In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains
In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains
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DOI：
10.1073/pnas.93.15.7464
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发表时间：
1996-07-23
影响因子：
11.1
通讯作者：
Alnemri, ES
中科院分区：
文献类型：
--
作者：
FernandesAlnemri, T;Armstrong, RC;Alnemri, ES
新出现的证据表明，由多种天冬氨酸特异性半胱氨酸蛋白酶 (ASCP) 组成的可放大蛋白酶级联是导致在经历程序性细胞死亡的哺乳动物细胞中观察到的细胞凋亡变化的原因。在这里，我们描述了从人类 Jurkat T 淋巴细胞中克隆两种新型 ASCP。与其他 ASCP 一样，名为 Mch4 和 Mch5 的新蛋白酶源自单链酶原。然而，它们的假定活性位点包含 QACQG 五肽，而不是所有已知 ASCP 中存在的 QACRG。此外，它们的 N 末端包含 FADD 样死亡效应结构域，表明可能与 FADD 相互作用。 Mch4 在大肠杆菌中的表达产生了一种活性蛋白酶，与其他 ASCP 一样，该蛋白酶可被四肽醛 DEVD-CHO 有效抑制 (K-i = 14 nM)。有趣的是，Mch4 和丝氨酸蛋白酶颗粒酶 B 均在保守的 IXXD-S 序列处切割重组 proCPP32 和 proMch3，以产生活性蛋白酶的大亚基和小亚基。粒酶 B 还在同源 IXXD-A 加工序列处切割 proMch4，以产生成熟的 Mch4。这些观察结果表明，CPP32 和 Mch3 是凋亡细胞中成熟 Mch4 蛋白酶的靶标。Mch4 和 Mch5 中 FADD 样结构域的存在表明这些蛋白酶在 Fas 凋亡途径中发挥作用。此外，这些蛋白酶可以参与颗粒酶B凋亡途径。
Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate-specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death, Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in all known ASCPs, Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (K-i = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells, The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathway.