Project 1- Reconstitution of SNAR-mediated Fusion with Native and Artificial Mem

项目 1 - 重建 SNAR 介导的与天然和人工 Mem 的融合

• 批准号: 8376187
• 负责人: Reinhard Jahn
• 金额: $ 17.6万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8376187
• 关键词: Achievement; Address; Affect; Alcoholism; Artificial Membranes; Attention Deficit Disorder; Binding; Binding Sites; Biological Assay; C-terminal; Cell membrane; Collaborations; Complex; Consumption; Cryoelectron Microscopy; Distal; Docking; Exocytosis; Fluorescence; Funding; In Vitro; Instruction; Kinetics; Liposomes; Measures; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Mutation; N-terminal; Neurons; Pathway interactions; Peripheral; Protein Binding; Proteins; Reaction; Resolution; S-nitro-N-acetylpenicillamine; SNAP receptor; Schizophrenia; Secretory Vesicles; Shapes; Site; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Transmembrane Domain; Vesicle; Work; artificial vesicle; genetic regulatory protein; in vivo; insight; nervous system disorder; novel therapeutic intervention; presynaptic; programs; protein protein interaction; reaction rate; receptor; reconstitution; stoichiometry; synaptotagmin; synaptotagmin I; syntaxin; vesicle-associated membrane protein

Exocytotic membrane fusion of synaptic vesicles is driven by the SNAREs syntaxin, SNAP-25, and synaptobrevin/VAMP, which assemble in trans between the membranes in a zipper-like fashion, thus pulling the membranes together and overcoming the energy barrier for fusion. In addition, regulatory proteins including synaptotagmin, Munc18, and complexin interact both with the SNAREs and the participating membranes, thus controlling SNARE reactivity and shaping the specific features of cacium-dependent exocytosis of synaptic vesicles. While the zippering model is intuitively satisfying and confirmed by a large body of evidence, the precise sequence of protein-protein interactions and the site of action of the regulatory proteins along the fusion pathway is still only partially understood and controversially discussed. Here we use in-vitro fusion of native secretory vesicles and liposomes reconstituted with purified proteins to isolate partial reactions of the fusion pathway, to understand the structure, dynamics and stoichiometries of the intermediate states of the participating proteins, and to determine the parameters affecting kinetics of each reaction. In the previous funding period, we solved the ci7stal structure of the neuronal SNARE complex with its linkers and transmembrane domains and characterized the interaction of synaptotagmin with SNARE-containing liposomes. Moreover, we developed refined assays for measuring fusion intermediates including fluorescence cross-correlation and cryo electron microscopy, studied the fusion of synaptic vesicles with SNARE-containing liposomes, and interfered with SNARE assembly at the partially or fully zippered state, thus gaining access to intermediate states of the fusion pathway. We now propose to take advantage of these achievements to characterize the intermediate steps in SNARE-mediated fusion and to determine the precise step at which the regulatory proteins synaptotagmin 1, complexin 1, and Munc18-1 operate on membrane fusion catalyzed by neuronal SNAREs. In the first specific aim, we plan to elucidate how SNARE assembly and zippering is connected to membrane docking, hemifusion, and fusion using native and artificial vesicles. In the second specific aim, we will determine the steps in SNARE nucleation and zippering that are acted upon by the regulatory proteins synaptotagmin, MunclS, and complexin. The program will be carried out in close collaboration with Projects 2 and 3 in which complementary approaches are pursued. We expect to obtain essential mechanistic information about the molecular mechanism of neuronal exocytosis that is not obtainable by other approaches..

突触囊泡的胞吐膜融合是由SNARESSTAXIN，SNAP-25和 Synaptobrevin/Vamp，以拉链般的方式组装在膜之间的trans，从而拉动 膜在一起并克服融合的能量障碍。另外，调节蛋白 包括Synaptotagmin，Munc18和Complextin都与贪食和参与 膜，从而控制圈反应性并塑造cacium依赖性的特定特征 突触囊泡的胞吐作用。虽然拉链模型直观地满足并通过大型 证据体，蛋白质蛋白相互作用的精确序列和调节的作用部位 沿融合途径的蛋白质仍然仅部分理解和有争议。 在这里，我们使用与纯化蛋白重构的天然分泌囊泡和脂质体的体外融合 分离融合途径的部分反应，以了解 参与蛋白的中间状态，并确定影响动力学的参数 每个反应。在上一个资金期间，我们解决了神经元的CI7STAL结构 复合物与其接头和跨膜结构域，并表征了Synaptotagmin的相互作用 与含糖的脂质体。此外，我们开发了测量融合的精制测定法 包括荧光互相关和冷冻电子显微镜在内的中间体研究了融合 具有含糖脂质体的突触囊泡，并在部分或 完全拉链状态，从而获得融合途径的中间状态。我们现在建议 利用这些成就来表征网罗介导的融合中的中间步骤 并确定调节蛋白突触1，复合蛋白1和 MUNC18-1在神经元幼犬催化的膜融合上运行。在第一个特定目标中，我们计划 阐明军鼓组装和拉链如何连接到膜对接，半分裂和融合 使用天然和人造囊泡。在第二个特定目标中，我们将确定军鼓的步骤 由调节蛋白突触蛋白，MUNCL和MUNCL和 复合素。该计划将与项目2和3密切合作，其中 采用互补方法。我们希望获得有关 神经元胞吐作用的分子机制，其他方法无法获得。