A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro
A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro
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DOI：
10.1074/jbc.272.20.13432
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发表时间：
1997-05-16
影响因子：
4.8
通讯作者：
Wyche, JH
中科院分区：
文献类型：
--
作者：
Han, ZY;Hendrickson, EA;Wyche, JH
凋亡半胱氨酸蛋白酶 caspase-3 在细胞中表达为无活性的 32 kDa 前体，在凋亡过程中通过蛋白水解产生成熟 caspase-3 的 17 kDa (p17) 和 12 kDa (p12) 亚基。 两个氨基酸序列，ESMD 向下箭头 S（氨基酸 25-29）和 IETD 向下箭头 S（氨基酸 172-176），前体中的 已被定义为产生 p17 和 p12 亚基的切割位点，使用无细胞测定系统，我们证明 caspase-3 前体似乎首先在 IETD 向下箭头 S 位点处被切割，产生 p12 亚基和 20-kDa (p20) 肽。随后，p20 在 ESMD 向下箭头 S 位点处切割，生成成熟的 p17 亚基， IETD 向下箭头 S 位点的裂解需要蛋白酶活性，该活性可被肽 Ac-IETD-CHO（乙酰基-IETD-醛）和其他蛋白酶抑制剂选择性抑制，例如牛痘病毒丝氨酸蛋白酶抑制剂 CrmA 和 N-α-甲苯磺酰基-L-苯丙氨酸氯甲基酮。在 ESMD/S 位点催化裂解的蛋白酶被另一种肽 Ac-ESMD-CHO（乙酰基-ESMD-醛）选择性抑制。更有趣的是，caspase-3 抑制剂 Ac-DEVD-CHO（而非 caspase-1 抑制剂 Ac-YVAD-CHO）也选择性抑制在 ESMD 向下箭头 S 位点处裂解的蛋白酶活性。这表明 ESMD 向下箭头位点的裂解要么是自催化的，要么需要类似 caspase-3 的活性。总之，我们证明 p17:p12 形式的 caspase-3 的生产是一个连续的两步过程，并且似乎需要两种不同的酶活性。
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 down arrow S (amino acids 25-29) and IETD down arrow S (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 down arrow S site, producing the p12 subunit and a 20-kDa (p20) peptide, Subsequently, the p20 is cleaved at the ESMD down arrow S site, generating the mature p17 subunit, The cleavage at the IETD down arrow S 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 down arrow S site. This indicated that the cleavage at the ESMD down arrow 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.