Common role of Sec1p/Munc18 proteins in SNARE assembly and membrane fusion

Sec1p/Munc18 蛋白在 SNARE 组装和膜融合中的常见作用

• 批准号: 9311137
• 负责人: Yongli Zhang
• 金额: $ 35.76万
• 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: 2017-08-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9311137
• 关键词: Binding; Binding Proteins; Biological Assay; C-terminal; Cancerous; Cells; Collaborations; Complex; Coupled; Crystallization; Cultured Cells; Cytotoxin; Detection; Diabetes Mellitus; Disease; Docking; Epilepsy; Exocytosis; Family; Fluorescence; Fluorescence Microscopy; Image; Immune System Diseases; Immune response; Immunologic Deficiency Syndromes; Impairment; Individual; Insulin; Intellectual functioning disability; Intracellular Membranes; Kinetics; Length; Lipids; Liposomes; Lymphocyte; Measures; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Mutation; N-ethylmaleimide-sensitive protein; N-terminal; Pathology; Pathway interactions; Play; Process; Protein Family; Research; Resolution; Role; SNAP receptor; Silicon Dioxide; Specificity; Structure; Synapses; Temperature; Testing; Vacuole; base; complex R; disease-causing mutation; experimental study; fungus; glucose uptake; human disease; insight; killings; laser tweezer; nanodisk; neurotransmitter release; receptor; reconstitution; single molecule; syntaxin binding protein 1; tool

Our objective is to uncover the common mechanism by which Sec1p/Munc18-family (SM) proteins enhance SNARE assembly and membrane fusion. SM and SNARE proteins constitute the evolutionarily and structurally conserved core machinery for membrane fusion. Numerous mutations in SM and SNARE proteins have been identified that cause intellectual disability, epilepsy, immune disorders, and other disorders. In SNARE assembly, three or four SNAREs located on two membranes join into a parallel four- helix bundle like a zipper, forcing the membranes to fuse. However, SNAREs alone assemble slowly with limited specificity and accuracy. Despite extensive research in the past two decades, it remains unclear what mechanistic roles that SM proteins play in SNARE assembly, why they are essential for membrane fusion, and how they go wrong in diseases. Based on latest progresses, we hypothesize that SM proteins promote the rate and specificity of SNARE assembly, thereby enhancing membrane fusion. To test this hypothesis, we will examine several models. In particular, SM proteins bind SNARE motifs in register and act as a template to nucleate SNARE assembly. To test this and other models, we will measure the intermediates, energetics and kinetics of the coupled SNARE assembly and membrane fusion in the absence and presence of SM proteins. We will further elucidate the effects of disease-causing mutations in the process. Our primary tool is high-resolution optical tweezers combined with single-molecule fluorescence detection, which has successfully been applied to characterize assembly of cytoplasmic SNARE domains. Specifically, we will (1) measure the intermediates, energy, and kinetics of coupled trans-SNARE assembly and membrane fusion; (2) elucidate effects of SM proteins on trans-SNARE assembly and membrane fusion; (3) understand the molecular mechanisms of diseases or malfunctions caused by SNARE and SM mutations. Our research will reveal a common mechanism of SM-SNARE proteins and their pathologies.

我们的目标是揭示Sec 1 p/Munc 18家族（SM）蛋白在细胞内表达的共同机制。 增强SNARE组装和膜融合。SM和SNARE蛋白在进化上构成了 和结构上保守的膜融合核心机制。SM和SNARE中的许多突变 已经鉴定出导致智力残疾、癫痫、免疫紊乱和其他疾病的蛋白质。 紊乱在SNARE组件中，位于两个膜上的三个或四个SNARE连接成平行的四个- 螺旋束像拉链一样，迫使膜融合。然而，SNARE单独组装缓慢， 有限的特异性和准确性。尽管在过去的二十年里进行了广泛的研究，但仍不清楚 SM蛋白在SNARE组装中发挥的机械作用，为什么它们对膜融合至关重要， 以及它们如何在疾病中出错。根据最新进展，我们假设SM蛋白促进 SNARE组装的速率和特异性，从而增强膜融合。为了验证这个假设， 我们将研究几种模型。特别是，SM蛋白结合SNARE基序，并作为一种免疫调节剂。 模板成核SNARE组装。为了测试这个模型和其他模型，我们将测量中间体， 在不存在和存在下耦合SNARE组装和膜融合的能量学和动力学 SM蛋白质我们将进一步阐明致病突变在这一过程中的作用。我们 主要工具是结合单分子荧光检测的高分辨率光镊， 其已成功地应用于表征细胞质SNARE结构域的组装。 具体来说，我们将（1）测量耦合反式SNARE组装的中间体，能量和动力学 （2）阐明SM蛋白对反式SNARE组装和膜融合的影响 （3）了解SNARE和SM引起疾病或功能障碍的分子机制 突变。我们的研究将揭示SM-SNARE蛋白及其病理学的共同机制。