FREE RADICALS, PROTEIN AGGREGATES & PARKINSON'S DISEASE

描述(摘自调查人员摘要) 拟议工作的广泛的长期目标是理解 帕金森氏症中的环境毒素。这种疾病会影响1%的 在美国，每年诊断出40岁以上的人和50,000个新病例 各州。接触除草剂百草枯与增加 患帕金森氏症的风险。路易小体--黄斑狼疮的病理标记物 疾病，是在实质细胞中发现的异常蛋白质聚集体 黑。主要的假设是百草枯诱导的活性氧 导致路易体的形成。拟议的体外研究重点放在 在路易中发现的活性氧物种、过氧化氢和两种蛋白质 小体、细胞色素C和α-突触核蛋白。具体的假设是 测试表明，有四个步骤将过氧化氢的形成与 路易小体的形成：1)过氧化氢诱导酪氨酸自由基 关于细胞色素C；2)细胞色素C自由基转移到酪氨酸 α-突触核蛋白上的残基；3)这些自由基反应形成共价 以及，4)共价交联链加速α-突触核蛋白 聚合。每一步都是一个特定的目标。目标1是量化氢气 过氧化氢诱导的细胞色素C表面自由基。目的2是为了 量化酪氨酸自由基从细胞色素c转移到α- 突触核蛋白。目的3是证明细胞色素C和α-突触核蛋白 自由基反应形成共价交联键。目标4是要证明 交联会加速α-突触核蛋白的聚集。 自旋捕获将被用来稳定自由基。质谱学将 被用来计算自由基位置。电子顺磁共振将是 用于识别部首的类型。这两种蛋白质的位点特异性变体 将用于定位初级结构中的基团。质谱学 和氨基酸分析将被用来证明交联性，和SDS-PAGE 将用于评估聚合。

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.