before [ ] . hiv is a type of retrovirus. it can enter uninfected cells to replicate itself. inhibiting viral maturation and viral reverse transcription are then the major methods so far for treating hiv-infected patients. two groups of inhibitors have since been developed. one group aims to stop protease cleavage activities and is referred to as the protease inhibitor . the other aims to stop reverse transcriptase cleavage activities and is referred to as the reverse transcriptase inhibitor . however, hiv often develops resistance to the drugs applied. drug-resistant mutants of hiv- protease limit the long-term effectiveness of current antiviral therapy [ ] . the emergence of drug resistance remains one of the most critical issues in treating hiv- -infected patients [ ] . the genetic reason for drug resistance is the high mutation rate along with a very high replication rate of the virus. these two factors work together, leading to the evolution of drug-resistant variants and consequently resulting in therapy failure. the resistance to hiv- protease inhibitors can be analyzed at a molecular level with a genotypic or a phenotypic method [ , ] . the genotypic method is used to scan a viral genome for some mutation patterns for potential resistance. as an alternative, the viral activity can be measured in cell culture assays. yet another alternative, phenotypic testing is based on clinic observations, ithat is, directly measuring viral replication in the presence of increasing drug concentration. genotypic assays have been widely used as tools for determining hiv- drug resistance and for guiding treatment. the use of such tools is based on the principle that the complex impact of amino acid substitutions in hiv reverse transcriptase or protease on the phenotypic susceptibility or clinical response to the available antiretroviral agents is observable [ ] . genotypic assays are used to analyze mutations associated drug resistance or reduced drug susceptibility. however, this method is problematic, because various mutations and mutational patterns may lead to drug resistance [ ] , and therefore the method occasionally fails to predict the effects of multiple mutations [ ] . in addition, genotypic assays provide only indirect evidence of drug resistance [ ] . although hiv- genotyping is widely accepted for monitoring antiretroviral therapy, how to interpret the mutation pattern associated with drug resistance to make accurate predictions of susceptibility to each antiretroviral drug is still challenging [ ] . phenotypic assays directly measure drug resistance [ ] , where drug resistance can be experimentally evaluated by measuring the ratio of free drug bound to hiv- protease molecules. however, this procedure is generally expensive and time consuming [ , ] . to deal with the difficulties encountered in genotypic assay analysis and phenotypic evaluation, a combination of inhibitor flexible docking and molecular dynamics simulations was used to calculate the protein-inhibitor binding energy. from this, an inhibitory constant is calculated for prediction [ ] . later, some statistical models were established for predicting drug resistance. for instance, a statistical model was proposed to analyze the viral and immunologic dynamics of hiv infection, taking into account drug-resistant mutants and therapy outcomes [ ] . the factors causing the increase in cd cell count and the decrease in viral load from baseline after six months of haart (highly active antiretroviral therapy) were analyzed. note that cd stands for cluster of differentiation , which is a molecule expressed on the surface of t helper cells. in addition to the baseline viral load and cd cell count, which are known to affect response to therapy, the baseline cd cell count and resistance characteristics of detectable strains are shown to improve prediction accuracy. note that cd stands for cluster of differentiation , which is a membrane glycoprotein found primarily on the surface of cytotoxic t cells. a logistic regression model was built for the prediction of the odds of achieving virology suppression after weeks of haart in antiretroviral-naive patients starting haart according to their week viral load [ ] . a regression model was built on a set of matched genotype-phenotype pairs for the prediction of phenotypic drug resistance from genotypes [ ] . as it was observed that the range of resistance factors varies considerably among drugs, two simple scoring functions were derived from different sets of predicted phenotypes. the scoring functions were then used for discrimination analysis [ ] . in addition, machine learning algorithms were used. decision trees as a method for mimicking human intelligence were implemented to predict drug resistance [ ] . a fuzzy prediction model was built based on a clinical data set of patients failing highly active antiretroviral therapy (haart) and starting salvage therapy with baseline resistance genotyping and virological outcomes after three and six months [ ] . in the model, a set of rules predicting genotypic resistance was initially derived from an expert and implemented using a fuzzy logic approach. the model was trained using the virological outcomes data set, that is, stanford's hiv drug-resistance database (stanford hivdb). expert systems were also used for this purpose [ ] . neural networks as a type of powerful nonlinear modelers were utilized to predict hiv drug resistance for two protease inhibitors, indinavir and saquinavir [ ] . other than using sequence information for prediction, a structure-based approach was proposed to predict drug resistance [ ] . models of wt complexes were first produced from crystal structures. mutant complexes were then built by amino acid substitutions in the wt complexes with subsequent energy minimization of the ligand (a small molecule binding to a protein or receptor) and pr (protease receptor) binding site residues. a computational model was then built based on the calculated energies. the data set studied was obtained from an online relational database, the hiv rt and protease database ( http://hivdb.stanford.edu ) [ ] . the susceptibility of the data to five protease inhibitors were determined, saquinavir (sqv), indinavir (idv), ritonavir (rtv), nelfinavir (nfv) and amprenavir (apv). we obtained genotype-phenotype pairs for each of the protease inhibitor, except for apv, for which we obtained pairs. phenotypic resistance testing measures in-vitro viral replication of the wild type and the viral sample in increasing drug concentrations [ ] . the resistance factor, defined as the ratio of % inhibitory concentration (ic ) of the respective viral sample to ic of the nonresistant reference strain, reports the level of resistance as the fold change in susceptibility to the drug as compared to a fully susceptible reference strain. while genotypic resistance testing is done by scanning the viral genome for resistance-associated mutations. the major and minor mutations observed in a resistant hiv protease can be seen in figure in [ ] . hiv protease is an aspartyl protease with residues. in this study, we considered major and minor mutation sites, obtaining a window size of residues for each drug. we divided the sample set into two classes by attaching to each peptide a label or - for resistant (functional) and susceptible (nonfunctional), respectively. this division depended on whether the resistance factor of a sample exceeded the cutoff value or not. based on previously published datasets and studies, we assumed the cutoff value for the resistant factor of each sample with respect to each protease inhibitor to be . . we used matthews correlation coefficient [ ] the larger the matthews correlation coefficient, the better the model fits the data. if the value of the matthews correlation coefficient is , it represents a complete correlation. if the value is , the prediction is completely random. if the value is negative, the prediction is on the opposite side of the target. the evaluation is carried out based on the simulation of fivefold cross-validation for each drug and each algorithm. figure . shows the comparison between vector machines and peptide machines. it can be seen that the peptide machines outperformed the vector machines. we also constructed neural networks models for this task, their performance is far worse than vector machines (data are not shown). in building a computer model for a real application, there are two important issues for model validation in addition to model parameter estimation. the first is how to evaluate a model. the second is how to select a model based on the evaluation. there are always some hyperparameters for determination when using neural networks or machine learning algorithms. for instance, the number of hidden neurons in feedforward neural networks is such a hyperparameter, and the determination of the optimal number of hidden neurons is not an easy task. using the training data for the evaluation will not deliver meaningful result, as an optimization process can easily overfit the data, leading to poor generation capability. the generalization capability should measure how well a built model works with novel data. based on this requirement, we then need to consider how to use the available data for proper model validation. there are three commonly used methods for this purpose: cross-validation, resampling, and jackknife. all these methods use the same principle, that the validation data must not involve any process of model parameter estimation. this means that the available data must be divided into two parts. one is for model parameter estimation, which is commonly referred to as training . the other is for model evaluation (validation) and selection. the difference between these three methods is the strategy used for the division of a given data set. with the resampling method, we normally randomly sample a certain percentage of data for training and the rest for validation. such a process is commonly repeated many times. suppose there are n data points and we repeat the sampling process for m times. there will be m validation models, each of which has different training and validation data with some possible overlap. note that all these validation models use the same hyperparameters; for instance, they all use h hidden neurons. the parameters of the i th validation model are estimated using the i th training data set with k i < n data points. the i th validation model is validated on the i th validation data set with n -k i data points. because we use different training data sets each time, it is expected that the parameters of the i th validation model will be different from those of the j th validation model. the validation performance of the i th validation model is certainly different from that of the j th validation model as well. we denote by ω i the evaluated performance for the i th validation model. the evaluation statistic of the model with designed hyperparameters can follow to determine the proper values for the hyperparameters so as to select a proper model, we can vary the values assigned to the hyperparameters. if we have g hyperparameters for selection, the selection will be taken in a g -dimensional space, where each grid is a combination of hyperparameters. suppose we need to determine only the number of hidden neurons, we then have a series of m and s . the best model can be selected through it should be noted that, for the resampling method, some data points may be used for multiple times in training or validation. in cross-validation, we normally randomly divide a data set into m folds. each fold contains distinctive data points. if we denote by w i as the set of data points in i th fold, we have w i Ç w j = φ, meaning that two sets have no elements in common. every time, we select one fold as the validation set and the remaining m - folds are used as the training set for model parameters estimate. such a process is repeated for m times, until each fold has been used for validation once. this means that there are m validation models. again, all these validation models use the same hyperparameters. the i th validation model is trained using the folds except for the i th fold and validated on the i th fold. the parameters of the i th validation model also is different from those of the j th validation model, and the validation performance of different validation models will vary. note that each data point ise validated only once. we denote by when data size is not too large, one commonly prefers using the jackknife (often called leave-one-out cross-validation ) method. in using the jackknife method, we normally pick up one data point for validation and use the remaining data points for training. such a process is repeated for n times until each data point has been exhausted for validation. this means that there are n validation models for n data points. obviously, all these validation models use the same hyperparameters. the i th validation model is trained using all the data points except for the i th data point and validated on the i th data point. equation can be used for evaluation. the best model can be selected through the use of eq. . model validation can help us evaluate models and select models with a proper setting of hyperparameters. however, such evaluation values cannot be regarded as the final model evaluation, which can be delivered to users. the evaluated performance on validation data may not be able to reflect the true performance, because the validation data has been used to tune hyperparameters. as a consequence, the evaluated performance on validation data commonly overestimates the true model accuracy. based on this understanding, a proper peptide classification methodology must contain a blind test stage. this means that, after model evaluation and model selection, we may need to test the "best" model using another data set, which has never been used for estimating model parameters and tuning model hyperparameters or selecting models [ ] . a very important issue must be discussed here, especially for peptide classification. many peptide classification tasks deal with functional site predictions. within each protein sequence, there might be a few functional sites, such as protease cleavage sites, protein interaction sites, or protein posttranslational modification sites. based on the substrate size, we can extract a peptide around one functional site. such a peptide is commonly regarded as a functional peptide. to conduct proper peptide classification, we have to have a set of nonfunctional peptides. each nonfunctional peptide has no functional site at the desired residue(s). we normally use a sliding window with a fixed length to scan a protein sequence from the n-terminal to the c-terminal, one residue by one residue to generate no-functional peptides. a commonly used method is to combine both functional and nonfunctional peptides to produce a data set. suppose we use the cross-validation method, the data set is then randomly divided into m folds for cross-validation. one then builds m validation models for model evaluation and model selection. now a question arises. when we use such kind of models for testing on novel whole protein sequences, the performance is commonly not as expected. this means that the model is some where overfitted. if model parameter estimation follows a proper procedure, the most probable problem is the data used for training and validation. we mentioned previously that we must maintain the independence of a validation data set from a training data set. when we check the method as described, we can clearly see two interesting facts. first, a training peptide and a validation peptide may come from the same protein. if one protein has conserved amino acids, the validation peptides generated this way may not be independent of the training peptides. second, a training peptide and a validation peptide may have many identical residues if they are extracted from neighboring sliding widows. based on this analysis, we proposed to use protein-oriented validation [ ] . suppose we have n proteins, we may divide these n proteins into m folds. we then generate validation peptides using one fold and generate training peptides from the remaining folds. the validation models are constructed using the training peptides scanned from the sequences of the training proteins and verified on the validation peptides scanned from the sequences of the validation proteins. we always have an important issue when using machine learning approaches for peptide classification, that is, if the model is correct forever. the answer is no, as a peptide data set collected at a certain time can be far less than complete. based on this understanding, the models built using an incomplete set of peptides may not be able to generalize well forever. when new experimentally determined peptides have been collected, the exiting models must be corrected so that they can conduct classification tasks well. in this section, we investigate an interesting peptide classification topic, where we can use a model built on published peptides for peptide classification, and we can use a model built on both published peptides and newly submitted sequences in ncbi (www.ncbi.nih.gov) for peptide classification. we found there is a significant difference between the two. the case studied in this session is about hepatitis c virus (hcv), which is a member of the flaviviridae family [ ] and is the major agent of parenterally transmitted non-a/non-b hepatitis [ , ] . the nomenclature of schechter and berger [ ] is applied to designate the cleavage sites on the peptides, p -p -p -p -p -p -p ' -p ' -p ' -p ' , the asymmetric scissile bond being between p and p ' . two resources are available for modeling. first, research articles have published experimentally determined peptides, cleaved and noncleaved. second, some databank like ncbi has collected many new submissions. each submission contains a whole protein sequence with a number of experimentally determined cleavage sites. in terms of this, there are two strategies for modeling. if we believe that the published peptides are able to represent all the protease cleavage specificity, we can select indicator peptides from these published peptides for modeling. we refer to a model constructed this way as a type-i model . in fact, viruses mutate very rapidly; hence, the published peptides may not be able to represent the cleavage specificity in the recent submissions to ncbi. we can then select more indicator peptides, which show more viral mutation information from the new sequences downloaded from ncbi to build a more informative model referred to as a type-ii model. from published papers (data are not shown), we collected experimentally determined peptides. they are referred to as published peptides in this study. among them, are cleaved, while are noncleaved. twenty-five whole protein sequences were downloaded from ncbi. they are aaa , aaa , aaa , aab , baa , baa , baa , baa , baa , baa , baa , baa , bab , caa , caa , cab , gnwvtc, jc , np_ , np_ , p , p , p , pc , and s . within these whole protein peptides (data are not shown) are cleavage sites. the cleavage sites with notes of potential, by similarity , or probable were removed. first, we built type-i models using the leave-one-out method. each model is built on published peptides and tested on remaining published peptide. the models are then evaluated in terms of the mean accuracy, which is calculated on leaveone-out testes. the best model is selected and tested on new sequences downloaded from ncbi, which are regarded as the independent testing data. the simulation shows that the noncleaved, cleaved, and total accuracies are %, %, and %, respectively. shown in fig. . are the prediction results on five whole protein sequences, where the horizontal axis indicates the residues in whole protein sequences and the vertical axis the probability of positive (cleavage). if the probability at a residue is larger than . , the corresponding residue is regarded as the cleavage site p . the simulation shows that there were too many false positives. the average of false positive fraction was %, or misclassified noncleavage sites. second, we built type-ii models. each model is built and validated using all the published peptides ( ) plus the peptides scanned from newly downloaded sequences. in this run, the protein-oriented leave-one-out method is used. with this method, of new sequences is removed for validation on a model built using published peptides and the peptides scanned from the remaining new sequences. this is repeated times and the performance is estimated on the predictions on new sequences. the best model is selected and tested on the peptides on the remaining new sequence. these peptides are regarded as the independent testing peptides. the noncleaved, cleaved, and total accuracies are %, %, and %, respectively. it can be seen that the prediction accuracy has been greatly improved compared with the type-i models. shown in fig. . is a comparison between the type-i and the type-ii models. it shows that the type-ii models greatly outperformed the type-i models in terms of the performance for noncleaved peptides. more important, the standard deviation in the type-ii models is much smaller, demonstrating high robustness. shown in fig. . are the prediction results on five protein sequences using the type-ii models, where the horizontal axis indicates the residues in whole protein tpf total type-i type-ii fig. . a comparison between the type-i and type-ii models. it can be seen that the type-ii models performed much better than the type-i models in terms of the increase of specificity (truenegative fraction). therefore, the false-positive fraction (false alarm) has been significantly reduced. tnf and tpf stand for true-negative fraction (specificity) and true-positive fraction (sensitivity). reproduced from [ ] with permission fig. . the probabilities of cleavage sites among the residues for five whole protein sequences for the type-ii models. the horizontal axes indicate the residues in whole sequences and the vertical axes the probabilities of positives (cleavage sites). the numbers above the percentages are the ncbi accession numbers and the percentages are the false positive fractions and the true positive fractions. the simulation shows that the type-ii models demonstrated very small falsepositive fractions compared with the type-i models. it can be seen that the probability of positives are very clean . reproduced from [ ] with permission sequences and the vertical axis the probability of positive (cleavage). the simulation shows fewer false positives than the type-i models. the reason is that many of the newly downloaded sequences were published after the reports with the published peptides. the published peptides may not contain complete information for all these new sequences. this chapter introduced the state-of-the-art machines for peptide classification. through the discussion, we can see the difference between the peptide machines and other machine learning approaches. each peptide machine combines the feature extraction process and the model construction process to improve the efficiency. because peptide machines use the novel bio-basis function, they can have biologically well-coded inputs for building models. this is the reason that the peptide machines outperformed the other machine learning approaches. the chapter also discussed some issues related with peptide classification, such as model evaluation, protein-oriented validation, and model lifetime. each of these issues is important in terms of building correct, accurate, and robust peptide classification models. for instance, the blind test can be easily missed by some new bioinformatics 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