Structure-Function Studies of Tight Junction Membrane Proteins

紧密连接膜蛋白的结构-功能研究

• 批准号: 8152114
• 负责人: Alan S Yu
• 金额: $ 47.3万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8152114
• 关键词: Structure; Function; Studies; Tight; Junction

DESCRIPTION (provided by applicant): Tight junctions have three critically important functions in both epithelia and endothelia. Tight junctions form the paracellular barrier that separates body compartments, they act as a fence that maintains apical-basolateral polarity of the cell, and they have paracellular pores that allow selective permeation of ions and small molecules across the cell monolayer. Three integral membrane proteins are thought to play a central role in these functions of the tight junction and hence are of high biological interest: claudin (a multigene family with 24 members), occludin, and tricellulin. The long-term goal of this proposal is to solve the structure of each of these proteins at atomic resolution and to use structure-guided mutagenesis to elucidate the structural mechanisms underlying their unique functions. The PI has already assembled a multidisciplinary team of investigators from Los Angeles, Pittsburgh, Boston, and Berlin to tackle the challenging problem of unraveling the function of claudins. This team has unique expertise in assaying paracellular pore function by electrophysiological methods, visualizing tight junction structure by freeze fracture electron microscopy, and hybrid molecular/Brownian dynamics modeling of pore function. The PI previously established a collaboration with the laboratory of Dr. Robert Stroud to express claudin proteins with the goal of crystallization, and has made significant progress. Thus, the applicants constitute a team that is uniquely poised to partner with a PSI Biology Network Center for Membrane Protein Structure Determination to tackle the structure determination and functional characterization of tight junction membrane proteins. Aim 1. Solve the structure of claudin by X-ray crystallography Aim 2. Determine the molecular basis of the pore functions of claudin Aim 3. Determine the structural basis of the barrier and fence functions of claudin Aim 4. Structure-function comparisons of tight junction membrane proteins PUBLIC HEALTH RELEVANCE: Tight junctions are junctions between cells that are needed for epithelia (e.g. skin, kidney, intestine) to act as barriers. This proposal would determine the structure of proteins in the membrane of the tight junction and use this information to elucidate the mechanism by which they function. This work has widespread significance for understanding diseases due to epithelial barrier dysfunction.

描述（由申请人提供）：紧密连接在上皮和内皮中具有三个至关重要的功能。紧密连接形成了分离人体隔室的细胞屏障，它们充当围栏，可维持细胞的顶质外侧极性，并且具有细胞细胞孔，可以在整个细胞单层中选择性渗透离子和小分子。人们认为，三种整体膜蛋白在紧密连接的这些功能中起着核心作用，因此具有很高的生物学兴趣：克劳丁（Claudin）（一个具有24个成员的多基因家族），occludin和tricellulin。该提案的长期目标是在原子分辨率下解决这些蛋白质的结构，并使用结构引导的诱变来阐明其独特功能的结构机制。 PI已经组建了来自洛杉矶匹兹堡的跨学科调查员团队 波士顿和柏林解决了揭开克劳丁斯功能的具有挑战性的问题。该团队具有通过电生理方法测定细胞细胞孔功能的独特专业知识，通过冻结裂缝电子显微镜和混合分子/布朗动力学可视化紧密的连接结构 孔功能的建模。 PI先前与Robert Stroud博士实验室建立了合作，以表达Claudin蛋白的目的是结晶，并取得了重大进展。因此， 申请人构成一个独特的团队，该团队准备与PSI生物学网络中心合作 膜蛋白结构确定以应对结构测定和功能 紧密连接膜蛋白的表征。 AIM 1。通过X射线晶体学解决Claudin的结构 目标2。确定克劳丁孔功能的分子基础 目标3。确定克劳丁的障碍和围栏功能的结构基础 AIM 4。紧密连接膜蛋白的结构功能比较 公共卫生相关性：紧密的连接是上皮（例如皮肤，肾脏，肠道）所需的细胞之间的连接。该建议将确定紧密连接的膜中蛋白质的结构，并使用此信息阐明其发挥作用的机制。这项工作对于由于上皮屏障功能障碍而导致的疾病具有广泛的意义。