Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. VSV-induced membrane fusion occurs at a very narrow pH range, between 6.2 and 5.8, suggesting that His protonation is required for this process. To investigate the role of His in VSV fusion, we chemically modified these residues using diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited membrane fusion mediated by VSV in a concentration-dependent manner and that the complete inhibition of fusion was fully reversed by incubation of modified virus with hydroxylamine. Fluorescence measurements showed that VSV modification with DEPC abolished pH-induced conformational changes in G protein, suggesting that His protonation drives G protein interaction with the target membrane at acidic pH. Mass spectrometry analysis of tryptic fragments of modified G protein allowed the identification of the putative active His residues. Using synthetic peptides, we showed that the modification of His-148 and His-149 by DEPC, as well as the substitution of these residues by Ala, completely inhibited peptide-induced fusion, suggesting the direct participation of these His in VSV fusion.

Background: Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading oligopeptidases which have been investigated.Results: Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-oligopeptidase inhibitor, JA-2. Thimet oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited byo-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor enzyme in the perivascular microenvironment is suggested based on the observed rTOP inhibition by an S-nitrosothiol NO donor.Conclusion: Data show that melanoma cells secrete endo-oligopeptidases which have an important role in tumor proliferation in vitro and in vivo. rTOP inhibited growth of subcutaneously injected B16F10-Nex2 cells in mice. TOP from tumor cells and bradykinin in endothelial cells are two antagonist factors that may control angiogenesis essential for melanoma growth. A regulatory role of NO or S-nitrosothiols is suggested.

The inhibition of human cysteine cathepsins B, L, S and K was evaluated by a set of hypervalent tellurium compounds (telluranes) comprising both organic and inorganic derivatives. All telluranes studied showed a time- and concentration-dependent irreversible inhibition of the cathepsins, and their second-order inactivation rate constants were determined. The organic derivatives were potent inhibitors of the cathepsins and clear specificities were detected, which were parallel to their known substrate specificities. In all cases, the activity of the tellurane-inhibited cathepsins was recovered by treatment of the inactivated enzymes with reducing agents. The maximum stoichiometry of the reaction between cysteine residues and telluranes were also determined. The presented data indicate that it is possible to design organic compounds with a tellurium(IV) moiety as a novel warhead that covalently modifies the catalytic cysteine, and which also form strong interactions with subsites of cathepsins B, L, S and K, resulting in more specific inhibition.