Protein Radical, Protein Nitration, Mitochondria, and AD

描述(摘自调查人员摘要) 阿尔茨海默病(AD)的致病因素包括年龄、遗传和 暴露在环境中。在神经元层面上，能量代谢的缺陷 与线粒体细胞色素C氧化酶(CcO)缺乏症显著相关 在AD大脑中。一氧化碳氧化损伤引起的线粒体异常 已被认为是公元后因果事件的一部分。长期的 这项拟议研究的目的是确定分子机制。 与AD相关线粒体CcO的氧化损伤有关。抗议者加入了进来 用ESR自旋捕捉技术检测到的CCO中的自由基(S)是 通过与过氧化氢等过氧化氢反应而形成的有机 过氧化物质和过氧化脂质。现有证据表明，一种特定的 线粒体细胞色素C中的酪氨酸可能被硝化形成3- 硝基酪氨酸。候选人假设一种特定的氨基酸 CCOO中的残基(S)参与了自由基的形成，3- 当纯化的CcO暴露于活性物质中时，可以检测到硝基酪氨酸 氮物种(RN)，如一氧化氮(NO)或过氧亚硝酸盐。这个 放射性同位素标记的自旋陷阱，[14C]标记的2-甲基-2-亚硝基丙烷(MNP)， 将被合成并用于捕获一氧化碳中以蛋白质为中心的自由基(S)。 与自旋陷阱相关的CcO亚单位(位置)将可视化 通过十二烷基硫酸钠-PAGE和放射性同位素分布。将使用NO和过氧亚硝酸盐 目的：检测精制一氧化碳中酪氨酸的硝化反应。一氧化碳的硝化将 用3-硝基酪氨酸抗体进行免疫印迹鉴定。 将使用多肽化学/图谱和质谱学来鉴定 与上述氧化事件相关的特定氨基残基(S) 损坏。上述自由基形成所涉及的功能结构域和 硝化作用将被确认。已确定领域的基本作用 将通过免疫化学进行研究。研究结果将为我们提供基本的 关于神经退行性变的病理相关性的信息 与线粒体功能障碍有关的疾病，如阿尔茨海默病 氧化损伤。

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.