Kang Patrick T, Chen Chwen-Lih, Chen Yeong-Renn
Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA.
Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA.
Free Radic Biol Med. 2015 Feb;79:56-68. doi: 10.1016/j.freeradbiomed.2014.11.016. Epub 2014 Nov 28.
In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS(•)) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 and 75-kDa subunits was detected under the conditions of (•)O2(-) production, and mass spectrometry confirmed that formation of Complex I PrS(•) mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS(•) (Kang et al., Free Radical Biol. Med.52:962-973; 2012). In this investigation, we tested our hypothesis in the murine heart of eNOS(-/-). The eNOS(-/-) mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS(-/-) myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in (•)O2(-) generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS(-/-) murine heart. When Complex I from the mitochondria of the eNOS(-/-) murine heart was analyzed by immunospin trapping and probed with anti-GSH antibody, both PrS(•) and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS(•), supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS(•) is a unique pathway for the redox regulation of mitochondrial function in vivo.
作为对氧化应激的反应，线粒体复合物I会发生可逆的S-谷胱甘肽化。我们推测，复合物I的蛋白质S-谷胱甘肽化（PrSSG）是由一种动力学机制介导的，该机制涉及体内活性蛋白硫基自由基（PrS(•)）和谷胱甘肽（GSH）。先前的研究表明，在体外，在产生超氧阴离子（•）O2(-)的条件下，可检测到分离的复合物I在51 kDa和75 kDa亚基上发生S-谷胱甘肽化，并且质谱分析证实复合物I PrS(•)的形成介导了PrSSG。将心肌细胞暴露于甲萘醌会导致复合物I PrSSG和PrS(•)增加（Kang等人，《自由基生物学与医学》52:962 - 973；2012年）。在本研究中，我们在eNOS(-/-)小鼠心脏中验证了我们的假设。已知eNOS(-/-)小鼠患有高血压，并会出现进行性心脏肥大的病理表型。从eNOS(-/-)心肌中分离出的线粒体表现出明显的功能障碍，状态3呼吸受损、呼吸控制指数下降以及电子传递链（ETC）成分的酶活性降低。进一步的生化分析和电子顺磁共振（EPR）测量表明乌头酸酶活性存在缺陷、超氧阴离子（•）O2(-)生成活性显著增加以及生理环境更加氧化。这些结果表明eNOS(-/-)小鼠心脏线粒体中的促氧化活性增加以及随后的氧化应激增加。当通过免疫自旋捕获分析eNOS(-/-)小鼠心脏线粒体中的复合物I并用抗GSH抗体进行检测时，复合物I的PrS(•)和PrSSG均显著增强。在小鼠心脏中过表达超氧化物歧化酶2（SOD2）显著降低了检测到的PrS(•)，支持了氧化应激诱导的PrS(•)介导复合物I PrSSG是体内线粒体功能氧化还原调节的独特途径这一结论。
