Structural studies of tight junction proteins

描述（由申请人提供）：紧密连接（TJ）是在内皮细胞和上皮细胞中形成选择性细胞旁屏障的基本细胞结构，包括血脑屏障、血视网膜屏障、肺上皮和肠上皮。TJ的屏障特性调节代谢物通量、流体流动和药物吸收进入这些组织。在大量人类疾病中观察到TJ屏障性质的功能障碍，包括癌症（例如脑肿瘤）、中风、视网膜病、肾病、肠易激综合征、阿尔茨海默病和血管性痴呆以及哮喘。此外，TJ组分被一系列病原体用作细胞受体。因此，进一步了解TJ的基本结构和功能生物学将对人类健康产生广泛的影响。具体而言，这些信息可能导致改进的药物输送方法，用于调节疾病中TJ渗透性和预防某些感染的新协议。目前，对TJ屏障特性的分子基础以及它们是如何调节的知之甚少。在这个建议中，我们描述了一个组合的结构，生物化学和细胞和分子的方法来阐明闭合蛋白（OCC），跨膜组件的TJ，这已牵连在TJ屏障特性的调节功能。这些研究建立在我们以前发表和未发表的数据，涉及OCC，及其结合伙伴ZO-occludens 1（ZO-1）蛋白，在调节由TJ赋予的屏障特性。计划中的研究分为三个具体目标。在Aim 1中，描述了使用细胞生物学方法确定Occ（S471）中丝氨酸471磷酸化对TJ细胞结构功能影响的分子机制的实验。在Aim 2中，我们描述了确定ZO-1/Occ蛋白结合核心（包括S471）及其功能结合复合物的结构细节的实验。在Aim 3中，我们扩展了这些方法，使用全长Occ和二聚体ZO-1构建体确定S471磷酸化Occ对其与ZO-1相互作用的贡献。总之，这三个目标的结果将提供一个结构和生化基础，了解OCC功能和起点，为开发新的策略，调节TJ屏障性能的目标ZO-1/OCC复合物。

DESCRIPTION (provided by applicant): Tight junctions (TJs) are essential cellular structures that form the selective paracellular barriers in endothelial and epithelial cells, including the blood-brain, and blood-retinal barriers, lung and gut epithelium. The barrier properties of TJs regulate metabolite flux, fluid flow and drug uptake into these tissues. Dysfunction in TJ barrier properties is observed in a large number of human diseases including cancer (e.g. brain tumors), stroke, retinopathies, kidney disorders, irritable bowel syndrome, Alzheimer's disease and vascular dementia, and asthma. Moreover, TJ components are used as cellular receptors by a range of pathogens. Thus, an improved understanding of the basic structural and functional biology of TJs will have wide ranging impact for human health. Specifically, this information may lead to improved drug delivery methods, novel protocols for regulating TJ permeability in disease and prevention of some infections. Currently, little is known about the molecular basis for TJ barrier properties and how they are regulated. In this proposal, we describe a combined structural, biochemical and cellular and molecular approach to illuminate the function of occludin (Occ), a transmembrane component of TJs, which has been implicated in the regulation of TJ barrier properties. These studies build upon our previously published and unpublished data implicating Occ, and its binding partner zona occludens 1 (ZO-1) protein, in regulating barrier properties conferred by TJs. The planned studies are organized into three specific aims. In Aim1, experiments are described, using a cell biological approach to determine the molecular mechanism(s) for the effects of phosphorylation of Serine 471 in Occ (S471) on TJ cellular structure function. In Aim2, we describe experiments to determine the structural details of the ZO-1/Occ protein-binding core, which includes S471, and with their functional binding complexes In Aim 3, we extend these approaches to determine the contribution of S471 phosphorylation of Occ to its interaction with ZO-1 using full length Occ and dimeric ZO-1 constructs. Together, the results of these three aims will provide a structural and biochemical basis for understanding Occ function and starting point for developing novel strategies for modulating TJ barrier properties that target the ZO-1/Occ complex.