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-突触核蛋白是一种小蛋白质，富含于整个大脑的突触前神经末梢。虽然主要存在于细胞质中，但该蛋白质对磷脂膜也具有高亲和力。这种膜结合能力很可能对于蛋白质的正常功能至关重要，其中包括调节突触小泡的释放。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) 的神经变性有关。拟议研究的长期目标是确定作为帕金森病有效药物靶点的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重点关注磷脂膜是否在神经毒性α-突触核蛋白聚集体的形成中发挥作用的问题。据推测，膜充当“平台”来触发有害的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3片的α-突触核蛋白聚集体的形成；（2）确定真核细胞中α-突触核蛋白是否形成膜结合的、潜在有毒的聚集体；（3）检查α-突触核蛋白氧化对膜结合形成的影响。 聚合体。将通过全内反射荧光显微镜和衰减全反射傅立叶变换红外光谱监测蛋白质在支持的脂质双层上的聚集。膜上α-突触核蛋白寡聚体的形成也将在试管模型、酵母或多巴胺神经元中通过（i）差速离心结合蛋白质印迹分析来监测； (ii) 使用环境敏感荧光团进行荧光测量； (iii) 荧光寿命成像显微镜。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毒性的诱导相关。这些方法还将用于确定α-突触核蛋白的氧化是否影响膜结合的形成 试管模型或真核细胞中的α-突触核蛋白寡聚物。这些研究的结果将提供关于α-突触核蛋白在膜表面的聚集是否与帕金森病的神经毒性有关的线索。膜结合α-突触核蛋白寡聚体是有效药物靶标的证据将有助于开发筛选测定法以鉴定新的治疗方法。