Han Z, Hendrickson E A, Bremner T A, Wyche J H
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
J Biol Chem. 1997 May 16;272(20):13432-6. doi: 10.1074/jbc.272.20.13432.
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.
凋亡性半胱氨酸蛋白酶caspase-3在细胞中以无活性的32 kDa前体形式表达，在凋亡过程中，成熟caspase-3的17 kDa（p17）和12 kDa（p12）亚基通过蛋白水解从该前体产生。前体中的两个氨基酸序列ESMD↓S（第25 - 29位氨基酸）和IETD↓S（第172 - 176位氨基酸）已被确定为产生p17和p12亚基的切割位点。使用无细胞检测系统，我们证明caspase-3前体似乎首先在IETD↓S位点被切割，产生p12亚基和一个20 kDa（p20）肽段。随后，p20在ESMD↓S位点被切割，产生成熟的p17亚基。在IETD↓S位点的切割需要一种蛋白酶活性，该活性被肽Ac-IETD-CHO（乙酰-IETD-醛）以及其他蛋白酶抑制剂选择性抑制，如牛痘病毒丝氨酸蛋白酶抑制剂CrmA和N-α-甲苯磺酰-L-苯丙氨酸氯甲基酮。催化ESMD/S位点切割的蛋白酶被另一种肽Ac-ESMD-CHO（乙酰-ESMD-醛）选择性抑制。更有趣的是，caspase-3抑制剂Ac-DEVD-CHO，而不是caspase-1抑制剂Ac-YVAD-CHO，也选择性抑制在ESMD↓S位点切割的蛋白酶活性。这表明在ESMD↓S位点的切割要么是自催化的，要么需要一种类似caspase-3的活性。总之，我们证明caspase-3的p17:p12形式的产生是一个连续的两步过程，似乎需要两种不同的酶活性。
