Proteasome-mediated degradation of Smac during apoptosis:: XIAP promotes Smac ubiquitination in vitro
Proteasome-mediated degradation of Smac during apoptosis:: XIAP promotes Smac ubiquitination in vitro
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DOI：
10.1074/jbc.m200317200
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发表时间：
2002-09-27
影响因子：
4.8
通讯作者：
Cohen, GM
中科院分区：
文献类型：
--
作者：
MacFarlane, M;Merrison, W;Cohen, GM
在细胞凋亡过程中，Smac（第二类caspase衍生激活剂）/DIABLO（一种IAP（凋亡抑制蛋白）结合蛋白）从线粒体释放，并通过解除IAP对caspase的抑制来增强细胞凋亡。我们证明，MCF-7细胞暴露于死亡诱导配体，TRAIL（肿瘤坏死因子相关凋亡诱导配体），导致Smac从线粒体中快速释放，这发生在细胞色素c丢失之前或与之平行。Smac释放被Bcl-2/Bcl-xL或泛半胱天冬酶抑制剂抑制，表明该事件是半胱天冬酶依赖性的，并由Bcl-2家族成员调节。释放后，Smac被蛋白酶体迅速降解，这是一种通过与蛋白酶体抑制剂共处理而抑制的效应。由于XIAP的RING指结构域具有泛素-蛋白连接酶活性，并且XIAP与成熟Smac紧密结合，因此进行体外泛素化测定，其显示XIAP在Smac的泛素化中充当泛素-蛋白连接酶（E3）。XIAP与Smac的结合以及XIAP的RING指结构域对于泛素化都是必不可少的，这表明XIAP的泛素-蛋白连接酶活性可能促进细胞释放的Smac的快速降解。因此，除了其在抑制半胱天冬酶活性方面的充分表征的作用外，XIAP还可以通过靶向促凋亡分子进行蛋白酶体降解来保护细胞免受意外的线粒体损伤。
During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.