Photoreceptor Mitochondrial Protein Nitration in Uveitis

描述（由申请人提供）：葡萄膜炎致盲是由感光细胞变性引起的。这种退化归因于巨噬细胞浸润和随后产生的各种炎症介质，包括一氧化氮衍生的高活性氧化剂过氧亚硝酸盐，已知过氧亚硝酸盐通过酪氨酸残基的硝化引起光感受器损伤。然而，我们的初步数据显示，在巨噬细胞浸润之前，硝化作用开始于EAU的早期阶段。此外，我们发现硝化作用选择性地涉及线粒体蛋白。这些新发现暗示光感受器细胞线粒体蛋白硝化是光感受器变性起始的中心分子事件。这种硝化作用肯定不是由巨噬细胞介导的。

DESCRIPTION (provided by applicant): Blindness in uveitis results from degeneration of photoreceptor cells. This degeneration is attributed to macrophage infiltration and the subsequent generation of various inflammatory mediators, including the nitric oxide-derived highly reactive oxidant peroxynitrite, which is known to cause photoreceptor damage via nitration of tyrosine residues. Our preliminary data, however, shows the nitration commences in the early phase of EAU, prior to infiltration by macrophages. Moreover, we found that the nitration selectively involves the mitochondrial proteins. These novel findings implicate photoreceptor cell mitochondrial protein nitration as a central molecular event in the initiation of photoreceptor degeneration. This nitration is certainly not mediated by macrophages. We propose a hypothesis: "Photoreceptor degeneration in autoimmune uveitis begins with selective mitochondrial protein nitration as a consequence of reactive nitric oxide species generation in the photoreceptor mitochondria." We will test this hypothesis with the following specific aims in animals with early EAU, prior to macrophage infiltration of the retina. 1. Evaluate nitric-oxide reactive species generation in the photoreceptor mitochondria (immunohistochemistry, immunoblotting, and mass spectrometry). 2. Evaluate mitochondrial protein nitration in the photoreceptors (immunohistochemical localization of nitrated proteins, immunoblotting of photoreceptor proteins, and capillary liquid chromatography-tandem mass spectrometry). 3. Evaluate functions of the photoreceptor mitochondria (oxygen consumption and activities of electron transport chain complexes, and mitochondrial transmembrane potential). To assure successful completion of the specific aims, we have assembled a multidisciplinary team of experts. Professor Terry Lee is an accomplished chemist with expertise in identifying nitrated proteins, and their site of nitration by mass spectrometry and other novel methods. Dr. Guey-Shuang Wu has expertise in PCR, Western blot, and separation of biological molecules. The principal investigator is experienced in the field of EAU, immunohistochemistry, free radical biology, and reactive nitric oxide species. Understanding the role of mitochondria in the pathogenesis of retinal degenerations could lead to the development of specific therapy to minimize mitochondrial generation of peroxynitrite.