key: cord-0294456-css0yhoy authors: He, Hongjian; Guo, Jiaqi; Xu, Jiashu; Wang, Jiaqing; Liu, Shuang; Xu, Bing title: The Dynamic Continuum of Nanoscale Peptide Assemblies Facilitates Endocytosis and Endosomal Escape date: 2021-03-18 journal: bioRxiv DOI: 10.1101/2021.03.18.435896 sha: 1e3fff58080ce08e379132905c0b5baad7cc6a5a doc_id: 294456 cord_uid: css0yhoy Considerable number of works have reported alkaline phosphatase (ALP) enabled intracellular targeting by peptide assemblies, but little is known how these substrates of ALP enters cells. Here we show that the nanoscale assemblies of phosphopeptides, as a dynamic continuum, cluster ALP to enable caveolae mediated endocytosis (CME) and eventual endosomal escape. Specifically, fluorescent phosphopeptides, as substrates of tissue nonspecific alkaline phosphatase (TNAP), undergo enzyme catalyzed self-assembly to form nanofibers. As shown by live cell imaging, the nanoparticles of phosphopeptides, being incubated with HEK293 cells overexpressing red fluorescent protein-tagged TNAP (TNAP-RFP), cluster TNAP-RFP in lipid rafts to enable CME, further dephosphorylation of the phosphopeptides form the peptide nanofibers for endosomal escape inside cells. Inhibiting TNAP, cleaving the membrane anchored TNAP, or disrupting lipid rafts abolishes the endocytosis. Moreover, decreasing the formation of peptide nanofibers prevents the endosomal escape. As the first study establishing a dynamic continuum of supramolecular assemblies for cellular uptake, this work not only illustrates an effective design for enzyme responsive supramolecular therapeutics, but also provides mechanism insights for understanding the dynamics of cellular uptakes of proteins or exogenous peptide aggregates at nanoscale. continuum of supramolecular assemblies for cellular uptake, this work not only illustrates an effective design for enzyme responsive supramolecular therapeutics, but also provides mechanism insights for understanding the dynamics of cellular uptakes of proteins or exogenous peptide aggregates at nanoscale. Because many drug targets identified by molecular cell biology are inside cells, considerable efforts have focused on engineering molecules for intracellular delivery of various cargo. [1] [2] [3] Besides the use of cationic molecules for enhancing cellular uptake of therapeutics, 4-6 one of the most explored approaches for intracellular delivery is to engineer molecules to be responsive to chemical or physical stimuli, such as redox, 7-10 pH, [11] [12] [13] enzymes, [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] or light. 24, 25 Among enzymatic approach, alkaline phosphatases (ALP) instructed self-assembly of peptides is particularly effective to facilitate the cellular uptake of the peptide assemblies. [14] [15] [16] [17] [18] [26] [27] [28] However, the mechanism of this phenomenon is largely unknown. Coincidently, viruses also use responsive motif for cell entry. For example, the life cycle of virus (e.g., SARS-CoV-2) 29 begins at the attachment to the plasma membrane of host cells 30 to clustering plasma membrane-bound receptors, followed by viral proteolytic priming (VPP) 31, 32 for endocytosis and endosomal escape. Prompted by the mechanism of viral cell entry, we hypothesize that the assemblies of phosphopeptides, as the substrate of ALP, act as a dynamic continuum 33 to cluster the ALP to facilitate endocytosis and to undergo enzymatic morphological transition that enables the endosomal escape (Scheme 1). This hypothesis is also based on several additional rationales: (i) ALP is a GPI-anchor protein, and its clustering enables caveolae mediated endocytosis; 30 (ii) ALP, locating in lipid rafts, plays a key role in the cellular uptake of aberrant protein aggregates; and (iii) ALP catalyzed dephosphorylation enables morphological transition (e.g., nanoparticles to nanofibers) and generates artificial intracellular filaments. 17 To prove our hypothesis, we synthesize a phosphopeptide, which, above its critical micelle concentration (CMC), self-assembles to form nanoparticles, which transform into nanofibers upon the dephosphorylation catalyzed by ALP. The phosphopeptide molecules initially aggregate on the plasma membrane to cluster tissue nonspecific alkaline phosphatase (TNAP) in lipid rafts of HEK293 cells that overexpress TNAP, then enable caveolae-mediate endocytosis (CME). The assemblies of the phosphopeptides gradually changes their morphology from nanoparticles to nanofibers depending on the extent of dephosphorylation. Abolishing the interaction between the TNAP and the phosphopeptides significantly decrease the endocytosis. The TNAP, remained in endosome, catalyzes the further dephosphorylation of the phosphopeptides, generating peptide nanofibers to facilitate endosomal escape. As the first study that illustrates the intracellular peptide assemblies resulted from dynamic peptide assemblies rather than a monomeric peptide or static assemblies, this work not only provides insights for understanding the cellular uptakes of proteins or exogenous peptide aggregates, but also offers the guidance for designing enzyme responsive supramolecular assemblies as intracellular targeting therapeutics. Figure S1 ). Upon the addition of ALP for catalytically dephosphorylating NBD-1p, the nanoparticles formed by NBD-1p transform into short nanofibers (21.9±3.7 nm in diameter and about 300-1000 nm in length) made of NBD-1 ( Figure 1A and Scheme S1). This result confirms that NBD-1p undergoes enzyme-instructed self-assembly. 34 Scheme 1. Illustration of a dynamic continuum of peptide assemblies for endocytosis and endosomal escape We choose HEK293 cells for testing our hypothesis because of their constitutively low expression of ALP, reliable growth, and propensity for transfection. 35 Treating HEK293 cells with the polyethyleneimine (PEI) 36 Figure 2B and 2C) . These results further support that the interaction between TNAP and the nanoparticles of NBD-1p is important for signaling CME. Below the CMC, the individual NBD-1p molecules neither aggregate on the plasma membrane of HEK293_TNAP-RFP cells ( Figure S5 and Movie S3) nor enter the cells efficiently ( Figure 2D ), although the monomeric phosphopeptides may still associate with the TNAP on cell surface as the substrate of TNAP ( Figure 2E ). These results indicate that the clustering of TNAP in lipid rafts by the nanoparticles formed by the self-assembly of NBD-1p is essential for signaling the CME of the assemblies of NBD-1p ( Figure 2E ). Figure 3C ). Dephosphorylation assay reveals that ALP dephosphorylates NBD-1p much faster than NBD-(D)Sp at the same concentration ( Figure 3D ), indicating that ALP exhibits higher affinity for NBD-1p than NBD-(D)Sp. The results above suggest that the interaction between ALP and its substrate is necessary for the CME of the phosphopeptides. Moreover, a sequence-scrambled phosphopeptide derivative of NBD-1p, denoted as NBD-2p (with the sequence of D-Phe-D-Phe-D-Lys( -NBD)-D-pTyr, Scheme S1), also enters the HEK293_TNAP-RFP cells more efficiently than the non-transfected HEK293 cells ( Figure S6 ). This result indicates that the cellular uptake of the phosphopeptide assemblies containing D-pTyr depends more on the interactions between TNAP and the enzyme triggers than on the binding between receptors and specific peptide sequences. [44] [45] [46] confirms the dephosphorylation of the peptide inside cells ( Figure S7 ). Additionally, TEM images of liposomes encapsulating NBD-1p and ALP exhibit the generation of nanofibers that rupture the liposomes ( Figure 4A ). These results imply that the TNAP in endocytic vesicles convert the NBD-1p nanoparticles to nanofibers (NBD-1), which break the membrane of endocytic vesicles for escaping into cytosol. More TEM images reveal that the NBD-1p assemblies mixed with ALP initially remain as nanoparticles until certain dephosphorylation ratio that triggers the particle-to-fiber phase transition ( Figure S8 and 3D ). This result implies that although dephosphorylation begins once TNAP binds to NBD-1p assemblies on plasma membrane, the assemblies likely maintain as nanoparticles (which consist of mainly NBD-1p and small amount NBD-1) during internalization until being further dephosphorylated in endosomal vesicles where nanofibers form for endosome rupturing. This observation also agrees with the reports that filamentous nanostructures have much reduced/limited cellular uptake ability relative to their spherical counterparts. 48, 49 for endosomal rupturing after partial dephosphorylation by ALP ( Figure 4C and 3D) . Thus, the remained phosphopeptides, after releasing into cytosol, get further dephosphorylation by intracellular TNAP. This process produces networks of peptidic nanofibers that encapsulate intracellular TNAP ( Figure 1D and 3A, TNAP colocalizes with NBD). However, the co-assemblies of NBD-1p and NBD-(D)Sp (1:1 or 1:3) mixed with ALP mostly remain as nanoparticles ( Figure 4D and S9B) because NBD-(D)Sp is a poor substrate of ALP ( Figure 3D ). When the mixture has the ratio of NBD-1p and NBD-(D)Sp in 3 to 1, the co-assemblies behave similarly to the assemblies of NBD-1p ( Figure S9 ). Moreover, a phosphopeptide derivative (NBD-3p) carrying D-pTry and pSer (Scheme S1) also ends up in the endosomal compartments of HEK293_TNAP-RFP cells after incubation ( Figure S11 ). Like the co-assemblies of NBD-1p and NBD-(D)Sp with 1:1 or 1:3 ratio, NBD-3P, being treated by ALP, inefficiently undergoes the nanoparticle-to-nanofiber transition ( Figure S12 ). These results suggest that the particle-tofiber transition of the phosphopeptide assemblies catalyzed by the ALP in endosome is critical for the endosomal escape of the peptide assemblies, likely via rupturing the endosome by the formation of nanofibers. In conclusion, this work demonstrates the continuous morphological transformation of peptide assemblies, catalyzed by ALP, is responsible for the endocytosis, mainly through caveolaemediated endocytic pathway, and endosomal escape of the peptide assemblies. Although other endocytosis pathways, such as macropinocytosis, may contribute to the cellular uptake of phosphopeptide assemblies, they are less likely act as the main path for the phosphopeptide assemblies ( Figure S13 ). Here, TNAP catalytically controls a dynamic morphological transition, thus enables endocytosis and endosomal escape of peptide assemblies, in an analogy to the cell entry of virus. Because NBD-1p mainly exists as nanoparticles at the concentration above CMC, the effects of monomeric NBD-1p likely are insignificant. We suggest this type of cell uptake as "Enzyme Primed Endocytosis (EPE)". While the contribution of VPP in the cell entry of virus has been extensively studied, little research elucidates how EPE controls the cellular uptake of other molecular assemblies. In analogy to the ligand-receptor mediated endocytosis that require tight binding, the clustering of enzymes in lipid rafts and the enzymatic reaction of supramolecular assemblies require rapid enzyme kinetics because NBD-(D)Sp hardly enter the cells. This study also provides a mechanistic understanding of the role of peptide assemblies for context-dependent signaling. 50, 51 Although this work focuses on ALP and peptides, the mechanism revealed likely is applicable for the endocytosis of the substrates of esterase 52 or proteases, 53 including non-peptide substrates of enzymes. The Supporting Information is available free of charge at: https://pubs.acs.org/doi/xxx. NBD-3p; (Figure S12) TEM images of NBD-3p incubated with ALP Confocal fluorescence images of HEK293_TNAP-RFP cells incubated with NBD-1p in the presence of macropinocytosis inhibitor; and (Figure S14) Mass spectrum. 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The authors thank NIH (R01CA142746) and NSF (DMR-2011846) for their support.