Guy S. Salvesen

Yama/CPP32β, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase

Abstract Although the mechanism of mammalian apoptosis has not been elucidated, a protease of the CED-3/ICE family is anticipated to be a component of the death machinery. Several lines of evidence predict that this protease cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis, is inhibitable by the CrmA protein, and is distinct from ICE. We cloned a ced-3/ICE -related gene, designated Yama , that encodes a protein identical to CPP32β. Purified Yama was a zymogen that, when activated, cleaved PARP to generate the 85 kDa apoptotic fragment. Cleavage of PARP by Yama was inhibited by CrmA but not by an inactive point mutant of CrmA. Furthermore, CrmA blocked cleavage of PARP in cells undergoing apoptosis. We propose that Yama may represent an effector component of the mammalian cell death pathway and suggest that CrmA blocks apoptosis by inhibiting Yama.

Zinc is a potent inhibitor of the apoptotic protease, caspase-3. a novel target for zinc in the inhibition of apoptosis

The prevention of apoptosis by Zn2+ has generally been attributed to its inhibition of an endonuclease acting in the late phase of apoptosis. In this study we investigated the effect of Zn2+ on an earlier event in the apoptotic process, the proteolysis of the “death substrate” poly(ADP-ribose) polymerase (PARP). Pretreatment of intact Molt4 leukemia cells with micromolar concentrations of Zn2+caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide. Using a cell-free system consisting of purified bovine PARP as a substrate and an apoptotic extract or recombinant caspase-3 as the PARP protease, Zn2+ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn2+ on PARP, a protein with two zinc finger domains, we used recombinant caspase-3 and a chromogenic tetrapeptide substrate containing the caspase-3 cleavage site. In this system, Zn2+ inhibited caspase-3 with an IC50 of 0.1 μm. These results identify caspase-3 as a novel target of Zn2+ inhibition in apoptosis and suggest a regulatory role for Zn2+ in modulating the upstream apoptotic machinery.

In vivo catabolism of α1-antichymotrypsin is mediated by the Serpin receptor which binds α1-proteinase inhibitor, antithrombin III and heparin cofactor II

The in vivo catabolism of 125I-labeled α1-antichymotrypsin was studied in our previously described mouse model. Native α1-antichymotrypsin cleared with an apparent t12 of 85 min, but α1-antichymotrypsin in complex with chymotrypsin or cathepsin G cleared with a t12 of 12 min. Clearance of the complex was blocked by a large molar excess of unlabeled complexes of proteinases with either α1-antichymotrypsin or α1-proteinase inhibitor. These studies indicate that the clearance of α1-antichymotrypsin-proteinase complexes utilizes the same pathway as complexes with the homologous inhibitor α1-proteinase inhibitor. Previous studies have demonstrated that this pathway is also responsible for the catabolism of two other serine proteinase inhibitors, antithrombin III and heparin cofactor II. This pathway is thus responsible for removing several proteinases involved in coagulation and inflammation from the circulation, thereby decreasing the likelihood of adventitious proteolysis.

Inhibition of Cysteine and Serine Proteinases by the Cowpox Virus Serpin CRMA

Interleukin ls converting enzyme (ICE) is the founding member of a family of cysteine proteinases active in the metabolism of intracellular proteins [1]. Other members of this family include Ich-1, CPP32, Ced-3, all of which have been implicated in apoptosis (programmed cell death) [2–4]. Though ICE itself may also be involved in apoptosis, its main role is probably to activate pro-interleukin-ls (proIL-ls) via specific cleavage following Asp residues [1,5].

Expression of a functional α-macroglobulin receptor binding domain in Escherichia coli

We have expressed receptor‐binding domains of human α2‐macroglobulin in Escherichia coli. Expression levels of both recombinants were quite high, but the human one was insoluble, probably forming inclusion bodies. The rat domain, which lacks the human disulfide, was produced in a soluble form and readily purified by two simple chromatographic steps. Purified recombinant rat α1‐macroglobulin receptor‐binding domain was fully functional in binding to the α‐macroglobulin receptor on human fibroblasts. This 142 residue domain should serve as an excellent template for analyzing the structural requirements for α‐macroglobulin receptor ligation and dissecting the varied biological functions resulting from such ligation.

In Vitro Expression of Serpins

Publisher Summary This chapter describes the in vitro expression of serpins. All serpins do not inhibit proteinases but those that do show a distinctive unfolding tendency at relatively low denaturant concentration. Upon cleavage in the reactive site loop (RSL), the conformation is dramatically stabilized, which is unusual because proteolysis usually has the opposite effect on most proteins. This transition from an unstable to a stable conformation is almost certainly caused by the formation of a unit of secondary structure known as the A-sheet. Inhibitory serpins are conformationally unstable in the virgin form but stabilized by proteolysis in the RSL. Checks of conformational stability before and after RSL cleavages are, thus, a useful tool in assessing serpin integrity. Many serpins form complexes with proteinases that resist dissociation under conditions that are expected to unravel proteinases. This is a characteristic not shared by other natural active-site-directed proteinase inhibitors.

Polypeptide Chain Structure of Inter-α-Trypsin Inhibitor and Pre-α-Trypsin Inhibitor: Evidence for Chain Assembly by Glycan and Comparison with other “Kunin”-Containing Proteins

Proteins structurally related to the proteinase inhibitor aprotinin, systematically known as pancreatic trypsin inhibitor (Kunitz), occur in animals from a variety of orders including mammals, moluscs and coelenterates.1 The wide distribution of these proteins suggests that the ancestral gene is very old, at least as old as the radiation of multicellular animals.2