Structural Analysis of Membrane Tethering and Fusion Proteins

膜束缚和融合蛋白的结构分析

• 批准号: 10369677
• 负责人: FREDERICK M HUGHSON
• 金额: $ 37.53万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10369677
• 关键词: Architecture; Attention; Awareness; Binding; Biological Process; Carrier Proteins; Cell membrane; Cells; Chimeric Proteins; Closure by clamp; Coated vesicle; Collaborations; Complex; Coupled; Cryoelectron Microscopy; Disease; Eukaryotic Cell; Exocytosis; Foundations; Future; Goals; In Vitro; Infection; Lasso; Lipids; Lysosomes; Mediating; Membrane; Membrane Fusion; Modification; Molecular Chaperones; Mutation; Neurons; Organelles; Pathway interactions; Pattern; Process; Protein Sortings; Proteins; Proteomics; Reaction; Reporting; Research; Role; SNAP receptor; Sorting - Cell Movement; Structure; Surface; Synapses; System; Therapeutic Intervention; Thermodynamics; Vacuole; Vesicle; Work; X-Ray Crystallography; Yeasts; base; biochemical tools; design; experimental study; falls; flexibility; human disease; improved; in vivo; laser tweezer; late endosome; membrane assembly; nanomachine; neurotransmitter release; programs; protein function; reconstitution; single molecule; syntaxin binding protein 1; trafficking; vesicle transport; virtual

PROJECT SUMMARY / ABSTRACT The traffic patterns established by transport vesicles are of fundamental importance for protein localization, modification, and function within eukaryotic cells. Cargo transported by these vesicles is delivered through the fusion of the vesicle with the membrane of a target organelle or, in the case of exocytosis, the plasma membrane. Membrane fusion is executed by SNARE complexes that bridge the vesicle and target membranes. The formation of these complexes requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. We hypothesize that SNARE complex assembly reactions in the cell are orchestrated by `topologically aware' chaperones called multisubunit tethering complexes (MTCs). We furthermore propose that the key task of catalyzing four-helix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with—and sometimes as integral subunits of—the MTCs. Therefore, the overarching goal of this proposal is to achieve an improved structural and mechanistic understanding of MTC and SM function, especially as they relate to one another, in the assembly of membrane fusogenic SNARE complexes. Aim 1 is focused on SM proteins with the goal of characterizing their precise catalytic role in SNARE complex assembly. Principally through the use of X-ray crystallography and complementary single-molecule optical tweezers experiments, we will determine the structures and thermodynamic stabilities of SM-bound SNARE assembly intermediates. In Aims 2 and 3, we broaden our focus to include MTCs. In Aim 2, we will investigate the simplest known MTC, the yeast Dsl1 complex, and its interactions with SNAREs and the SM protein Sly1. Cryo-EM studies of arrested SNARE assembly intermediates in complex with both the Dsl1 complex and Sly1 are designed to reveal how the Dsl1 complex and Sly1 collaborate. In Aim 3, we will turn our attention to the homotypic fusion and vacuole protein sorting (HOPS) complex, a well-studied MTC that is required for fusion at late endosomes and lysosomes/vacuoles. Importantly, HOPS contains an SM protein as an integral subunit, making it an ideal system for studying MTC–SM collaboration. In order to elucidate how HOPS organizes SNAREs for assembly, we will expand our ongoing cryo-EM studies of HOPS to include bound SNAREs and SNARE assembly intermediates. Overall, this research program has the potential to revolutionize our mechanistic understanding of chaperoned SNARE complex assembly, with potentially profound implications for elucidating diverse biological processes and their subversion during infection and disease. While the proposed work is more fundamental than applied, it will lay a foundation for efforts to manipulate trafficking and other processes entailing membrane fusion, with potential future applications to therapeutic intervention.

项目摘要 /摘要 运输蔬菜建立的交通模式对于蛋白质定位至关重要， 修饰和真核细胞内的功能。这些蔬菜运输的货物是通过 囊泡与靶细胞器的膜融合或在胞吞作用的情况下是血浆 膜。膜融合是由桥接囊泡和靶的军鼓复合物执行的 机制。这些配合物的形成要求四种不同 不同的机制，经历了耦合的折叠和装配反应，在此过程中，圈圈图案拉链 进入平行的四螺旋捆。这个复杂的过程在体外效率低下，肯定是 在体内的更多挑战，它必须与各种非认知和非同志的形成竞争 军鼓复合物。我们假设细胞中的圈套复合物组装反应由 “拓扑意识到”的伴侣称为多亚基链接复合物（MTC）。我们进一步提议 催化四螺旋束组的关键任务落到Sec1/munc18（SM）蛋白质上 以及MTC的组成子单位，有时也是MTC的组成子单位。因此，这是总体目标 建议是为了改善对MTC和SM功能的结构和机械理解， 尤其是当它们相互关联时，在膜融合式圈圈复合物的组装中。目标1是 专注于SM蛋白，目的是表征其在SNARE Compeffer中的精确催化作用 集会。主要通过使用X射线晶体学和完成单分子光学 Tweezers实验，我们将确定SM结合SNARE的结构和热力学系统 组装中间体。在目标2和3中，我们将重点扩大到包括MTC。在AIM 2中，我们将调查 最简单的MTC，酵母DSL1复合物及其与SNARES和SM蛋白Sly1的相互作用。 与DSL1复合物和SLY1均具有捕获的圈套组装中间体的冷冻EM研究 旨在揭示DSL1复合物和SLY1的合作方式。在AIM 3中，我们将把注意力转移到 同型融合和吸尘蛋白分选（HOPS）复合物，这是一种经过良好研究的MTC，是融合所需的 内体晚期和溶酶体/液泡。重要的是，啤酒花包含SM蛋白作为整体亚基， 使其成为研究MTC – SM协作的理想系统。为了阐明啤酒花如何组织 用于组装的圈套，我们将扩展我们正在进行的啤酒花的冷冻EM研究，以包括绑定的贪食和 军鼓装配中间体。总体而言，该研究计划有可能改变我们的 对Chaineeroned的SNARE COMPLECT组装的机械理解，对 阐明潜水员生物学过程及其在感染和疾病期间的颠覆。而建议 工作比应用的基础更为基础，它将为操纵贩运和其他的努力奠定基础 过程需要膜融合，并在治疗干预中潜在的未来应用。