FREE RADICALS, PROTEIN AGGREGATES & PARKINSON'S DISEASE

自由基、蛋白质聚集体

• 批准号: 6214750
• 负责人: GARY JOSEPH PIELAK
• 金额: $ 13.6万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6214750
• 关键词: Lewy body; Parkinson's disease; SDS polyacrylamide gel electrophoresis; alpha synuclein; covalent bond; crosslink; cytochrome c; electron spin resonance spectroscopy; environmental toxicology; free radical oxygen; free radicals; hydrogen peroxide; mass spectrometry; paraquat; substantia nigra; tyrosine

DESCRIPTION (Taken from the Investigator's Abstract) The broad long-term objective of the proposed work is to understand the role of environmental toxins in Parkinson's Disease. This disorder affects 1% of people over age 40 and 50,000 new cases are diagnosed per year in the United States. Exposure to the herbicide paraquat is associated with an increased risk of Parkinson's Disease. Lewy bodies, the pathological marker of the disease, are abnormal protein aggregates found in cells of the substantia nigra. The main hypothesis is that paraquat-induced reactive oxygen species lead to Lewy body formation. The proposed in vitro studies focus on the reactive oxygen species, hydrogen peroxide, and two proteins found in Lewy bodies, cytochrome C and alpha-synuclein. The specific hypothesis to be tested is that four steps link the formation of hydrogen peroxide to the formation of Lewy bodies: 1) hydrogen peroxide induces tyrosine free radicals on cytochrome C; 2) the cytochrome C radicals are transferred to tyrosine residues on alpha-synuclein; 3) these radicals react to form covalent crosslinks; and, 4) the covalent crosslinks accelerate alpha-synuclein aggregation. Each step is a specific aim. Aim 1 is to quantify the hydrogen peroxide-induced radicals on the surface of cytochrome C. Aim 2 is to quantify the tyrosine radicals transferred from cytochrome c to alpha- synuclein. Aim 3 is to show that the cytochrome C and alpha-synuclein radicals react to form covalent crosslinks. Aim 4 is to show that crosslinking accelerates alpha-synuclein aggregation. Spin trapping will be used to stabilize the radicals. Mass spectrometry will be used to count the radical sites. Electron paramagnetic resonance will be used to identify the type of radical. Site-specific variants of both proteins will used to locate the radicals in the primary structure. Mass spectrometry and amino acid analysis will be used to demonstrate crosslinking, and SDS PAGE will be used to assess aggregation.

描述（取自研究者的摘要） 拟议工作的长期长期目标是了解角色 帕金森氏病中的环境毒素。 这种疾病影响了1％ 在联合 国家。 暴露于除草剂paraquat与增加有关 帕金森氏病的风险。 路易尸体，病理标记 疾病，是在田间细胞中发现的异常蛋白质聚集体 尼格拉。 主要假设是帕拉奎特诱导的活性氧 导致Lewy身体形成。 提出的体外研究重点是 活性氧，过氧化氢和两种蛋白质 身体，细胞色素C和α-核蛋白。 特定的假设为 测试的是四个步骤将过氧化氢的形成与 路易体的形成：1）过氧化氢诱导酪氨酸自由基 在细胞色素c上； 2）将细胞色素C自由基转移到酪氨酸 α-核蛋白的残留物； 3）这些自由基反应形成共价 交叉链接； ，4）共价交联加速α-核蛋白 聚合。 每个步骤都是一个特定的目标。 目标1是量化氢 细胞色素表面的过氧化物诱导的自由基的目标2是 量化从细胞色素C到α-转移的酪氨酸自由基 突触核素。 AIM 3是表明细胞色素C和α-核蛋白 自由基反应形成共价交联。 目标4是表明 交联加速α-核蛋白聚集。 自旋陷阱将用于稳定自由基。 质谱将 用于计算激进位点。 电子顺磁共振将是 用于识别激进的类型。 两种蛋白质的位点特异性变体 将用于将自由基定位在主要结构中。 质谱法 和氨基酸分析将用于证明交联，SDS页面 将用于评估聚合。