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
2009
Jiang, Bimei | Wang, Kangkai | Liang, Pengfei | Xiao, Weimin | Wang, Haiyun | Xiao, Xianzhong
Hydrogen peroxide (H₂O₂) 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 H₂O₂. 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 H₂O₂ 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 H₂O₂-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.
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