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: 7312209
• 负责人: Reinhard Jahn
• 金额: $ 16.94万
• 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/7312209
• 关键词: artificial membranes; chemical kinetics; conformation; exocytosis; liposomes; membrane fusion; membrane proteins; molecular assembly /self assembly; nerve /myelin protein; neuroendocrine system; protein binding; protein reconstitution; protein structure function; synaptic vesicles; synaptotagmin; syntaxin

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.

军鼓蛋白在膜融合中起关键作用。介导神经元胞吐作用的网罗是最佳的特征，其中包括囊泡蛋白突触蛋白/录vin和质膜居民 - 居住蛋白的语法1和SNAP-25。贪食会经历与重大构象变化有关的组装隔离周期。人们认为，膜之间的网罗组装可以催化双层的融合。然而，用鼻累的脂质体的融合以缓慢的动力学进行，到目前为止，还没有从纯化的成分中重新构成了控制胞吐作用或其他细胞内融合反应已知的调节步骤。在这里，我们建议详细介绍蛋白质脂质体与神经元蛋白质的融合，或者要么与含有相应的网罗的脂质体或含有相应的网res作为内源蛋白的生物膜融合。已经开发了敏感测定法，可以测量囊泡对接和融合。初步结果表明，含糖的脂质体的融合速率很大程度上取决于膜中的鼻涕的构象状态。首先，我们将定义确定含有突触的脂质体的融合动力学的条件，该脂质体与含有语法素1和SNAP-25的脂质体的融合动力学，包括构象和低聚态和寡聚蛋白的影响，以及诸如Synaptototagagmin 1，复合蛋白，复合蛋白和Munc-18的调节蛋白的影响。其次，我们将深入研究语法L/SNAP-25脂质体与突触囊泡的融合，重点是确定导致初步实验中观察到的增强融合动力学的因素。第三，我们将研究含突触的脂质体与等离子体的倒流的结合和融合 源自神经内分泌细胞的膜。