title: 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.
abstract: 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.
authors.affiliation: Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China.
authors.forename: Bimei
authors.initials: B
authors.lastname: Jiang
authors.forename: Kangkai
authors.initials: K
authors.lastname: Wang
authors.forename: Pengfei
authors.initials: P
authors.lastname: Liang
authors.forename: Weimin
authors.initials: W
authors.lastname: Xiao
authors.forename: Haiyun
authors.initials: H
authors.lastname: Wang
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Xiao
mesh_terms: 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*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; D000255:Adenosine Triphosphate; D053453:Caspase 9; D020169:Caspases