Membrane binding and aggregation of alpha-synuclein

α-突触核蛋白的膜结合和聚集

• 批准号: 7576792
• 负责人: JEAN-CHRISTOPHE ROCHET
• 金额: $ 22.16万
• 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 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7576792
• 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; 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

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 associatedwith 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 doparnine 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 doparnine 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.

α-突触核蛋白是在整个脑中的突触前神经末梢中富集的小蛋白质。虽然 主要是胞质的，该蛋白对磷脂膜也具有高亲和力。这个膜- 结合能力很可能是蛋白质正常功能所必需的，包括调节蛋白质的结合能力。 突触囊泡的释放。神经病理学和遗传学数据表明，聚集（寡聚） α-突触核蛋白的种类与帕金森病（PD）的神经变性有关。长期 所提议的研究的目的是鉴定作为有效药物的a-突触核蛋白的聚集形式。 PD中的靶点，并表征导致这些聚集体形成的分子相互作用， 患病的神经元在本申请中描述的工作集中在磷脂是否与磷脂结合的问题上。 膜在神经毒性α-突触核蛋白聚集体的形成中起作用。它是假设 细胞膜充当触发有害α-突触核蛋白寡聚体形成的“平台”。该项目将 解决这一假设与以下具体目标：（1）以确定是否膜结合 促进试管模型中β-片层富集的α-突触核蛋白聚集体的形成;（2）确定是否 α-突触核蛋白在真核细胞中形成膜结合的、潜在毒性的聚集体;（3）检测α-突触核蛋白在真核细胞中的表达。 α-突触核蛋白氧化对膜结合聚集体形成的影响。的聚集 通过全内反射荧光显微镜监测支持的脂质双层上的蛋白质， 衰减全反射傅里叶变换红外光谱a-突触核蛋白寡聚体的形成 也将在试管模型、酵母或多巴酚丁胺神经元中通过（i）微分 离心结合Western印迹分析;（ii）荧光测量，使用环境- 敏感的荧光团;和（iii）荧光寿命成像显微镜。细胞活力研究将 进行以确定膜结合聚集体的形成是否与诱导 多巴胺神经元的毒性。这些方法也将被用来确定是否氧化的a- 突触核蛋白影响试管模型或真核生物中膜结合的α-突触核蛋白寡聚体的形成 细胞这些研究的结果将提供线索，是否聚集的α-突触核蛋白上， 膜表面与PD中的神经毒性有关。有证据表明膜结合的α-突触核蛋白寡聚体 是有效的药物靶点将促进筛选测定的发展以鉴定新的治疗剂。