Protein Radical, Protein Nitration, Mitochondria, and AD

蛋白质自由基、蛋白质硝化、线粒体和 AD

• 批准号: 6779207
• 负责人: YEONG-RENN CHEN
• 金额: $ 10.8万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-13 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779207
• 关键词: Alzheimer' s disease; SDS polyacrylamide gel electrophoresis; cytochrome oxidase; electron spin resonance spectroscopy; enzyme activity; hydrogen peroxide; immunochemistry; lipid peroxides; mass spectrometry; mitochondria; mitochondrial disease /disorder; neural degeneration; nitric oxide; oxidative stress; peroxynitrites; proteins; tyrosine; western blottings

DESCRIPTION (Taken from the Investigator's Abstract) The causative factors of Alzheimer's disease (AD) involve aging, genetics, and environmental exposures. At the neuronal level, a defect of energy metabolism associated with mitochondrial cytochrome C oxidase (CcO) deficiency was marked in AD brain. The mitochondrial abnormality caused by oxidative damage of CcO has been suggested as a part of causative events in AD. The long-term objective of this proposed research is to identify the molecular mechanism related to oxidative damage of AD-associated mitochondrial CcO. The proteincentered radical(s) in CcO, as detected with ESR spin-trapping technique, is formed by reacting with peroxides, such as hydrogen peroxide, organic peroxides, and lipid peroxides. Available evidence indicates that a specific tyrosine in mitochondrial cytochrome C may be nitrated to form 3- nitrotyrosine. The candidate hypothesizes that a specific amino acid residue(s) in CcO is involved in the radical formation and that 3- nitrotyrosine can be detected when the purified CcO is exposed to a reactive nitrogen species (RNS) such as nitric oxide (NO) or peroxynitrite. The radioisotope-labeled spin trap, [14C]-labeled 2-methyl-2-nitrosopropane (MNP), will be synthesized and used to trap the protein-centered radical(s) in CcO. The subunit (location) of CcO associated with the spin trap will be visualized by SDS-PAGE and radioisotope distribution. NO and peroxynitrite will be used to test the nitration of tyrosine in purified CcO. The nitration of CcO will be characterized by immunoblotting using the antibody against 3-nitrotyrosine. Peptide chemistry/mapping and mass spectrometry will be used to identify specific amino residue(s) associated with the above events of oxidative damage. The functional domains involved in the above radical formation and nitration will be identified. The essential role of the identified domains will be studied by immunochemistry. The results will provide fundamental information concerning the pathological relevance of neurodegenerative disease, such as AD, associated with mitochondrial dysfunction caused by oxidative damage.

描述（摘自研究者摘要） 阿尔茨海默氏病（AD）的致病因素涉及衰老、遗传和 环境暴露。在神经元水平上，能量代谢的缺陷 与线粒体细胞色素C氧化酶（CcO）缺乏有关 在AD脑中。CcO氧化损伤引起的线粒体异常 已被认为是AD的病因事件的一部分。长期 这项研究的目的是确定分子机制， 与AD相关的线粒体CcO的氧化损伤有关。蛋白质中心 用ESR自旋捕获技术检测到的CcO中的自由基， 通过与过氧化物，如过氧化氢，有机 过氧化物和脂质过氧化物。现有证据表明， 线粒体细胞色素C中的酪氨酸可被硝化形成3- 硝基酪氨酸候选人假设一种特定的氨基酸 CcO中的残基参与自由基的形成， 当纯化的CcO暴露于反应性物质时，可以检测到硝基酪氨酸 氮物质（RNS），如一氧化氮（NO）或过氧亚硝酸盐。的 放射性同位素标记的自旋阱，[14 C]标记的2-甲基-2-亚硝基丙烷（MNP）， 将被合成并用于捕获CcO中以蛋白质为中心的自由基。 与自旋陷阱相关的CcO亚基（位置）将被可视化 通过SDS-PAGE和放射性同位素分布。将使用NO和过氧亚硝酸盐 以测试纯化的CcO中酪氨酸的硝化。CcO的硝化将 通过使用抗3-硝基酪氨酸的抗体的免疫印迹来表征。 肽化学/图谱和质谱法将用于鉴定 与上述氧化反应事件相关的特定氨基残基 损害参与上述自由基形成的功能结构域和 将识别硝化。已确定领域的重要作用 将通过免疫化学进行研究。结果将提供基本的 关于神经退行性疾病的病理学相关性的信息 与线粒体功能障碍相关的疾病，如AD， 氧化损伤