Structure-Function Studies of Tight Junction Membrane Proteins

描述(申请人提供)：紧密连接在上皮细胞和内皮细胞中都有三个至关重要的功能。紧密的连接形成了分隔身体隔间的细胞旁屏障，它们起到了保持细胞顶端-基底侧极性的屏障的作用，它们有细胞旁孔，允许离子和小分子选择性地渗透到细胞单层。三种完整的膜蛋白被认为在紧密连接的这些功能中发挥核心作用，因此具有很高的生物学意义：claudin(一个有24个成员的多基因家族)、occludin和tricellin。这一提议的长期目标是在原子分辨率下解决每一种蛋白质的结构，并使用结构导向突变来阐明其独特功能的结构机制。 私家侦探已经组建了一个由洛杉矶、匹兹堡、 波士顿和柏林，以解决分解笨拙的功能这一具有挑战性的问题。该团队在电生理学方法分析细胞旁毛孔功能、冷冻断口电子显微镜显示紧密连接结构以及分子/布朗混合动力学方面拥有独特的专业知识。 孔道函数的建模。 PI之前与Robert Stroud博士的实验室建立了合作关系，以结晶为目标表达Claudin蛋白，并取得了重大进展。因此， 申请者组成了一个独特的团队，准备与PSI生物网络中心合作 膜蛋白结构测定要解决的结构确定和功能 紧密连接膜蛋白的特性。 目的1.用X-射线单晶衍射法确定克拉丁的结构 目的2.确定克拉丁类药物孔功能的分子基础 目的3.确定克拉丁屏障和栅栏功能的结构基础 目的4.紧密连接膜蛋白的结构与功能比较 与公共卫生相关：紧密连接是上皮细胞(如皮肤、肾脏、肠道)作为屏障所需的细胞之间的连接。这一建议将确定紧密连接膜中蛋白质的结构，并利用这一信息来阐明它们发挥作用的机制。这项工作对于理解上皮屏障功能障碍引起的疾病具有广泛的意义。

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.