Molecular Mechanisms of Intestinal Epithelial Tight Junction Regulation

描述（由申请方提供）：上皮屏障功能障碍有助于肠道和全身性疾病的进展。然而，有一个根本的差距，分离的临床病理意义的分子理解的机制，负责屏障调节。在过去的二十年中，已经鉴定了形成细胞旁屏障的紧密连接的主要组分，并分为三大类：支架蛋白，例如ZO-1;跨膜结构蛋白，例如闭合蛋白;和孔形成蛋白，例如封闭蛋白。 这些蛋白质如何相互作用以调节屏障尚不完全清楚。因此，紧密连接生物学正处于一个十字路口，需要从蛋白质的发现过渡到确定必要的调控机制。这些必须考虑的上下文中的不同组件的细胞旁渗透性，定义流量的大溶质或小离子和水。我们最近已经表明，这些方面的屏障功能的差异调节的病理相关的细胞因子TNF和IL-13，分别。这些研究的长期目标是从分子角度了解紧密连接结构和调节，并利用这些知识开发调节特定屏障成分和改善健康的方法。本申请的目的是使用新开发的体外和体内方法来定义调节动态蛋白质行为和屏障功能的紧密连接组分之间的相互作用。 我们的中心假设是，负责紧密连接蛋白锚定和运输的相互作用是细胞旁屏障功能的主要决定因素。这一假设是根据目前供资周期提供的有力的初步数据提出的。该项目的基本原理是，它将提供前所未有的洞察到调节屏障功能的分子相互作用，反过来，将允许操纵这些过程以获得治疗益处。该假设将通过特定的目的进行检验：1）确定特定ZO-1结构域对运输、锚定和紧密连接屏障调节的贡献; 2）确定调节闭合蛋白运输、蛋白质间相互作用以及体外和体内屏障功能的结构元件和磷酸化事件;和3）鉴定claudin结构域调节孔组装和打开的结构元件和功能相互作用。虽然每个目标都集中在一个关键的紧密连接蛋白或蛋白质家族，这些之间的相互作用将允许紧密连接结构和调控的统一模型的整合和发展。该提案具有创新性，因为它探索了蛋白质相互作用的动态调节控制屏障功能的新想法，这标志着我们对紧密连接生物学的理解发生了重大转变。这项研究意义重大，因为它将增强我们对屏障功能障碍的理解，并将屏障丧失的特定机制与疾病联系起来。开发的概念和工具将使开发针对不同屏障成分的药物成为可能，并最终治疗上皮和内皮屏障疾病。

DESCRIPTION (provided by applicant): Epithelial barrier dysfunction contributes to progression of intestinal and systemic disease. However, there is a fundamental gap that separates clinicopathologic significance from molecular understanding of the mechanisms responsible for barrier regulation. The major components of the tight junction, which forms the paracellular barrier, have been identified over the past two decades and fall into three major groups; scaffold proteins, e.g. ZO-1; transmembrane structural proteins, e.g. occludin; and pore-forming proteins, e.g. claudins. How these proteins interact to regulate the barrier is incompletely understood. Thus, tight junction biology is at a crossroads that requires a transition from protein discovery to identification of essential regulatory mechanisms. These must be considered in the context of distinct components of paracellular permeability that define flux of either large solutes or small ions and water. We have recently shown that these aspects of barrier function are differentially regulated by the pathologically-relevant cytokines TNF and IL-13, respectively. The long term goal of these studies is to understand tight junction structure and regulation in molecular terms and to leverage this knowledge to develop approaches to modulate specific barrier components and improve health. The objective of this application is to define the interactions among tight junction components that regulate dynamic protein behavior and barrier function using newly-developed in vitro and in vivo approaches. Our central hypothesis is that the interactions responsible for tight junction protein anchoring and trafficking are the primary determinants of paracellular barrier function. This hypothesis has been formulated on the basis of strong preliminary data produced with support of the current funding cycle. The rationale for this project is that it will provide unprecedented insight into the molecular interactions that regulate barrier function and, in turn, will allow manipulation of these processes for therapeutic benefit. The hypothesis will be tested via specific aims: 1) To define the contributions of specific ZO-1 domains to trafficking, anchoring, and tight junction barrier regulation; 2) To determine the structural elements and phosphorylation events that regulate occludin trafficking, interprotein interactions, and in vitro and in vivo barrier function; and 3) To identify the structural elements and functional interactions by which claudin domains regulate pore assembly and opening. While each aim focuses on a critical tight junction protein or protein family, interactions among these will allow integration and development of a unified model of tight junction structure and regulation. The proposal is innovative because it explores the novel idea that dynamic regulation of protein interactions controls barrier function, which signals a major shift in our understanding of tight junction biology. The proposed research is significant because it will enhance our understanding of barrier dysfunction and link specific mechanisms of barrier loss to disease. The concepts and tools developed will make it possible to develop agents that target distinct barrier components and, ultimately, to treat diseases of epithelial and endothelial barriers.