Membrane binding and aggregation of alpha-synuclein

α-突触核蛋白的膜结合和聚集

• 批准号: 7171572
• 负责人: JEAN-CHRISTOPHE ROCHET
• 金额: $ 22.31万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171572
• 关键词: Address; Affect; Affinity; Attenuated; Behavior; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Brain; Cell Fraction; Cell Survival; Cell model; Cells; Centrifugation; Characteristics; Coupled; Data; Development; Drug Delivery Systems; Environment; Eukaryotic Cell; Fluorescence; Fluorescence Microscopy; Fractionation; Genes; Genetic; Goals; Image; Label; Lead; Link; Lipid Bilayers; Lipids; Measurement; Membrane; Membrane Lipids; Methionine; Methods; Microscopy; Modeling; Molecular Weight; Monitor; Mutation; Nerve; Nerve Degeneration; Neurons; Organelles; Parkinson Disease; Pathogenesis; Patients; Phospholipids; Play; Post-Translational Protein Processing; Proteins; Rate; Research; Role; Screening procedure; Solutions; Spectroscopy, Fourier Transform Infrared; Surface; Synaptic Vesicles; Testing; Toxic effect; Tube; Vesicle; Western Blotting; Work; Yeast Model System; Yeasts; alpha synuclein; dopaminergic neuron; early onset; fluorophore; neurotoxic; neurotoxicity; novel therapeutics; oxidation; presynaptic; protein aggregate; protein aggregation; self assembly; synuclein

DESCRIPTION (provided by applicant): a-Synuclein is a small protein enriched in presynaptic nerve terminals throughout the brain. Though predominantly cytosolic, the protein also has a high affinity for phospholipid membranes. This membrane- binding ability is most likely essential for the protein's normal function, which consists of modulating the release of synaptic vesicles. Neuropathological and genetic data suggest that aggregated (oligomeric) species of a-synuclein are associated with neurodegeneration in Parkinson's disease (PD). The long-term objectives of the proposed research are to identify aggregated forms of a-synuclein that are valid drug targets in PD and to characterize the molecular interactions that lead to the formation of these aggregates in diseased neurons. The work described in this application is focused on the problem of whether phospholipid membranes play a role in the formation of neurotoxic a-synuclein aggregates. It is hypothesized that membranes act as a 'platform' to trigger the formation of harmful a-synuclein oligomers. The project will address this hypothesis with the following specific aims: (1) to determine whether membrane binding promotes the formation of (3-sheet-rich a-synuclein aggregates in test-tube models; (2) to determine whether a-synuclein forms membrane-bound, potentially toxic aggregates in eukaryotic cells; (3) to examine the effects of a-synuclein oxidation on the formation of membrane-bound aggregates. The aggregation of the protein on supported lipid bilayers will be monitored by total internal reflection fluorescence microscopy and attenuated total reflection Fourier transform infrared spectroscopy. The formation of a-synuclein oligomers on membranes will also be monitored in test-tube models, yeast, or dopamine neurons via (i) differential centrifugation combined with Western blot analysis; (ii) fluorescence measurements, using an environment- sensitive fluorophore; and (iii) fluorescence lifetime imaging microscopy. Cell viability studies will be conducted to determine whether the formation of membrane-bound aggregates correlates with the induction of toxicity in dopamine neurons. These methods will also be used to determine whether the oxidation of a- synuclein affects the formation of membrane-bound a-synuclein oligomers in test-tube models or eukaryotic cells. The results of these studies will provide clues as to whether the aggregation of a-synuclein on membrane surfaces is linked to neurotoxicity in PD. Evidence that membrane-bound a-synuclein oligomers are valid drug targets would facilitate the development of screening assays to identify novel therapeutics.

描述（由申请人提供）：α-突触核蛋白是一种在整个大脑的突触前神经末梢中富集的小蛋白。虽然主要是胞质，蛋白质也有一个高亲和力的磷脂膜。这种膜结合能力很可能是蛋白质正常功能所必需的，包括调节突触囊泡的释放。神经病理学和遗传学数据表明α-突触核蛋白的聚集（寡聚）种类与帕金森病（PD）中的神经变性相关。所提出的研究的长期目标是鉴定作为PD中有效药物靶标的α-突触核蛋白的聚集形式，并表征导致患病神经元中形成这些聚集体的分子相互作用。本申请中描述的工作集中于磷脂膜是否在神经毒性α-突触核蛋白聚集体的形成中起作用的问题。假设膜充当“平台”以触发有害的α-突触核蛋白寡聚体的形成。该项目将通过以下具体目标来解决这一假设：（1）确定膜结合是否促进试管模型中富含β-片层的α-突触核蛋白聚集体的形成;（2）确定α-突触核蛋白是否在真核细胞中形成膜结合的潜在毒性聚集体;（3）检查α-突触核蛋白氧化对膜结合聚集体形成的影响。将通过全内反射荧光显微镜和衰减全反射傅里叶变换红外光谱法监测蛋白质在支持的脂质双层上的聚集。还将在试管模型、酵母或多巴胺神经元中通过以下方式监测α-突触核蛋白寡聚体在膜上的形成：（i）差速离心结合蛋白质印迹分析;（ii）使用环境敏感性荧光团的荧光测量;和（iii）荧光寿命成像显微术。将进行细胞活力研究，以确定膜结合聚集体的形成是否与多巴胺神经元中毒性的诱导相关。这些方法也将用于确定α-突触核蛋白的氧化是否影响试管模型或真核细胞中膜结合的α-突触核蛋白寡聚体的形成。这些研究的结果将提供关于α-突触核蛋白在膜表面上的聚集是否与PD中的神经毒性相关的线索。膜结合的α-突触核蛋白寡聚体是有效的药物靶标的证据将促进筛选测定的发展以鉴定新的治疗剂。