Venkatraman Aparna, Landar Aimee, Davis Ashley J, Ulasova Elena, Page Grier, Murphy Michael P, Darley-Usmar Victor, Bailey Shannon M
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Am J Physiol Gastrointest Liver Physiol. 2004 Apr;286(4):G521-7. doi: 10.1152/ajpgi.00399.2003. Epub 2003 Dec 11.
Redox modification of mitochondrial proteins is thought to play a key role in regulating cellular function, although direct evidence to support this hypothesis is limited. Using an in vivo model of mitochondrial redox stress, ethanol hepatotoxicity, the modification of mitochondrial protein thiols was examined using a proteomics approach. Specific labeling of reduced thiols in the mitochondrion from the livers of control and ethanol-fed rats was achieved by using the thiol reactive compound (4-iodobutyl)triphenylphosphonium (IBTP). This molecule selectively accumulates in the organelle and can be used to identify thiol-containing proteins. Mitochondrial proteins that have been modified are identified by decreased labeling with IBTP using two-dimensional SDS-PAGE followed by immunoblotting with an antibody directed against the triphenylphosphonium moiety of the IBTP molecule. Analyses of these data showed a significant decrease in IBTP labeling of thiols present in specific mitochondria matrix proteins from ethanol-fed rats compared with their corresponding controls. These proteins were identified as the low-K(m) aldehyde dehydrogenase and glucose-regulated protein 78. The decrease in IBTP labeling in aldehyde dehydrogenase was accompanied by a decrease in specific activity of the enzyme. These data demonstrate that mitochondrial protein thiol modification is associated with chronic alcohol intake and might contribute to the pathophysiology associated with hepatic injury. Taken together, we have developed a protocol to chemically tag and select thiol-modified proteins that will greatly enhance efforts to establish posttranslational redox modification of mitochondrial protein in in vivo models of oxidative or nitrosative stress.
线粒体蛋白的氧化还原修饰被认为在调节细胞功能中起关键作用，尽管支持这一假设的直接证据有限。利用线粒体氧化还原应激的体内模型——乙醇肝毒性，采用蛋白质组学方法研究了线粒体蛋白硫醇的修饰情况。通过使用硫醇反应性化合物（4-碘丁基）三苯基鏻（IBTP），实现了对对照大鼠和乙醇喂养大鼠肝脏线粒体中还原型硫醇的特异性标记。该分子选择性地积聚在细胞器中，可用于鉴定含硫醇的蛋白质。使用二维SDS-PAGE，然后用针对IBTP分子三苯基鏻部分的抗体进行免疫印迹，通过IBTP标记减少来鉴定已被修饰的线粒体蛋白。对这些数据的分析表明，与相应对照相比，乙醇喂养大鼠特定线粒体基质蛋白中存在的硫醇IBTP标记显著减少。这些蛋白质被鉴定为低Km醛脱氢酶和葡萄糖调节蛋白78。醛脱氢酶中IBTP标记的减少伴随着该酶比活性的降低。这些数据表明，线粒体蛋白硫醇修饰与长期饮酒有关，可能有助于与肝损伤相关的病理生理学。综上所述，我们开发了一种化学标记和选择硫醇修饰蛋白的方案，这将大大加强在氧化或亚硝化应激体内模型中建立线粒体蛋白翻译后氧化还原修饰的研究工作。
