RECONSTITUTION OF SNARE-MEDIATED FUSION WITH NATIVE AND ARTIFICIAL MEMBRANES

圈套介导的天然膜和人工膜融合的重建

• 批准号: 7797456
• 负责人: Reinhard Jahn
• 金额: $ 23.36万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7797456
• 关键词: Amino Acids; Artificial Membranes; Binding; Biological; Biological Assay; Calcium; Cell membrane; Cells; Complex; Data; Docking; Event; Exocytosis; Goals; Indium; Kinetics; Laboratories; Liposomes; Measures; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Modeling; Molecular Conformation; Nature; Neuroendocrine Cell; Neurons; PC12 Cells; Play; Preparation; Proteins; Reaction; Recombinant Proteins; Role; SNAP receptor; Secretory Vesicles; Solutions; Stretching; Surface; Synaptic Vesicles; System; VAMP-2; Vesicle; conformational conversion; genetic regulatory protein; presynaptic; proteoliposomes; reconstitution; research study; synaptotagmin; synaptotagmin I; syntaxin; syntaxin 1; syntaxin 1A; vesicle-associated membrane protein

SNARE proteins play a key role in membrane fusion. The SNAREs mediating neuronal exocytosis are among the best characterized, they include the vesicle protein synaptobrevin/VAMP and the plasma membrane-resident proteins syntaxin 1 and SNAP-25. SNAREs undergo an assembly-disassembly cycle that is associated with major conformational changes. Assembly of SNAREs between membranes is thought to catalyze the fusion of bilayers. However, fusion of liposomes reconstituted with SNAREs proceeds with slow kinetics, and so far none of the regulatory steps known to control exocytosis or other intracellular fusion reactions has been reconstituted from purified components. Here we propose to characterize in detail the fusion of proteoliposomes with neuronal SNARE proteins either with liposomes containing the corresponding SNAREs or with biological membranes containing the corresponding SNAREs as endogenous proteins. Sensitive assays have been developed allowing for measuring both vesicle docking and fusion. Preliminary results have shown that the fusion rate of SNARE-containing liposomes strongly depends on the conformational state of the SNAREs in the membrane. First, we will define the conditions that determine the fusion kinetics of liposomes containing synaptobrevin with those containing syntaxin 1 and SNAP-25, including the influence of conformational and oligomeric states and of regulatory proteins such as synaptotagmin 1, complexins, and Munc-18. Second, we will study in depth the fusion of syntaxin l/SNAP-25 liposomes with synaptic vesicles, with an emphasis on identifying the factors responsible for the enhanced fusion kinetics observed in preliminary experiments. Third, we will investigate the binding and fusion of synaptobrevin-containing liposomes with inverted lawns of plasma membranes derived from neuroendocrine cells.

SNARE蛋白在膜融合中起关键作用。介导神经元胞吐作用的SNARE是最具特征的，它们包括囊泡蛋白突触泡蛋白/VAMP和质膜驻留蛋白突触融合蛋白1和SNAP-25。SNARE经历与主要构象变化相关的组装-拆卸循环。膜之间SNARE的组装被认为催化双层的融合。然而，用SNARE重构的脂质体的融合以缓慢的动力学进行，并且迄今为止，已知控制胞吐作用或其他细胞内融合反应的调节步骤中没有一个是从纯化的组分重构的。在这里，我们建议详细描述蛋白脂质体与神经元SNARE蛋白的融合，无论是含有相应SNARE的脂质体，还是含有相应SNARE作为内源性蛋白的生物膜。已经开发了允许测量囊泡对接和融合的灵敏测定。初步结果表明，含有SNARE的脂质体的融合速率强烈依赖于膜中SNARE的构象状态。首先，我们将定义的条件，确定含有突触泡蛋白与那些含有突触融合蛋白1和SNAP-25的脂质体的融合动力学，包括构象和寡聚状态和调节蛋白，如突触结合蛋白1，复合蛋白，和Munc-18的影响。第二，我们将深入研究突触融合蛋白1/SNAP-25脂质体与突触囊泡的融合，重点是确定在初步实验中观察到的增强的融合动力学的因素。第三，我们将研究含突触小泡蛋白的脂质体与血浆倒草坪的结合和融合 神经内分泌细胞膜。