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.
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.
Jiang
Bimei
B
Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China.
Wang
Kangkai
K
Liang
Pengfei
P
Xiao
Weimin
W
Wang
Haiyun
H
Xiao
Xianzhong
X
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D053453:Caspase 9 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; D000255:Adenosine Triphosphate; D053453:Caspase 9; D020169:Caspases