Molecular Biology of the Synapse

突触的分子生物学

• 批准号: 6887098
• 负责人: EILEEN M. LAFER
• 金额: $ 33.76万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6887098
• 关键词: Caenorhabditis elegans; binding sites; clathrin; endocytosis; molecular biology; neural transmission; phosphoproteins; protein binding; protein protein interaction; protein structure function; receptor binding; squid; synapses; synaptic vesicles; synaptotagmin; tissue /cell culture

DESCRIPTION (provided by applicant): The long-term goals of this project are to contribute to the determination of the molecular mechanisms which underlie neurotransmission. Synaptic transmission is a cycle of exo- and endocytosis. During the previous project period, we established that the clathrin pathway is essential for synaptic vesicle endocytosis, even during low physiological rates of stimulation. Furthermore, we elucidated a number of the molecular interactions which are required for synaptic vesicle recycling in vivo. In order to take this work to the next level, we believe two types of approaches are required. One is to utilize biophysical methods including X-ray crystallography, surface plasmon resonance, site-directed mutagenesis and solution biochemistry to elucidate mechanisms by which these complex interactions lead to vesicle assembly and uncoating. The other is to utilize electrophysiological methods to determine the temporal order of assembly of this macromolecular complex in living synapses. Therefore the proposal is organized around the following specific aims: Aim 1: Determine the time-course of a core set of protein-protein interactions that underlie neuronal endocytosis in living synapses. Aim 2: Characterize mechanisms of clathrin coated vesicle assembly and its regulation. We will evaluate the hypothesis that the large subunits of the adaptors contain multiple clathrin binding sites, evaluate the controversial hypothesis that the FxDxF and DPF sequence motifs that are involved in direct interactions with AP-2 are also involved in direct interactions with clathrin, determine where on clathrin TD the various different types of clathrin binding sites interact, and evaluate the hypothesis that Eps15 is a ruler that sets vertex-vertex/edge-edge distances in an assembling clathrin lattice. Aim 3: Characterize mechanisms of clathrin coated vesicle uncoating. We will determine how ATP hydrolysis in the ATPase domain of Hsc70 transmits a conformational change to the substrate binding domain which causes it to either bind or release substrate, determine the nature of the interaction between auxilin and Hsc70, elucidate the mechanism by which the J-domain stimulates the ATPase activity of Hsc70, and determine the role of the 'lid' opening and closing in the Hsc70 mechanism. Successful completion of these studies will allow us to achieve a deeper level of understanding of the fundamental processes of endocytosis and neurotransmission.

描述（由申请人提供）： 该项目的长期目标是有助于确定神经传递的分子机制。突触传递是外吞作用和内吞作用的循环。在之前的项目期间，我们确定网格蛋白途径对于突触小泡内吞作用至关重要，即使在低生理刺激率下也是如此。此外，我们阐明了体内突触小泡回收所需的许多分子相互作用。为了将这项工作提升到一个新的水平，我们认为需要两种方法。一是利用生物物理方法，包括 X 射线晶体学、表面等离振子共振、定点诱变和溶液生物化学来阐明这些复杂的相互作用导致囊泡组装和脱壳的机制。另一种是利用电生理学方法来确定这种大分子复合物在活突触中组装的时间顺序。因此，该提案围绕以下具体目标进行组织： 目标 1：确定构成活突触中神经元内吞作用的一组核心蛋白质-蛋白质相互作用的时间进程。目标 2：表征网格蛋白包被的囊泡组装机制及其调节。我们将评估接头的大亚基包含多个网格蛋白结合位点的假设，评估参与与 AP-2 直接相互作用的 FxDxF 和 DPF 序列基序也参与与网格蛋白直接相互作用的有争议的假设，确定其中在网格蛋白 TD 上，各种不同类型的网格蛋白结合位点相互作用，并评估 Eps15 是在装配中设置顶点-顶点/边-边距离的标尺的假设网格蛋白晶格。目标 3：表征网格蛋白包被的囊泡脱壳的机制。我们将确定 Hsc70 ATP 酶结构域中的 ATP 水解如何将构象变化传递至底物结合结构域，从而导致其结合或释放底物，确定辅助素和 Hsc70 之间相互作用的性质，阐明 J-结构域刺激 Hsc70 的 ATP 酶活性，并决定 Hsc70 机制中“盖子”打开和关闭的作用。这些研究的成功完成将使我们对内吞作用和神经传递的基本过程有更深入的了解。