key: cord-1000093-9qetwea4
authors: Sigalov, Alexander B.
title: SCHOOL Model and New Targeting Strategies
date: 2008
journal: Multichain Immune Recognition Receptor Signaling
DOI: 10.1007/978-0-387-09789-3_20
sha: bc0859f34bfb9b1e75220c42362947892dfdb6f7
doc_id: 1000093
cord_uid: 9qetwea4

Protein-protein interactions play a central role in biological processes and thus are an appealing target for innovative drug design and development. They can be targeted by small molecule inhibitors, peptides and peptidomimetics, which represent an alternative to protein therapeutics that carry many disadvantages. In this chapter, I describe specific protein-protein interactions suggested by a novel model of immune signaling, the Signaling Chain HOmoO Ligomerization (SCHOOL) model, to be critical for cell activation mediated by multichain immune recognition receptors (MIRRs) expressed on different cells of the hematopoietic system. Unraveling a long-standing mystery of MIRR triggering and transmembrane signaling, the SCHOOL model reveals the intrareceptor transmembrane interactions and interreceptor cytoplasmic homointeractions as universal therapeutic targets for a diverse variety of disorders mediated by immune cells. Further, assuming that the general principles underlying MIRR-mediated transmembrane signaling mechanisms are similar, the SCHOOL model can be applied to any particular receptor of the MIRR family. Thus, an important application of the SCHOOL model is that global therapeutic strategies targeting key protein-protein interactions involved in MIRR triggering and transmembrane signal transduction may be used to treat a diverse set of immune-mediated diseases. This assumes that clinical knowledge and therapeutic strategies can be transferred between seemingly disparate disorders, such as T-cell-mediated skin diseases and platelet disorders, or combined to develop novel pharmacological approaches. Intriguingly, the SCHOOL model unravels the molecular mechanisms underlying ability of different human viruses such as human immunodeficiency virus, cytomegalovirus and severe acute respiratory syndrome coronavirus to modulate and/or escape the host immune response. It also demonstrates how the lessons learned from viral pathogenesis can be used practically for rational drug design. Application of this model to platelet collagen receptor signaling has already led to the development of a novel concept of platelet inhibition and the invention of new platelet inhibitors, thus proving the suggested hypothesis and highlighting the importance and broad perspectives of the SCHOOL model in the development of new targeting strategies.

Specific protein-protein interactions are responsible for the function of numerous processes in the cell and constitute the foundation for the majority of cell recognition, proliferation, growth, differentiation, programmed cell death and signal transduction in health and disease.^"^ It seems that almost every important pathway includes and is critically influenced by protein-protein interactions.^ Because of the ubiquitous nature of these interactions and the knowledge that inappropriate protein-protein binding can lead to disease, the specific and controlled inhibition and/or modulation of these interactions provides a promising novel approach for rational drug design, as revealed by recent progress in the design of inhibitory antibodies, peptides and small molecules. A number of recent reviews have addressed this topic.^'^^ Thus, revealing information about specific protein-protein interactions in any particular pathway (i.e., transmembrane signaling) can provide targets for a generation of new drugs.

Multichain immune recognition receptors (MIRRs) recognize foreign antigens and initiate a variety of biological responses. Examples of MIRRs include the T-cell receptor (TCR) complex, the B-cell receptor (BCR) complex, Fc receptors (e.g., FceRI, FcaRI, FcyRI and FcyRIII), NK receptors (e.g., NKG2D, CD94/NKG2C, KIR2DS, NKp30, NKp44 and NKp46), immunoglobulin (Ig)-like transcripts and leukocyte Ig-like receptors (ILTs and LIRs, respectively), signal regulatory proteins (SIRPs), dendritic cell immunoactivating receptor (DCAR), myeloid DNAX adapter protein of 12 kD (DAP12)-associating lectin 1 (MDL-1), novel immune-type receptor (NITR), triggering receptors expressed on myeloid cells (TREMs) and the platelet collagen receptor, glycoprotein VI (GPVI). MIRR-mediated transmembrane (TM) signal transduction plays an important role in health and disease^^'^^ making these receptors attractive targets for rational intervention in a variety of immune disorders. Thus, future therapeutic strategies depend on our detailed understanding of the molecular mechanisms underlying the MIRR triggering and subsequent TM signal transduction.

All members of the MIRR family are multisubunit complexes formed by the association of recognition subunits with signal-transducing subunits that contain in their cytoplasmic (CYTO) domains the immunoreceptor tyrosine-based activation motif (ITAM) or the YxxM motif, found in the DAP-10 CYTO domain (see Chapter 12) . This association in resting cells is mostly driven by the noncovalent TM interactions between recognition and signaling components and plays a key role in receptor assembly and integrity (see also .^8,21-26 Crosslinkingof the receptors after ligand binding results in phosphorylation of the ITAM/YxxM tyrosines, which triggers the elaborate intracellular signaling cascade. The extracellular (EC) recognition of an antigen/ligand and the sequence of biochemical events that ensues after the phosphorylation of ITAMs/YxxM are understood in significant detail. However, the molecular mechanism linking EC antigen/ ligand-induced clustering of MIRR ligand-binding subunits to intracellular phosphorylation of signaling subunits has been a long-standing unsolved mystery. It was also unknown how this putative mechanism can explain the intriguing ability of immune cells to discern and differentially respond to slightly different ligands. This impeded our advance understanding of the immune response, the development of novel pharmacological approaches and even more important, the potential transfer of clinical knowledge, experience and therapeutic strategies between seemingly disparate immune disorders.

Despite numerous models of MIRR-mediated TM signal transduction suggested for particular MIRRs (e.g., TCR, BCR, FcRs, NK receptors, etc.), no current model fully explains how ligand-induced TM signal transduction commences at the molecular level. As a consequence, these models are mostly descriptive and do not reveal clinically important potential points of therapeutic intervention. In addition, no general model of MIRR-mediated immune cell activation has been suggested, thus preventing the potential transfer of therapeutic strategies between seemingly disparate immune disorders.

A recendy developed novel mechanistic model, the SCHOOL model,^^^° describes the crucial protein-protein interactions underlying the molecular mechanism of MIRR triggering and TM signaling (Fig. 1 , see also Chapter 12) . In this chapter, I describe these specific interactions as new . The model proposes that formation of competent MIRR signaling subunit oligomers driven by the homooligomerization of signaling subunits is necessary and sufficient to trigger the receptors and induce transmembrane (TM) signal transduction and downstream sequence (see Chapter 12 for detail). All interchain interactions in this intermediate are shown by light gray arrows reflecting their transition state. Immunoreceptor tyrosine-based activation motifs (ITAMs) are shown as gray rectangles. Circular arrow indicates ligand-induced receptor reorientation. Phosphate groups are shown as gray circles. Small solid black arrows indicate specific intersubunit hetero-and homointeractions between TM and cytoplasmic (CYTO) domains, respectively. Within the model, MIRR triggering and signaling is an outcome of the ligand-induced interplay between three key protein-protein interactions: antigen/ligand-MIRR interactions, intrareceptor TM interactions and interreceptor CYTO homointeractions (B). Two of these interactions can be considered as new therapeutic targets (C): 1) TM interactions between MIRR antigen-recognizing and signal-transducing subunits (target I) that play an important role in receptor assembly and integrity on resting cells; and 2) CYTO homointeractions between MIRR signaling subunits (target II) that represent a main driving force of MIRR triggering/signaling. therapeutic targets revealed by the model for the treatment of diverse immune and other disorders mediated by MIRRs. Assuming that the similar structural architecture of the MIRRs dictates similar mechanisms of MIRR triggering and subsequent TM signal transduction, the model su^ests that these targets are similar in seemingly unrelated diseases. This builds the structural basis for the development of novel pharmacological approaches as well as the transfer of clinical knowledge, experience and therapeutic strategies between various immune disorders. In addition, it significantly improves our understanding of the immunomodulatory activity of human viruses such as human immunodeficiency virus (HIV), human cytomegalovirus (CMV) and severe acute respiratory syndrome coronavirus (SARS-CoV), human T-cell leukemia type 1 virus (HTLV-1) and assumes that the lessons learned from viral pathogenesis can be used for the development of new therapeutic approaches. An important application of this hypothesis is that a general pharmaceutical approach may be used to treat diverse immune-mediated diseases.

antigen or anti-MIRR antibodies (e.g., anti-CD3£ and anti-TCRp for TCR or anti-]g(3 antibodies for BCR) leads to receptor clustering coupled with a multi-step structural reorganization driven by the homooUgomerization of MIRR signaling subunits (Fig. lA) . Ligand-induced MIRR clustering leads to receptor reorientation and formation of a dimeric/oligomeric intermediate in which signaling chains from different receptor units start to trans-homointeract and form signaling oligomers (Fig. lA, stages 1 and 2 ). Upon formation of signaling oUgomers, protein tyrosine kinases phosphorylate the tyrosine residues in the ITAMs located on the CYTO tails of MIRR signaling subunits, leading to the generation of intracellular activation signal(s), dissociation of signaling oligomers and internalization of the engaged MIRR hgand-binding subunits (Fig. lA, stages 2  and 3) . Then, signaling oligomers interact with the signaling subunits of nonengaged receptors resulting in formation of higher-order signaling oligomers, thus propagating and amplifying the activation signal and resulting in internalization of the non-engaged MIRR recognition subunits (Fig. lA, stages 4 

Introducing the homotypic interactions between MIRR signahng subunits as one of the key interactions involved in MIRR tri^ering and TM signahng, the plausible and easily testable SCHOOL model defines this process as an outcome of the interplay between three major driving forces (Table 1 1) Antigen/Ugand-MIRR interactions. These interactions cluster two or more MIRRs in sufficient proximity and correct (permissive) relative orientation to initiate homointeractions between particular MIRR signaling subunits. 2) Intrareceptor TM interactions. These interactions stabilize and maintain receptor integrity in resting cells and balance opposing interactions, the interreceptor CYTO homointeractions, in stimulated cells, thus helping to discriminate Ugands/antigens in their functional ability to trigger MIRRs and induce a cellular activation signal. 3) Interreceptor homointeractions. These interaaions between the CYTO domains of MIRR signaling subunits lead to the formation of oUgomeric signaling structures, thus triggering phosphorylation of ITAMs and initiating the signaling cascade. Thus, the SCHOOL model reveals the last two key interactions of MIRR triggering/signaling as new therapeutic targets (Fig. IC) .

Antigen/ligand-MIRR interactions are generally of low affinity (micromolar range) and have rapid association and dissociation kinetics (reviewed, for example, for TCR in 35). This low affinity binding in combination with fast kinetics allows immune cells to recognize and discriminate a variety of antigens/ligands with high specificity, selectivity and sensitivity in order to respond with a variety ofbiological responses. Considering that EC and TM regions of MIRRs are well-ordered receptor segments while MIRR signaling CYTO domains have been recendy shown to represent a novel class of intrinsically disordered proteins,^^'^^ an important and intriguing question is raised: how do MIRRs transduce highly ordered information about antigen recognition/discrimination from outside the cell through the cell membrane into intracellular biochemical events, thus triggering specific pathways and resulting in a specific functional outcome?

Despite intensive studies of MIRR-mediated TM signal transduction, the only model that can answer this question and even more important, mechanistically explain how this signahng starts, is the SCHOOL model (see also Chapter 12) .^^'^^ Intriguingly, all three protein-protein interactions, namely antigen/Ugand-MIRR EC interactions as well as intrareceptor TM heterointeractions and interreceptor CYTO homointeractions (Fig. IB, Table 1 ), fall within the similar micromolar affinity range and are characterized by relatively rapid kinetics.^^'^' "^^ This conjugated and well-balanced system of interprotein interactions provides the ideal basis to explain the molecular mechanisms of the abihty of MIRRs to transduce the extracellular information about recognition of different ligands/antigens through the cell membrane and translate it into different activation signals, thus triggering different intracellular pathways and resulting in different cell responses. Within the model, the MIRR-generated intracellular activation signals are combinatorial in nature and involve multiple components such as formation of different competent MIRR signaUng subunit oligomers (see also Chapter 12)^^^^ and different ITAM Tyr phosphorylation patterns."^^'^^ This system also explains mechanistically high specificity, selectivity and sensitivity of immune cells in recognition and discrimination of different antigens/ligands and how this recognition/discrimination results in different functional outcomes. This is particularly important for the TCR^^ that has four different signaling subunits, namely t, and CD38, CD36 and CDSy, known to play different roles in T-cell biology (see Chapters 1 and 12). In addition, in contrast to other MIRR signaling subunits, t, has three ITAMs that can provide differential tyrosine phosphorylation patterns in response to different ligands, initiating different intracellular signaling pathways. Thus, within the model, TCR-mediated signaling and cell activation has the highest combinatorial potential as compared to other MIRRs, explaining a high variability of distinct TCR-triggered intracellular signaling pathways and therefore distinct T-cell functional responses depending on the nature of the stimulus (see also Chapter 12) .^^'^^

Interactions between TM helices of recognition and signaling MIRR subunits maintain receptor integrity in unstimulated cells and determine the relative positions of these subunits in the recep-tor complex (angles, distances, etc.), thus dictating the overall geometry and topology of MIRRs. Within the SCHOOL model, the overall structural architecture (i.e., geometry and topology) of MIRRs that is dictated and maintained by TM interactions between MIRR recognition and signaling subunits (Fig. 1 , see also Chapter 12),^^'^ in combination with the requirement to initiate interreceptor homointeractions between MIRR signaling subunits ( Fig. 1) , impose several restraints for multivalent antigen/ligand-induced MIRR triggering (Table 2, see also Chapter 12):^^'^° • sufficient interreceptor proximity in MIRR dimers/oligomers • correct (permissive) relative orientation of the receptors in MIRR dimers/oligomers • long enough duration of the MIRR-ligand interaction that generally correlates with the strength (affinity/avidity) of the ligand • sufficient lifetime of an individual receptor in MIRR dimers/oUgomers The importance of these factors for productive MIRR triggering and TM signaling is strongly supported by a growing body of evidence and described in detail in Chapter 12 of this book. Briefly, it should be noted that the restraints imposed by the model play an especially important role during the first stage of MIRR triggering (Fig. 1) . At this point, these spatial, structural and temporal requirements (correct relative orientation, sufficient proximity, long enough duration of the MIRR-ligand interaction and lifetime of MIRR dimers/oligomers) should be fulfilled to favor initiation of trans-homointeractions between MIRR signaling subunits and formation of competent signaling subunit oligomers. If these requirements are not fulfilled at this "final decision-making" point, the formed MIRR dimers/oligomers may dissociate from the ligand and remain signaling-incompetent and/or break apart to its initial monomeric receptor complexes. Also, at this stage, sUghdy different ligands may bring two or more MIRRs in different relative orientations that favor homointeractions between different signaling subunits and result in formation of different signaling oligomers or their combinations, thus initiating distinct signaling pathways. This mechanism can explain the abiUty of MIRRs to differentially activate a variety of signaling pathways depending on the nature of the stimulus.

The SCHOOL model is fundamentally different from those numerous models that have been previously suggested for particular MIRRs and has several important advantages (see also Chapter 12):^^'^° • This is the first general mechanistic model for aU MIRRs known to date, including TCR, BCR, Fc receptors, NK receptors, ILTs, LIRs, SIRPs, DCAR, MDL-1, NITR, TREMs, GPVI and others and for those that will be discovered in the future. Assuming the general principles underlying MIRR triggering and TM signaling mechanisms are similar for all MIRRs, the SCHOOL model can easily be apphed to any particular receptor of the MIRR family, • This is the first model that is based on specific protein-protein interactions-biochemical processes that can be influenced and controlled^^^'^^*^^-and specific inhibition and/or modulation of these interactions provides a promising novel approach for rational drug design, as revealed by recent progress in the design of inhibitory antibodies, peptides and small molecules.^'^'^'^^ • Introducing the CYTO homointeractions between MIRR signaling subunits as one of the key elements of MIRR tri^ering and signaling, the SCHOOL model imposes functionally important restraints (Table 2 , see also Chapter 12) and suggests molecular mechanisms for the vast majority of imexplained immunological observations accumulated to date (see also Chapter 12) .^^-3o • Unraveling the molecular mechanisms underlying MIRR triggering and subsequent TM signaling, the model suggests unique and powerful tools to study the immune response and a means to control and/or modulate it (see also Chapter 12) .^^'°'^ • Based on specific protein-protein interactions, the SCHOOL model reveals new therapeutic targets ( Fig. 1) for the treatment of a variety of disorders mediated by immune

• An important apphcation of the SCHOOL model is that similar therapeutic strategies targeting key protein-protein interactions involved in MIRR triggering and TM signal transduction may be used to treat diverse immune-mediated diseases. This assumes that clinical knowledge, experience and therapeutic strategies can be transferred between seemingly disparate immune disorders or used to develop novel pharmacological approaches and that a general pharmaceutical approach may be used to treat diverse immune disorders.

As mentioned previously ( (3) interreceptor CYTO homointeractions. The SCHOOL model reveals these specific protein-protein interactions as points of intervention to inhibit and/or modulate MIRR-mediated TM signaling, thus inhibiting and/or modulating the immune response. While antigen/ligand-receptor interactions are a well-known target for drug design and development (see also Chapters 15, [17] [18] [19] ,^^'^^ the last two protein-protein interactions that are critically involved in MIRR tri^ering/signaling, represent promising novel therapeutic targets as revealed by the model (Fig. 1C) .^^'^'^'^ As suggested by the model, controlled inhibition/modulation of these particular interactions represents a means to inhibit/modulate MIRR-mediated TM signaling and specific downstream signaling pathways, thus inhibiting/modulating the immune response. Ihis can be used in rational drug design and the development of novel strategies for the treatment of a variety of diseases and medical conditions that involve MIRR-mediated signaling. Importantly, unraveling the molecular basis of MIRR triggering and signaling and reveaUng specific protein-protein interactions that play a critical role in MIRR-mediated TM signal transduction and cell activation, the SCHOOL model suggests invaluable and unique powerful tools to dissect mechanisms of the related cell functional outcomes in response to antigen/ligand and to study many important aspects of viral pathogenesis (see also Chapter 22) .^^'°'^'^ In this Chapter, I demonstrate how the SCHOOL model, together with the lessons learned fi-om viral pathogenesis, can be used practically for rational drug design and the development of new therapeutic approaches to treat a variety of seemingly unrelated disorders, such as T-cell-mediated skin diseases and platelet disorders.

Since it was first published in 2004,^^ the SCHOOL model has revealed intra-MIRR TM interactions as important therapeutic targets as well as points of great interest to study the molecular mechanisms underlying the MIRR-mediated cell response in health and disease (Figs. I and 2).^^'°'^*^^ Notably, the model has provided a mechanistic explanation at the molecular level for specific processes behind "outside-in" MIRR signaling that were unclear (see also Chapter 22) 27-30, 57. 58 £xamples include molecular mechanisms of action of the therapeutically important TCR TM peptides^"^^ first introduced by ManoHos et al in 1997^^ and the mechanism underlying HIV-1 fusion peptide (FP)-induced inhibition of antigen-dependent T-ceU activation.^^ The relevance of the latter mechanism has since been confirmed experimentally.^^ Within the SCHOOL model, upon antigen/ligand stimulation, the intra-MIRR TM interactions balance opposing interactions, the inter-MIRR CYTO homointeractions and represent one of three major driving forces of MIRR triggering that helps to discriminate ligands/antigens in their functional ability to tri^er MIRRs and induce a cellular activation signal (Table 1 , Fig.  Figure2 . Target I. Transmembrane interactions between MIRR recognition and signaling subunits. This is a simplified graphical illustration of the molecular mechanisms underlying proposed intervention by transmembrane-targeted agents (peptides and their derivatives, small molecule disruptors of protein-protein interactions, etc). Within the SCHOOL model, specific blockade of transmembrane interactions between recognition and signaling subunits is proposed to result in "predissociation" of the receptor complex, thus preventing formation of competent signaling oligomers and inhibiting antigen-dependent immune cell activation (A). In contrast, stimulation of these "predissociated" MIRRs with cross-linking antibodies to signaling subunit should not effect, according to the model, on receptor triggering and cell activation (B). It is noteworthy that the proposed strategies can be used not only to inhibit but also to modulate MIRR-mediated transmembrane signal transduction, thus modulating the immune response (see main text for details). Abbreviations and symbols as in Figure 1 . Reprinte from Trends Pharmacol Sci, 27, Sigalov AB, Immune cell signaling: a novel mechanistic model reveals new therapeutic targets, 518-524, copyright 2006 with permission from Elsevier. IB). As suggested by the model (Figs. IC and 2),^^'°-^^'^ specific blockade or disruption of the TM interactions between MIRR recognition and signaling subunits causes a physical and functional disconnection of the subunits. Peptides and their derivatives, small molecule disruptors of protein-protein interactions, site-specific mutations and other similar agents/modifications can be used to affect the MIRRTM interactions. It should be noted that in this context, a physical disconnection means "predissociation" rather than full dissociation of the subunits because in the absence of stimulus, they can still remain together. Antigen/ligand stimulation of these "predissociated" receptors leads to reorientation and clustering of the recognition but not signaUng subunits. As a result, signaling oligomers are not formed, ITAM Tyr residues do not become phosphorylated and the signaling cascade is not initiated (Fig. 2A) . In contrast, this "predissociation" does not prevent the formation of signaling oUgomers when signaling subunits are clustered by specific antibodies that trigger cell activation, e.g., anti-MIRR signaling antibodies (Fig. 2B ) such as anti-CD3 for TCR and anti-IgP antibodies for BCR, or anti-TCRP antibodies for TCR (not illustrated).

Our current understanding of the MIRR structure and the nature and specificity of TM interactions between receptor recognition and signaling subunits not only allows us to block or disrupt these protein-protein interactions but also to modulate the interactions by sequence-based approach with using corresponding peptides and/or their derivatives. Strengthening/weakening and/or selective disruption of the association between particular recognition and signaling subunits might allow us not to inhibit, but rather to modulate the ligand-induced cell response. In addition, selective functional disconnection of particular signaUng subunits from their recognition partner represents an invaluable tool in studies of MIRR-mediated TM signaling and cell activation. It should also be noted that methods of computational design, synthesis and optimization of TM peptides and peptidomimetics, as well as high-throughput screening techniques to search for the relevant TM mutations or small molecule disruptors, are currently developed and well-established,^'^^'^"^'^^'^^'^ making the proposed powerful approach both feasible and of great fundamental and clinical value.

I suggest that the TM interactions between recognition and signaling MIRR subunits represent extremely important points of control in MIRR triggering and cell activation. Since we can now use the SCHOOL model to design the TM-targeted agents effective in inhibition and/or modulation of MIRR-mediated TM signaling (Figs. 1 and 2, see also Chapter 12),^^^^' 5^' 58 we have a powerful and well-controlled influence upon MIRR-mediated cell activation and control the immune response. The relevant TM-targeted agents for any particular member of MIRR family can be readily designed using the SCHOOL model and our knowledge about structural organization of this receptor. Examples include the TM peptides of TCR,^"^^^'^'^ NK receptors^^ and GPVI^^ tested to inhibit/modulate the relevant receptor-mediated cell response. Importandy, the SCHOOL model unravels the TM-targeted molecular mechanisms underlying ability of different human viruses such as human immunodeficiency virus, cytomegalovirus and severe acute respiratory syndrome coronavirus to modulate and/or escape the host immune response.^^'^^'^^ It also demonstrates how the lessons learned from viral pathogenesis can be used practically for rational drugdesign.^^*^^ These and other examples that successfully prove the main concept of the SCHOOL model-driven TM strategy are considered in detail below.

Obviously, allowing us to effectively control MIRR signaling and therefore the immune response, the MIRRintrareceptor TM interaaions represent an important target of pharmacological intervention as first revealed and suggested by the SCHOOL model in 2004.^^ It further assumes that a general therapeutic strategy aiming to disrupt/modulate these interactions in the MIRRs may be used in the existing and future treatment of seemingly unrelated immune diseases. In other words, according to the main concept of the SCHOOL model, specific therapeutic agent(s) that target particular MIRR(s) involved in pathogenesis of the relevant immune disorder can be readily designed using basic principles of structural assembly of this receptor and the SCHOOL model as applied to this particular member of MIRR family. An exciting and promising example of using the SCHOOL model-driven TM approach for both fundamental and clinical applications has been recendy demonstrated by CoUier et al,^ as covered in more detail below.

The SCHOOL model is the first model to clearly explain molecular mechanisms of action of TCR TM peptides (see also Chapter 16) and extend the concept of their action through these mechanisms to any other TM peptides of MIRRs and to the MIRR-mediated processes involved in viral pathogenesis.^^'^^'^^'^^ Selected agents si^ested or predicted by the SCHOOL model to affect MIRR TM interactions, thus inhibiting or modulating MIRR-mediated immune cell activation, are listed in Table 3 .

TM peptides capable of inhibiting MIRR-mediated cell activation were first reported in 1997 for antigen-stimulated TCR-mediated T-cell activation by Manolios et al7^ Since that time, despite extensive basic and clinical studies of these and several other TM peptides (see also Chapter 16),^^''^''^'^^'^°^ the molecular mechanisms of action of these clinically relevant peptides have not been elucidated until 2004 when the SCHOOL model was first introduced.^^

The vast majority of basic and clinical findings were reported for the TCR TM core peptide (TCR CP), or TCR mimic peptide, which represents a synthetic peptide corresponding to the sequence of the TM region of the ligand-binding TCRa chain critical for TCR assembly and function. This TM region has been shown to interact with the TM domains of the signaling CD36e and ^ subunits,^^'^^ thus maintaining the integrity of the TCR in resting T-cells.

Briefly, as suggested by the SCHOOL model (Figs. 2 and 3, Table 3 , see also Chapter 22)^27-30,57 ^g TCR CP competes with the TCRa chain for binding to CD36e and ^ hetero-and homodimers, respectively, thus resulting in disconnection/predissociation of the signaling subunits from the remaining receptor complex (Fig. 3 ). This leads to inhibition of antigen-but not antibody-mediated TCR triggering and cell activation (Figs. 2 and 3). It should be highUghted that the proposed mechanism is the only mechanism consistent with all experimental and clinical data reported up to date for TCR and other MIRR TM peptides and their lipid and/or sugar COnjUgateS.58'^4'^5.77.92-100

Recendy, new experimental evidence supporting the proposed mechanism of inhibitory action of TCRa CP has been reported.^^ This study has clearly shown that this peptide does not affect TCR assembly and cell surface expression.^^ Most strikingly, Kurosaka et al^°° have demonstrated that TCRa CP coprecipitates with CD36e. Ihis finding perfectly fits the molecular explanation of its inhibitory action suggested for the first time in 2004 by applying the SCHOOL modeP^ and later developed further.^^'^^'^^ Again, within the model, competing with the TCRa chain, TCRa CP binds to CD36e and ^ signaling subunits, preventing an antigen-induced formation of the relevant competent signaling oUgomers and thus inhibiting an antigen-dependent T-cell response (Figs. 2 and 3, Table 3 , see also Chapter 12) .

The SCHOOL model predicts that the same mechanisms of inhibitory action can be applied to MIRR TM peptides corresponding to the TM regions of not only the MIRR recognition subunits but to the corresponding signaling subunits as well.^^'^° This was recently confirmed experimen-tally^'^^ by showing that the synthetic peptides corresponding to the sequences of the TM regions of the signaling CD3 (6, E, or y) and ^ subunits are able to inhibit the immune response in vivo (CD3 TM peptides) and NK cell cytolytic activity in vivo (^ TM peptide) (Table 3) . Interestingly, the model suggests a molecular explanation for the intriguing phenomenon recendy reported by Collier et al^ and interpreted by the authors as a discrepancy in CD3 TM peptide activity between in vitro and in vivo T-cell inhibition. It has been shown that the CD36 and CD3Y TM peptides do not impact T-cell function in vitro (the CD3s TM peptide has not been used in the reported in vitro experiments because of solubility issues) but that all three CD3 TM peptides decrease signs of inflammation in the adjuvant-induced arthritis rat model in vivo and inhibit an Figure 3 . A proposed molecular mechanism of action of the T-cell receptor core peptide (CP) and HIV-1 gp41 fusion peptide (FP). Considering the close similarity in patterns of inhibition of T-cell activation and immunosuppressive activity observed for CP and FP, the SCHOOL model reasonably suggests a similar molecular mechanism of action for both peptides. Within the SCHOOL model, these peptides compete with the TCRa chain for binding to the CD36E and ^ signaling subunits, thus disrupting the transmembrane (TM) interactions between these subunits and resulting in disconnection and predissociation of the relevant signaling subunits from the remaining receptor complex (also shown in the inset as a simplified axial view). This prevents formation of signaling oligomers upon multivalent antigen stimulation, thus inhibiting antigen-mediated T-cell activation. In contrast, stimulation of these "predissociated" MIRRs with cross-linking antibodies to signaling subunit should still lead to receptor triggering and cell activation. The model predicts that the same mechanisms of inhibitory action can be applied to TCR TM peptides corresponding to the TM regions of not only the TCRap recognition subunits butthecorrespondingCD3E,CD36,CD3Yand^signaling subunits as well, in addition, similar mechanisms are proposed to be used by other viruses, such as cytomegalovirus and severe acute respiratory syndrome-associated coronavirus, in their pathogenesis to modulate the host immune response.

immune response,'^ Within the SCHOOL model, these data do not reveal any discrepancy between in vivo and in vitro experiments. Instead, they can be considered, in fact, as the first direct experimental evidence of our ability to selectively modulate the MIRR-mediated TM signaling and the immune response, as predicted by the model.^^'^° In this context, the CD38 and CD3Y TM peptides disconnect the corresponding signaling subunits (CD36 and CD3Y, respectively) from the remaining receptor complex (Table 3) . Therefore, antigen stimulation does not result in formation of the relevant competent CD38 or CD3Y signaling oligomers and phosphorylation of their ITAM tyrosine residues, preventing initiation of the corresponding signaUng pathways and cell responses. Further, in their in vitro experiments, the authors^^ used an interleukin 2 (IL-2) production assay and T-cell proliferation as markers of T-cell activation. However, the previously reported in vitro activation studies with T-cells lacking CD3Y and/or CD38 cytoplasmic domains clearly indicate that antigen-stimulated induction of cytokine secretion and T-cell proliferation in these cells are intact,^°^'^°^ explaining the absence of inhibitory effect of the CD38 and CD3YTM peptides in the in vitro activation assays used.^ However, in vivo deficiency either of CD38 or CD3Y results in severe immunodeficiency disorders.^"^'^^ This can explain the inhibitory effect observed in the in vivo studies for all three CD3 TM peptides.^ These experimental data^ successfully proved that our ability to selectively physically disconnect specific signaling subunits using the MIRR TM peptides in hne with the SCHOOL model can result in their selective functional disconnection and thus provide a powerful tool to study MIRR functions and immune cell signaling.^^'^^ Even more importandy, it also confirms that as prediaed using the SCHOOL model,^^'^^ agents targeted specific intra-MIRR TM interactions can be designed not only to inhibit but also specifically modulate the immune response and therefore result in the development of novel therapeutic strategies for a variety of inmiune disorders.

Similar molecular mechanisms of action are su^ested by the SCHOOL model for other MIRR TM peptides and describe and/or predict their inhibitory/modulatory effect on MIRR-mediated cell activation (Table 3) . Recently, the SCHOOL model-driven TM-targeted strategy has been successfully applied to develop a novel concept of platelet inhibition and resulted in the invention of a new class of platelet inhibitors (Fig. 4 , Table 3 , see also Chapter 12) .^^'°^ This issue will be covered in more detail below.

In summary, considering the high therapeutic potential of the MIRR TM peptides illustrated by the clinical results for the TCR CP (Table 4 ) and the promising results for other peptides (Table  ^^58 .108 jj^g SCHOOL model represents an invaluable tool in further development of this novel pharmacological approach targeting MIRR TM interactions.

In contrast to MIRRs, single-chain receptors (SRs) can be characterized in the structural context as receptors with extracellular recognition domains and intracellular signaling domains located on the same protein chain. Examples include receptor tyrosine kinases (RTKs) that are TM glycoproteins consisting of a variable extracellular N-terminal domain, a single membrane spanning domain and a large cytoplasmic portion composed of a juxtamembrane domain, the highly conserved tyrosine kinase domain and a C-terminal regulatory region. Ligand binding is believed to stimulate monomeric receptor dimerization and trans-autophosphorylation at defined tyrosine residues through intrinsic kinase activity.^^'^ ^ ^ Further, the basic principles of SR signaling, namely the ligand-induced receptor dimerization/oligomerization and trans-autophosphorylation Table 4 

Number of Tyr residues in CYTO signaling domains, are considered to represent common mechanisms of triggering and TM signal transduction for the vast majority of various receptors.^^'^*^^^'^^ Dimerization of SRs is known to be mosdy driven by the homointeractions between receptor TM domains.^^'^^'^^'^^'^^'^^ These findings reveal the interreceptor TM protein-protein interactions as an attractive point of pharmacological intervention. At present, there is a groAvingline of experimental evidence indicating that an application of TM-targeted strategy to inhibit/modulate SR signaling might represent a promising therapeutic strategy.^^'^^'^^'^"^^^"

It should be noted that despite apparent similarities in using TM peptides in both SR-and MIRR-targeted TM strategies, the basic principles of the molecular mechanism underlying inhibition and/or modulation of SR and MIRR signaling by using the TM agents are totally different. As established in the field of SR signaling, the SR-targeted TM peptides/agents block/disrupt/ modulate interreceptor TM interaaions crucial for antigen/ligand-induced receptor oligomerization. This prevents the formation of competent receptor oUgomers, whereas MIRR-targeted TM peptides/agents, as suggested by the SCHOOL model (see also Chapter 12)^^'°' 5^' 5^ affect intra-MIRR TM interactions between recognition and signaling subunits, thus preventing upon antigen/ligand stimulation, formation of competent MIRR signaling oligomers but not MIRR oligomers/clusters in terms of MIRR recognition subunits and those signaUng subunits that are not affected by the TM agents. Because of this fundamental difference in molecular mechanisms of action of SR-and MIRR-targeted TM peptides/agents, I consider the SR-related findings as indirect evidence for the fundamental and clinical relevance of the MIRR TM-targeted strategy suggested by the SCHOOL model. Several examples of using TM peptides to inhibit SR signaling are described in more detail below.

Ligand binding-induced association of the TM domains has been proposed to favor productive dimerization of intracellular kinase domains to promote trans-autophosphorylation.^^^ Studies with the epidermal growth factor (EOF) and ErbB2 receptors have shown that synthetic peptides encompassing the TM domains of these receptors inhibit the autophosphorylation and signaling pathway of their cognate receptor. ^^^'^^ These peptides are thought to block/disrupt specific TM interactions, thereby inhibiting receptor dimerization and activation.^^"^'^^ Using differential epitope tagging, it has been demonstrated that P2-adrenergic receptors form homodimers and TM domain VI of the receptor may represent part of an interface for receptor dimerization.^^^ As shown, a peptide derived from this domain inhibits both dimerization and P-adrenergic agonist-promoted stimulation of adenylyl cyclase activity.^^^ In contrast, a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (DIDR) has been found to specifically inhibit D IDR binding and function without affecting receptor oligomerization.^^^ One possible explanation for this finding is that in addition to Ugand-stimulated dimerization of receptors, the correct (permissive) relative orientation in the receptor dimers formed can also play an important role in D1DR signaling. The importance of the relative orientation has been shown for other SRs such as, for example, EGF receptors,^^^ Epo receptor,^ ^"^'^^'^"^ toll-like receptors (TLRs) ^^^ and the integral membrane receptor LuxPQ.^^^ The presence of the TM peptide bound to the D IDR TM domain is likely to prevent Ugand-induced formation of receptor dimers with correct intermolecular orientation, thus preventing generation of the activation signal.

Another example of SR-targeted TM inhibitory peptides, the short peptide sequences corresponding to the Neu RTK TM domain, have been also reported to independently fold in membranes, interact with the full-length receptor and inhibit transformation of cells in vitro and m VIVO.

G-protein-coupled receptors (GPCR) are characterized by the presence of seven TM domains and represent a superfamily of proteins that mediate the function of neurotransmitters and peptide hormones and are involved in viral entry and perception of light, smell and taste. Structural analogs of individual TM domains of GPCRs have been reported to serve as potent and specific receptor inhibitors.^^^ Peptide sequences corresponding to the TM domains of chemokine receptors, CXCR4, also called fusin, an alpha-chemokine receptor specific for stromal-derived-factor-1 and CCR5, the chemokine receptor which HIV uses as a coreceptor to gain entry into macrophages.

have been demonstrated to specifically inhibit receptor signaling and the in vitro replication of HIV-l.^^^ Similarly, peptides mimicking the TM domains of cholecystokinin receptor A, have been found to abolish ligand binding and signahng through the receptor.^^^ Thus, the sequence-based blockade of the interreceptor TM protein interactions as applied to SR signaling provides indirect evidence for the importance and clinical significance of the intra-MIRR TM-targeted strategy suggested by the SCHOOL model.

In general terms, viral pathogenesis is the process by which viral infection leads to disease. The consequences of a viral infection depend on a number of viral and host factors that affect pathogenesis. Infection of host cells by enveloped viruses requires fusion of the viral membrane with the host cell membrane. This fusion is mediated by viral glycoproteins (gp), the proteins that are anchored to the viral membrane. The fusion glycoproteins of enveloped viruses, typically type-I integral membrane proteins, are known to contain in their sequences a short region called the "fusion peptide" (FP), which is required for mediating membrane fusion.^^^'^^ This region interacts with the host cell membrane at an early stage of the membrane fusion process. Despite advances in our understanding of the major principles of viral fusion mediated by the fusion glycoproteins,^^^^^^ litde is known about their role in functional modulation of MIRR-mediated TM signal transduction.

In this section, I focus on MIRR signaling-related immunomodulatory activity recendy reported for HIV and CMV. As suggested by the SCHOOL model, the molecular mechanisms underlying this activity affect MIRR TM interactions and can be also used by other viruses. To illustrate this point, I describe an application of the model in the pathogeneses of two other viruses, SARS-CoV and HTLV-1.1 also demonstrate how the SCHOOL model-driven TM strategy, together with the lessons learned from viral pathogenesis, can be used practically for rational drug design and the development of new therapeutic approaches. 

The FP found in the N terminus of the HIV envelope glycoprotein gp41 functions together with other gp4l domains to fuse the virion with the host cell membrane.^^'^"^^ Surprisingly, this peptide has been recently shown to have not only a fusogenic activity but also a T-cell-targeted immunomodulatory activity: it colocalizes with CD4 and TCR molecules, coprecipitates with the TCR and inhibits antigen-stimulated T-cell proliferation and proinflammatory cytokine secretion in vitro.^^ These effects are specific, T-cell activation via PMA/ionomycin or mitogenic antibodies to CD3 is not affected by FP and FP does not interfere with antigen-presenting cell function.^^ In mice, HIV FP shows immunosuppressive activity, inhibiting the activation of arthritogenic T-cells in the autoimmune disease model of adjuvant arthritis and reducing the disease-associated interferon-y (IFN-y) response.^^ The close match between these findings^^ and the experimental data generated for TCR CP^^'^'^ suggests a mechanistic similarity underlying the TCR-targeted HIV FP and TCR CP activities.

However, as with TCR CP, despite ongoing studies of HIV gp41 FP,^° the molecular mechanisms of immunomodulatory action of this peptide have not been elucidated until 2006 when the SCHOOL model was first applied to this area.^^ Considering the close similarity in patterns of inhibition of T-cell activation and immunosuppressive activity observed for FP^^ and CP,^^'^'^ the SCHOOL model reasonably suggests a similar molecular mechanism of action for TCR TM peptides and HIV gp41 FP (Fig. 3, Tables 3 and 5 ).27^0' 57 Primary sequence analysis of these two peptides ( Table 6 ) shows different primary sequences but a similarity in charged or polar residue distribution patterns with two positively charged residues spaced apart by 4 (CP) or 8 (FP) amino acids. For CP, Arg and Lys residues are known to mediate the interaction between recognition TCRa subunit and signaling CD38E and ^ subunits.^^ Importandy, for FP, both arginines are located in the C-terminal half, su^esting that this sequence could be important for the interaction with the TCR. Figure 3 shows a potential mode of action of CP and FP as proposed by the SCFIOOL model (see also Tables 3 and 5 ). Briefly, CP and FP compete with the TCRa chain for binding to CD388 and ^ hetero-and homodimers, respectively, thus resulting in TM disconnection/predissociation of the signaUngsubunits from the remaining receptor complex (Fig. 3) . This mechanism of FP action su^ests the existence of an interaction interface in the C-terminal half of the peptide. Within the model,^^'^^'^^ the peptide prevents formation of CD38e and ^ signaling oligomers and thus inhibits antigen-dependent T-cell activation (Fig. 3, Table 5 ), acting similarly in this respect to TCR CP (Fig. 3, Tables 3 and 4) .^^'°'^ However, stimulation with anti-CD3 antibodies of these "predissociated" TCRs still should result^^'^^'^^ and results^^'^^ in receptor triggering and cell activation. The model suggests that clinically relevant antibodies (OKT3) could be used to modulate the affected T-cell response during HIV infection. Recendy, OKT3 antibodies have been used successfully in HIV therapy to augment immune activation.^"^^ More recent studies^^ have confirmed the predicted molecular mechanism of immunomodulatory activity of the HIV FP. Finally, it should be noted that the proposed mechanism is the only mechanism consistent with all experimental data on immunomodulatory action of HIV gp41 FP reported up to date.^^'^° A highly specific natural inhibitor of HIV-1 gp41 FP has been recently reported to block HIV-1 entry.^"^^'^^ This agent that has been isolated from human hemofiltrate and designated VIRus Inhibitory Peptide (VIRIP),^^° represents a 20-residue peptide, corresponding to the C-proximal region of a 1-antitrypsin. Importandy, it has been shown that VIRIP direcdy interacts with the gp41 FP and a few amino acid changes increase its antiretroviral activity potency by two orders of magnitude, thus demonstrating the usability and efficiency of rational peptide design approaches.^^^

According to the SCHOOL model, the TCR TM interactions represent not only important therapeutic targets for immune-mediated diseases but also a point of HIV intervention. The molecular mechanisms revealed by the model can be used in rational antiviral drug design and the development of novel antiviral therapies.

Another application of the SCHOOL model to HIV pathogenesis is related to the molecular mechanisms of action of the HIV pathogenicity factor Nef, a key protein in viral replication and progression of disease. Several studies have shown that this protein interacts with the TCR ^ chain and mediates downmodulation of TCR-CD3 complex. ^^^'^^ Notably, Nef lowers the threshold of CD4-I-T-ceU activation.^^"^'^^ Other study showed that Nef induces transcription of an array of genes almost identical to that triggered upon exogenous stimulation of TCR.^^^ Nef has been also reported to affect T-cell activation events through its interactions within the lipid raft microenvironment,^^^ induce signal transduction via the recruitment of a signaling machinery, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade^^^ and, finally, to form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T-cell activation. ^^^ Thus, the extent of T-cell activation imprinted by expression of Nef is a matter of controversy. In addition, although we know that Nef binds the TCR ^ chain,^^^'^^ the role of this interaction and the mechanism used by Nef to modulate T-cell activation remain unknown.

Importandy, similar to ^,^^'^ Nef exists in several discrete oligomeric species, namely monomers, dimers and trimers.^^^ Within the model,^^'^^ natively oligomeric Nef may crosslink homodimeric ^ chains, leading to the formation of multivalent TCR complexes that have been shown to be responsible for sensing low concentrations of antigen.^^^ This mechanism could explain the observed activation sensitization in T-cells by Nef^^"^'^^ On the other hand, Nef dimers may crosslink ^ homodimers in the "permissive" relative orientation and promote formation of competent signaling ^ oligomers, generating an activation signal A (Fig. 3 , see also Chapter 12)^^'^ and resulting in Table 5 .

HIV gp41 FP TCR Colocalizes u'ith CD4 and TCR molecules, coprecipi- dissociation of the ^ signaling oligomers from the remaining receptor complex with its subsequent internalization. The SCFIOOL model suggests that the oligomer interfaces of ^ and/or Nef are involved in the molecular mechanisms underlying the immunomodulatory effects of Ne£^^'^^ As recendy shown, ^^^ a Nef mutant carrying a mutation targeted to the conserved residue D123, in addition to losing the ability to oligomerize, is defective for major histocompatibility complex class I (MHCT) downmodulation and enhancement of viral infectivity, suggesting that the oligomerization of Nef may be critical for its multiple functions. In this regard, I suggest that both proposed mechanisms may take place in vivo and selection between these two alternative pathways may possibly depend on the type of cells infected and/or on the cell membrane lipid content. Thus, CYTO heterointeractions at the Nef-^ interface and CYTO homointeractions in Nef and t, oligomers may represent attractive targets for the design of antiviral agents.

The usability and efficiency of this SCFIOOL model-driven CYTO approach have been later demonstrated for Nef-mediated internalization of surface CD80 or CD86 that is dependent on the binding of Nef to the CYTO domain of the target CD80 or CD86 molecule, respectively.^^ This issue will be covered in more detail below.

The coronavirus SARS CoV is the etiological agent of SARS that represents the life-threatening disease associated with a mortality of about 10%.^^^ In recent studies, in which a total of 38 patients with SARS were enrolled, have shown that CD4+ and CD8+ T-lymphocyte levels were reduced in 100% and 87% of patients, respectively.^^^ Thus, one can suggest that the virus can have an immunomodulatory activity and this activity is TCR-targeted.

In the traditional view of FlIV disease course, acute HW infection is characterized by massive and rapid CD4+ T-cell loss, whereas chronic infection is characterized by persistent immune activation that drives viral replication and further CD4+ T-cell depletion.^^^'^^^ Thus, HIV infection has been thought of as a relatively indolent disruption of CD4+ T-cells eventually leading to collapse of immune function. As with SARS CoV,^^^ this notion has been largely based on measurements of CD4+ T-cell counts in peripheral blood.^^^'^^ Despite the lack of direct evidence, it is reasonable to suggest that SARS-CoV has a TCR-targeted immunomodulatory activity. More specifically, as with HIV, this activity might be especially important during virus entry to suppress the host response to virus infection. Like other enveloped viruses encoding class I viral fusion proteins such as HIV^^^ and Ebola and avian sarcoma viruses,^^^ SARS-CoV is presumed to use membrane fusion mechanisms for viral entry.^'^''^ It has been shown that the SARS-CoV viral spike (S) protein 2 (S2) is a class I viral fusion protein and is responsible for driving viral and targeT-cell membrane fusion.^^^ Recendy, inhibitory peptides derived from the membrane-proximal heptad repeat region (HR2) of the S2 protein have been suggested as an attractive basis for the development of therapeutics for SARS.^^^ The putative SARS-CoV FP has also been identified at the N terminus of the SARS-CoV S2 subunit.^^^ As shown by using synthetic peptides,^^^ the fusogenic activity of the SARS-CoV FP appears to be dependent on its amino acid sequence, as scrambling the peptide renders it unable to partition into large unilamellar vesicles (LUVs), assume a defined secondary structure, or induce both fusion and leakage of LUV Primary sequence analysis of the SARC-CoV FP and TCRcx TM domain (or TCR CP) shows different primary sequences but reveals a similarity in charged or polar residue distribution patterns with two positively charged residues spaced apart by 4 amino acids (Table 6 ). These two positively charged residues are critical for TCR assembly and function. Within the SCHOOL model,^^'^^'^^ die RILLLK and RSMTLTVQAR motifs in TCR CP and HIV FP (Table 6) , respectively play an important role in mimicking the TCRa TM region and therefore in an inhibitory activity of these peptides (Fig. 3, Tables 3 and 5) . Intriguingly, SARS CoV FP has a structural motif KTPTLK that is strikingly similar to that of TCR CP (Table 6 ). Considering the common structural features of three peptides (TCR CP, SARS-CoV FP and HIV FP) as well as functional similarities between HIV FP and SARS FP in the context of their T-cell-targeted activities, I suggest that like TCR CP and HIV FP, SARS-CoV FP should mimic the TCRa TM domain and therefore exhibit an inhibitory effect on the antigen-mediated TCR TM signaling (Table 5 ). In the context of the SCHOOL model, molecular mechanisms of this inhibitory action of SARS-CoV FP are similar to those suggested for TCR CP and HIV FP (Fig. 3, Tables 3 

As hypothesized, the TCR TM interactions might represent a point of SARS-CoV intervention. If true, the molecular mechanisms revealed can be used in rational antiviral drug design and the development of novel antiviral therapies. I believe that future studies will experimentally prove this hypothesis.

HTLV-l is a type C complex retrovirus. It infects and immortalizes human CD4-I-T-cells in vitro and is associated with the development of adult T-cell leukemia/lymphoma (ATL).^^^'^^° Recent observations demonstrate an immunomodulatory ability of the HTLV-1 regulatory protein p22i8i.i82 ^j^j suggest roles that T-cell activation may play in the pathogenesis of HTLV-1-induced disease.^^'^^1-^^^ Below, I consider similarities between the HIVgp4l and HTLV-l gp21 FPs and the Nef and HTLV-1 p 12 proteins, respectively and describe my structural and functional predictions related to potential TCR-targeted activities of HTLV-l FP and pl2. Currendy, there is no experimental evidence for these predicted activities. Despite this, I beUeve that future studies will prove the hypotheses made by using the SCHOOL model.

Similarly to HIVgp41 protein, ^"^'"^^ the ectodomain of HTLV-l TM protein (gp21) contains an N-terminally located fusion peptide, a sequence that inserts into target cellular membranes and is well-known to be critical for membrane fusion activity.^^"^'^^ However, in contrast to the HIV FP, there has been no report to date of an immunomodulatory activity of the HTLV-l FP.

Primary sequence analysis of these two FPs (Table 6 ) indicates different sequences but reveals an interesting similarity in charged or polar residue distribution patterns with two positively charged residues spaced apart by 8 (HIV FP) or 7 (HTLV-l FP) amino acids. Considering the structural similarities of both FPs and the fact that T-cells are main target for both viruses, it is reasonable to suggest a TCR-targeted immunomodulatory activity for the HTLV-1 FP. As proposed by the SCHOOL for HIV FP and TCR CP (Fig. 3, Tables 3 and S) ,^^-^^^^ a potential mode of action of HTLV-l can involve the TM competition with the TCRa subunit for binding to CD368 and t, subunits, thus resulting in TM disconnection/predissociation of the signaling subunits from the remaining receptor complex (Fig. 3 , Table 3 and 5). As with the HIV FP, this mechanism of HTLV-l FP action su^ests the existence of an interaction interface in the C-terminal half of the peptide. Within the model, the peptide should prevent formation of signaling-competent CD368 and t, oligomers and thus inhibit antigen-dependent T-cell activation, acting similarly in this respect to both TCR CP and HIV FP (Fig. 3, Tables 3,4 and 5) .^^-3o.57.75 However, stimulation with anti-CD3 antibodies of these "predissociated" TCRs should still result (Fig. 3 )^^ 30. 57 ^^ receptor triggering and cell activation. As with HIV infection, the model suggests that clinically relevant antibodies (i.e., OKT3) could be used to modulate the affected T-cell response during HTLV-l infection.

In summary, I propose a new hypothesis that considers the largely unexplored immunomodulatory role of the FP in the HTLV-l infection and pathogenesis of ATL. If true, this hypothesis will generate new therapeutic targets and opportunities. I also suggest that our current and future clinical knowledge, experience and therapeutic strategies can be potentially transferred in this respect between the HIV-and HTLV-l-related medical conditions.

The pl2 protein of HTLV-1 is a small oncoprotein that has been shown to have multiple functions. Expression of pl2 has been demonstrated to induce nuclear factor of activation of T-cells (NF-AT), increase calcium release and transcriptional factor Stat 5 activation in T-cells suggest-ing that pl2 may alter T-cell signaling.^^^^^^ Interestingly, pl2 is important for viral infectivity in quiescent human peripheral blood lymphocytes (PBLs) and the establishment of persistent infection in rabbits.^^^'^^^ Despite the distinct structures, both retroviral accessory proteins HTLV-l pl2 and HIVNef are able to modulate TCR-mediated signaling and play a critical role in enhancing viral infectivity in primary lymphocytes and infected animals. It has been recently reported that pl2 could complement for effects of Nef on HIV-1 infection of Magi-CCR5 cells, which express CD4, CXCR4 and CCR5 on the surface, or macrophages.^^^ Also, the clones of Jurkat cells expressing the highest levels of p 12 have been found to exhibit a more rapid rate of cell proliferation than the parental cells.^^^ Similarly to HIVNef, the pl2 protein, upon engagement of the TCR, relocalizes to the interface between T-ceUs and antigen-presenting cells, defined as the immunological synapse (IS).^^^ Both Nef and pl2 are recruited to the IS, but Nef potentiates TCR signaling^^^ while pl2 dampens it.^^^ In summary, targeting TCR-mediated signaling seems to be a shared feature of both HIV and HTLV-1 viruses, reflecting probably their similar evolutionary pathway towards their adaptation to the host immune response. Thus, it is possible that similar molecular mechanisms maybe involved in TCR-targeting strategies used by Nef and p 12 to modulate TCR-mediated signaUng pathways. If true, this hypothesis will generate new therapeutic targets (i.e., protein-protein interactions at the interface of pl2 and its potential TCR-related partners) and opportunities, similar to those suggested for HIV Nef

To escape from NK cell-mediated surveillance, human CMV interferes with the expression of NKG2D ligands in infected cells. In addition, the virus may keep NK inhibitory receptors engaged by preserving human leukocyte antigen (HLA) class I molecules that have a limited role in antigen presentation.^^^ Despite considerable progress in the field, a number of issues regarding the involvement of NK receptors in the innate immune response to human CMV remain unresolved.

Recently, a direct interaction between the human CMV tegument protein pp65 and the NK cell activating receptor NKp30 has been reported.^^^ It has been shown that the binding of pp65 to NKp30 is specific and functional. Surprisingly, the recognition of pp65 by NKp30 does not lead to NK cell activation but instead results in a general inhibition mediated by the dissociation of the signaling ^ subunit from the NKp30-^ receptor complex. ^^^ This results in the diminishing of activating signals and loss in the ability of NK cells to kill normal, tumor and virus-infected cells.19^

Within the context of SCHOOL model,^^^^ the reported action of the human CMVpp65 protein may be due to its potential impact on the TM interactions between NKp30 and ^, leading to disconnection and dissociation of the ^ subunit.^^ This would prevent the formation of signaling-competent ^ oligomers upon ligand stimulation and consequently, inhibit NK cell cytolytic activity (Fig. 2, Table 5 ) in a manner similar in this respect to the inhibitory action of TCR CP (Fig. 3, Table 3 ). Primary sequence analysis of the N-terminal end of pp65 shows the existence of multiple positively and negatively charged amino acid residues (Table 6 ). This pp65 region possibly contains the sequence that mimics the NKp30 or ^ TM domain with the Arg or Asp residues, respectively, that are known to mediate the interaction between recognition NKp30 chain and signaling ^ subunit (see also Chapter 4) .^^ However, further experimental studies are needed to confirm the proposed mechanism.

General issues related to viral pathogenesis in the context of MIRR TM signaUng are covered in more detail in Chapter 22. In this section, I briefly describe several important lessons that we can learn from the SCHOOL model-revealed similarity of the molecular mechanisms underlying viral pathogenesis and MIRR signaling-targeted immunomodulatory viral activity important for viral immune escape. I also consider the striking similarities of the molecular mechanisms and basic structural principles that are suggested by the model to explain immunomodulatory effects of viral fusion and accessory proteins and synthetic agents affecting intra-or inter-MIRR protein-protein interactions in the TM or CYTO miUeus, respectively.

It seems that in general, viruses use TM-targeted immunomodulatory activity of their fusion proteins mosdy during virus entry to suppress the host immune response, whereas modulation of CYTO interactions by using accessory proteins such as HIV Nef and HTLV-1 p 12 plays a role in viral repUcation and enhancing viral infectivity in the host. Thus, our improved understanding of MIRR signaling-targeted immunomodulatory viral activity might allow us to reveal novel targets at these stages of viral pathogenesis.

I beUeve that lessons that we can learn from viral pathogenesis in the context of the SCHOOL model of immune signaling are very important for our further understanding of the molecular mechanisms used by viruses to infect the host and escape its inmiune response. I also believe that these lessons are of both fundamental and clinical value. Why?

1. Now we know the molecular mechanisms of inhibitory action of MIRR TM peptides such as TCR TM peptides,^^'^'^^'^^'^^'^^-^ NK TM peptides^^ and GPVI TM peptide,^^ as su^ested by the SCHOOL model.^^'^^'^^ We also know that the same mechanisms are very likely to be used in vivo by HIV gp41 FP and also, as predicted by the SCHOOL model, by fusion proteins of other viruses, such as SARS-CoV and HTLV-1 FPs, to suppress the host immune response.^^'^°'^^ Considering the high specificity and efficiency of viral agents in inhibition of immune receptors in combination with our current knowledge of the protein-protein interactions underlying this process, we can now use modern well-established computational, bioinformatic and synthetic methodologies^^'^^ to design and produce highly specific and effective TM-targeted agents that are able to affect specific TM interactions of a targeted MIRR and suppress and/or modulate the MIRR-mediated immune response. These agents would be of great fundamental and clinical value. Similar conclusions can be drawn for our ability to use the SCHOOL model of immune signaling and the lessons learned from our current knowledge of the CYTO-targeted viral strategies to design and produce efficient and specific CYTO-targeted agents. 2. AccordingtotheSCHOOLmodel,TCRCP,HIVgp41 FP and, as predicted, SARS-CoV FP and HTLV-1 FP, affect simUar TCR TM interactions (Fig. 3 , Tables 3 and 5) .'^ ^^^^ Primary sequence analysis of these peptides (Table 6) shows different primary sequences but a striking similarity in charged or polar residue distribution patterns, suggesting that a computational approach combined with the molecular mechanisms of action of these peptides revealed by the SCHOOL model, can and should be used in the rational design of effective immunomodulatory TM-targeted peptides. General well-known principles of designing TM peptides with an ability to insert into the membrane might be readily used at this stage.^^ 3. As suggested by the SCHOOL model (Fig. 2) , TM-targeted agents should inhibit MIRR-mediated cell activation induced only by antigen/ligand but not antibodies to MIRR signaling subunits. Indeed, it has been shown for TCR CP^^ and HIV FP^^ that these TM peptides inhibit only antigen-mediated T-cell activation, whereas stimulation with anti-CD3 antibodies in the presence of the peptides still results in functional cell response. Entirely similar considerations can be applied for other viral FPs such as SARS-CoV FP and HTLV-1 FR Thus, the SCHOOL model suggests that antibodies to MIRR signaling subunits can be used as immunotherapeutics to modulate the affected immune cell response during viral infection. 4. For TCR, considering our selective ability to physically and more importantly, functionally disconnect any particular CD3 and/or ^ signaling subtmits from the remaining receptor by using the relevant TM peptides and basic principles of the SCHOOL model of TCR signaling (Fig. 3) , we can design, synthesize and use these peptides as a powerful tool to dissect fine molecular mechanisms of viral pathogenesis in the context of TCR signaling.

5. Two unrelated enveloped viruses, HIV and human CMV, use a similar mechanism to modulate the host immune response mediated by two functionally different MIRRs-TCR and NKp30. As predicted by the SCHOOL model, SARS-CoV and HTLV-1 can also use similar mechanisms during virus infection. Intriguingly, as shown in Table 6 , similar positively charged residue distribution pattern with two Arg and/or Lys residues spaced apart by 8 amino acids is observed for the FPs of seemingly unrelated viruses such as HIV, Lassa virus (LASV),^^^ lymphocytic choriomeningitis virus (LCMV),^^"^ Mopeia virus (MOPV) ^^^ and Tacaribe virus (TACV).^^"^ Thus, it is very likely that similar general immunomodulatory mechanisms can be or are used by other viral and possibly nonviral pathogens (see also Chapter 22) . In addition, as with HIV gp41 FP,^'^'^^ it is promising to apply a similar strategy to block viral entry by using the agents able to interact directly with FPs of other viruses.

Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix, which triggers platelet adhesion and aggregation. ^^^'^^ The consequence of this process is the formation of a thrombus, which prevents blood loss at sites of injury or leads to occlusion and irreversible tissue damage or infarction in diseased vessels.^^^ Despite intensive research efforts in antithrombotic drug discovery and development, uncontrolled hemorrhage still remains the most common side effect associated with antithrombotic drugs that are currendy in use.

The major physiological function of platelets is hemostasis, prevention of bleeding and the effect of aspirin has established that they are also involved in its pathological variant, thrombosis.^^ Platelets also play a critical role in coronary artery disease and stroke, as evidenced by the well-documented benefits of antiplatelet therapy. ^^^ Platelet adhesion, aggregation and activation induced by collagen is critically dependent upon the engagement and clustering of GPVI, a type I transmembrane platelet glycoprotein of about 62 kDa and the major signalling receptor for collagen on platelets (see also Chapter 5) .2i,i96,i98-2oo GPVI has no intrinsic signaling capacity and signaling is achieved through the association with its signaling partner, the FcRy chain.^^ The selective inhibition of GPVI and/or its signaling is thought by most experts in the field to inhibit thrombosis without affecting hemostatic plug formation, thus providing new therapeutical strategies to fight platelet-mediated diseases (see also Chapter 5^ 21, 201204 jj^ contrast to antithrombotic drugs that are currently in use, GPVI receptor-specific inhibitors represent an ideal class of clinically suitable antithrombotics. However, despite intensive studies of the CPVI-FcRy receptor complex,^^'^^'^^^'^^ the mechanism of GPVI signaling was not known until very recently when the SCHOOL model was introduced and applied to GPVI triggering and TM signal transduction.'^^'^^-^^ This resulted in the development of a novel concept of platelet inhibition and the invention of new platelet inhibitors within this promising antithrombotic strategy.^^'^^^ The invented inhibitors are proposed to be useful in the prevention/treatment of thrombosis and other medical conditions involving collagen-induced platelet activation and aggregation as well as in the production of drug-coated medical devices.^^'^^^ Within the SCHOOL model, GPVI-mediated platelet activation is a result of the interplay between CPVI-FcRy TM interactions, the association of two TM Asp residues in the FcRy homodimer with the TM Arg residue of GPVI,^^^ that maintain receptor integrity in platelets under basal conditions and homointeractions between FcRy subunits, leading to initiation of a signaling response (Table 3 , Fig. 4A ). Binding of the multivalent collagen ligand to two or more GPVT-FcRy receptor complexes pushes the receptors to cluster, rotate and adopt an appropriate orientation relative to each other (Fig. 4A, step 1) , at which point the trans-homointeractions between FcRy molecules are initiated. Upon formation of FcRy signaling oligomers, the Src-family kinases Fyn or Lyn phosphorylate the tyrosine residues in the FcRy ITAM that leads to TM transduction of the activation signal (Fig. 4A, step 2) and dissociation of FcRy oligomers and downmodulation of the engaged GPVI subunits (Fig. 4A, step 3) . Later, the dissociated oligomeric FcRy chains can interact with FcRy subunits of the non-engaged GPVI-FcRy complexes, resulting in formation of higher-order signaling oligomers and their subsequent phosphorylation, thus providing lateral signal propagation and amplification (not shown).

For the preformed oligomeric receptor complexes described by Berlanga et al,^°^ this model suggests that under basal conditions, the overall geometry of the receptor dimer keeps FcRy chains apart, whereas stimulation by collagen results in breakage of GPVI-GPVI extracellular interactions and reorientation of signaling FcRy homodimers, thus bringing them into a close proximity and an appropriate relative orientation permissive of initiating the FcRy homointeractions (Fig. 4A) . Thus, the SCHOOL model highUghts a striking similarity between the data on the coexistence of mono-and multivalent TCRs^^^ or GPVIs^^^ in resting T-cells or nonstimulated platelets, respectively and su^ests a similar molecular explanation to answer an important and intriguing question raised in these studies: why does the observed basal TCR or GPVI oligomerization not lead to receptor triggering and subsequent T-cell or platelet activation, respectively, whereas agonist-induced receptor crosslinking/clusteringdoes? See also Chapter 12.^^'°'^ Suggesting how binding to collagen triggers the GPVI-mediated signal cascade at the molecular level, the SCHOOL model of collagen-induced GPVI signaling reveals GPYI-FcRy TM interactions as a novel therapeutic target for the prevention and treatment of platelet-mediated thrombotic events.^^'^°'^^ Specific blockade or disruption of these interactions causes a physical and functional disconnection of the subunits (Fig. 4B, Table 3 ). Antigen stimulation of these "predissociated" receptor complexes leads to clustering of GPVI but not FcRy subunits. As a result, FcRy signaling oligomers are not formed, ITAM Tyr residues do not become phosphorylated and the signaling cascade is not initiated. Agents that target GPVl-FcRy TM interactions may thus represent a novel class of platelet inhibitors. These include, but are not limited to, peptides, peptide derivatives and compositions and nonpeptide small molecule inhibitors. Preliminary experimental results^^'^^^ provided support for this novel concept of platelet inhibition and demonstrated that incubation of whole blood samples with a peptide corresponding to the TM domain of GPVI (Gly-Asn-Leu-Val-Arg-Ile-Cys-Leu-Gly-Ala-Val) at a final concentration of 100 ^M prior to addition of collagen (10 and 20 |ig/ml) or convubdn (10 ng/ml) leads to a 30-60% reduction in both the percentage of P-selectin-positive platelets and the expression of the platelet activation markers, P-selectin and PAC-1 (Sigalov AB, Barnard, MR, Frelinger AL, Michelson AD, unpubhshed results). This effect is specific: platelet activation via ADP (20 [>iM) is not affected by the peptide. As assumed by the SCHOOL model, this peptide penetrates the platelet membrane and competitively binds to the FcRy TM domain, thus replacing GPVI receptor from its interaction with the signaling FcRy subunit and resulting in "predissociation" of the GPVI-FcRy receptor complex (Fig. 4B) . Notably, a control peptide containing a single amino acid substitution (Arg to Ala) does not display inhibitory activity, a phenomenon predicted by the SCHOOL model since this peptide cannot compete with GPVI for binding with FcRy in the TM milieu.

In conclusion, a combination of basic principles of the SCHOOL model with a recendy reported computational design of peptides that target TM helixes in a sequence-specific manner^^ and other well-established techniques to search for the relevant TM mutations or small molecule disruptors as well as to synthesize and optimize TM peptides and peptidomimetics^'^^'^'^^'^^'^^ opens up a new avenue for designing novel platelet inhibitors, making the proposed strategy both feasible and of great fundamental and cUnical value. Combining breakthrough scientific ideas and advances in different fields^^'^^'^^'^^'^^ and the high market potential,^^^ the suggested technology opens new perspectives in innovative antithrombotic drug discovery and development.

As another interesting application of the SCHOOL model that challenges its predictive power, I describe my prognosis related to the use of TM-and possibly CYTO-targeted agents as therapeutics to treat inflammatory bowel diseases (IBDs). Briefly, intestinal inflammation in colitic severe combined immunodeficiency (SCID) mice has been recendy shown to be characterized by significant increase of CD4+NKG2D+ T-cells.^^° As also demonstrated,^^^ neutralizing anti-NKG2D mAb treatment prevents or ameUorates the development of colitis primarily by inhibiting the expansion and/or infiltration of pathogenic T-cells in the colon and secondarily by inhibiting the development of pathogenic Thl cells. The authors concluded that targeting of NKG2D signaling in NKG2D-expressing pathogenic CD4+ T-cells may be a useful strategy for the treatment of Thl-mediated chronic intestinal inflammation such as Crohn's disease.^^^ I surest that TM-targeted agents such as the TM peptides designed by using the SCHOOL model-driven TM-targeted strategy should have specific NKG2D-inhibitory activity and therefore can be used as promising therapeutics to prevent and/or treat IBDs (Table 4) . Future studies will prove or disprove this hypothesis.

As mentioned above, the CYTO domains of the vast majority of MIRR signaling subunits, namely, CD3e, CD3d, CD3g, C> Igot» IgP and FcRy, have been recently shown to represent a new class of intrinsically disordered proteins (IDPs, see also Chapter 12) .^^'^^ By definition, IDPs (or natively unfolded, or intrinsically unstructured) are proteins that lack a well-defined ordered structure under physiological conditions in vitro, i.e., neutral pH and room temperature.^^ ^ A highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling.^^^^^^ In addition, intrinsically disordered regions of human plasma membrane proteins have been very recendy demonstrated to preferentially occur in the cytoplasmic segment.^^^ Finally, it has been suggested that protein phosphorylation, one of the critical and obligatory events in cell signaling, occurs predominandy within intrinsically disordered protein regions.^^^ My major assumption is that a flexible, random-coil conformation of the MIRR signaling subunit CYTO domains plays an important role in MIRR triggering and TM signaling.^^^^*^^^^ I also suggest that the CYTO domains of those MIRR signaling subunits that have not been studied so far (e.g., DAP 12, DAP 10 and FceRip), are IDPs as well. Future studies will prove or disprove this hypothesis.

Surprisingly, all intrinsically disordered CYTO domains studied exist under physiological conditions as specific oligomers (mostly, dimers), as I discovered in 2001 and published in 2004^^ and even more interestingly, these IDPs do not undergo a transition between disordered and ordered states upon dimerization.^^^^ This specific dimerization is distinct from nonspecific aggregation behavior seen in many systems. These findings oppose the generally accepted view on the behavior of IDPs, providing first evidence for the existence of specific dimerization interactions for IDP species and thus opening a new Hne of research in this new and quickly developing field of IDPs. The unusualness and uniqueness of the discovered biophysical phenomenon that was found to be a general phenomenon with all CYTO domains studied in this work,^^ led me to hypothesize that the homointeractions between MIRR signaling subunits represent the missing piece in the puzzle of MIRR triggering and TM signal transduction and to develop the SCFiOOL model (see also Chapter I2).^«-^«' 5^-5«

Since it was first published in 2004,^^ the SCHOOL model has revealed inter-MIRR CYTO homointeractions as important therapeutic targets as well as points of great interest to study molecular mechanisms underlying the MIRR-mediated cell response in health and disease (Figs. 1 and 5^ 27 30, 57, 58 ^it}^in thc modcl, upon antigen/Ugand stimulation, these interactions represent one of three major driving forces of MIRR triggering signal (Table 1 , Fig. IB , see also Chapter 12) . As suggested by the SCHOOL model, specific blockade of the interreceptor CYTO homointeractions between MIRR signaling subunits by CYTO-targeted agents or site-specific point mutations within the dimerization/oligomerization interfaces prevents formation of competent signaling oligomers ( Figs. 1 and 5 ) and initiation of a MIRR-mediated cell response. Similar to Target I, the intra-MIRR TM interactions, modulation of the inter-MIRR homointeractions between particular signaling cytoplasmic domains might allow us to modulate the ligand-induced cell response. In addition, our ability to selectively prevent the formation of signaling oligomers of particular subunit(s) might also prove to be an important tool in functional studies of MIRRs. Peptides and their derivatives, small molecule disruptors of protein-protein interactions, site-specific mutations and other similar agents/modifications can be used to affect the MIRR CYTO interactions. As mentioned above. Importandy, in contrast to TM-targeted agent-affected MIRRs that can be still activated by specific antibodies (Fig. 2B) , stimulation of CYTO-targeted agent-affected MIRRs with specific antibodies that trigger cell activation should not result in MIRR triggering and generation of the activation signal (Fig. 5) .

Thus, I suggest that like intra-MIRR TM interactions, the interreceptor CYTO homointeractions between MIRR signaling subunits represent extremely important points of control in MIRR triggering and cell activation. Since now we can use the SCHOOL model to design the CYTO-targeted agents effective in inhibition and/or modulation of MIRR-mediated TM signaling (Figs. 1 and 5, see also Chapter 12)^^'^ and have a powerful and well-controlled influence upon MIRR-mediated cell activation, thus controlling the immune response. The relevant CYTO-targeted agents for any particular member of MIRR family can be readily designed using the SCHOOL model and our knowledge about structural organization of this receptor.

Since homooligomerization of the MIRR signaling subunit CYTO domains was discovered^^ and these CYTO homointeractions were suggested to represent an important therapeutic target,^^'^^ no direct experimental evidence has been reported to support this hypothesis. However, there is a growing line of indirect evidences indicating the importance of CYTO domains in functionally relevant homooligomerization of other receptors in vivo and demonstrating that the SCHOOL model-driven MIRR CYTO-targeted strategy using a variety of CYTO-targeted agents and/or mutations (Fig. 5) is technologically feasible and can be readily applied in both fundamental and cUnical applications. These findings are mostly related to the field of SR triggering and TM signaling and will be described below.

Fas (CD95, APO-1, TNFRSF6) is a tumor necrosis factor (TNF) receptor superfamily member that direcdy triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity.^^^ Although Fas-associated death domain (FADD) and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain (DD) leads to activation of caspase-8 in the receptor signaling complex.^^^'^^ Recendy, Ugand-induced formation of surface receptor oligomers has been reported for Fas receptor.^^^ A cytoplasmic DD of this SR, upon ligand stimulation, binds to the homologous DD of the adaptor protein FADD and homooligomerizes, thus initiating the caspase signaling cascade (Fig. 6A) . Interestingly, an autoimmune lymphoproliferative syndrome-linked mutation in Fas cytoplasmic domain (T225K) impairs receptor oligomerization and inhibits Fas-mediated signaling but retains the ability to interact with FADD (Fig. 6A) .^^^ This suggests that homointeractions between signaling cytoplasmic tails themselves play an important role in ligand-induced surface receptor oligomerization and subsequent signaling.

This interesting finding supports the proposed MIRR CYTO-targeted strategy and provides a promising direction for future research. One can also hypothesize that similar mutations located in the CYTO domains of MIRR signaling subunits might occur naturally in MIRR-mediated disorders and disturb the homooligomerization interface(s), thus preventing formation of competent signaling subunit oligomers and MIRR triggering.

There is growing line of evidence indicating that CYTO peptides and peptidomimetics can be successfiilly used to target CYTO hetero-or homointeractions between entire protein molecules or the CYTO domains of TM proteins. ^^"^'^^^^^ Ihis means that once we can identify a new promising therapeutic CYTO target, it is technologically feasible to design, synthesize and use the relevant peptide-based agents, peptidomimetics and small molecules (or screen for the appropriate agents by using high throughput screening assays). Selected examples of CYTO-targeted agents used to inhibit CYTO protein-protein interactions, thus modifying the functional response, are considered in more detail below.

Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein that recruits signaling proteins to TLR/IL-1 receptor (IL-IR) superfamily and thus plays a crucial role in the signaling pathways triggered by these receptors in innate host defense.^^^'^^ A critical event in MyD88-trgiggered signaling pathway is homodimerization of MyD88 mediated by its TLR/ IL-IR translation initiation domain (TIR) that is able to heterodimerize with the receptor and homodimerize with another MyD88 molecule (Fig. 6B) .^^^'^^'^^ Dimerization of MyD88 favors the recruitment of downstream signaling molecules such as two IL-lR-associated kinases (IRAKs): IRAKI and IRAK4 (Fig. 6B) . Recently eptapeptides that mimic the BB-loop region of the conserved TIR domain of MyD88, have been shown to effectively inhibit homodimerization with either the isolated TIR or full-length MyD88 (Fig. 6B) .^^^ The authors also demonstrated that a cell permeable analog of MyD88 eptapeptide inhibits homodimerization of MyD88 TIR domains in an in vitro cell system and significandy reduces IL-1 signaling, indicating that the MyD88 homodimerization interface is a good target for specific inhibition of MyD88-mediated signaling in vivo.^^^ Importandy, a synthetic peptidomimetic compound modeled after the structure of a heptapeptide in the BB-loop of the MyD88-TIR domain has been shown very recently to inhibit MyD88 dimerization in coimmunoprecipitation experiments.^^^ This effect is specific for homodimerization of the TIR domains and does not affect homodimerization of the DDs. Ihe agent causes inhibition of IL-l(3-mediated activation of NF-KB transcriptional activity.^^^ After oral administration, the compound results in dose-dependent inhibition of IL-l(3-induced production of IL-6 in treated mice.^^^ In addition, it suppresses B-cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling.^^^ These data indicate that the peptidomimetic compound studied blocks IL-IR/TLR signaling by interfering with MyD88 homodimerization. This suggests that inhibition of MyD88 homodimerization in the CYTO milieu by peptide-based agents or peptidomimetics may have therapeutic potential in treatment of chronic inflammatory diseases.^^^ These findings strongly support basic principles of the SCHOOL model-suggested strategy with its new point of intervention to inhibit/modulate MIRR triggering and the immune response, the MIRR signaling subunit CYTO homointeractions. As with TM-targeted strategy, our current understanding of MIRR structure and the nature and specificity of antigen/ligand-induced homointeractions between receptor signaling subunits not only allows us to inhibit these protein-protein interactions but also to modulate the interactions by a sequence-based approach using corresponding peptides and/or their derivatives. Peptidomimetics and small molecules can be used for these purposes, as well. Strengthening/weakening and/or selective inhibition of the association between particular signaling subunits might allow us not to inhibit, but rather to modulate the ligand-induced cell response. In addition, selective functional inhibition of particular signaling subunits represents an invaluable tool in studies of MIRR-mediated TM signaling and cell activation.

As another example, the processes by which Nef mediates the redistribution of CD80 and CD86 in human monocytic cells can be considered.^^ The endocytic mechanism used to trigger internaUzation of CD80 and CD86 is known to involve Nef binding to the CYTO tails of these target proteins.^^ In an inhibition assay, synthetic peptides corresponding to the CYTO domains of CD80 or CD86 have been demonstrated to inhibit Nef binding to the same peptides immobilized on polystyrene plates. ^^ Introduction of these CYTO peptides into Nef-expressing U937 cells using the Chariot reagent at 4° C causes substantial reduction in the loss of CD80 or CD86, respectively, from the cell surface of Nef-expressing cells,^^ thus proving the principal feasibility and the utihty of the CYTO-targeted strategy suggested by the SCHOOL model.

Interestingly, unlike wild-type Nef, the Nef D123G mutant has been shown to lose its ability to mediate efficient internalization of cell-surface CD80 or CD86, or bind to the CYTO peptides of CD80 or CD86.^^ As mentioned before, mutation of a conserved D123 residue affects the ability of Nef to form dimers and results in impairment of Nef biological frmctions such as MHC class I downmodulation and enhancement of viral infectivity, indicating that the oUgomerization of Nef may be critical for its multiple fimctions.^^^ In this regard, I suggest that the impaired function of the Nef D123G mutant with regard to downmodulation of CD80/CD86 can be explained by its inability to form oUgomers. If true, this means that the rational design of antiviral agents that are able to target CYTO homointeractions in Nef oUgomers may represent an attractive target in the CYTO miUeu, not only with regard to Nef-mediated modulation of TCR triggering and TM signaUng, but also with respect to other Nef biological frmctions.

Peptide-based CYTO-targeted strategy has been also successfully applied to modulate outside-in TM signaling mediated by the platelet receptors such as GPIb/IX/V,^^^ GPIIb^^^ and the megakaryocyte-and platelet-specific integrin allbp3.^^^

The platelet GPIb/IX A^ receptor plays a key role in platelet adhesion at sites of vascular damage through its interaction with subendotheUal-bound von WiUebrand factor (VWF).^^^'^^ However, despite the crucial role that the GPIb/IXA^ receptor complex plays in hemostasis, the molecular mechanisms of its signaUng are not completely understood. The GPIb/IXA'^ complex consists of four subunits, namely, GPIba, GPIbp, GPIX and GPV. An amino acid sequence in the CYTO domain of the GPIbp subunit between residues R151 and A161 has been shown to be highly conserved across species and plays an important physiological role.^^^ It has been also reported^^^ that a synthetic CYTO-targeted agent, the ceU-permeable palmitylated peptide corresponding to this sequence, completely inhibits low-dose thrombin-and ristocetin-induced aggregation in washed platelets, significantly reduces thromboxane (TXA) production in platelets stimulated by thrombin compared with coUagen, substantiaUy decreases activation of the integrin allbp3 in response to thrombin and significantly reduces the adhesion of washed platelets to VWF under static conditions and the velocity of platelets rolUng on VWF. This demonstrates an effective impact of this peptide-based CYTO-targeted agent on platelet frmction in terms of roUing velocity, adhesion, spreading, signaUng to allbp3 and a^egation.

The integrin allbp3 plays an important role in hemostasis mediating platelet adhesion, aggregation and bidirectional signaling.^^^*^^^ Litde is known about the molecular mechanisms underlying the regulation of allb-mediated outside-in signaling. Recendy, it has been shown that this signaling is enhanced in platelets of a patient lacking the terminal 39 residues of the P3 CYTO domain, as detected by thromboxane production and granule secretion and requires Ugand cross-linking of allbp3 and platelet aggregation.^^^ A synthetic CYTO-targeted agent, the ceU-permeable palmitylated P3 peptide corresponding to the CYTO sequence R724-R734, has been demonstrated to effectively and specifically inhibit this outside-in signaling,^^^ thus supporting basic principles and feasibility of the SCHOOL model-suggested CYTO-targeted strategy.

All integrin a subunits are known to contain a highly conserved KXGFFKR motif in their CYTO domains that plays a crucial role in the regulation of integrin affinity for their Ugands.^^^'^^'^^ A synthetic CYTO-targeted agent, the palmitylated peptide corresponding to the K989-R995 sequence of the CYTO domain of the platelet integrin GPIIb (allb) subimit has been shown to specificaUy induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin.^^^ The authors conclude that this Upid-modified peptide imitates the CYTO domain of GPIIb and, in a highly specific and effective manner, initiates paraUel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA2 synthesis and secretion.^^^ An example of using a synthetic peptide to inhibit protein-protein homointeractions in the intracellular milieu has been recently reported in studies of Ebola virus (EBOV), a filovirus that causes sporadic outbreaks of a fatal hemorrhagic fever in Africa.^^^'^^ Viral protein 30 (VP30), one of seven structural proteins of this enveloped virus,^""^ is the constituent of the nucleocapsid and represents an EBOV-specific transcription activation factor.^"^^ The essential role of homooligomerization for the function of VP30 and the significance of the self-assembly of VP30 for viral transcription and propagation have been recently reported.^^^ Interestingly, it has been also shown that the homooligomerization of VP30 can be dose dependently inhibited by a 25-mer peptide derived from the presumed oligomerization interface region.^^° Importantly, when this peptide is transfected into EBOV-infected cells, the peptide inhibits viral replication, suggesting that inhibition of VP30 oligomerization represents a target for EBOV antiviral drugs.^^° This confirms that, as proposed by the SCHOOL model for MIRR-mediated TM signaling and cell activation,^^^^ protein-protein homodimerization/homooligomerization interface(s) can represent an important point of intervention in the CYTO milieu and be targeted by synthetic peptides, their derivatives and peptidomimetics.

Another potential application of the CYTO-targeted strategy involves the use of CYTO-targeted agents to modulate TLR4 signaling. This receptor is activated by monophosphoryl lipid A, derived from the active moiety (lipid A) of bacterial endotoxin (lipopolysaccharide, LPS). As recently demonstrated,^^^ LPS binds to a secreted glycoprotein MD-2, which in turn binds to TLR4 and induces aggregation and signal transduction. It has been also shown that TLR4 can form homodimers.^"^^ Despite both TLR4 monomers and dimers are able to activate NF-KB, this activation is significantly enhanced upon homodimerization.^^^ However, NF-KB activation by TLR4 monomer, but not homodimer, is completely inhibited by dominant negative MyD88, suggesting that TLR4 homodimers and monomers can activate NF-KB through different mechanisms.^^^ Using the protein complementation assay, a novel method to detect protein-protein interactions in vivo,^^ the TLR4 homodimerization has been shown to be mediated by the TLR4 CYTO domain.^^^ I suggest that, similar to other applications mentioned above, CYTO-targeted agents can be used to modulate TLR4-mediated signaling and cell activation, thus modulating the host immune response to LPS.

Despite numerous models of MIRR signaling suggested for particular MIRRs and a growing interest in targeting MIRR signaling as a potential treatment strategy for many immune disorders, the molecular mechanisms that underlie MIRR triggering and subsequent TM signal transduction were unknown for a long time, preventing our improved understanding of these fundamentally important processes and therefore the development of novel pharmacological approaches.

Discovery of an unusual and unique biophysical phenomenon, the existence of specific homointeractions between the intrinsically disordered CYTO domains of MIRR signaling subunits,^^'^^ defined the last piece in the puzzle of MIRR triggering and TM signaling and led me to the development of a general model of MIRR-mediated immune cell activation, the SCHOOL model.^^^^'^'^ Suggesting MIRR triggering as an outcome of ligand-induced interplay between three major driving forces represented by well-defined protein-protein interactions that strikingly fall within the similar micromolar affinity range and are characterized by relatively rapid kinetics, the model finally unravels a long-standing mystery of MIRR-mediated TM signal transduction. Importandy, assuming that the molecular mechanisms underlying TM signaling and cell activation mediated by all receptors that belong to the MIRR family are similar, the SCHOOL model can be readily applied to any particular member of this receptor family. In doing so, the model suggests molecular mechanisms for the vast majority of unexplained immunological observations accumulated to date (see also Chapter 12) and reveals novel universal therapeutic targets for a diverse variety of disorders mediated by immune cells, thus opening new horizons in both funda-mental and clinical research in different fields such as immunology, structural biology, virology, hematology and others.

My central hypothesis is that the similar struaural architecture of the MIRRs dictates similar mechanisms of MIRR tri^ering and subsequent signaUng and cell activation and therefore su^ests the existence of similar therapeutic targets in seemingly unrelated diseases. This makes possible the development of novel pharmacological approaches as well as the transfer of clinical knowledge, experience and therapeutic strategies between various immune disorders. In addition, this hypothesis significantly improves our understanding of the immunomodulatory activity of many human viruses. Thus, the lessons learned from the SCHOOL model and viral pathogenesis indicate that a general drug design approach may be used to treat a variety of different and seemingly unrelated inmiune diseases. The model unraveled the striking similarity of the molecular mechanisms underlying immunomodulatory aaivities of the TCR TM peptides first introduced by Manolios et al in 1997^^ and viral fusion peptides that appeared to be used by different viruses not only to entry target cells but also to modulate and escape the host immune response. This si^ests the possibility to design, synthesize and apply highly specific and effective therapeutic agents and strongly supports the feasibility, utility and both fundamental and clinical importance of the TM-targeted strategy suggested by the SCHOOL model. Application of this model to the platelet collagen receptor GPVI has already resulted in the development of a novel concept of platelet inhibition and the invention of novel platelet inhibitors. Importandy, the similar basic principles based on our current knowledge of the structural assembly of MIRRs and the molecular mechanisms of MIRR signaling suggested by the SCHOOL model were used to explain immunomodulatory activity of TCR TM peptides and to design, synthesize and apply new GPVI-targeted platelet inhibitors. Again, within the model, a similar approach can be applied to any particular receptor of the MIRR family and therefore to any disease or medical conditions mediated by this receptor. This is not only a comprehensive example of the usability and predictive power of the SCHOOL model but also supports my central hypothesis in the context of our ability to develop general pharmacological approaches and transfer clinical knowledge, experience and therapeutic strategies between seemingly disparate immune-mediated diseases.

In summary, I would like to highlight that the SCHOOL model (described in Chapter 12 in more detail) provides a set of basic principles underlying MIRR-mediated signaling and indicates that a general drug design approach could be used to treat many different, seemingly unrelated, immune diseases. Considering the multipUcity and diversity of the MIRRs involved in the pathogenesis of numerous human diseases, the proposed model can contribute significantly to the improvement of existing therapies and the design of new therapeutic strategies for malignancies, diverse immune system disorders, including those with infections caused by various viruses and other MIRR-mediated medical conditions.

Protein-protein interactions as targets for small molecule drug discovery

Protein-protein interactions in human disease

Inhibition of protein-protein association by small molecules: Approaches and progress

Targeting protein-protein interactions by rational design: Mimicry of protein surfaces

Scaffolds for blocking protein-protein interactions

Disruption of protein-protein interactions: Towards new targets for chemotherapy

Design and structure of peptide and peptidomimetic antagonists of protein-protein interaction

Development of small molecules designed to modulate protein-protein interactions

Modulation of protein-protein interactions with small organic molecules

Protein-protein interactions as a target for drugs in proteomics

Protein-protein interactions: Mechanisms and modification by drugs

Emerging classes of protein-protein interaction inhibitors and new tools for their development

Disabled receptor signaling and new primary immunodeficiency disorders

Fc receptors and their role in immune regulation and autoimmunity

Fc receptors: their diverse functions in immunity and immune disorders

IgA and IgA-specific receptors in human disease: structural and functional insights into pathogenesis and therapeutic potential

Fcepsilon-and Fcgamma-receptor signaling in diseases

Human natural killer cell activating receptors

Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis

Platelet receptors and their role in diseases

Platelet glycoprotein VI: Its structure and function

Transmembrane helical interactions and the assembly of the T-cell receptor complex

The organizing principle in the formation of the T-cell receptor-CD3 complex

Mutations within the NH2-terminal transmembrane domain of membrane immunoglobulin (Ig) M alters Ig alpha and Ig beta association and signal transduction

Fc receptor biology

Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells

Multi-chain immune recognition receptors: Spatial organization and signal transduction

Multichain immune recognition receptor signaling: Different players, same game?

Immune cell signaling: A novel mechanistic model reveals new therapeutic targets

Transmembrane interactions as immunotherapeutic targets: Lessons from viral pathogenesis

Homooligomerization of the cytoplasmic domain of the T-cell receptor zeta chain and of other proteins containing the immunoreceptor tyrosine-based activation motif

Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits

Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form

Principles of protein-protein interactions

Ligand recognition by alpha beta T-cell receptors

Intramembrane helix-helix association in oligomerization and transmembrane signaling

The stability of transmembrane helix interactions measured in a biological membrane

Role of the T-cell receptor ligand affinity in T-cell activation by bacterial superantigens

Alphabeta T-cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization

An unusual mechanism for ligand antagonism

Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen

The CD3 gammaepsilon/deltaepsilon signaling module provides normal T-cell functions in the absence of the TCR zeta immimoreceptor tyrosine-based activation motifs

T-cell receptor signal transmission: Who gives an ITAM?

The avian B-cell receptor complex: Distinct roles of Igalpha and Igbeta in B-ccU development

The Ig-alpha ITAM is required for efficient diffisrentiation but not proliferation of preB-cells

Ig beta tyrosine residues contribute to the control of B-cell receptor signaling by regulating receptor internalization

The Fc(epsilon)RIbeta subunit functions as an amplifier of Fc(epsilon)RIgamma-mediated cell activation signals

Signal transduction by immunoglobulin Fc receptors

Diffisrential Src family kinase activity requirements for CD3 zeta phosphorylation/ZAP70 recruitment and CD3 epsilon phosphorylation

Disruption of extracellular interactions impairs T-cell receptor-CD3 complex stability and signaling

Dissociation of intracellular signaling pathways in response to partial agonist ligands of the T-cell receptor

Qualitatively distinct signaling through T-cell antigen receptor subunits

Qualitatively differential regulation of T-cell activation and apoptosis by T-cell receptor zeta chain ITAMs and their tyrosine residues

Reciprocal Regulation of SH3 and SH2 Domain Binding via Tyrosine Phosphorylation of a Common Site in CD3{epsilon}

How TCRs bind MHCs, peptides and coreceptors

Protein-protein interactions and cancer: Small molecules going in for the kill

Interaction between HIV gp41 fusion peptide and T-cell receptor: Putting the puzzle pieces back together

More on: glycoprotein VI oUgomerization: A novel concept of platelet inhibition

Antiplatelet therapy: In search of the 'magic bullet

New approaches to allergen immunotherapy

New developments in FcepsilonRI regulation, function and inhibition

Platelets and anti-platelet therapy

Soluble T-cell receptors: Novel immunotherapies

Novel treatments for food allergy

CD3-specific antibodies: A portal to the treatment of autoimmunity

New reagents on the horizon for immune tolerance

The recombinant T-cell receptor strategy: Insights into structure and function of recombinant immunoreceptors on the way towards an optimal receptor design for cellular immunotherapy

Inhibition of collagen-induced platelet reactivity by DGEA peptide

Antigen-specific blockade of T-cells in vivo using dimeric MHC peptide

The beta subunit of the type I Fcepsilon receptor is a target for peptides inhibiting IgE-mediated secretory response of masT-cells

From T-cell activation signals to signaling control of anti-cancer immunity

Immune receptors: targets for therapy of leukemia/lymphoma, autoimmune diseases and for the prevention of allograft rejection

T-cell receptor mimic peptides and their potential application in T-cell-mediated disease

T-cell antigen receptor peptides inhibit signal transduction within the membrane bilayer

Lipidation and glycosylation of a T-cell antigen receptor (TCR) transmembrane hydrophobic peptide dramatically enhances in vitro and in vivo function

Discrepancy in CD3-transmembrane peptide activity between in vitro and in vivo T-cell inhibition

T-cell antigen receptor transmembrane peptides modulate T-cell function and T-cell-mediated disease

HIV-1 fusion peptide targets the TCR and inhibits antigen-specific T-cell activation

T-Cell inactivation and immunosuppressive activity induced by HIV gp4l via novel interacting motif

T-Cell antigen receptor-alpha chain transmembrane peptides: Correlation between structure and function

Polar residue tagging of transmembrane peptides

Transmembrane interactions in the activation of the Neu receptor tyrosine kinase

Optimizing synthesis and expression of transmembrane peptides and proteins

Computational design of peptides that target transmembrane helices

Designing transmembrane alpha-helices that insert spontaneously

Bioinformatic discovery of novel bioactive peptides

A shortcut to peptides to modulate platelets

Visual detection of specific, native interactions between soluble and microbead-tethered alpha-helices from membrane proteins

Synthetic peptides as models for intrinsic membrane proteins

Immunoreceptor transmembrane peptides and their effect on natural killer (NK) cell cytotoxicity

Biophysical studies of a transmembrane peptide derived from the T-cell antigen receptor

Transmembrane T-cell receptor peptides inhibit B-and natural killer-cell function

T-cell antigen receptor peptide-lipid membrane interactions using surface plasmon resonance

D-enantiomer peptide of the TCRalpha transmembrane domain inhibits T-cell activation in vitro and in vivo

Therapeutic application of T-cell receptor mimic peptides or cDNA in the treatment of T-cell-mediated skin diseases

Peptides in the treatment of inflammatory skin disease

Hydrophobic transmembrane-peptide lipid conjugations enhance membrane binding and functional activity in T-cells

T-cell antigen receptor (TCR) transmembrane peptides colocalizc with TCR, not Upid rafts, in surface membranes

T-cell antigen receptor assembly and cell surface expression is not affected by treatment with T-cell antigen receptor-alpha chain transmembrane Peptide

A structurally altered D,L-amino acid TCRalpha transmembrane peptide interacts with the TCRalpha and inhibits T-cell activation in vitro and in an animal model

Role of CD3 delta in surface expression of the TCR/CD3 complex and in activation for killing analyzed with a CD3 delta-negative cytotoxic T-lymphocyte variant

Role of CD3gamma and CD3delta cytoplasmic domains in cytolytic T-lymphocyte functions and TCR/CD3 down-modulation

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T-cell function

Contributions of the T-cell receptor-associated CD3gamma-ITAM to thymocyte selection

Severe combined immunodeficiency caused by deficiency in either the delta or the epsilon subunit of CD3

Inhibiting collagen-induced platelet aggregation and activation with reptide variants. US 12/001,258 and PCT PCT/US2007/025389 patent applications were filed on 12/11/2007 and 12/12/2007, respectively, claiming a priority to US provisional patent application 60/874

Dimerization of cell surface receptors in signal transduction

Structural analysis of receptor tyrosine kinases

Switching signals on or off by receptor dimerization

Dimerization as a regulatory mechanism in signal transduction

Transmembrane signaling: the joy of aggregation

Receptor signaling: When dimerization is not enough

The HER receptor family: A rich target for therapeutic development

The kiss of death: Promises and failures of death receptors and ligands in cancer therapy

Identification of amino acid residues crucial for chemokine receptor dimerization

Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex

Regulation of signal transduction and signal diversity by receptor oligomerization

The power of two: Protein dimerization in biology

Signal transduction and oncogenesis by ErbB/HER receptors

The single transmembrane domains of ErbB receptors self-associate in cell membranes

Two tumour necrosis factor receptors: Structure and f\inction

Transmembrane peptides as inhibitors of ErbB receptor signaling

A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation

A transmembrane domain-derived peptide inhibits Dl dopamine receptor function without affecting receptor oUgomerization

Activation of platelet alphallbbeta3 by an exogenous peptide corresponding to the transmembrane domain of alphallb

Inhibition of G-protein-coupled receptor function by disruption of transmembrane domain interactions

Inhibition by transmembrane peptides of chimeric insulin receptors

Structure and function of the platelet integrin alphallbbeta3

An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor

Efficiency of signalling through cytokine receptors depends critically on receptor orientation

An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation

Will any dimer do?

Toll-like receptors as molecular switches

Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing

Specific short transmembrane sequences can inhibit transformation by the mutant neu growth factor receptor in vitro and in vivo

What studies of fusion peptides tell us about viral envelope glycoprotein-mediated membrane fusion (review)

Peptides and membrane fusion: towards an understanding of the molecular mechanism of protein-induced fusion

Virus membrane fusion

Fusion peptides and the mechanism of viral fusion

Mechanisms of viral membrane fusion and its inhibition

Lipids as modulators of membrane fusion mediated by viral fusion proteins

Modulation of the immunological synapse: A key to HIV-1 pathogenesis?

HIV-1 pathogenesis

Identification of the fusion peptide of primate immunodeficiency viruses

Detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus

OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T-cell depletion

Targeting the sticky fingers of HIV-1

Discovery and optimization of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide

Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation

The T-cell receptor zeta chain contains two homologous domains with which simian immunodeficiency virus Nef interacts and mediates down-modulation

The conserved process of TCR/CD3 complex down-modulation by SIV Nef is mediated by the central core, not endocytic motifs

Modulation of specific surface receptors and activation sensitization in primary resting CD4+ T-lymphocytes by the Nef protein of HIV-1

HIV-1 Nef increases T-cell activation in a stimulus-dependent manner

Nef triggers a transcriptional program in T-cells imitating single-signal T-cell activation and inducing HIV virulence mediators

HIV type 1 Nef increases the association of T-cell receptor (TCR)-signaling molecules with T-cell rafts and promotes activation-induced raft fusion

Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts

Induction of Fas ligand expression by HIV involves the interaction of Nef with the T-cell receptor zeta chain

Cooperative interactions of simian immunodeficiency virus Nef, AP-2 and CD3-zeta mediate the selective induction of T-cell receptor-CD3 endocytosis

Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein

Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response

Mutation of a conserved residue (D123) required for oUgomerization of himian immunodeficiency virus type 1 Nef protein abolishes interaction with human thioesterase and results in impairment of Nef biological functions

A two-pronged mechanism for HIV-1 Nef-mediated endocytosis of immune costimulatory molecules CD80 and CD86

The Immunobiology of SARS*

Expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome

HIV disease progression: immune activation, microbes and a leaky gut

T-cell depletion in HIV-1 infection: How CD4+ T-cells go out of stock

A general model for the transmembrane proteins of HIV and other retroviruses

Similar structural models of the transmembrane proteins of Ebola and avian sarcoma viruses

Crystal structure of severe acute respiratory syndrome coronavirus spike protein fiision core

Following the rule: Formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors

Structural characterization of the fiision-active complex of severe acute respiratory syndrome (SARS) coronavirus

Functional analysis of an epitope in the S2 subunit of the murine coronavirus spike protein: involvement in fusion activity

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein

Human T-lymphotropic virus 1: Recent knowledge about an ancient infection

Adult T-cell leukemia: Antigen in an ATL cell line and detection of antibodies to the antigen in human sera

Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 use diflFerent receptor complexes to enter T-cells

Inhibition of T-cell receptor signal transduction and viral expression by the linker for activation of T-cells-interacting pl2(I) protein of human T-cell leukemia/lymphoma virus type 1

Substitution of HIV Type 1 Nef with HTLV-1 pl2

Activation of human T-cell leukemia virus type 1 LTR promoter and cellular promoter elements by T-cell receptor signaling and HTLV-1 Tax expression

The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function

Evidence that the transmembrane domain proximal region of the human T-cell leukemia virus type 1 fusion glycoprotein gp21 has distinct roles in the prefusion and fusion-activated states

Activation of nuclear factor of activated T-cells by human T-lymphotropic virus type 1 accessory protein pl2(I)

Human T-cell lymphotropic virus type 1 pl2(I) expression increases cytoplasmic calcium to enhance the activation of nuclear factor of activated T-cells

HTLV-1 pi2(1) protein enhances STAT5 activation and decreases the interleukin-2 requirement for proliferation of primary human peripheral blood mononuclear cells

Human T-lymphotropic virus type 1 open reading frame I pi2(1) is required for efficient viral infectivity in primary lymphocytes

Selective ablation of human T-cell lymphotropic virus type 1 pi21 reduces viral infectivity in vivo

Nef is physically recruited into the immunological synapse and potentiates T-cell activation early after TCR engagement

NK cell receptors involved in the response to human cytomegalovirus infection

Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus

Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity

Platelet physiology and abnormalities of platelet function (first of two parts)

Glycoprotein VI but not alpha2betal integrin is essential for platelet interaction with collagen

Platelet inhibitor therapy: mechanisms of action and clinical use

Structural basis for the platelet-collagen interaction: The smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproiine triplets

Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain

Platelet-collagen interaction: Is GPVI the central receptor?

The Fab fragment of a novel anti-GPVI monoclonal antibody, OM4, reduces in vivo thrombosis without bleeding risk in rats

GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism

Platelet collagen receptors: A new target for inhibition?

Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo

Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium

Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors

Glycoprotein VI oligomerization in cell lines and platelets

Antithrombotic drug market

Blockade of NKG2D signaling prevents the development of murine CD4+ T-cell-mediated cohtis

Why are "natively unfolded" proteins unstructured under physiologic conditions?

Intrinsically unstructured proteins and their functions

Intrinsic disorder and protein function

Intrinsic disorder in cell-signaling and cancer-associated proteins

Intrinsically disordered regions of human plasma membrane proteins preferentially occur in the cytoplasmic segment

The importance of intrinsic disorder for protein phosphorylation

Exploring biology with small organic molecules

Nature-inspired' drug-protein complexes as inhibitors of Abeta aggregation

Targeting protein-protein interactions for cancer therapy

Screening for peptide drugs from the natural repertoire of biodiverse protein folds

Peptide microarrays for the detection of molecular interactions in cellular signal transduction

Death receptor-induced cell killing

The Fas signaling pathway: More than a paradigm

Signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane

A palmitylated peptide derived from the glycoprotein lb beta cytoplasmic tail inhibits platelet activation

A sequence within the cytoplasmic tail of GpIIb independently activates platelet aggregation and thromboxane synthesis

The beta3 subunit of the intcgrin alphallbbeta3 regulates alphallb-mediated outside-in signaling

Pivotal Advance: Inhibition of MyD88 dimerization and recruitment of IRAKI and IRAK4 by a novel peptidomimetic compound

Peptide-mediatcd interference of TIR domain dimerization in MyD88 inhibits interleukin-1-dependent activation of NF-{kappa}B

Oligomerization of Ebola virus VP30 is essential for viral transcription and can be inhibited by a synthetic peptide

Toll-like receptor signalling

The role of MyD88-like adapters in Toll-like receptor signal transduction

The vascular biology of the glycoprotein Ib-IX-V complex

Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von WiUebrand factor

Platelet glycoprotein Ilb/IIIa receptors in cardiovascular medicine

Integrin signaling: The platelet paradigm

Inducible interaction of integrin alpha 2 beta 1 with calreticulin. Dependence on the activation state of the integrin

Breaking the integrin hinge. A defined structural constraint regulates integrin signaling

Integrin alpha chain cytoplasmic tails regulate "antibody-redirected" cell adhesion, independently of ligand binding

Ebolavirus and other filoviruses

Comparison of the transcription and repUcation strategies of marburg virus and Ebola virus by using artificial replication systems

Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction

Integrin-nucleated Toll-like receptor (TLR) dimerization reveals subcellular targeting of TLRs and distinct mechanisms of TLR4 activation and signaling

Detection of protein-protein interactions by protein fragment complementation strategies

Cytoplasmic domain-mediated dimerizations of toll-like receptor 4 observed by beta-lactamase enzyme fragment complementation

I would like to thank Walter M. Kim for his critical reading of this manuscript.