Photoreceptor Mitochondrial Protein Nitration in Uveitis

葡萄膜炎中的光感受器线粒体蛋白硝化

• 批准号: 7117201
• 负责人: NARSING A RAO
• 金额: $ 35.31万
• 依托单位: DOHENY EYE INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7117201
• 关键词: autoimmune disorder; disease /disorder etiology; electron transport; electrospray ionization mass spectrometry; free radicals; gas chromatography mass spectrometry; immunocytochemistry; laboratory rat; laser capture microdissection; liquid chromatography; mass spectrometry; membrane potentials; mitochondria; nitration; nitric oxide; oxygen consumption; polymerase chain reaction; protein structure function; two dimensional gel electrophoresis; uveitis; visual photoreceptor; western blottings

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.

描述（由申请人提供）：葡萄膜炎的失明是由于感光细胞退化的。这种退化归因于巨噬细胞浸润以及随后的各种炎症介质（包括一氧化氮衍生的高反应性氧化剂过氧硝酸盐），已知通过氮残留物的硝酸化引起感光受体损害。然而，我们的初步数据显示了巨噬细胞渗透之前的EAU早期硝化作用。此外，我们发现硝化选择性涉及线粒体蛋白。这些新发现的结果暗示了感光细胞线粒体蛋白硝化作用是在感光体变性的开始中的中央分子事件。这种硝化当然不是巨噬细胞介导的。 我们提出了一个假设：“自身免疫性葡萄膜炎中的感光受体变性始于选择性线粒体蛋白硝化，这是由于感光受体线粒体中反应性一氧化氮物种产生的结果。”我们将在视网膜巨噬细胞浸润之前，在早期EAU的动物中以以下特定目标检验这一假设。 1。评估光感受器线粒体（免疫组织化学，免疫印迹和质谱法）中的一氧化物反应性物种产生。 2。评估光感受器中的线粒体蛋白硝化作用（硝化蛋白的免疫组织化学定位，光感受器蛋白的免疫印迹和毛细血管液相色谱量表量表质谱法）。 3。评估光感受器线粒体的功能（电子传输链复合物的氧和活性以及线粒体跨膜电位）。 为了确保成功完成具体目标，我们组建了一个多学科的专家团队。特里·李（Terry Lee）教授是一位有成就的化学家，具有识别硝化蛋白质的专业知识，及其质​​谱和其他新方法的硝化作用。 Guey-Shuang Wu博士在PCR，Western印迹和生物分子的分离方面具有专业知识。主要研究者在EAU，免疫组织化学，自由基生物学和反应性一氧化氮物种方面经验丰富。了解线粒体在视网膜变性的发病机理中的作用可能会导致特定疗法的发展，以最大程度地减少过氧亚硝酸盐的线粒体产生。