A preliminary characterization is provided of a naturally occurring cyclic peptide with interesting and potent biological activity. A 31-residue cyclic peptide, designated cyclopsychotride A [1], was obtained from the organic extract of the tropical plant, Psychotria longipes. Compound 1 inhibited [125I] neurotensin (NT) binding to HT-29 cell membranes (IC50 3 microM) and also stimulated increased levels of cytosolic Ca2+ in two unrelated cell lines that do not express NT receptors. The peptide was found to dose-dependently increase intracellular Ca2+ at concentrations ranging from 3 to 30 microM, and this response was not blocked by a known NT antagonist. Cyclopsychotride A [1] possesses three disulfide linkages and is thought to be the largest cyclic peptide isolated from a natural source. Both 1H-nmr and cd spectroscopy showed 1 to be highly structured.

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K m (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50 -70 nM), whereas the K m for the thrombin-or thrombin:thrombomodulin-catalyzed reaction is 10 -40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus ␣ 2 -antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k cat /K m ). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.

␤-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid ␤ peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4 Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k cat /K m of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4 Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/ renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

No abstract

We have built a model of the specificity pocket of the protease of hepatitis C vfrus on the basis of the known structures of trypsin-like serine proteases and of the conservation pattern of the protease sequences among various hepatitis C strains. The model allowed us to predict that the substrate of this proteas should have a cysteine residue in position P1. This hypothesis was subsequently proved by N-terminal sequencing of two products of the protease. The success of this "blind" test increases our confidence in the overall correctness of our proposed alment of the enzyme sequence with those of other proteases of known ure and constitutes a first step in the construction of a complete model of the viral protease domain.Hepatitis C virus (HCV) is the major cause of parentally transmitted non-A non-B hepatitis and is also implicated as the cause in many sporadic cases ofnon-A non-B hepatitis (1, 2). Chronic infection with HCV has also been linked to the development of hepatocellular carcinoma (3-5).HCV is an enveloped virus with a positive-strand RNA genome of -9.5 kb (6-13). This virus is probably related to flaviviruses and pestiviruses in view of the similarity of the deduced amino acid sequences and hydropathy profile of the viral proteins (14). As for pestiviruses and flaviviruses, the HCV genomic RNA includes a single open reading frame encoding a precursor polyprotein that is cleaved co-or posttranslationally into mature viral polypeptides. The putative viral structural proteins are encoded by the 5' quarter of the genome, whereas the remaining part encodes the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) that are believed to be components of the viral replication apparatus (15).The gene order in the HCV genome has been shown to be the following: 5'-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3' (16,17 (14,25). Consistent with this prediction, when the proposed catalytic Ser-1165 of the polyprotein was mutated into an Ala, processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites was abolished (16, 21). Interestingly, cleavage at three of the four sites of the mutated polyprotein could be restored by supplying a functional NS3 in trans, while processing at the NS3-NS4A only occurred in cis (16). This finding indicates that the cleavage of the latter site is an intramolecular event.The comparative analysis of the three-dimensional structure of serine proteases obtained so far, with or without bound inhibitors, has shed light on the way diverse cleavage specificities are generated by the substrate binding sites. Structural information on the protease domain of HCV NS3, and in particular on the architecture of its substrate binding pocket, would be extremely helpful in making predictions on the specificity of the enzyme, as well as in designing structure-based inhibitors that could be used as therapeutic agents for treatment of HCV infections. To this end, we decided to undertake the modeling of the threedimensional structure of the enzyme by taking advantage of the wealth of st...