Direct single-molecule observation ofregulated SNARE assembly

调节 SNARE 组装的直接单分子观察

• 批准号: 9256820
• 负责人: ALEKSANDER REBANE
• 金额: $ 2.87万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9256820
• 关键词: Action Potentials; Address; Affect; Ataxia; Binding; Biological Assay; Biological Process; C-terminal; Calcium; Cis Tests; Closure by clamp; Complex; Disease; Epilepsy; Event; Exocytosis; Hormones; Human; Impairment; Intellectual functioning disability; Kinetics; Length; Light; Lipids; Measurement; Measures; Mechanics; Mediating; Membrane; Membrane Fusion; Mental disorders; Methods; Modeling; Molecular; Molecular Conformation; Mutation; N-terminal; Nervous System Physiology; Neurologic; Neurons; Neuropathy; Neurotransmitters; Non-Insulin-Dependent Diabetes Mellitus; Parkinson Disease; Pathway interactions; Positioning Attribute; Process; Proteins; Psyche structure; Regulation; Research; Resolution; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Schizophrenia; Structure; Sum; Synapses; Synaptic Transmission; Synaptic Vesicles; Testing; Transmembrane Domain; Vesicle; base; controlled release; disability; disease-causing mutation; experimental study; falls; insight; insulin secretion; interest; laser tweezer; membrane reconstitution; millisecond; nanodisk; nanometer; nervous system disorder; neurotransmitter release; new therapeutic target; receptor; reconstitution; response; single molecule; synaptotagmin; vesicular release

Project Summary – Aleksander A. Rebane Imbalances in Ca2+-triggered exocytosis of hormones and neurotransmitters cause severe diseases, including type 2 diabetes and various neurological disorders such as Parkinson’s disease, schizophrenia, and epilepsy. Yet it remains poorly understood how Ca2+ triggers exocytosis. Of particular interest is synaptic exocytosis of neurotransmitters, which occurs in response to the local influx of Ca2+, triggered by the arrival of an action potential at the axonal terminal. The core machinery responsible for membrane fusion in regulated exocytosis consists of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), complexin, and synaptotagmin. SNAREs form a broad class of molecular fusion machines that associate by forcefully zippering into a coiled-coil four-helix structure, thus drawing opposing membranes into close proximity for fusion. Additional components are required to turn fusion on and off to regulate synaptic transmission. This task is achieved by complexin and synaptotagmin, which suppress unwanted spontaneous synaptic exocytosis by suspending SNARE zippering halfway as a clamp. Controlled release occurs when calcium removes the clamp to resume SNARE zippering and induce neurotransmitter release. However, the exact constituents of the clamp and the mechanism of clamping and de-clamping are poorly understood. It has been difficult to observe the molecular events along the regulated SNARE assembly pathway using traditional ensemble-based methods. These events are inherently transient and occur solely in the presence of the membrane’s repulsive force. We address these difficulties by using optical tweezers to apply precisely known pulling forces on single SNARE complex molecules to mimic membrane repulsion and to stabilize the partially assembled intermediates, while using molecular extension measurements to determine the structures on millisecond timescale and at nanometer resolution. We analyze these measurements with state-of-the-art methods to derive the conformations, energies, and kinetics of SNARE complex assembly intermediates. In Aim 1, we will analyze the effect of two mutations in the SNARE complex, SNAP-25 I67T and I67N, which cause severe neuropathy, including ataxia and intellectual disability. We will use optical tweezers to measure how these mutations change the energetics, kinetics, and pathways of SNARE zippering. We will then employ reconstituted membrane-fusion assays to compare how the observed changes in SNARE zippering affect SNARE-mediated membrane fusion. In Aim 2, we will directly observe the effect of complexin, synaptotagmin, and calcium on SNARE assembly. We will investigate by what mechanism SNARE assembly is clamped by complexin, and how synaptotagmin and Ca2+ release this clamp. Our research will reveal how SNARE mutations may cause neuropathy and how complexin and synaptotagmin regulate Ca2+-dependent release. Our research will provide concrete molecular mechanisms to act as new drug targets for neurological disease.

项目摘要 – Aleksander A. Rebane Ca2+ 触发的激素和神经递质胞吐作用失衡会导致严重疾病，包括 2 型糖尿病和各种神经系统疾病，如帕金森病、精神分裂症和癫痫症。 然而，人们对 Ca2+ 如何触发胞吐作用仍知之甚少。特别令人感兴趣的是突触胞吐作用 神经递质，响应局部 Ca2+ 流入而发生，由动作到来触发 轴突末端的电位。负责调节胞吐作用中膜融合的核心机制 由可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE)、复合蛋白和 突触结合蛋白。 SNARE 形成了一类广泛的分子融合机器，通过强力连接 拉链成卷曲螺旋四螺旋结构，从而将相对的膜拉得很近 融合。需要额外的组件来打开和关闭融合以调节突触传递。这 任务是通过复合蛋白和突触结合蛋白来实现的，它们抑制不需要的自发突触胞吐作用 通过将 SNARE 拉链悬挂在中间作为夹子。当钙去除 夹子恢复 SNARE 拉链并诱导神经递质释放。然而，其具体成分 人们对夹具以及夹紧和松开的机制知之甚少。已经很难 使用传统的基于集成的方法观察沿着受调控的 SNARE 组装途径的分子事件 方法。这些事件本质上是短暂的，并且仅在存在膜排斥力的情况下发生 力量。我们通过使用光镊在单个光镊上施加精确已知的拉力来解决这些困难 SNARE 复杂分子模拟膜排斥并稳定部分组装 中间体，同时使用分子延伸测量在毫秒内确定结构 时间尺度和纳米分辨率。我们用最先进的方法分析这些测量结果 推导出 SNARE 复杂组装中间体的构象、能量和动力学。在目标 1 中，我们将 分析 SNARE 复合体 SNAP-25 I67T 和 I67N 中两种突变的影响，这两种突变会导致严重的 神经病，包括共济失调和智力障碍。我们将使用光镊来测量这些 突变改变了 SNARE 拉链的能量学、动力学和途径。然后我们将雇用 重建膜融合测定，以比较观察到的 SNARE 拉链变化如何影响 SNARE 介导的膜融合。在目标2中，我们将直接观察复合蛋白、突触结合蛋白、 和 SNARE 组件上的钙。我们将研究 SNARE 组件是通过什么机制夹紧的 复合蛋白，以及突触结合蛋白和 Ca2+ 如何释放这个钳位。我们的研究将揭示 SNARE 如何 突变可能导致神经病变以及复合蛋白和突触结合蛋白如何调节 Ca2+ 依赖性释放。 我们的研究将提供具体的分子机制作为神经系统疾病的新药物靶点。