ZO-1 domain interactions in tight junction structure and barrier function

描述(申请人提供)：上皮屏障丧失是许多肠道和其他器官疾病的致病成分。多蛋白质紧密连接复合体形成相邻细胞之间的细胞旁屏障，并限制大小溶质通过细胞旁途径的流量。紧密连接蛋白之间的许多相互作用已被描述。外周膜支架蛋白闭锁带-1(ZO-1)通过不同的结构域直接结合，介导了其中的几种相互作用。据报道，ZO-1与20多种蛋白质相互作用，包括Claudins、ZO-2和ZO-3、连接黏附分子(JAM)、G112、1连环蛋白、occludin、F-肌动蛋白、皮质蛋白和cinglin。我们实验室最近的研究表明，即使在稳定状态下，紧密连接也是高度动态的。已发表的研究和我的初步数据表明，ZO-1相互作用的细微变化可以导致紧密连接蛋白的动态性质和屏障功能的显着变化。进一步的证据表明，ZO-1可能是屏障功能的关键决定因素，在用细胞因子TNF处理上皮细胞后，ZO-1被不适当地重新分布，这是克罗恩病和实验性炎症性肠病屏障丧失和发病机制的核心。然而，ZO-1参与屏障功能的确切机制却知之甚少。因此，这项建议的目的是确定ZO-1和其他紧密连接蛋白之间的特定分子相互作用对ZO-1的动态行为、屏障功能和屏障调节的贡献。基于大量已发表的和初步的数据，我的中心假设是ZO-1和其他蛋白质之间的相互作用定义了稳态和对外界刺激的反应的正常运输和动态行为。为了验证这一假设，我将确定特定的ZO-1结构域缺失如何影响ZO-1和其他紧密连接蛋白的运输和动态交换，以调节上皮屏障功能(目标1)，并确定特定的ZO-1结构域在响应生理和病理生理相关刺激时的作用，包括肿瘤坏死因子(目标2)。在这些目标中，我将第一次使用创新的工具和技术来全面评估ZO-1相互作用对上皮屏障结构和功能的影响。这些研究意义重大，因为这些数据有望确定与不同的疾病相关屏障缺陷相关的ZO-1结构域，通过提供针对特定ZO-1相互作用、恢复屏障功能和防止疾病进展所需的信息，将有益于人类健康。

DESCRIPTION (provided by applicant): Epithelial barrier loss is a pathogenic component of numerous diseases of the intestines and other organs. The multi-protein tight junction complex forms the paracellular barrier between adjacent cells and limits the flux of large and small solutes across the paracellular pathway. Many interactions between tight junction proteins have been described. The peripheral membrane scaffolding protein, zonula occludens-1 (ZO-1) mediates several of these interactions by direct binding through distinct domains. ZO-1 has been reported to interact with over 20 proteins, including claudins, ZO-2 and ZO-3, junctional adhesion molecule (JAM), G112, 1 catenin, occludin, F-actin, cortactin, and cingulin. Recent studies from our laboratory have shown that the tight junction is highly dynamic, even at steady state. Published studies and my preliminary data suggest that subtle changes in ZO-1 interactions can result in marked changes in the dynamic properties of tight junction proteins and barrier function. As further evidence that ZO-1 may be a critical determinant of barrier function, ZO-1 is inappropriately redistributed following treatment of epithelia with the cytokine TNF, which is central to barrier loss and pathogenesis in Crohn's and experimental inflammatory bowel disease. The precise mechanisms of ZO-1 involvement in barrier function are poorly understood, however. Thus, the objective of this proposal is to define the contributions of specific molecular interactions between ZO-1 and other tight junction proteins to ZO- 1 dynamic behavior, barrier function, and barrier regulation. Based on strong published and preliminary data, my central hypothesis is that interactions between ZO-1 and other proteins define normal trafficking and dynamic behaviors at steady state and in response to exogenous stimuli. To test this hypothesis, I will determine how specific ZO-1 domain deletions impact the trafficking and dynamic exchange of ZO-1 and other tight junction proteins to regulate epithelial barrier function (Aim 1) and define the roles of specific ZO-1 domains in response to physiologically- and pathophysiologically-relevant stimuli, including TNF (Aim 2). Within these aims, I will use innovative tools and techniques to comprehensively assess, for the first time, the impact of ZO-1 interactions on epithelial barrier structure and function. These studies are significant because the data, which are expected to identify ZO-1 domains that are linked to distinct disease-associated barrier defects, will benefit human health by providing information necessary to target specific ZO-1 interactions, restore barrier function, and prevent disease progression.