Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of bid by multiple papain-like lysosomal cathepsins
Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of bid by multiple papain-like lysosomal cathepsins
复制标题
DOI：
10.1074/jbc.m308347200
复制
发表时间：
2004-01-30
影响因子：
4.8
通讯作者：
Turk, B
中科院分区：
文献类型：
--
作者：
Cirman, T;Oresic, K;Turk, B
越来越多的证据表明，溶酶体蛋白酶积极参与细胞凋亡。使用HeLa细胞作为模型系统，我们表明，选择性溶酶体破坏与L-亮氨酰-L-亮氨酸甲酯的结果在细胞凋亡，其特征在于易位的溶酶体蛋白酶到胞质溶胶和裂解的促凋亡Bcl-2-家族成员投标。细胞凋亡和Bid裂解，但不是溶酶体蛋白酶易位到胞质溶胶，可以防止15 μ M L-反式环氧琥珀酰（OEt）-亮氨酸-3-甲基丁酰胺，木瓜蛋白酶样半胱氨酸蛋白酶的抑制剂。用15 μ M N-苯甲酰氧羰基-VAD-氟甲基酮孵育细胞可阻止凋亡，但不能阻止Bid裂解，表明该系统中组织蛋白酶介导的凋亡是半胱天冬酶依赖性的。在中性pH下进行的体外实验表明，木瓜蛋白酶样组织蛋白酶B、H、L、S和K主要在Arg（65）或Arg（71）处切割Bid。用组织蛋白酶C和X或天冬氨酸蛋白酶组织蛋白酶D没有观察到Bid裂解。用组织蛋白酶B、H、L和S处理的全长Bid的孵育导致细胞色素c从分离的线粒体中快速释放。因此，Bid可能是溶酶体破坏诱导的细胞凋亡的重要介质。
Increasing evidence suggests that lysosomal proteases are actively involved in apoptosis. Using HeLa cells as the model system, we show that selective lysosome disruption with L-leucyl-L-leucine methyl ester results in apoptosis, characterized by translocation of lysosomal proteases into the cytosol and by the cleavage of a proapoptotic Bcl-2-family member Bid. Apoptosis and Bid cleavage, but not translocation of lysosomal proteases to the cytosol, could be prevented by 15 muM L-trans-epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 muM N-benzoyloxycarbonyl-VAD-fluoromethyl ketone prevented apoptosis but not Bid cleavage, suggesting that cathepsin-mediated apoptosis in this system is caspase-dependent. In vitro experiments performed at neutral pH showed that papain-like cathepsins B, H, L, S, and K cleave Bid predominantly at Arg(65) or Arg(71). No Bid cleavage was observed with cathepsins C and X or the aspartic protease cathepsin D. Incubation of full-length Bid treated with cathepsins B, H, L, and S resulted in rapid cytochrome c release from isolated mitochondria. Thus, Bid may be an important mediator of apoptosis induced by lysosomal disruption.