Benderdour Mohamed, Charron Guy, DeBlois Denis, Comte Blandine, Des Rosiers Christine
Department of Nutrition, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
J Biol Chem. 2003 Nov 14;278(46):45154-9. doi: 10.1074/jbc.M306285200. Epub 2003 Sep 6.
Mitochondrial NADP+-isocitrate dehydrogenase activity is crucial for cardiomyocyte energy and redox status, but much remains to be learned about its role and regulation. We obtained data in spontaneously hypertensive rat hearts that indicated a partial inactivation of this enzyme before hypertrophy development. We tested the hypothesis that cardiac mitochondrial NADP+-isocitrate dehydrogenase is a target for modification by the lipid peroxidation product 4-hydroxynonenal, an aldehyde that reacts readily with protein sulfhydryl and amino groups. This hypothesis is supported by the following in vitro and in vivo evidence. In isolated rat heart mitochondria, enzyme inactivation occurred within a few minutes upon incubation with 4-hydroxynonenal and was paralleled by 4-hydroxynonenal/NADP+-isocitrate dehydrogenase adduct formation. Enzyme inactivation was prevented by the addition of its substrate isocitrate or a thiol, cysteine or glutathione, suggesting that 4-hydroxynonenal binds to a cysteine residue near the substrate's binding site. Using an immunoprecipitation approach, we demonstrated the formation of 4-hydroxynonenal/NADP+-isocitrate dehydrogenase adducts in the heart and their increased level (210%) in 7-week-old spontaneously hypertensive rats compared with control Wistar Kyoto rats. To the best of our knowledge, this is the first study to demonstrate that mitochondrial NADP+-isocitrate dehydrogenase is a target for inactivation by 4-hydroxynonenal binding. Furthermore, the pathophysiological significance of our finding is supported by in vivo evidence. Taken altogether, our results have implications that extend beyond mitochondrial NADP+-isocitrate dehydrogenase. Indeed, they emphasize the implication of post-translational modifications of mitochondrial metabolic enzymes by 4-hydroxynonenal in the early oxidative stress-related pathophysiological events linked to cardiac hypertrophy development.
线粒体NADP⁺-异柠檬酸脱氢酶活性对心肌细胞的能量和氧化还原状态至关重要，但其作用和调节机制仍有许多有待研究之处。我们在自发性高血压大鼠心脏中获得的数据表明，在心肌肥大发展之前，该酶存在部分失活。我们检验了以下假设：心脏线粒体NADP⁺-异柠檬酸脱氢酶是脂质过氧化产物4-羟基壬烯醛修饰的靶点，4-羟基壬烯醛是一种能与蛋白质巯基和氨基快速反应的醛类物质。以下体外和体内证据支持了这一假设。在分离的大鼠心脏线粒体中，与4-羟基壬烯醛孵育几分钟后酶就会失活，同时伴有4-羟基壬烯醛/NADP⁺-异柠檬酸脱氢酶加合物的形成。添加其底物异柠檬酸或一种硫醇（半胱氨酸或谷胱甘肽）可防止酶失活，这表明4-羟基壬烯醛与底物结合位点附近的一个半胱氨酸残基结合。通过免疫沉淀方法，我们证明了心脏中4-羟基壬烯醛/NADP⁺-异柠檬酸脱氢酶加合物的形成，并且与对照Wistar Kyoto大鼠相比，7周龄自发性高血压大鼠中该加合物水平增加了210%。据我们所知，这是第一项证明线粒体NADP⁺-异柠檬酸脱氢酶是4-羟基壬烯醛结合导致失活的靶点的研究。此外，我们这一发现的病理生理学意义得到了体内证据的支持。综上所述，我们的结果所具有的意义超出了线粒体NADP⁺-异柠檬酸脱氢酶本身。事实上，它们强调了4-羟基壬烯醛对线粒体代谢酶的翻译后修饰在与心肌肥大发展相关的早期氧化应激相关病理生理事件中的作用。
