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

描述（由申请人提供）：上皮屏障丧失是许多肠道和其他器官疾病的致病成分。多蛋白紧密连接复合物在相邻细胞之间形成细胞旁屏障，并限制大小溶质通过细胞旁通路的通量。许多紧密连接蛋白之间的相互作用已经被描述。外周膜支架蛋白，闭塞带-1 （ZO-1）通过不同结构域的直接结合介导了这些相互作用。据报道，ZO-1可与20多种蛋白相互作用，包括claudin、ZO-2和ZO-3、连接粘附分子（JAM）、G112、1 catenin、occludin、F-actin、cortinn和cingulin。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能导致紧密连接蛋白的动态特性和屏障功能的显著变化。进一步证明ZO-1可能是屏障功能的关键决定因素，在细胞因子TNF治疗上皮后，ZO-1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尚不清楚。因此，本提案的目的是确定ZO-1与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决定了在稳态和响应外源刺激时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（目的1），并定义特定的ZO-1结构域在响应生理和病理生理相关刺激（包括TNF）中的作用（目的2）。在这些目标中，我将首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.