Structural studies of tight junction proteins

描述(申请人提供)：紧密连接(TJ)是基本的细胞结构，在血管内皮细胞和上皮细胞(包括血-脑、血-视网膜屏障、肺和肠道上皮)中形成选择性的细胞旁屏障。TJ的屏障特性调节代谢物流量、液体流动和药物进入这些组织的摄取。在包括癌症(如脑肿瘤)、中风、视网膜病变、肾脏疾病、肠易激综合征、阿尔茨海默病和血管性痴呆以及哮喘在内的大量人类疾病中观察到TJ屏障特性的功能障碍。此外，TJ组分被一系列病原体用作细胞受体。因此，更好地了解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。具体地说，这些信息可能导致改进药物输送方法、调节疾病中TJ渗透性的新方案以及预防某些感染。目前，人们对TJ屏障特性的分子基础以及它们是如何调节的知之甚少。在这项建议中，我们描述了一个结构，生化和细胞和分子相结合的方法来阐明阻滞素(OCC)的功能，它是TJS的一个跨膜成分，参与了TJ屏障特性的调节。这些研究建立在我们之前发表和未发表的数据的基础上，这些数据表明OCC及其结合伙伴ZO-1蛋白在调节TJ赋予的屏障属性中发挥作用。计划中的研究被组织成三个具体目标。在AIM1中，描述了用细胞生物学方法确定OCC(S471)丝氨酸471磷酸化对TJ细胞结构功能影响的分子机制(S)的实验。在AIM2中，我们描述了确定包括S471在内的ZO-1/OCC蛋白结合核心的结构细节的实验，以及在AIM 3中利用它们的功能结合复合体，我们扩展了这些方法，以确定S471的OCC磷酸化对其与ZO-1相互作用的贡献，使用全长OCC和二聚体ZO-1结构。综上所述，这三个目标的结果将为理解OCC功能提供结构和生化基础，并为开发针对ZO-1/OCC复合体的TJ屏障特性调节新策略提供起点。

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 These studies build upon our previously published and unpublished data implicating Occ, and its binding partner zona occludens 1 (ZO-1) protein, in regulating barrier properties conferred by TJs.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.