Molecular basis for the regulation of SNARE assembly in neuronal exocytosis

神经元胞吐作用中 SNARE 组装调节的分子基础

• 批准号: 10202630
• 负责人: DAVID S CAFISO
• 金额: $ 37.26万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202630
• 关键词: Automobile Driving; Binding; Biochemical; Biochemistry; Biophysics; C2 Domain; Calcium; Cell membrane; Collaborations; Complex; Data; Docking; Electron Spin Resonance Spectroscopy; Electrons; Electrostatics; Environment; Event; Exocytosis; Goals; Heterogeneity; Length; Lipids; Magnetic Resonance; Measures; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microscopy; Modeling; Molecular; Molecular Conformation; Molecular Structure; NMR Spectroscopy; Nerve; Neuraxis; Neurobiology; Neurons; Physiologic pulse; Physiological; Positioning Attribute; Procedures; Process; Proteins; Regulation; Resolution; Role; SNAP receptor; Site; Structure; Synaptic Vesicles; System; Testing; Time; Total Internal Reflection Fluorescent; Vesicle; base; controlled release; design; genetic regulatory protein; imaging modality; innovation; millisecond; nervous system disorder; neurotransmitter release; phosphatidylinositol phosphate, PtdIns(4,5)P2; presynaptic; presynaptic neurons; programs; protein complex; reconstitution; restraint; sensor; spectroscopic imaging; stoichiometry; structural biology; synaptotagmin; synaptotagmin I; syntaxin; vesicle-associated membrane protein

PROJECT SUMMARY The overall goal of this program project is to elucidate the precise molecular mechanism and regulation of the fusion machine that drives exocytosis for the controlled release of neurotransmitter at nerve terminals. The assembly of SNARE molecules residing in the synaptic vesicle and presynaptic plasma membrane takes center stage and provides the driving energy for this process. Even though we know the structure of the fully assembled cis-SNARE complex after fusion in atomic detail and have detailed conformational models for several of the SNAREs before fusion, we do not precisely know how (i) they are conditioned with regulatory proteins such as Munc18 and Munc13 to form an active acceptor complex on the plasma membrane, (ii) how this acceptor SNARE complex engages with the synaptic vesicle SNARE upon encounter, and (iii) how this high-energy trans-SNARE complex is ultimately triggered by the synaptic vesicle protein synaptotagmin and calcium to proceed to full assembly and fusion. Three projects led by three expert leaders in the biochemistry, structural biology, and biophysics of neuronal exocytotic membrane fusion are designed to jointly unravel the precise molecular interactions that drive the neuronal fusion machine through the vesicle docking, priming, and fusion steps with the highest possible structural and time resolution. The team will seek to define the structures and configurations of the active presynaptic acceptor SNARE complex and the fusion-restricted trans-SNARE complex between two membranes, and the team will strive to uncover the molecular mechanism, by which calcium-synaptotagmin engages with the membranes and/or complex to release their fusion-restriction. To achieve this goal the team will use a unique combination of approaches ranging from highly innovative biochemical procedures to reconstitute the relevant proteins, EPR, DEER, and NMR spectroscopy to characterize the pertinent structures in membrane environments, and FLIC and single vesicle TIRF microscopy to measure membrane topology and read out fusion on the millisecond timescale.

项目摘要 该项目的总体目标是阐明细胞凋亡的精确分子机制和调控。 在神经末梢驱动胞吐作用以控制神经递质释放的融合机器。的 位于突触囊泡和突触前质膜中的SNARE分子的组装 并为这一过程提供驱动能量。即使我们完全知道 在原子细节上融合后组装的顺式SNARE复合物，并具有详细的构象模型， 在融合之前的几个陷阱，我们不知道（i）他们是如何与调节条件 蛋白质如Munc 18和Munc 13在质膜上形成活性受体复合物，（ii）如何 这种受体陷阱复合物与突触囊泡陷阱相遇时接合，以及（iii）这种受体陷阱复合物如何与突触囊泡陷阱相遇。 高能反式SNARE复合物最终由突触囊泡蛋白synaptotagmin触发， 钙进行完全组装和融合。 三个项目由三位神经元生物化学、结构生物学和生物物理学方面的专家领导 胞吐膜融合的目的是共同解开精确的分子相互作用，驱动 神经元融合机通过囊泡对接、预充和融合步骤，以尽可能高的速率进行 结构和时间分辨率。该小组将寻求确定的结构和配置的积极 突触前受体SNARE复合物和两个之间的融合限制性反式SNARE复合物 细胞膜，研究小组将努力揭示钙突触结合蛋白的分子机制， 与膜和/或复合物接合以释放它们的融合限制。 为了实现这一目标，该团队将使用一种独特的方法组合，从高度创新的 生化程序，以重建相关的蛋白质，EPR，DEER，和NMR光谱， 表征膜环境中的相关结构，以及FLIC和单囊泡TIRF显微镜 来测量细胞膜的拓扑结构，并在毫秒级的时间尺度上读出融合。