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Apoptosis is a regulated form of cell death essential to the removal of unwanted cells. At its core, a family of cysteine peptidases named caspases cleave key proteins allowing cell death to occur. To do so, each caspase catalytic pocket recognizes preferred amino acid sequences resulting in proteolysis, but some also use exosites to select and cleave important proteins efficaciously. Such exosites have been found in a few caspases, notably caspase-7 that has a lysine patch (K38KKK) that binds RNA, which acts as a bridge to RNA-binding proteins favoring proximity between the peptidase and its substrates resulting in swifter cleavage. Although caspase-7 interaction with RNA has been identified, in-depth characterization of this interaction is lacking. In this study, using in vitro cleavage assays, we determine that RNA concentration and length affect the cleavage of RNA-binding proteins. Additionally, using binding assays and RNA sequencing, we found that caspase-7 binds RNA molecules regardless of their type, sequence, or structure. Moreover, we demonstrate that the N-terminal peptide of caspase-7 reduces the affinity of the peptidase for RNA, which translates into slower cleavages of RNA-binding proteins. Finally, employing engineered heterodimers, we show that a caspase-7 dimer can use both exosites simultaneously to increase its affinity to RNA because a heterodimer with only one exosite has reduced affinity for RNA and cleavage efficacy. These findings shed light on a mechanism that furthers substrate recognition by caspases and provides potential insight into its regulation during apoptosis.

Significance During apoptosis, hundreds of intracellular proteins are cleaved by caspases. In addition to recognizing optimized motifs in the primary structure of its substrates, we show that caspase-7 possesses an exosite to enhance poly(ADP-ribose) polymerase 1 (PARP-1) recognition via the mutual binding of RNA. We also demonstrate that caspase-7 binds RNA and that PARP-1, via 2 RNA-binding domains, likely binds the same RNA molecule, bringing itself near caspase-7 for prompt inactivation. This mechanism is conserved in the mouse ortholog. We have also validated the use of RNA for the efficient cleavage of 6 other RNA-BPs, demonstrating that this distinctive mode of proteolysis enhancement by caspase-7 is an essential feature of substrate recognition during apoptosis., To achieve swift cell demise during apoptosis, caspases cleave essential proteins for cell survival and removal. In addition to the binding of preferred amino acid sequences to its substrate-binding pocket, caspase-7 also uses exosites to select specific substrates. 4 lysine residues (K38KKK) located in the N-terminal domain of caspase-7 form such an exosite and promote the rapid proteolysis of the poly(ADP-ribose) polymerase 1 (PARP-1), but the mechanism of recognition remains mostly unknown. In this study, we show that the overall positive charge of the exosite is the critical feature of this evolutionarily conserved binding site. Additionally, interaction with the caspase-7 exosite involves both the Zn3 and BRCT domains of PARP-1 and is mediated by RNA. Indeed, PARP-1 proteolysis efficacy is sensitive to RNase A and promoted by added RNA. Moreover, using affinity chromatography and gel shift assays, we demonstrate that caspase-7, but not caspase-3 or a caspase-7 with a mutated exosite, binds nucleic acids. Finally, we show that caspase-7 prefers RNA-binding proteins (RNA-BPs) as substrates compared to caspase-3 and that RNA enhances proteolysis by caspase-7 of many of these RNA-BPs. Thus, we have uncovered an unusual way by which caspase-7 selects and cleaves specific substrates.

Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, caspase-7 is more proficient at cleaving poly(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure–activity relationship analyses using mutagenesis of the caspase-7 NTD and kinetics show that residues 36–45 of caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD142↓G), which is far from the optimal sequence, we tested all res...

During apoptosis, caspases feast on several hundreds of cellular proteins to orchestrate rapid cellular demise. Indeed, caspases are known to get a taste of every cellular process in one way or another, activating some, but most often shutting them down. Thus, it is not surprising that caspases proteolyze proteins involved in intracellular trafficking with particularly devastating consequences for this important process. This review article focuses on how caspases target the machinery responsible for smuggling goods within and outside the cell.

Advanced glycation end-products (AGE) and the receptor for AGE (RAGE) have been linked to numerous diabetic vascular complications. RAGE activation promotes a self-sustaining state of chronic inflammation and has been shown to induce apoptosis in various cell types. Although previous studies in vascular smooth muscle cells (VSMC) showed that RAGE activation increases vascular calcification and interferes with their contractile phenotype, little is known on the potential of RAGE to induce apoptosis in VSMC. Using a combination of apoptotic assays, we showed that RAGE stimulation with its ligand CML-HSA promotes apoptosis of VSMC. The formation of stress granules and the increase in the level of the associated protein HuR point toward RAGE-dependent endoplasmic reticulum (ER) stress, which is proposed as a key contributor of RAGE-induced apoptosis in VSMC as it has been shown to promote cell death via numerous mechanisms, including up-regulation of caspase-9. Chronic NF-κB activation and modulation of Bcl-2 homologs are also suspected to contribute to RAGE-dependent apoptosis in VSMC. With the goal of reducing RAGE signaling and its detrimental impact on VSMC, we designed a RAGE antagonist (iRAGE) derived from the primary amino acid sequence of HSA. The resulting CML peptide was selected for the high glycation frequency of the primary sequence in the native protein in vivo. Pretreatment with iRAGE blocked 69.6% of the increase in NF-κB signaling caused by RAGE activation with CML-HSA after 48h. Preincubation with iRAGE was successful in reducing RAGE-induced apoptosis, as seen through enhanced cell survival by SPR and reduced PARP cleavage. Activation of executioner caspases was 63.5% lower in cells treated with iRAGE before stimulation with CML-HSA. To our knowledge, iRAGE is the first antagonist shown to block AGE-RAGE interaction and we propose the molecule as an initial candidate for drug discovery.

Caspases and their substrates are key mediators of apoptosis and strongly implicated in various physiological processes. As the serine/threonine kinase family is involved in apoptosis and serine/threonine kinase 3 (STK3) is a recently identified caspase-6 substrate, we assessed the expression and cleavage of STK3 in murine peripheral organs and brain regions during the aging process. We also assessed caspase-3, -6, -7, and -8 expression and activity in order to delineate potential mechanism(s) underlying the generation of the STK3 fragments observed and their relation to the apoptotic pathway. We demonstrate for the first time the cleavage of STK3 by caspase-7 and show that STK3 protein levels globally increase throughout the organism with age. In contrast, caspase-3, -6, -7, and -8 expression and activity vary significantly among the different organs analyzed suggesting differential effects of aging on the apoptotic mechanism and/or nonapoptotic functions of caspases throughout the organism. These results further our understanding of the role of caspases and their substrates in the normal aging process and highlight a potential role for STK3 in neurodegeneration.

During apoptosis, initiator caspases (8, 9 and 10) activate downstream executioner caspases (3, 6 and 7) by cleaving the IDC (interdomain connector) at two sites. Here, we demonstrate that both activation sites, site 1 and site 2, of caspase 7 are suboptimal for activation by initiator caspases 8 and 9 in cellulo, and in vitro using recombinant proteins and activation kinetics. Indeed, when both sites are replaced with the preferred motifs recognized by either caspase 8 or 9, we found an up to 36-fold improvement in activation. Moreover, cleavage at site 1 is preferred to site 2 because of its location within the IDC, since swapping sites does not lead to a more efficient activation. We also demonstrate the important role of Ile195 of site 1 involved in maintaining a network of contacts that preserves the proper conformation of the active enzyme. Finally, we show that the length of the IDC plays a crucial role in maintaining the necessity of proteolysis for activation. In fact, although we were unable to generate a caspase 7 that does not require proteolysis for activity, shortening the IDC of the initiator caspase 8 by four residues was sufficient to confer a requirement for proteolysis, a key feature of executioner caspases. Altogether, the results demonstrate the critical role of the primary structure of caspase 7's IDC for its activation and proteolytic activity.

Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.

To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently reported that YopH acutely inhibits T cell antigen receptor signaling by dephosphorylating the Lck tyrosine kinase. Here, we show that prolonged presence of YopH in primary T cells or Jurkat T leukemia cells causes apoptosis, detected by annexin V binding, mitochondrial breakdown, caspase activation, and internucleosomal fragmentation. YopH also causes cell death when expressed in HeLa cells, and this cell death was inhibited by YopH-specific small molecule inhibitors. Cell death induced by YopH was also prevented by caspase inhibition or co-expression of Bcl-xL. We conclude that YopH not only paralyzes T cells acutely, but also ensures that the cells will not recover to induce a protective immune response but instead undergo mitochondrially regulated programmed cell death.

The first step in caspase activation is transition of the latent zymogen to an active form. For the initiator caspases, this occurs through dimerization of monomeric zymogens at an activating complex. Recent studies have suggested that FLIPL [FLICE-like inhibitory protein, long form; FLICE is FADD (Fas-associated death domain protein)-like interleukin-1β-converting enzyme], previously thought to act solely as an inhibitor of caspase-8 activation, can under certain circumstances function to enhance caspase activation. Using an in vitro induced-proximity assay, we demonstrate that activation of caspases-8 and -10 occurs independently of cleavage of either the caspase or FLIPL. FLIPL activates caspase-8 by forming heterodimeric enzyme molecules with substrate specificity and catalytic activity indistinguishable from those of caspase-8 homodimers. Significantly, the barrier for heterodimer formation is lower than that for homodimer formation, suggesting that FLIPL is a more potent activator of caspase-8 than is caspase-8 itself.

Central to the execution phase of apoptosis are the two closely related caspase-3 and -7. They share common substrate specificity and structure, but differ completely in the sequence of their respective N-terminal regions including their N-peptides, a 23-28 residue segment that are removed during zymogen activation. We show that the N-peptide of caspase-7 plays no role in the fundamental activation or properties of the active protease in vitro. However, the N-peptide modifies the properties of caspase-7 in vivo. In ectopic expression experiments, caspase-7 constructs with no N-peptide are far more lethal than constructs that have an uncleavable peptide. Moreover, the N-peptide of caspase-7 must be removed before efficient activation of the zymogen can occur in vivo. These disparate requirements for the N-peptide argue that it serves to physically sequester the caspase-7 zymogen in a cytosolic location that prevents access by upstream activators (caspase-8, -9, and -10). The N-peptide must first be removed, probably by caspase-3, before efficient conversion and activation of the zymogen can occur in vivo.

One of the most valuable tools that have been developed for the study of apoptosis is the availability of recombinant active caspases. The determination of caspase substrate preference, the design of sensitive substrates and potent inhibitors, the resolution of caspase structures, the elucidation of their activation mechanisms, and the identification of their substrates were made possible by the availability of sufficient amounts of enzymatically pure caspases. The current chapter describes at length the expression, purification, and basic enzymatic characterization of apoptotic caspases.

A series of mutants incorporating furin recognition sequences within the P6-P1 region of the reactive site loop of alpha(1)-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild type (P6)LEAIPM(P1) sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k(ass) and k'(ass)), or to inhibit furin-dependent proteolysis of a model precursor in vivo. Each variant abolished processing of pro-von Willebrand factor in transfected hEK293 cells. The k(ass) of all variants were found to be similar (1.1-1.7 x 10(6) m(-1) s(-1)) except for one mutant, RERIRR, which had a k(ass) of 3.3 x 10(5) m(-1) s(-1). However, the stoichiometry of inhibition varied with values ranging from 2.9 to >24, indicating rapid formation of the acyl-enzyme intermediate (high k'(ass)). Moreover, those variants having high stoichiometry of inhibition values were accompanied by the rapid formation of cleaved forms of the inhibitors. The data suggest that the rate of conversion of the acyl-enzyme (EI') into the highly stable complex (EI*) was affected by replacement of specific residues within the reactive site loop. Taken together, the results reveal how furin recognition sequences within the context of the biochemical properties of serpins will play a role in the capacity of the protein to follow either the inhibitory or the substrate pathway.

SPC1 (furin/PACE), an enzyme belonging to the S8 group of serine endoproteases, is a type I integral membrane protein that catalyzes the processing of a multitude of precursor proteins. We report here the use of transfected Drosophila melanogaster Schneider 2 cells to produce milligram amounts of two forms of recombinant human SPC1. In order to investigate the role of the cysteine-rich region (CRR) of SPC1, we compared the biochemical and enzymatic properties of hSPC1/714 that has the C-terminal tail and transmembrane region of the native enzyme removed with that of hSPC1/585 which had, in addition, the CRR deleted. Two stable cell lines were established. The S2-hSPC1/714 line secreted a major form of apparent molecular weight of 83 kDa and a minor form of 80 kDa whereas the S2-hSPC1/585 line secreted a single 59-kDa protein. PNGase F treatment of the different forms demonstrated that the enzymes were glycosylated. Automated NH 2 -terminal sequencing revealed that all purified forms resulted from processing at the expected zymogen activation site. Removal of the CRR resulted in a broadening of the enzyme's pH range, a shift of K 0.5 for Ca 2+ , and a shorter enzymatic half-life when compared to the longer form, which suggest that the CRR of hSPC1 may help in stabilizing the enzyme's proteolytic activity. The use of this high-level expression system will meet the demand for material necessary to perform biochemical and structural studies that are needed to further our understanding of this and other SPCs at the molecular level.

PACE4 is one of the neuroendocrine-specific mammalian subtilisin-related endoproteases believed to function in the secretory pathway. The biosynthesis and secretion of PACE4 have been studied using transfected neuroendocrine and fibroblast cell lines, as well as primary pituitary cultures. ProPACE4 (approx. 106 kDa) is cleaved intracellularly before secretion of PACE4 (approx. 97 kDa); the N-terminal propeptide cleavage is accelerated in a truncated form of PACE4 lacking the Cys-rich C-terminal region (PACE4s). Neither PACE4 nor PACE4s is stored in regulated neuroendocrine secretory granules, whereas pro-opiomelanocortin-derived peptides and prohormone convertase 1 enter the regulated secretory pathway efficiently. The relatively slow cleavage of the proregion of proPACE4 in primary anterior pituitary cells, followed by rapid secretion of PACE4, is similar to the results for proPACE4 in transfected cell lines. The enzyme activity of PACE4 is distinct from furin and prohormone convertases, both in the marked sensitivity of PACE4 to inhibition by leupeptin and the relative insensitivity of PACE4 to inhibition by Ca2+ chelators and dithiothreitol; PACE4 is not inhibited by the α1-antitrypsin Portland variant that is very potent at inhibiting furin. The unique biosynthetic and enzymic patterns seen for PACE4 suggest a role for this neuroendocrine-specific subtilisin-like endoprotease outside the pathway for peptide biosynthesis.

One of the most exciting breakthroughs of the 90's in the fields of biochemistry, cell biology and neuroendocrinology is the identification of a novel family of proteolytic enzymes called mammalian subtilisin-like convertases. This family is comprised so far of seven distinct endoproteases responsible for the proteolytic excision of biologically active polypeptides from inactive precursor proteins. Six years after the initial observation of a structural conservation between a characterized yeast enzyme (kexin) and a human gene product (furin), it is now well accepted that one of these convertases, furin, has the enzymatic capabilities to efficiently and correctly process a great variety of precursors. Furin's ability to cleave precursors within both the exocytic and endocytic pathways will require sustained efforts in order to delineate all of its physiological roles.

The biologically inactive precursor proendothelin-1 (proET-1) is initially processed intracellularly to the intermediate big endothelin peptide (big ET-1) before its conversion to the endothelin-1 (ET-1) peptide by the endothelin-converting enzyme (ECE). We recently demonstrated that purified furin, a calcium-dependent serine endoprotease belonging to the family of mammalian convertases, cleaved proET-I in vitro and hence produced the physiologically relevant big ET-1 peptide. Therefore, furin becomes a candidate proET-1-cleaving enzyme responsible for the initial biosynthetic processing steps of this precursor. In this study we examined the inhibitory properties of two convertase inhibitors, i.e., the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) and the α 1 -antitrypsin Portland (AT-PDX) on proET-I processing. When purified furin or PACE4, another mammalian convertase, was incubated in the presence of dec-RVKR-cmk, proET-I processing was completely abolished. In the presence of purified AT-PDX, furin-related cleavage of proET-I was abolished but not PACE4 processing. In addition, incubation of endothelial cells with dec-RVKR-cmk inhibited production of ET-1 but did not significantly alter the levels of the prostanoid PGI 2 . These results indicate that convertase-related processing of proET-I can be inhibited in vitro and in vivo, and that convertase-specific processing may be prevented with AT-PDX.

Recently it was documented that furin, a calcium-dependent serine endoprotease, cleaves many protein precursors at pairs of basic amino acids, thus liberating the biologically active peptides. The endothelin precursors follow a biosynthetic pathway similar to these proteins, where the precursor is initially processed to the intermediate, big endothelin (big ET) before its conversion to the endothelin (ET) peptide. Analysis of the amino acid sequence of the endothelin pro-proteins shows that they are susceptible to processing by endoproteases that cleave at pairs of basic amino acids. For example, human endothelin-1 (ET-1) precursor possesses a typical furin cleavage site motif (Arg-X-Lys/Arg-Arg) at the following residues: Arg32-Ser33-Lys34-Arg35 and Arg72-Ser73-Lys74-Arg75. We have isolated mRNA from cultured bovine endothelial cells and, using a human furin cRNA probe, shown that a furin mRNA of 4.5 kb is present in these cells. We propose that furin, a novel endoprotease belonging to the mammalian subtilisin family of serine proteases, may be implicated in the processing of pro-endothelin precursors, liberating big ET.

Caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.

Caspase-8 is the initiator caspase of the extrinsic apoptosis pathway and also has a role in non-apoptotic physiologies. Identifying endogenous substrates for caspase-8 by using integrated bioinformatics and biological approaches is required to delineate the diverse roles of this caspase. We describe a number of novel putative caspase-8 substrates using the Prediction of Protease Specificity (PoPS) program, one of which is histone deacetylase 7 (HDAC7). HDAC7 is cleaved faster than any other caspase-8 substrate described to date. It is also cleaved in primary CD4+CD8+ thymocytes undergoing extrinsic apoptosis. By using naturally occurring caspase inhibitors that have evolved exquisite specificity at concentrations found within the cell, we could unequivocally assign the cleavage activity to caspase-8. Importantly, cleavage of HDAC7 alters its subcellular localization and abrogates its Nur77 repressor function. Thus we demonstrate a direct role for initiator caspase-mediated proteolysis in promoting gene transcription.

Proteolytic processing at the end of the G(1) phase generates a CUX1 isoform, p110, which functions either as a transcriptional activator or repressor and can accelerate entry into S phase. Here we describe a second proteolytic event that generates an isoform lacking two active repression domains in the COOH terminus. This processing event was inhibited by treatment of cells with synthetic and natural caspase inhibitors. In vitro, several caspases generated a processed isoform that co-migrated with the in vivo generated product. In cells, recombinant CUX1 proteins in which the region of cleavage was deleted or in which Asp residues were mutated to Ala, were not proteolytically processed. Importantly, this processing event was not associated with apoptosis, as assessed by terminal dUTP nick end labeling assay, cytochrome c localization, poly(ADP-ribose) polymerase cleavage, and fluorescence-activated cell sorting. Moreover, processing was observed in S phase but not in early G(1), suggesting that it is regulated through the cell cycle. The functional importance of this processing event was revealed in reporter and cell cycle assays. A recombinant, processed, CUX1 protein was a more potent transcriptional activator of several cell cycle-related genes and was able to accelerate entry into S phase, whereas mutants that could not be processed were inactive in either assay. Conversely, cells treated with the quinoline-Val Asp-2,6-difluorophenoxymethylketone caspase inhibitor proliferated more slowly and exhibited delayed S phase entry following exit from quiescence. Together, our results identify a substrate of caspases in proliferating cells and suggest a mechanism by which caspases can accelerate cell cycle progression.

Caspase-7 is an obligate dimer of catalytic domains, with generation of activity requiring limited proteolysis within a region that separates the large and small chains of each domain. Using hybrid dimers we distinguish the relative contribution of each domain to catalysis by the whole molecule. We demonstrate that the zymogen arises from direct dimerization and not domain swapping. In contrast to previous conclusions, we show that only one of the catalytic domains must be proteolyzed to enable activation. The processed domain of this singly cleaved zymogen has the same catalytic activity as a domain of fully active caspase-7. A transient intermediate of singly cleaved dimeric caspase-7 can be found in a cell-free model of apoptosis induction. However, we see no evidence for an analogous intermediate of the related executioner caspase-3. Our study demonstrates the efficiency by which the executioner caspases are activated in vivo.

CrmA is a cowpox virus serpin required for full host infectivity and virulence. Residues 51–56 (DKNKDD), the only region that differs significantly from related viral serpins, were investigated for functional importance. A 1.6 A X-ray structure reported here showed that this region can adopt either structured or unstructured conformations. Three variants were expressed, one with the region 51–56 deleted, one substituted by alanines, and one in which this region was replaced by the sequence encoded in smallpox virus. NMR showed that the region is an exposed, flexible loop that can be deleted without perturbing the serpin. The region is also very susceptible to proteolysis. Significantly, inhibition of caspases 1 and 8 was unaffected by the mutations, and each of the variants was as effective as wild-type CrmA in promoting survival from apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Thus, although the 51–56 region of CrmA is unique, and is exposed and highly susceptible to proteolysis, any in vivo role must involve a function other than proteinase inhibition or cell sparing.

Furin, a member of the subtilisin-like pro-protein convertase family, is a type I membrane protein that undergoes ectodomain shedding. Metabolic labeling of cells stably expressing furin demonstrated that the shed form of furin is detected after 30 min. Moreover, sequence analysis revealed that specific residues of the cysteine-rich region of furin aligned with those of tumor necrosis factor receptor, which is also shed. Introduction within furin’s cysteine-rich region of mutations that impair TNFR1 shedding also abolished furin shedding. Our results show that shedding of furin occurs rapidly and further suggest that specific cysteine residues may impart a conformation to the enzyme, thereby affecting its susceptibility to proteolysis.

This unit describes a protocol to obtain milligram amounts of enzymatically active pure recombinant caspases. Specific details for the expression, purification of caspase-3, -6, -7, -8, -9 and -10 are discussed along with strategies to obtain particular forms (e.g., the zymogen) of some of them.

Caspases are a family of cysteine proteases with a strict specificity for aspartate residues involved in inflammatory process and programmed cell death. This overview unit provides basic information on their structure, enzymatic activity, substrate specificity, activation,inhibition and their implication in pathologies. It is intended to be an overview for investigators that are unfamiliar with this family of enzymes but it is also applicable to scientists pursuing research in this field.

Production of endothelin-1 is thought to be a three-step process consisting of an initial proteolytic cleavage of the proendothelin-1 precursor to big endothelin-1-Lys-Arg, C-terminal trimming by a carboxypeptidase and further processing of the big endothelin-1 peptide to endothelin-1 by endothelin-converting enzyme (ECE). To further clarify the mechanism of processing in the biosynthesis of endothelin-1, we introduced a point mutation into endothelin-1 cDNA to replace the Arg in the -4 position of the recognition motifs of furin-like convertase in human preproendothelin-1 (Arg49 or Arg89) by Gly. When mutant cDNAs were expressed in Chinese hamster ovary (CHO)-K1 cells, they failed to be processed at the mutated processing signal, suggesting that the Arg-Ser-Lys-Arg motifs of preproendothelin-1 are recognized by CHO-K1 furin-like convertase. Co-transfection with ECE-1 cDNA revealed that cleavage at Arg52 is not essential for cleavage by ECE-1, but that cleavage at Arg92 is critical. Although a high-molecular-mass form of endothelin-1 is produced by processing by ECE-1 without cleavage at Arg52, it did not evoke Ca2+ transient in endothelinA-receptor-expressing cells. In conclusion, prior cleavage at Arg92 by furin-like convertase is absolutely necessary for cleavage by ECE-1 at Trp73 to produce mature endothelin-1.

Endothelin-1 (ET-1) is the most potent vasoactive peptide known to date. The peptide is initially synthesized as an inactive precursor (proET-1) which undergoes proteolysis at specific pairs of basic amino acids to yield bigET-1. Production of ET-1 then proceeds by cleavage of bigET-1 by the endothelin converting enzyme (ECE). Here, we demonstrate that the in vitro cleavage of proET-1 by furin, a mammalian convertase involved in precursor processing, produced bigET-1. Upon further processing, bigET-1 was converted to biologically active ET-1. Furthermore, we demonstrate that the furin inhibitor, decanoyl-Arg-ValLys-Arg chloromethylketone, abolished production of ET-1 in endothelial cells.

Endothelial cells (ECs) secrete numerous bioactive peptides that are initially synthesized as inactive precursor proteins. One of these, proendothelin-1 (proET-1), undergoes proteolysis at specific pairs of basic amino acids. Here, we wished to examine the role of mammalian convertases in this event. Northern blot analysis shows that only furin and PC7 are expressed in ECs. In vitro cleavage of proET-1 by furin or PC7 demonstrated that both enzymes efficiently and specifically process proET-1. These data reveal that furin and PC7 have similar specificities towards proET-1 and suggest that both enzymes may participate in the maturation of proET-1 in ECs.

Recent studies have demonstrated that a serpin variant, alpha1-antitrypsin Portland (AT-PDX), can inhibit the mammalian convertase furin. Here, we examine the mechanism by which this inhibition takes place. We find that furin, which does not belong to the trypsin-like serine protease family, the usual targets of serpins, forms an SDS-heat denaturation-resistant complex with AT-PDX both in vitro and in vivo. AT-PDX inhibited furin with an association rate constant (k(ass)) of 1.5 x 10(6) M(-1) s(-1) which is similar to k(ass) values reported for serpins with trypsin-like enzymes. These results illustrate that AT can be modified to act essentially as a suicide inhibitor of furin, an enzyme of the subtilase superfamily of serine proteases.