A Sequential Two-Step Mechanism for the Production of the Mature p17:p12 Form of Caspase-3 in Vitro

Abstract

The apoptotic cysteine protease, caspase-3, is expressed in cells as an inactive 32-kDa precursor from which 17 kDa (p17) and 12 kDa (p12) subunits of the mature caspase-3 are proteolytically generated during apoptosis. Two amino acid sequences, ESMD2S (amino acids 25-29) and IETD2S (amino acids 172-176), in the precursor have been defined as the cleavage sites for the production of the p17 and p12 subunits. Using a cell-free assay system, we demonstrate that the caspase-3 precursor appears to be cleaved first at the IETD2S site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD2S site, generating the mature p17 subunit. The cleavage at the IETD2S site required a protease activity that was selectively inhibited by the peptide, Ac-IETD-CHO (acetyl-IETD-aldehyde), and other protease inhibitors, such as the cowpox viral serine protease inhibitor, CrmA, and N-␣-tosyl-L-phenylalanine chloromethyl ketone. The protease that catalyzed the cleavage at the ESMD/S site was selectively inhibited by another peptide, Ac-ESMD-CHO (acetyl-ESMD-aldehyde). More interestingly, the caspase-3 inhibitor, Ac-DEVD-CHO, but not the caspase-1 inhibitor, Ac-YVAD-CHO, also selectively inhibited the protease activity that cleaves at the ESMD2S site. This indicated that the cleavage at the ESMD2S site was either autocatalytic or that it required a caspase-3-like activity. In summary, we demonstrate that production of the p17:p12 form of caspase-3 is a sequential two-step process and appears to require two distinct enzymatic activities.In recent years, evidence has accumulated that the degradation of certain proteins by members of the caspase family is a general biochemical event taking place in cells undergoing apoptosis (for reviews, see Refs. 1-3). One caspase family member, caspase-3 (4 -6), has been studied extensively. In particular, several of the cellular protein targets of caspase-3 have been identifed. These include the DNA repair enzyme poly-(ADP-ribose) polymerase (PARP) 1 (5, 7), the 70-kDa protein component of the U1 small nuclear ribonucleoprotein (8), and the catalytic subunit of DNA-dependent protein kinase (DNA-PK cs ) (9 -11). Interestingly, inhibition of caspase-3 or caspase-3-like proteases in various cells has been shown to block apoptosis (6,(12)(13)(14)(15)(16)(17). In addition, the functional inactivation of the caspase-3 gene in knock-out mice results in the profound absence of apoptosis in certain tissues and lethality shortly after birth (18). Therefore, caspase-3 appears to be an extremely biologically relevant apoptotic protease.All caspase family members are initially synthesized as inactive precursors and require proteolytic processing themselves to generate the two subunits that form the active protease. This suggests that the apoptotic machinery may be regulated, in part, by a proteolytic cascade (for review, see Ref. 19). In the case of caspase-3, the mature enzyme is formed from 17 kDa (p17) and 12 kDa (p12) subunits, which are produced from a 32-kDa precurs...

A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis

“…We next attempted to determine the limiting factors for caspase-3 activation in HL-60 and HCW-2 cells using S-100 cytosolic extracts and an in vitro caspase-3 activation assay (Liu et al, 1996;Han et al, 1997;Li et al, 1997). The addition of cytochrome c to the S-100 cytosolic extract from HL-60 cells induced proteolytic activation of the endogen- Unexpectedly, caspase-3 processing also occurred with HCW-2 extracts (Figure 3).…”

“…The mature caspase-3 consists of 17 kDa (p17) and 12 kDa (p12) subunits, which are produced from a 32 kDa (p32) precursor (FernandesAlnemri et al, 1994;Nicholson et al, 1995;Tewari et al, 1995a;Liu et al, 1996;Han et al, 1997). Some aspects of the mechanism by which the caspase-3 precursor is converted into the active enzyme are known and in particular endogenous caspase-3 in membrane-free cytosolic extracts from various cell lines can be activated by the addition of exogenous cytochrome c and dATP (Liu et al, 1996;Li et al, 1997) or exogenous cytochrome c alone (Han et al, 1997) under appropriate in vitro assay conditions. Since caspase precursors are present in the cytosol of cells whereas active cytochrome c is located in the mitochondria, these observations suggested that the efflux of cytochrome c from mitochondria into the cytosol during the apoptotic induction process was a key regulatory event in the activation of caspase-3.…”

Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway

“…25 In this study, we have examined the specificity of M-791 to inhibit caspases in cells, as assessed by caspase processing and substrate cleavage (Figure 1). M-791 caused a concentrationdependent inhibition of the autoprocessing of caspase-3 36 and the cleavage of two caspase-3 substrates, XIAP and cleavage of caspase-9 at D330. 18,37 Furthermore, M-791 inhibited neither the processing of caspase-6, -7 and -8 nor the initial processing of caspase-9 and -2.…”

p27Kip1 induces drug resistance by preventing apoptosis upstream of cytochrome c release and procaspase-3 activation in leukemic cells

“…When U937 cells overexpressing p27 Kip1 were treated in similar conditions, both procaspase-3 and procaspase-2L remained highly expressed in the cells 24 h after the beginning of cell treatment ( Figure 4a). Activation of caspase-3 involves its cleavage in a p20 and a p12 fragments, then the cleavage of the p20 into a p17 fragment that heterodimerizes with the p12 subunit to form the active caspase (Han et al, 1997). Using a polyclonal antibody that identi®es the p20 and the p17 fragments of caspase-3, we observed that these fragments appeared later and with a lower intensity in p27 Kip1 overexpressing cells compared to mock-transfected cells (Figure 4a).…”