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, ESMD downward arrowS (amino acids 25-29) and IETD downward arrowS (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 IETD downward arrowS site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD downward arrowS site, generating the mature p17 subunit. The cleavage at the IETD downward arrowS 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-alpha-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 ESMD downward arrowS site. This indicated that the cleavage at the ESMD downward arrowS 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.
Highlights
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
We have recently shown that when the human promyelocytic leukemia HL-60 cell line was induced to undergo apoptosis by exposure to staurosporine (STS), one of the early biochemical events was the conversion of the 32-kDa caspase-3 precursor into the mature protease that was responsible for the subsequent proteolysis of DNA-PKcs [9]
We studied the effect of cytochrome c on the status of the endogenous 32-kDa caspase-3 precursor present in cytosolic extracts derived from HL-60 cells
Summary
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). 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). We have recently shown that when the human promyelocytic leukemia HL-60 cell line was induced to undergo apoptosis by exposure to staurosporine (STS), one of the early biochemical events was the conversion of the 32-kDa caspase-3 precursor into the mature protease that was responsible for the subsequent proteolysis of DNA-PKcs [9]. DNA-PK, DNA-dependent protein kinase; DNA-PKcs, catalytic subunit of DNA-PK; TPCK, N-␣-tosyl-L-phenylalanine chloromethyl ketone; CHO, aldehyde; STS, staurosporine
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