Moon Kwan-Hoon, Hood Brian L, Kim Bong-Jo, Hardwick James P, Conrads Thomas P, Veenstra Timothy D, Song Byoung J
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, USA.
Hepatology. 2006 Nov;44(5):1218-30. doi: 10.1002/hep.21372.
Increased oxidative/nitrosative stress is a major contributing factor to alcohol-mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol-induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S-nitrosylated mitochondrial proteins and to use a biotin-N-maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol-inducible CYP2E1. The biotin-N-maleimide-labeled oxidized and/or S-nitrosylated mitochondrial proteins from pair-fed controls or alcohol-fed rat livers were subsequently purified with streptavidin-agarose. The overall patterns of oxidized and/or S-nitrosylated proteins resolved by 2-dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy-nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3-ketoacyl-CoA thiolase involved in mitochondrial beta-oxidation of fatty acids was significantly inhibited in alcohol-exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion, our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol-mediated liver damage.
氧化/亚硝化应激增加是酒精介导的线粒体功能障碍的主要促成因素。然而，在酒精诱导的氧化/亚硝化应激下哪些线粒体蛋白会发生氧化修饰，目前了解甚少。本研究的目的是系统地研究氧化和/或S-亚硝基化的线粒体蛋白，并使用生物素-N-马来酰亚胺探针评估它们在大鼠酒精性脂肪肝中的失活情况。暴饮或长期酒精暴露显著提高了一氧化氮、诱导型一氧化氮合酶和乙醇诱导的CYP2E1水平。随后用链霉亲和素琼脂糖纯化来自配对喂养对照或酒精喂养大鼠肝脏的生物素-N-马来酰亚胺标记的氧化和/或S-亚硝基化线粒体蛋白。二维聚丙烯酰胺凝胶电泳解析的氧化和/或S-亚硝基化蛋白的总体模式在长期和暴饮酒精治疗组中非常相似。通过质谱鉴定出79种与对照大鼠相比斑点强度有差异的蛋白质。这些包括线粒体醛脱氢酶2（ALDH2）、ATP合酶、酰基辅酶A脱氢酶、3-酮酰基辅酶A硫解酶，以及许多参与伴侣活性、线粒体电子传递和离子转运的蛋白质。参与脂肪酸线粒体β氧化的3-酮酰基辅酶A硫解酶的活性在酒精暴露的大鼠肝脏中显著受到抑制，这与生化和组织学分析确定的肝脏脂肪积累一致。活性测定和免疫印迹结果表明，在大鼠酒精性脂肪肝中，ALDH2和ATP合酶也通过其半胱氨酸或酪氨酸残基的氧化修饰而受到抑制。总之，我们的结果有助于解释线粒体功能障碍以及对酒精介导的肝损伤易感性增加的潜在机制。
