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

描述（由申请人提供）：上皮屏障丧失是肠道和其他器官多种疾病的致病成分。多蛋白紧密连接复合物形成相邻细胞之间的细胞旁屏障，并限制大小溶质穿过细胞旁途径的通量。已经描述了紧密连接蛋白之间的许多相互作用。外周膜支架蛋白，闭锁小带-1（ZO-1）介导的直接结合通过不同的领域，这些相互作用中的几个。据报道，ZO-1与20多种蛋白质相互作用，包括claudins、ZO-2和ZO-3、连接粘附分子（JAM）、G112、1连环蛋白、闭合蛋白、F-肌动蛋白、皮质蛋白和扣带蛋白。我们实验室最近的研究表明，紧密连接是高度动态的，即使在稳态。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能导致紧密连接蛋白和屏障功能的动态特性发生显着变化。作为ZO-1可能是屏障功能的关键决定因素的进一步证据，ZO-1在用细胞因子TNF治疗上皮细胞后不适当地重新分布，这是克罗恩病和实验性炎症性肠病中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知之甚少。因此，本提案的目的是确定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动态行为、屏障功能和屏障调节的贡献。基于强有力的已发表和初步的数据，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响应外源刺激的正常贩运和动态行为。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），并定义特定的ZO-1结构域响应生理和病理生理相关刺激的作用，包括TNF（目标2）。在这些目标，我将使用创新的工具和技术，全面评估，第一次，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，因为这些数据有望识别与不同疾病相关屏障缺陷相关的ZO-1结构域，通过提供靶向特定ZO-1相互作用、恢复屏障功能和预防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.