Chua B T, Guo K, Li P
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore.
J Biol Chem. 2000 Feb 18;275(7):5131-5. doi: 10.1074/jbc.275.7.5131.
Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.
半胱天冬酶是一类独特的半胱氨酸蛋白酶家族，参与细胞因子激活和凋亡执行过程，可根据其前结构域的长度进行亚分组。长前结构域半胱天冬酶，如半胱天冬酶 -8 和半胱天冬酶 -9，被认为主要作为上游半胱天冬酶，切割下游短前结构域半胱天冬酶，如半胱天冬酶 -3 和 -7。我们在此报告，已鉴定出半胱天冬酶是钙激活蛋白酶（钙蛋白酶）的直接底物。钙蛋白酶在与上游半胱天冬酶不同的位点切割半胱天冬酶 -7，产生蛋白水解无活性的片段。半胱天冬酶 -8 和半胱天冬酶 -9 也可被钙蛋白酶直接切割。通过对切割产物进行 N 端测序，已鉴定出半胱天冬酶 -9 中的两个钙蛋白酶切割位点。钙蛋白酶切割半胱天冬酶 -9 产生截短的半胱天冬酶 -9，其在细胞裂解物中无法激活半胱天冬酶 -3。此外，钙蛋白酶直接切割半胱天冬酶 -9 可阻断 dATP 和细胞色素 c 诱导的半胱天冬酶 -3 激活。因此，我们的结果表明，钙蛋白酶可能通过有效使上游半胱天冬酶失活，而作为半胱天冬酶加工和凋亡的负调节因子。
