Single Molecule Studies of SNARE-Induced Vesicle Fusion

SNARE 诱导囊泡融合的单分子研究

• 批准号: 7098989
• 负责人: AXEL T BRUNGER
• 金额: $ 29.56万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-10 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7098989
• 关键词: PC12 cells; adenosinetriphosphatase; calcium flux; cholesterol; conformation; fatty acylation; fluorescence resonance energy transfer; intermolecular interaction; lipid bilayer membrane; liposomes; membrane fusion; model design /development; molecular assembly /self assembly; molecular biology; nanotechnology; nerve /myelin protein; palmitates; posttranslational modifications; protein structure function; synaptic vesicles; synaptotagmin; transcription factor

DESCRIPTION (provided by applicant): The maintenance of distinct organelles within the eukaryotic cytosol is essential for survival. The exchange of material between these organelles requires the merger of two phospholipids membranes. All known forms of intracellular membrane fusion, including synaptic vesicle fusion, involve a highly conserved family of proteins termed SNAREs (Soluble N-ethyl maleimide sensitive factor Attachment Protein Receptors). Auxiliary SNARE binding proteins are known to regulate membrane fusion events, such as the Ca2+ binding protein synaptotagmin. Crystal structures of some of the key players involved in this process have been solved, such as the synaptic SNARE complex and the cytoplasmic domain of synaptotagmin. In vitro bulk liposome-liposome fusion experiments have established that SNARES and synaptotagmin constitute a minimal, albeit inefficient, fusion machinery, but they have done little to reveal the underlying molecular mechanism, both in terms of sequential and spatial interactions between proteins and lipids during fusion. We propose to study the molecular mechanism of Ca2+-triggered synaptic vesicle fusion by single molecule fluorescence methods. Our previous work has provided the framework for the proposed studies. Specifically, we propose to study correlations between protein-protein, protein-lipid interactions and fusion, to study the effect of post-translational modifications of SNARE proteins and of the lipid/cholesterol composition on fusion, to study the interactions of SNAREs and synaptotagmin at the interface between docked membranes, and to study the molecular mechanism of SNARE complex disassembly by the ATPase N- ethylmalemeide-sensitive factor (NSF). These in vitro studies will be complemented by in vivo studies using PC12 cells. We anticipate that our in vitro system can be extended to include other factors in order to obtain a reconstituted system that may eventually approach the properties of the synaptic vesicle fusion machinery in the neuron. Such a system could serve as an efficient model system for novel drug discovery.

描述（由申请人提供）：真核细胞质内独特细胞器的维持是生存所必需的。这些细胞器之间的物质交换需要两个磷脂膜的合并。所有已知形式的细胞膜融合，包括突触囊泡融合，都涉及一个高度保守的蛋白家族，称为SNAREs（可溶性n -乙基马来酰亚胺敏感因子附着蛋白受体）。已知辅助SNARE结合蛋白调节膜融合事件，如Ca2+结合蛋白synaptotagmin。一些参与这一过程的关键分子的晶体结构已经被解决，如突触SNARE复合体和synaptotagmin的细胞质结构域。体外大体积脂质体-脂质体融合实验已经证实，SNARES和synaptotagmin构成了一个最小的、尽管效率低下的融合机制，但它们几乎没有揭示潜在的分子机制，无论是在融合过程中蛋白质和脂质之间的顺序和空间相互作用方面。我们提出用单分子荧光方法研究Ca2+触发突触囊泡融合的分子机制。我们以前的工作为拟议的研究提供了框架。具体而言，我们拟研究蛋白与蛋白、蛋白与脂质相互作用与融合之间的相关性，研究SNARE蛋白翻译后修饰和脂质/胆固醇组成对融合的影响，研究SNAREs和synaptotagmin在对接膜界面上的相互作用，研究atp酶N-乙基丙烯酰胺敏感因子（NSF）分解SNARE复合物的分子机制。这些体外研究将通过使用PC12细胞的体内研究进行补充。我们预计我们的体外系统可以扩展到包括其他因素，以获得一个重建的系统，最终可能接近神经元突触囊泡融合机制的特性。该系统可作为新药发现的有效模型系统。