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-突触核蛋白是一种富含突触前神经末端的小蛋白。尽管主要是胞质的，但该蛋白质对磷脂膜也具有很高的亲和力。这种膜结合能力对于蛋白质的正常功能很可能是必不可少的，该功能包括调节突触囊泡的释放。神经病理学和遗传数据表明，A-突触核蛋白的聚集（寡聚）种与帕金森氏病（PD）的神经变性有关。拟议研究的长期目标是鉴定A-核蛋白的聚集形式，这些核蛋白是PD中有效的药物靶标，并表征导致患病神经元中这些骨料形成的分子相互作用。本应用中描述的工作集中在磷脂膜是否在神经毒性A-核蛋白聚集体的形成中起作用的问题。假设膜起着触发有害A核蛋白低聚物形成的“平台”。该项目将以以下特定目的解决这一假设：（1）确定膜结合是否促进了测试管模型中（3片富含A-Synclein的核糖蛋白聚集体的形成；（2）以确定A-Synuclein形成膜结合的，潜在的，潜在的氧化物氧化物中的氧化物中的氧化物；总体反射荧光显微镜将监测蛋白质的聚集。 （II）使用环境敏感的荧光团；这些方法还将用于确定A-突触核蛋白的氧化是否会影响结合膜结合的A-突触核蛋白在试管模型或真核细胞中的形成。这些研究的结果将提供有关A-突触核蛋白在膜表面的聚集的线索，与PD中的神经毒性有关。膜结合的A-核蛋白低聚物是有效的药物靶标的证据，可以促进筛查测定法以鉴定新的治疗剂。