Structural studies of the exocyst

外囊的结构研究

基本信息

批准号：
8132863
负责人：
Neil Vasan
金额：
$ 2.59万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/8132863
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 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

项目摘要

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）：分泌囊泡从反式高尔基体芽，并沿细胞骨架元素移动到活跃分泌的位点。外囊肿是〜800 kDa的杂项复合物，被认为在囊泡及其靶膜之间识别时起作用，因为囊泡在膜融合之前停靠到质膜。外囊肿与沿外环细胞途径的许多其他蛋白质相互作用，因此对其结构/结构的知识将在理解外胞胞菌病的分子机制方面至关重要。试图隔离或表达整个外囊肿的尝试没有成功。取而代之的是，我们将研究外囊肿子复合体，这应该更容易隔离结构研究所需的数量，但应得出有关亚基安排的信息（AIM 1）。确定的任何子复合物将受到凝胶过滤的溶解度的监测，并进行结晶试验（AIM 2）。天然晶体和/或重原子取代的晶体将用于相位。一旦确定了结构，我们将研究蛋白质如何与其他外囊亚基相互作用，以推断整体外囊肿结构（AIM 3）。将确定与子复合物的亚基结合伙伴，并通过域分析将结合伴侣相互作用区域缩小。突变分析将在与其他外囊亚基相互作用所需的子包中更精确地定义表面。此外，将进行类似的实验，以确定GTPases的激活形式如何与外囊肿亚基相互作用，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为外囊肿体系结构构建模型。公共卫生相关性：外囊肿途径或外囊肿的故障与多种心脏疾病有关，例如贫血，高血压和糖尿病。外囊肿对于转铁蛋白受体回收至关重要（在血红蛋白缺陷型贫血中受损），ANP和其他激素的分泌（高血压增加）以及胰岛素和GLUT4运输的分泌（在糖尿病中有缺陷）。对外囊肿的结构理解将有助于阐明囊泡束缚在囊泡束缚中的功能，并可能深入了解这些心脏和血液疾病的病因。