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: 6806772
• 负责人: NARSING A RAO
• 金额: $ 37.87万
• 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/6806772
• 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(申请人提供)：葡萄膜炎的失明是由于感光细胞退化所致。这种退化归因于巨噬细胞的渗透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，众所周知，过氧亚硝酸盐通过硝化酪氨酸残基导致光感受器损伤。然而，我们的初步数据显示，硝化作用开始于EAU的早期阶段，在巨噬细胞渗透之前。此外，我们还发现硝化作用选择性地涉及线粒体蛋白。这些新的发现暗示光感受器细胞线粒体蛋白硝化是光感受器退化的中心分子事件。这种硝化作用当然不是由巨噬细胞介导的。 我们提出了一个假说：“自身免疫性葡萄膜炎的光感受器退化始于选择性的线粒体蛋白硝化，这是光感受器线粒体产生反应性一氧化氮物种的结果。”我们将在早期EAU的动物身上，在巨噬细胞渗透到视网膜之前，用以下特定的目标来检验这一假说。 1.评价光感受器线粒体一氧化氮活性物质的生成(免疫组织化学、免疫印迹和质谱仪)。 2.评价线粒体蛋白在光感受器中的硝化作用(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管液相色谱-串联质谱仪)。 3.评价光感受器线粒体的功能(耗氧量和电子传输链复合体的活性，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·李教授是一位有成就的化学家，擅长用质谱学和其他新方法鉴定硝化蛋白质及其硝化部位。吴桂爽博士在聚合酶链式反应、蛋白质印迹和生物分子分离方面拥有专业知识。主要研究人员在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退行性变发病机制中的作用可能会导致开发专门的治疗方法，以最大限度地减少线粒体产生的过氧亚硝酸盐。