Structural studies of tight junction proteins

描述（由申请人提供）：紧密连接（TJs）是必不可少的细胞结构，可在内皮细胞和上皮细胞中形成选择性的细胞旁屏障，包括血脑、血视网膜屏障、肺和肠上皮。TJs的屏障特性调节代谢物通量、流体流动和药物进入这些组织。在许多人类疾病中，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、血管性痴呆和哮喘，都观察到TJ屏障特性的功能障碍。此外，TJ成分被一系列病原体用作细胞受体。因此，进一步了解TJs的基本结构和功能生物学将对人类健康产生广泛的影响。具体来说，这一信息可能导致改进药物递送方法，调节疾病中TJ通透性的新方案和预防某些感染。目前，对TJ屏障特性的分子基础及其调控机制知之甚少。在这篇文章中，我们描述了一种结合结构、生化、细胞和分子的方法来阐明occludin （Occ）的功能，Occ是TJ的跨膜成分，与TJ屏障特性的调节有关。这些研究建立在我们之前发表和未发表的数据基础上，这些数据表明Occ及其结合伙伴ZO-1蛋白可以调节TJs赋予的屏障特性。计划中的研究分为三个具体目标。在ai1中，实验描述了使用细胞生物学方法来确定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。在Aim2中，我们描述了确定ZO-1/Occ蛋白结合核心的结构细节的实验，其中包括S471及其功能结合复合物。在Aim 3中，我们扩展了这些方法，使用全长Occ和二聚体ZO-1构建物来确定Occ的S471磷酸化对其与ZO-1相互作用的贡献。总之，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复合物调节TJ屏障特性的新策略提供起点。

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 These studies build upon our previously published and unpublished data implicating Occ, and its binding partner zona occludens 1 (ZO-1) protein, in regulating barrier properties conferred by TJs.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