Molecular mechanisms of polarized endocytic traffic in epithelial cells

上皮细胞极化内吞运输的分子机制

• 批准号: 7805482
• 负责人: Rytis Prekeris
• 金额: $ 32.74万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7805482
• 关键词: Actins; Apical; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Calorimetry; Canis familiaris; Carrier Proteins; Cell membrane; Cells; Complex; Data; Defect; Disease; Drug Delivery Systems; Endosomes; Ensure; Epithelial; Epithelial Cells; Epithelium; Family; Glutathione; Goals; Guanosine Triphosphate Phosphohydrolases; Immunoprecipitation; In Vitro; KRP protein; Kidney; Laboratories; Lead; Lipids; Mediating; Membrane; Membrane Protein Traffic; Molecular; Mutagenesis; Nephrogenic Diabetes Insipidus; Pathway interactions; Process; Protein Family; Proteins; Publishing; Pulmonary Cystic Fibrosis; Recruitment Activity; Recycling; Role; Site-Directed Mutagenesis; Small Interfering RNA; Sorting - Cell Movement; Techniques; Testing; Tetanus Helper Peptide; Titrations; Transmembrane Transport; Work; Yeasts; base; basolateral membrane; cell motility; design; fluorescence imaging; in vivo; insight; kidney epithelial cell; knock-down; member; monolayer; nexin; novel therapeutic intervention; polarized cell; polymerization; protein complex; protein transport; public health relevance; rab GTP-Binding Proteins; research study; scaffold; small hairpin RNA; trafficking; yeast two hybrid system

DESCRIPTION (provided by applicant): Epithelia consist from polarized cells that are capable of selectively transporting substances across epithelial monolayer. This selective transport is achieved by the partitioning of the plasma membrane into distinct domains: apical and basolateral, with both of these plasma membrane compartments having distinct lipid and protein compositions. Since the fidelity of trans-epithelial protein transport is crucial to a variety of epithelial functions, epithelial cells have developed complicated mechanisms to ensure correct transport of proteins. Rab11 GTPases are the members of small monomeric GTPase super-family that has been implicated in regulating endocytic membrane transport. Rab GTPase work by recruiting various effector proteins to the distinct cellular compartments. Thus, understanding the role of these effector proteins is a key step in understanding the function of epithelial cell. In the last six years several Rab11-binding proteins have been identified, which include Rab11 family interacting proteins, also known as FIPs. Works from several laboratories, including ours, have shown that Rip11/FIP5 member of FIP family regulate polarized protein transport in epithelial cells. Furthermore, it was shown that FIPs act as scaffolding factors allowing the assembly of specific sorting/transport complexes required for the epithelial protein traffic. Based on recently published results and on preliminary data, we propose the following hypotheses. First, that Rip11/FIP5 mediates protein transport to apical plasma membrane. Second, that Rip11/FIP5 regulates apical protein transport by sequential recruiting of sortin nexin 18 (SNX18) and kinesin II to the endocytic membranes. Thus, the main goal of this proposal is to further characterize Rip11/FIP5-SNX18 and Rip11/FIP5-Kinesin II complex formation and determine their role on epithelial transport. I propose three different aims designed to test these hypotheses. In the aim #1 we will analyze the role of Rip11/FIP5 in regulating apical protein transport. We will use combination of tet-inducible protein knock-down, fluorescence imaging and in vivo transport assays. In the aim #2 and aim #3 we will characterize the binding of Rip11/FIP5 to SNX18 and Kinesin II. We will use the combination of immunoprecipitations, glutathione bead pull-down, yeast two-hybrid and isothermal calorimetry assays. In addition, in aim #2 and aim #3 we will determine the roles of Rip11/FIP5-binding proteins in regulating apical and basolateral membrane traffic. To that end we will use the combination of tet- inducible protein knock-down, fluorescence imaging, site-directed mutagenesis and in vivo transport assays. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the molecular mechanisms of Rab11-dependent polarized membrane traffic and define the membrane traffic steps that are regulated by each Rab11-Rip11/FIP5 protein complex. Furthermore, this project will also identify the molecular machinery mediating Rab11-dependent endocytic protein sorting. These data will advance our understanding, both conceptually and mechanistically, of the machinery governing protein targeting in epithelial cells. To elucidate the mechanisms of protein targeting is of major importance because the defects in this process cause a variety of pulmonary (cystic fibrosis) and renal (nephrogenic diabetes insipidus) disorders. Thus, new insights into the mechanisms of polarized membrane traffic may lead to the identification of new drug targets as well as new therapeutic approaches aimed at the identification and treatment of membrane traffic disorders.

描述（由申请人提供）：上皮由能够在上皮单层上选择性地运输物质的偏振细胞组成。这种选择性转运是通过将质膜划分为不同域的：顶部和基底外侧的，这两个质膜室都具有不同的脂质和蛋白质组成。由于跨上皮蛋白转运的保真度对于多种上皮功能至关重要，因此上皮细胞已经开发出复杂的机制，以确保蛋白质正确转运。 Rab11 GTPase是小型单体GTPase超级家庭的成员，与调节内吞膜转运有关。 RAB GTPase通过将各种效应蛋白募集到不同的细胞区室来起作用。因此，了解这些效应蛋白的作用是理解上皮细胞功能的关键步骤。在过去的六年中，已经确定了几种Rab11结合蛋白，其中包括Rab11家族相互作用的蛋白质，也称为FIP。来自包括我们的几个实验室的作品表明，FIP家族的RIP11/FIP5成员调节上皮细胞中的极化蛋白质转运。此外，结果表明，FIP充当脚手架因素，允许组装上皮蛋白质流量所需的特定分类/运输复合物。根据最近发布的结果和初步数据，我们提出了以下假设。首先，RIP11/FIP5介导蛋白质转运到顶端质膜。其次，该RIP11/FIP5通过顺序募集Nexin 18（SNX18）和驱动蛋白II到内吞膜来调节顶端蛋白质的转运。因此，该提案的主要目标是进一步表征RIP11/FIP5-SNX18和RIP11/FIP5-Kinesin II复合物的形成，并确定它们在上皮运输中的作用。我提出了三个旨在检验这些假设的不同目标。在目标＃1中，我们将分析RIP11/FIP5在调节顶端蛋白转运中的作用。我们将结合使用TET诱导的蛋白质敲低，荧光成像和体内转运测定法。在AIM＃2和AIM＃3中，我们将表征RIP11/FIP5与SNX18和Kinesin II的结合。我们将使用免疫沉淀，谷胱甘肽珠下拉，酵母两杂化和等温量热法测定的组合。此外，在AIM＃2和AIM＃3中，我们将确定RIP11/FIP5结合蛋白在调节顶端和基底外侧膜流量中的作用。为此，我们将使用TET-诱导蛋白敲低，荧光成像，定位诱变和体内转运测定的组合。公共卫生相关性：该项目的目的是了解RAB11依赖性膜流量的分子机制，并定义每个RAB11-RIP11/FIP5蛋白复合物调节的膜交通步骤。此外，该项目还将识别介导Rab11依赖性内吞蛋白排序的分子机械。这些数据将在概念和机械上都在上皮细胞中控制蛋白质靶向的机械的理解。阐明蛋白质靶向的机制至关重要，因为此过程中的缺陷会导致多种肺（囊性纤维化）和肾脏（肾脏基糖尿病）疾病。因此，对两极分化膜流量机制的新见解可能会导致鉴定新药物靶标以及旨在鉴定和治疗膜交通障碍的新治疗方法。