ATP-binding domain of heat shock protein 70 is essential for its effects on the inhibition of the release of the second mitochondria-derived activator of caspase and apoptosis in C2C12 cells
ATP-binding domain of heat shock protein 70 is essential for its effects on the inhibition of the release of the second mitochondria-derived activator of caspase and apoptosis in C2C12 cells
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DOI：
10.1111/j.1742-4658.2009.06989.x
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发表时间：
2009-05-01
期刊：
影响因子：
5.4
通讯作者：
Xiao, Xianzhong
中科院分区：
文献类型：
--
作者：
Jiang, Bimei;Wang, Kangkai;Xiao, Xianzhong
过氧化氢（H（2）O（2））是一种众所周知的氧化应激诱导剂，可引起许多细胞凋亡。以前，我们已经表明，热休克预处理阻断释放的第二个caspase（Smac）的细胞质和抑制C2 C12成肌细胞的细胞凋亡响应H（2）O（2）。本研究的目的是阐明潜在的机制，过表达的主要应激诱导蛋白，热休克蛋白（HSP）70，并表征由此产生的细胞变化。HSP 70过表达可显著抑制H2 O2处理的C2 C12细胞中Smac的释放，抑制caspase-9和caspase-3的激活和细胞凋亡。免疫共沉淀法未观察到HSP 70与Smac之间的直接相互作用。突变分析表明，HSP 70的ATP结合结构域，而不是肽结合结构域，是必不可少的这些观察到的HSP功能。综上所述，我们的研究结果提供了证据支持HSP 70在保护C2 C12细胞免受H（2）O（2）诱导和Smac促进的凋亡中的作用，通过阻止Smac从线粒体释放，从而抑制caspase-9和-3的活化。HSP 70的这种作用机制依赖于其ATP结合结构域，但不依赖于其与Smac蛋白的相互作用。
Hydrogen peroxide (H(2)O(2)) is a well known oxidative stress inducer causing apoptosis of many cells. Previously, we have shown that heat shock pretreatment blocked the release of the second mitochondria-derived activator of caspase (Smac) to the cytosol and inhibited apoptosis of C2C12 myoblast cells in response to H(2)O(2). The present study aimed to elucidate the underlying mechanism by over-expressing a major stress-inducible protein, heat shock protein (HSP) 70, and characterizing the resulting cellular changes. We demonstrate that HSP70 over-expression markedly inhibited the release of Smac and prevented the activation of caspases-9 and -3 and apoptosis in C2C12 cells under H(2)O(2) treatment. However, no direct interaction between HSP70 and Smac was observed by co-immunoprecipitation. Mutational analysis demonstrated that the ATP-binding domain of HSP70, rather than the peptide-binding domain, was essential for these observed HSP functions. Taken together, our results provide evidence supporting the role of HSP70 in the protection of C2C12 cells from H(2)O(2)-induced and Smac-promoted apoptosis by preventing the release of Smac from mitochondria, thereby inhibiting activation of caspases-9 and -3. This mechanism of HSP70 action is dependent on its ATP-binding domain but independent of its interaction with Smac protein.