Wu Guey-Shuang, Lee Terry D, Moore Roger E, Rao Narsing A
Doheny Eye Institute, University of South California, Keck School of Medicine, Los Angeles, CA 90033-1088, USA.
Invest Ophthalmol Vis Sci. 2005 Jul;46(7):2271-81. doi: 10.1167/iovs.04-1525.
In experimental autoimmune uveitis (EAU), phagocytes are thought to be the primary cells in the initiation and maintenance of pathologic tissue damage through the release of cytotoxic agents. Recently, the presence of nitric oxide synthase has been shown in mammalian mitochondria. In this study, the effect of mitochondrial peroxynitrite on the modification of cellular proteins was evaluated in the early phase of uveitis, before the infiltration of leukocytes.
Tyrosine nitration in proteins was detected by UV/Vis (visible) absorption and Western blot analysis. The identity of the nitrated protein was obtained by liquid chromatography-tandem mass spectrometry. The release of cytochrome c was assessed in whole retinal extract and in isolated mitochondria. The protein nitration in the inflamed retina was also localized by immunohistochemistry.
Before the leukocyte infiltration in the early phase of EAU, the mitochondria-originated peroxynitrite initiated the inflammatory insult by specifically nitrating three mitochondrial proteins. In vitro nitration of the control retina by peroxynitrite donor resulted in nonspecific nitration of all major retinal proteins. After nitration, cytochrome c was displaced from its original binding site in the respiratory chain. Further, the nitration appeared to commence in the early phase of inflammation, on postimmunization day 5, long before the peak of inflammation on day 14. Immunohistochemically, tyrosine-nitrated proteins were localized exclusively in the photoreceptor inner segments, which are known to be densely populated with mitochondria.
These data indicate that mitochondrial proteins are the prime targets of inactivation by the mitochondrial peroxynitrite and that photoreceptor mitochondria initiate the subsequent irreversible retinal damage in experimental uveitis.
在实验性自身免疫性葡萄膜炎（EAU）中，吞噬细胞被认为是通过释放细胞毒性因子引发和维持病理性组织损伤的主要细胞。最近，已证实在哺乳动物线粒体中存在一氧化氮合酶。在本研究中，在白细胞浸润之前的葡萄膜炎早期阶段，评估了线粒体过氧亚硝酸盐对细胞蛋白质修饰的影响。
通过紫外/可见（Vis）吸收和蛋白质印迹分析检测蛋白质中的酪氨酸硝化。通过液相色谱-串联质谱法确定硝化蛋白质的身份。在全视网膜提取物和分离的线粒体中评估细胞色素c的释放。还通过免疫组织化学对炎症视网膜中的蛋白质硝化进行定位。
在EAU早期白细胞浸润之前，线粒体来源的过氧亚硝酸盐通过特异性硝化三种线粒体蛋白引发炎症损伤。过氧亚硝酸盐供体对对照视网膜进行体外硝化导致所有主要视网膜蛋白的非特异性硝化。硝化后，细胞色素c从其在呼吸链中的原始结合位点被置换。此外，硝化似乎在炎症早期，即免疫后第5天开始，远早于第14天的炎症高峰期。免疫组织化学显示，酪氨酸硝化的蛋白质仅定位在光感受器内段，已知该部位线粒体密集。
这些数据表明线粒体蛋白是线粒体过氧亚硝酸盐失活的主要靶点，并且光感受器线粒体在实验性葡萄膜炎中引发随后不可逆的视网膜损伤。
