Membrane binding and aggregation of alpha-synuclein

α-突触核蛋白的膜结合和聚集

• 批准号: 7028831
• 负责人: JEAN-CHRISTOPHE ROCHET
• 金额: $ 23.03万
• 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/7028831
• 关键词: alpha synuclein; binding sites; drug screening /evaluation; fluorescence microscopy; gene complementation; gene dosage; gene duplication; gene environment interaction; infrared spectrometry; laboratory rat; membrane activity; molecular dynamics; neurons; oxidation; phospholipids; vesicle /vacuole; yeasts

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）：a- synuclein是一种小蛋白，富集于整个大脑的突触前神经末梢。虽然主要是细胞质，蛋白质也对磷脂膜有很高的亲和力。这种膜结合能力很可能是蛋白质正常功能所必需的，它包括调节突触囊泡的释放。神经病理学和遗传学数据表明，a-突触核蛋白的聚集（寡聚）物种与帕金森病（PD）的神经退行性变有关。拟议研究的长期目标是确定PD中有效药物靶点的a-突触核蛋白的聚集形式，并表征导致病变神经元中这些聚集物形成的分子相互作用。在本应用中描述的工作集中在磷脂膜是否在神经毒性a-突触核蛋白聚集体的形成中起作用的问题上。据推测，膜作为一个“平台”来触发有害的a-突触核蛋白低聚物的形成。该项目将通过以下具体目标来解决这一假设：(1)在试管模型中确定膜结合是否促进3-sheet-rich a-synuclein聚集体的形成；(2)确定a-synuclein是否在真核细胞中形成膜结合的潜在毒性聚集体；(3)研究a-synuclein氧化对膜结合聚集体形成的影响。利用全内反射荧光显微镜和衰减全反射傅立叶变换红外光谱技术监测蛋白质在支持的脂质双层上的聚集。在试管模型、酵母或多巴胺神经元中，也将通过(1)差速离心结合Western blot分析来监测膜上a-synuclein低聚物的形成；（ii）使用对环境敏感的荧光团进行荧光测量；（三）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a-synuclein的氧化是否会影响试管模型或真核细胞中膜结合的a-synuclein寡聚物的形成。这些研究结果将为a-突触核蛋白在膜表面的聚集是否与PD的神经毒性有关提供线索。膜结合的a-突触核蛋白寡聚物是有效的药物靶标的证据将促进筛选分析的发展，以确定新的治疗方法。