Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several mm in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.

Protein-protein interaction domains (PPIDs) are key elements in assembling functional protein complexes and controlling cellular activities. A major class of PPIDs is mediated by PDZ (for PSD-95, Dlg, ZO-1) domains [1][2][3], widespread scaffolding modules essential for regulating the localization and activity of numerous cellular effector proteins. Among the diverse protein interaction domains, PDZ domains are highly conserved in organisms from bacteria to humans [4]. They usually bind the C-terminus of their ligands.Consistent with their structural homology, PDZ domains exhibit overlapping recognition sequences, meaning that a given partner typically can interact with multiple domains. Some years ago, we proposed a general and efficient procedure for profiling PDZ-peptide interactions that provides a picture of specificity and selectivity covering the complete PDZligand sequence space by combining SPOT synthesis and K d prediction [5]. As expected, among the three PDZ domains that were analyzed (AF6, ERBIN and SNA1), the overlap of ligand sequences recognized at K d values between 50-100 μM was substantial. Recent studies have suggested that there is more diversity among PDZ sequence preferences than originally thought [6,7]. Nevertheless, the set of PDZ domains interacting with a given protein necessarily share overlapping binding motifs, and it remains challenging to develop a canonical peptide that will inhibit only a single PDZ domain out of this set.To address this issue, we focused on a set of five PDZ domains known to interact with the Cystic Fibrosis (CF) Transmembrane conductance Regulator (CFTR). The PDZ-containing proteins CAL (CFTR-Associated Ligand) [8,9] and its antagonists NHERF1 and NHERF2 (Na+/H+ Exchanger Regulatory Factor 1/2) [8,10], compete for the binding to CFTR. CAL contains one (CALP) and each NHERF protein two PDZ domains (N1P1, N1P2, N2P1 and N2P2) which control both the activity and the cell surface abundance of CFTR. NHERF family members increase CFTR activity at the apical membrane, whereas CAL promotes its lysosomal degradation. Thus, to explore novel therapeutic strategies for increasing the cellsurface abundance of CFTR, our goal was to design a selective inhibitor of the

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor (Kd=120 nM,MW=734Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

Current treatments for autoimmune disorders rely on non-specific immunomodulatory and global immunosuppressive drugs, which show a variable degree of efficiency and are often accompanied by side effects. In contrast, strategies aiming at inducing antigen-specific tolerance promise an exclusive specificity of the immunomodulation. However, although successful in experimental models, peptide-based tolerogenic “inverse” vaccines have largely failed to show efficacy in clinical trials. Recent studies showed that repetitive T cell epitopes, coupling of peptides to autologous cells, or peptides coupled to nanoparticles can improve the tolerogenic efficacy of peptides, suggesting that size and biophysical properties of antigen constructs affect the induction of tolerance. As these materials bear hurdles with respect to preparation or regulatory aspects, we wondered whether conjugation of peptides to the well-established and clinically proven synthetic material polyethylene glycol (PEG) might also work. We here coupled the T cell epitope OVA 323–339 to polyethylene glycols of different size and structure and tested the impact of these nano-sized constructs on regulatory (Treg) and effector T cells in the DO11.10 adoptive transfer mouse model. Systemic vaccination with PEGylated peptides resulted in highly increased frequencies of Foxp3 + Tregs and reduced frequencies of antigen-specific T cells producing pro-inflammatory TNF compared to vaccination with the native peptide. PEGylation was found to extend the bioavailability of the model peptide. Both tolerogenicity and bioavailability were dependent on PEG size and structure. In conclusion, PEGylation of antigenic peptides is an effective and feasible strategy to improve Treg-inducing, peptide-based vaccines with potential use for the treatment of autoimmune diseases, allergies, and transplant rejection.

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