Sirt3-catalyzed Deacetylation Of Mitochondrial Cytochrome C Oxidase Involved In Oxidative Stress Induced Cell Apoptosis

Mouse embryonal fibroblast cell line NIH/3T3 was treated with nicotinamide(NAM), a broad-spectrum sirtuin inhibitor. Cytochrome c oxidase was immunoprecipitated for subsequent detection of acetylation using an antibody that recognized internal acetyllysine residues. In the cells treated with NAM, the expression of Sirt3 was markedly reduced compared with control cells. Inhibition of sirtuin activity resulted in enhanced cytochrome c oxidase acetylation in NAM-treated cells as compared with control. si RNA knockdown and plasmid overexpression were also used to investigate the potential role of Sirt3 in regulating deacetylation of cytochrome c oxidase. Silencing of Sirt3 expression caused an increase in acetylation of cytochrome c oxidase subunit I. The acetylation of cytochrome c oxidase subunit I was markedly reduced in the Sirt3-overexpressed cells. Knockdown of Sirt3 resulted in a significant decrease in cytochrome c oxidase activity. The inhibited activity of cytochrome c oxidase lead to significant release of cytochrome c into the cytoplasm and caspase 3 was subsequently cleaved. When subunit I of cytochrome c oxidase was silenced, the release of cytochrome c to cytoplasm was promoted. Cleaved caspase 3 was also significantly expressed.AAPH, a ROS generator, were used to induce oxidative damage and apoptotic protein activation in NIH/3T3 cells. Protein expression of Sirt3, the activity and acetylation level of cytochrome c oxidase, and the regulatory mechanism of Sirt3 in apoptosis were studied. The oxidant AAPH(5, 7.5 m M, 12 h) could significantly increase ROS generation and reduce mitochondrial membrane potential. AAPH treatment(5, 7.5 m M, 12 h) also aroused an obvious release of cytochrome c into the cytoplasm and the expression of cleaved caspase 3 was markedly increased. The utimate effect of oxidative stress caused by AAPH treatment(5, 7.5 m M, 12 h) was the distinct decline of ATP content. The average protein expression level of Sirt3 in AAPH-treated NIH/3T3 cells was significantly lower than that of normal cells. A decline in cytochrome c oxidase activity was observed. The acetylation level of cytochrome c oxidase subunit I was markedly increased by AAPH.Brain ischemia/reperfusion model was developed by middle cerebral artery occlusion. Cerebral infarction was detected by TTC staining. ROS generation rate, the activity of cytochrome c oxidase and Sirt3 protein expression were also determined. An obvious infarct formation was noticed after 2 h of ischemia and 24, 48 and 72 h of reperfusion. ROS content in the cerebral cortex was significantly elevated after reperfusion. Cytochrome c oxidase activity was also decreased when compared to sham. The acetylation level of cytochrome c oxidase was also observed by immunoprecipitation. Cytochrome c oxidase subunit I acetylation was also increased.These results indicated that the subunit I of cytochrome c oxidase was a possible target of Sirt3 deacetylation. Silencing of Sirt3 led to an elevated acetylation of the subunit I of cytochrome c oxidase and decreased its enzyme activity. These caused a subsequent transportation of mitochondrial cytochrome c to cytoplasm and cleavage of caspase 3, which ultimatly induced cellular apoptosis. Oxidative stress arised by AAPH caused a decrease in Sirt3 expression and an increase in the acetylation of the subunit I of cytochrome c oxidase. Cerebral ischemia/reperfusion-induced oxidative stress down-regulated Sirt3 expression. The deficiency of Sirt3 plays an important role in the modulation of COX1 deacetylation, and subsequently results in the functional impairment of COX in ischemic rats.