Modeling SNARE-Mediated Membrane Fusion

SNARE 介导的膜融合建模

• 批准号: 10445738
• 负责人: Ben O'Shaughnessy
• 金额: $ 33.89万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445738
• 关键词: Action Potentials; Beta Cell; Biochemical; Catalogs; Cell fusion; Cell membrane; Cells; Chromaffin Cells; Closure by clamp; Communities; Complex; Computer Models; Core Protein; Coupled; Data; Dense Core Vesicle; Docking; Dynamin; Entropy; Event; Evoked Potentials; Evolution; Exocytosis; Family; Functional disorder; Funding; Goals; Grain; Growth; Hormones; Hot Spot; Impairment; Informal Social Control; Kinetics; Laboratories; Machine Learning; Measurement; Mechanics; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Modeling; Molecular; Mutate; Mutation; Neuraxis; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuroendocrine Cell; Neurons; Neurotransmitters; Pathway interactions; Physiological; Probability; Process; Property; Proteins; Regulation of Exocytosis; Reproducibility; Research; Respiratory Diaphragm; Rest; Running; S-nitro-N-acetylpenicillamine; SNAP receptor; Scheme; Secretory Vesicles; Shapes; Signal Transduction; Site; Stimulus; Structure; Study models; Synapses; Synaptic Cleft; Synaptic Vesicles; Testing; Thinness; Type 2 diabetic; Vesicle; experimental study; foot; hydrophilicity; in silico; insulin secretion; mathematical model; membrane model; millisecond; molecular dynamics; multi-scale modeling; nanodisk; neurotransmission; neurotransmitter release; novel; predictive modeling; reconstitution; response; sensor; simulation; synaptotagmin; synaptotagmin I; synaptotagmin II; syntaxin; target SNARE proteins; vesicular SNARE proteins; vesicular release

PROJECT SUMMARY Many basic processes rely on secretion of bioactive molecules by exocytosis, when membrane-enclosed vesicles containing neurotransmitters (NTs), hormones or other molecules fuse with the plasma membrane (PM) and release their contents through fusion pores. Neurotransmission relies on NT release at neuronal synapses, when a multicomponent machinery senses Ca!"influx triggered by an action potential and fuses small synaptic vesicles with the plasma membrane on sub-millisecond timescales, releasing NTs into the synaptic cleft to elicit a post-synaptic response. Other regulated exocytosis is slower, such as hormone release from neuroendocrine cells when a stimulus provokes large dense core vesicles to release contents after seconds or longer. In all cases, the membrane fusion step is accomplished by the SNARE proteins, when vesicle-associated VAMP (the v-SNARE) and two PM-associated t-SNAREs syntaxin and SNAP 25 zipper into a ternary complex, pulling the membranes together and fusing them. However, the mechanism of membrane fusion remains unclear. Other components in the machinery block (“clamp”) SNARE-mediated fusion, until the Ca!"signal releases the clamp. Synaptotagmin (Syt) is the Ca!"sensor for synchronous release, but the molecular identity of the clamp and the Ca!"-triggered unclamping mechanism are not established. Mutations in SNARE proteins and other NT release machinery components are associated with neurodevelopmental and neurodegenerative disorders, and impaired fusion pore dilation is associated with reduced insulin secretion by β-cells of type-2 diabetics. The proposed research aims to use mathematical modeling to establish the mechanisms of regulated membrane fusion and the mechanisms that regulate the vesicle and its pore for controlled contents release following fusion. From the previous funding period, we have working molecular dynamics (MD) simulations of SNARE-mediated membrane fusion and of the NT release machinery incorporating the core SNARE and Syt components. The simulations used sufficiently coarse-grained (CG) representations to achieve the computationally demanding millisecond physiological timescales of fusion and release. Aim 1 is to advance the SNARE-mediated fusion simulations with more realistic SNAREs, and to use machine learning to catalogue the pathways to membrane fusion as a function of the size of the fusing vesicles and other key variables. Mutated SNAREs will be simulated and compared to experiments by collaborators. Aim 2 is to use continuum mathematical modeling to establish the structure, energetics and evolution of the fused vesicle-PM complex and its fusion pore, and the mechanisms of SNARE- and Syt-mediated pore dilation. Aim 3 is to advance the MD simulations of the NT release machinery by introducing molecularly explicit representation of the membranes, and by incorporating additional components as their interactions become experimentally characterized, toward a long-term goal of “reconstituting” the machinery in silico. Simulations will test hypothesized Ca!"-triggered unclamping schemes, and will be run with mutations in the SNARE and Syt components that will be implemented experimentally by our collaborators.

项目摘要 许多基本过程依赖于当膜封闭时通过胞吐分泌生物活性分子。 含有神经递质（NT）、激素或其他分子的囊泡与质膜（PM）融合 并通过融合孔释放它们的内容物。神经传递依赖于神经元突触处的NT释放， 当一个多组件机器感知到Ca！“由动作电位触发的电流流入， 囊泡与质膜在亚毫秒的时间尺度上，释放NT到突触间隙中， 突触后反应其他受调节的胞吐作用较慢，如神经内分泌的激素释放， 当刺激物刺激大而致密的核心囊泡在数秒或更长时间后释放内容物时，细胞会发生反应。 在所有情况下，当囊泡相关的VAMP (the v-SNARE）和两个PM相关的t-SNARE突触融合蛋白和SNAP 25拉链成三元复合物， 将细胞膜融合在一起然而，膜融合的机制仍不清楚。其他 在机械块（“钳”）SNARE介导的融合，直到Ca！“信号释放夹钳。 Synaptotagmin（Syt）是Ca！“传感器同步释放，但分子身份的钳和 卡！“-触发松开机构未建立。SNARE蛋白突变和其他NT释放 机械组件与神经发育和神经退行性疾病有关， 受损的融合孔扩张与2型糖尿病患者的β细胞的胰岛素分泌减少有关。 这项研究的目的是利用数学模型来建立调节膜的机制 融合和调节囊泡及其孔以控制融合后内容物释放的机制。 从上一个资助期，我们有工作的分子动力学（MD）模拟SNARE介导的 膜融合和NT释放机制，包括核心SNARE和Syt组件。的 模拟使用足够粗粒度（CG）表示，以实现计算要求高的 毫秒级的融合和释放生理时间尺度。目的1是研究SNARE介导的融合 用更真实的SNARE模拟，并使用机器学习来编目膜的途径， 融合作为融合囊泡的大小和其他关键变量的函数。将模拟突变的SNARE 并与合作者的实验进行比较。目的二是利用连续介质数学模型建立 融合囊泡-PM复合物及其融合孔的结构、能量学、演化及其机理 SNARE和Syt介导的毛孔扩张。目的3是推进NT释放机制的MD模拟 通过引入膜的分子显式表示，并通过引入额外的组分， 随着它们的相互作用变得实验特征化，朝着“重建” 机器在silico。模拟将测试假设的Ca！“-触发的松开方案，并将使用 SNARE和Syt组件中的突变，将由我们的合作者进行实验。