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: 7283002
• 负责人: NARSING A RAO
• 金额: $ 35万
• 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/7283002
• 关键词: Animal Model; Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens; Apoptosis; Apoptotic; Autoimmune Diseases; Autoimmune Process; Biological; Biology; Blindness; Blood capillaries; Cells; Complex; Data; Development; Diffuse; Electron Transport; Event; Free Radicals; Generations; Immunoblotting; Immunohistochemistry; Infiltration; Inflammatory; Lead; Liquid Chromatography; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane Potentials; Methods; Microglia; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Organ; Oxidants; Oxygen Consumption; Pathogenesis; Peroxonitrite; Phagocytosis; Phase; Photoreceptors; Polymerase Chain Reaction; Principal Investigator; Process; Proteins; Recruitment Activity; Retina; Retinal; Retinal Degeneration; Role; Site; T-Lymphocyte; Testing; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Tyrosine; Uveitis; Western Blotting; autoimmune uveitis; autoreactive T cell; capillary; cytochrome c; cytokine; experience; human TNF protein; macrophage; multidisciplinary; nitrate; nitration; novel; photoreceptor degeneration; professor; research study; tandem mass spectrometry; uveoretinitis

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）：葡萄膜炎失明是由感光细胞退化引起的。这种变性归因于巨噬细胞浸润以及随后产生的各种炎症介质，包括一氧化氮衍生的高反应性氧化剂过氧亚硝酸盐，已知它会通过酪氨酸残基的硝化导致光感受器损伤。然而，我们的初步数据显示，硝化作用在 EAU 的早期阶段开始，在巨噬细胞渗透之前。此外，我们发现硝化选择性地涉及线粒体蛋白。这些新发现表明光感受器细胞线粒体蛋白硝化是光感受器变性启动的中心分子事件。这种硝化当然不是由巨噬细胞介导的。 我们提出一个假设：“自身免疫性葡萄膜炎中的光感受器变性始于选择性线粒体蛋白硝化，这是光感受器线粒体中反应性一氧化氮物质生成的结果。”我们将在巨噬细胞浸润视网膜之前患有早期 EAU 的动物中通过以下具体目标来检验这一假设。 1. 评估光感受器线粒体中一氧化氮反应物种的产生（免疫组织化学、免疫印迹和质谱）。 2. 评估光感受器中线粒体蛋白的硝化（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-串联质谱法）。 3. 评估光感受器线粒体的功能（耗氧量和电子传递链复合物的活性，以及​​线粒体跨膜电位）。 为了确保成功完成具体目标，我们组建了一支多学科专家团队。 Terry Lee 教授是一位卓有成就的化学家，擅长通过质谱和其他新方法识别硝化蛋白质及其硝化位点。吴桂双博士拥有 PCR、蛋白质印迹和生物分子分离方面的专业知识。主要研究者在 EAU、免疫组织化学、自由基生物学和活性一氧化氮领域具有丰富的经验。了解线粒体在视网膜变性发病机制中的作用可能有助于开发特异性疗法，以最大限度地减少线粒体产生的过氧亚硝酸盐。