Structure-Function Studies of Tight Junction Membrane Protein

紧密连接在上皮细胞和内皮细胞中具有三个至关重要的功能。紧密连接形成 作为分隔身体隔间的细胞旁屏障，它们充当维持 细胞的顶部-基底侧极性，并且它们具有允许选择性渗透的细胞旁孔。 离子和小分子穿过细胞单层。三种完整的膜蛋白被认为是发挥作用的。 在紧密连接的这些功能中起着中心作用，因此具有很高的生物学意义：claudin（a 具有24个成员的多基因家族）、闭合蛋白和三纤维素。该提案的长期目标是解决 这些蛋白质中的每一种的结构在原子分辨率下，并使用结构引导的诱变， 阐明其独特功能的结构机制。 PI已经组建了一个多学科的调查小组，来自洛杉矶，匹兹堡， 波士顿，和柏林来解决具有挑战性的问题，解开claudins的功能。这支球队有 在通过电生理方法测定细胞旁孔功能方面的独特专业知识， 冷冻断裂电子显微镜和混合分子/布朗动力学的结结构 孔隙功能建模PI先前与Robert博士的实验室建立了合作关系 Stroud以表达claudin蛋白结晶为目标，并取得了重大进展。因此，在本发明中， 申请人组成了一个独特的团队，准备与PShBiology网络中心合作， 膜蛋白结构测定解决结构测定和功能 紧密连接膜蛋白的表征。 目标1。用X射线晶体学方法解析claudin的结构 目标2.确定claudin孔功能的分子基础 目标3。确定封闭蛋白屏障和栅栏功能的结构基础 目标4。紧密连接膜蛋白的结构与功能比较

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 ofthe 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 ofthe 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 PShBiology 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