Single-molecule manipulation of proteins involved in membrane fusion, lipid exchange, and mechanosensation

参与膜融合、脂质交换和机械感觉的蛋白质的单分子操作

• 批准号: 10799300
• 负责人: Yongli Zhang
• 金额: $ 7.89万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10799300
• 关键词: Binding; Biological Assay; Calcium; Complex; Coupled; Diabetes Mellitus; Disease; Exocytosis; Family; Fluorescence Microscopy; Functional disorder; Goals; Immune System Diseases; Intracellular Membranes; Ion Channel; Ions; Link; Lipids; Location; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Methodology; Molecular; Molecular Machines; Munc18-1 protein; Neurons; Pathway interactions; Phosphorylation; Play; Process; Protein Family; Proteins; Research; Resolution; Role; SNAP receptor; Synapses; Time; alpha-SNAP; insulin secretion; laser tweezer; mechanical force; nanodisk; nervous system disorder; neurotransmission; optic tweezer; reconstitution; single molecule; synaptotagmin; syntaxin

SNARE proteins and Sec1p/Munc18-family (SM) proteins constitute the core molecular machines that mediate nearly all intracellular membrane fusion. Other proteins regulate the core machinery to enable fusion at the right time and location. Dysfunction of these proteins has been linked to neurological and immunological disorders, cancers, diabetes, and other diseases. Decades of research have established that SNAREs couple their ordered folding and assembly to membrane fusion in a SM protein-dependent manner. However, it remains unclear what mechanistic role SM proteins plays in SNARE assembly and how SNARE assembly is coupled to membrane fusion. Using high-resolution optical tweezers, we recently found that neuronal SM protein Munc18-1 catalyzes step-wise assembly of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle, a process essential for neurotransmission and insulin secretion. Importantly, Munc18-1 serves as a template to guide directional SNARE assembly along a new pathway. In this application, we plan to first establish that the template complex is a conserved intermediate for SNARE-SM fusion machineries and a key target for other proteins to regulate SNARE assembly and membrane fusion. We will examine effects of key regulators involved in calcium-triggered exocytosis (Munc13-1, complexin, synaptotagmin, NSF, and alpha-SNAP) and phosphorylation of SNARE and SM proteins on SNARE assembly and disassembly. Then, we will develop new assays to simultaneous detect step-wise SNARE assembly and state-wise membrane fusion using large nanodiscs and trapped GUVs. A major goal is to reconstitute the calcium-triggered membrane fusion under controlled experimental conditions and to understand their working mechanism. Finally, we will extend our methodologies to pinpoint the molecular mechanisms of membrane binding and lipid exchange by extended synaptotagmins (E- Syts) and of mechanosensation by ion channel NOMPC. Our long-term goal is to develop a general approach to elucidate stability, folding, and dynamics of membrane proteins.

SNARE蛋白和Sec1p/ munc18家族（SM）蛋白构成核心分子

项目成果

A dynamic template complex mediates Munc18-chaperoned SNARE assembly.

• DOI: 10.1073/pnas.2215124119
• 发表时间: 2022-12-06
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Yang, Jie;Jin, Huaizhou;Liu, Yihao;Guo, Yaya;Zhang, Yongli
• 通讯作者: Zhang, Yongli

Securing SNAREs for assembly.

固定 SNARE 以进行组装。

• DOI: 10.1074/jbc.h120.014815
• 发表时间: 2020
• 期刊: The Journal of biological chemistry
• 作者: Zhang,Yongli;Yang,Jie
• 通讯作者: Yang,Jie

Energetics, kinetics, and pathways of SNARE assembly in membrane fusion.

• DOI: 10.1080/10409238.2022.2121804
• 发表时间: 2022-08
• 期刊: Critical reviews in biochemistry and molecular biology
• 影响因子: 6.5