Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial Complex II in the post-ischemic myocardium
Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial Complex II in the post-ischemic myocardium
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DOI：
10.1074/jbc.m802691200
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发表时间：
2008-10-10
影响因子：
4.8
通讯作者：
Chen, Yeong-Renn
中科院分区：
文献类型：
--
作者：
Chen, Chwen-Lih;Chen, Jingfeng;Chen, Yeong-Renn
O-2(-) 和 NO 产生增加是心肌缺血/再灌注损伤中线粒体功能障碍的关键机制。线粒体电子传递链的一个关键部分是琥珀酸泛醌还原酶（SQR 或复合体 II）。在 SQR 中，70-kDa 黄素蛋白的氧化损伤和去谷胱甘肽作用发生在缺血后心脏中（Chen, Y. R.、Chen, C. L.、Pfeiffer, D. R. 和 Zweier, J. L. (2007) J. Biol. Chem. 282, 32640 -32654）。为了深入了解缺血后心肌中 70-kDa 蛋白的氧化修饰，我们使用 70-kDa 蛋白中已鉴定的 S-谷胱甘肽（VTK93 下的 (77)AAFGLSEAGFNTA (C)）作为嵌合表位，掺入“混杂”T 细胞表位，生成高效价多克隆抗体 AbGSC90。纯化的 AbGSC90 对分离的 SQR 显示出高结合亲和力。 AbGSC90 的抗体适度抑制 SQR 的电子转移和超氧化物生成活性。为了测试蛋白质硝化，对大鼠进行 30 分钟的冠状动脉结扎，然后再灌注 24 小时。使用 AbGSC90 对组织匀浆进行免疫沉淀，并使用 3-硝基酪氨酸抗体进行探测。在缺血后的心肌中检测到蛋白质酪氨酸硝化的增强。用过氧亚硝酸盐对分离的 SQR 进行体外蛋白质硝化，导致 70-kDa 多肽发生位点特异性硝化，并损害 SQR 电子转移活性。 SQR 的蛋白质硝化进一步损害了其与复合物 III 的蛋白质-蛋白质相互作用。液相色谱/串联质谱分析表明Tyr-56和Tyr-142参与了蛋白质酪氨酸硝化。当分离的SQR进行体外S-谷胱甘肽化时，过氧亚硝酸盐介导的氧化修饰和损伤显着减少，从而证实了S-谷胱甘肽化对硝化氧化损伤的保护作用。
Increased O-2(-) and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart (Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282, 32640 -32654). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide ((77)AAFGLSEAGFNTA (C) under bar VTK93) of the 70-kDa protein as a chimeric epitope incorporating a "promiscuous" T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.