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.

项目概要- Aleksander A.瑞贝恩 Ca 2+触发的激素和神经递质胞吐的不平衡导致严重的疾病，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。 然而，人们对Ca 2+如何触发胞吐作用仍知之甚少。特别感兴趣的是突触胞吐， 神经递质，其响应于由动作的到来触发的局部Ca 2+内流而发生 轴突末端的电位。调节胞吐中负责膜融合的核心机制 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形成了一大类分子融合机器，它们通过强有力的 拉链成卷曲螺旋四螺旋结构，从而将相对的膜拉得很近， 核聚变需要额外的组件来开启和关闭融合以调节突触传递。这 通过复合蛋白和突触结合蛋白完成任务，它们抑制不需要的自发突触胞吐作用 将SNARE拉链挂在中间作为夹子。当钙去除了 钳夹以恢复SNARE拉链并诱导神经递质释放。然而， 人们对夹持以及夹持和松开的机制知之甚少。一直难以 观察沿着调节SNARE组装途径的分子事件，使用传统的基于集合的 方法.这些事件本质上是短暂的，并且仅在膜的排斥性存在时发生。 力我们解决这些困难，通过使用光镊施加精确已知的拉力在单一的 诱捕复杂的分子，以模拟膜排斥，并稳定部分组装 中间体，同时使用分子延伸测量来确定毫秒上的结构 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复合物组装中间体的构象、能量和动力学。在目标1中，我们 分析SNARE复合物中的两个突变SNAP-25 I67 T和I67 N的影响，这两个突变导致严重的 神经病，包括共济失调和智力残疾。我们将使用光镊来测量这些 突变改变SNARE拉链的能量学、动力学和途径。然后我们将雇用 重组膜融合试验，以比较观察到的SNARE拉链的变化如何影响 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， 和钙对SNARE组装的影响。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Ca 2+如何释放这种钳。我们的研究将揭示 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Ca 2+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。