Jean-Bernard Denault

Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation

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.

Human caspase-7 activity and regulation by its N-terminal peptide.

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.

Processing of proendothelin-1 by human furin convertase.

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.

Furin/PACE/SPC1: A convertase involved in exocytic and endocytic processing of precursor proteins

One of the most exciting breakthroughs of the 90s 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. Furins ability to cleave precursors within both the exocytic and endocytic pathways will require sustained efforts in order to delineate all of its physiological roles.

Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis

During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K38KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7s N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.

Caspase-8 Cleaves Histone Deacetylase 7 and Abolishes Its Transcription Repressor Function

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.

Processing of proendothelin‐1 by members of the subtilisin‐like pro‐protein convertase family

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.

Presence of furin mRNA in cultured bovine endothelial cells and possible involvement of furin in the processing of the endothelin precursor

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.

The contribution of arginine residues within the P6-P1 region of alpha 1-antitrypsin to its reaction with furin.

A series of mutants incorporating furin recognition sequences within the P6–P1 region of the reactive site loop of α1-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild typeP6LEAIPMP1 sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k ass andk′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 × 106 m −1 s−1) except for one mutant, RERIRR, which had a k ass of 3.3 × 105 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 (highk′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.

Serpin‐like properties of α1‐antitrypsin Portland towards furin convertase

Recent studies have demonstrated that a serpin variant, α1‐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 1.5×106 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.