Regulation of Mitochondrial Glutathione Redox Status and Protein Glutathionylation by Respiratory Substrates
Regulation of Mitochondrial Glutathione Redox Status and Protein Glutathionylation by Respiratory Substrates
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DOI：
10.1074/jbc.m110.164160
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发表时间：
2010-12-17
影响因子：
4.8
通讯作者：
Cadenas, Enrique
中科院分区：
文献类型：
--
作者：
Garcia, Jerome;Han, Derick;Cadenas, Enrique
通过不连续Percoll梯度分离的脑和肝线粒体显示氧化的氧化还原环境，这反映在低GSH水平和高GSSG水平以及线粒体蛋白的显著谷胱甘肽化以及低NAD（P）H/NAD（P）值。根据GSH和GSSG浓度，通过不连续Percoll梯度法分离的脑线粒体的氧化还原电位计算为-171mV。免疫印迹和LC/MS/MS分析表明，琥珀酰辅酶A转移酶和ATP合酶（F-1复合物，α-亚基）被广泛谷胱甘肽化;这些蛋白质的S-谷胱甘肽化导致活性大幅降低。补充线粒体与复合物I或复合物II呼吸底物（苹果酸/谷氨酸或琥珀酸，分别）增加NADH和NADPH水平，导致还原谷胱甘肽水平的恢复，通过减少GSSG和脱谷胱甘肽线粒体蛋白。在这些条件下，脑线粒体的氧化还原电位计算为-291 mV。补充线粒体呼吸底物阻止GSSG的形成，因此，ATP合酶谷胱甘肽化响应H2 O2的挑战。ATP合酶似乎是氧化应激条件下谷胱甘肽化的主要线粒体蛋白。线粒体蛋白的谷胱甘肽化是氧化应激的主要结果，并且呼吸底物是通过维持线粒体NADPH水平的线粒体氧化还原状态（如通过硫醇/二硫键交换所反映的）的关键调节剂。
Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P) H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F-1 complex, alpha-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H2O2 challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.