Structural studies of the exocyst

外囊的结构研究

• 批准号: 7753268
• 负责人: Neil Vasan
• 金额: $ 4.62万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7753268
• 关键词: Active Sites; Affinity; Affinity Chromatography; Anemia; Architecture; Bacteria; Binding; Biochemistry; C-terminal; Cell membrane; Cleaved cell; Complex; Crystallization; Diabetes Mellitus; Docking; Electron Microscopy; Elements; Escherichia coli; Etiology; Exocytosis; GLUT4 gene; Gel Chromatography; Glutathione S-Transferase; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Heart; Heart Diseases; Hematological Disease; Hemoglobin; His-His-His-His-His-His; Hormones; Hypertension; Image; Individual; Knowledge; Length; Maps; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Sieve Chromatography; Monitor; N-terminal; Pathway interactions; Peptide Hydrolases; Phase; Proteins; Rattus; Relative (related person); Role; Saccharomyces cerevisiae; Secretory Vesicles; Selenomethionine; Solubility; Structure; Surface; Testing; Transferrin Receptor; Vesicle; Western Blotting; Yeasts; insight; insulin secretion; protein protein interaction; public health relevance; receptor recycling; research study; retinal rods; trafficking

DESCRIPTION (provided by applicant): Secretory vesicles bud from the trans-Golgi and move along cytoskeletal elements to sites of active secretion. The exocyst is an ~800 kDa hetero-octameric complex that is thought to function in recognition between the vesicle and its target membrane, as the vesicle is docking to the plasma membrane, prior to membrane fusion. The exocyst interacts with numerous other proteins along the exocytic pathway, so that knowledge of its architecture/structure will be central in understanding the molecular mechanisms underlying exocytosis. Attempts to isolate or express the entire exocyst have been unsuccessful. Instead we will study exocyst subcomplexes, which should be easier to isolate in quantities required for structural studies and which should nevertheless yield information regarding subunit arrangement (Aim 1). Any subcomplexes identified will be monitored for solubility on gel filtration and subject to crystallization trials (Aim 2). The native crystals and/or heavy atom substituted crystals will be used for phasing. Once the structure has been determined, we will study how the proteins interact with other exocyst subunits, to infer overall exocyst architecture (Aim 3). Subunit binding partners to the subcomplex will be identified, and binding partner interaction regions will be narrowed down, through domain analysis. Mutational analysis will be performed to more precisely define surfaces in the subcomplex required for interaction with other exocyst subunits. Additionally, similar experiments will be performed to determine how the activated form of GTPases interacts with exocyst subunits, which will give information regarding exocyst-membrane interactions. These functional studies will allow us to construct a model for exocyst architecture. Public Health Relevance: Malfunctions in the exocytic pathway or the exocyst are relevant to a number of cardiac diseases, such as anemia, hypertension, and diabetes. The exocyst is critical for transferrin receptor recycling (which is impaired in hemoglobin-deficient anemia), secretion of ANP and other hormones (which are increased in hypertension), and for secretion of insulin and GLUT4 trafficking (which are defective in diabetes). A structural understanding of the exocyst will help clarify the function of the exocyst in vesicle tethering, and might give insight into the etiologies of these heart and blood diseases.

描述（由申请人提供）：分泌囊泡从反高尔基体中出芽，并沿着细胞骨架元件移动到活跃分泌的位点。外囊是一种约 800 kDa 的异八聚体复合物，被认为在囊泡与其目标膜之间的识别中发挥作用，因为囊泡在膜融合之前与质膜对接。胞吐途径中的胞吐与许多其他蛋白质相互作用，因此对其结构/结构的了解对于理解胞吐作用的分子机制至关重要。分离或表达整个外囊的尝试并未成功。相反，我们将研究外囊亚复合体，它应该更容易分离结构研究所需的数量，并且应该产生有关亚基排列的信息（目标 1）。任何鉴定出的子复合物都将通过凝胶过滤监测溶解度并进行结晶试验（目标 2）。天然晶体和/或重原子取代的晶体将用于定相。一旦结构确定，我们将研究蛋白质如何与其他外囊亚基相互作用，以推断外囊的整体结构（目标 3）。通过域分析，将鉴定亚基与子复合物的结合伙伴，并缩小结合伙伴相互作用区域的范围。将进行突变分析，以更精确地定义与其他外囊亚基相互作用所需的子复合体中的表面。此外，还将进行类似的实验来确定 GTP 酶的激活形式如何与外囊亚基相互作用，这将提供有关外囊膜相互作用的信息。这些功能研究将使我们能够构建外囊结构的模型。公共健康相关性：胞吐途径或胞吐囊的功能障碍与许多心脏病有关，例如贫血、高血压和糖尿病。外囊对于转铁蛋白受体的再循环（在血红蛋白缺乏性贫血中受损）、ANP 和其他激素的分泌（在高血压中增加）以及胰岛素的分泌和 GLUT4 运输（在糖尿病中存在缺陷）至关重要。对外囊的结构了解将有助于阐明外囊在囊泡束缚中的功能，并可能有助于深入了解这些心脏和血液疾病的病因。