Structural and Functional Investigation of Tight Junction Membrane Proteins

描述（由申请人提供）：上皮细胞边界处的紧密连接对多细胞生物体中大多数组织的发育和功能至关重要，因为它们使上皮细胞能够在内部器官和腺体的分离、保护和成形中发挥作用。这些紧密连接充当物理和化学“屏障”，也充当“栅栏”，介导调节体液组成的大分子、溶质和离子的差异转运。紧密连接由几组蛋白质组成，但三类紧密连接整合膜蛋白（TJIMPs）：闭合蛋白（occludin）、密蛋白（claudins）和三纤维素（tricellulin）被认为在其结构和功能中起主导作用。TJIMP的破坏涉及几种人类疾病，例如肝炎和癌症，以及肾衰竭病症、眼部疾病和耳聋。紧密连接屏障功能可以提供操纵药物转运的靶点。但TJIMPs的功能仍然知之甚少。我们假设，选择域的TJIMPs决定“屏障”和“围栏”的功能，并在各种上皮细胞的紧密连接的多样性是由TJIMPs，构成他们。该提案旨在通过以下方式在分子水平上详细了解TJIMP的结构和功能：目的1：确定一种或多种选定的TJIMP的晶体结构，目的2：检查TJIMP的生理功能。生物信息学将与高通量克隆、表达和蛋白质分析技术相结合，以选择一个或多个具有成功X射线晶体结构测定的最佳概率的靶点。功能 分析将采用选择性诱变、细胞粘附测定、脂质标记策略、蛋白质定位、冷冻断裂电子显微镜和电生理学方法来评估原位“屏障”和“栅栏”功能。这项研究对于理解与TJIMPs破坏相关的疾病具有广泛的意义，并为治疗提供靶点，以及促进对药物转运的理解。

DESCRIPTION (provided by applicant): The tight junctions at the boundaries of epithelial cells are of critical importance to the development and function of most tissues in multicellular organisms because they enable epithelia to work in the separation, protection, and shaping of internal organs and glands. These tight junctions act as physical and chemical "barriers" and also as "fences", mediating differential transport of macromolecules, solutes, and ions that regulate body fluid composition. Tight junctions are composed of several groups of proteins, but three classes of tight junction integral membrane proteins (TJIMPs): occludin, claudins, and tricellulin; are thought to play a leading role in their architecture and function. Disruptions of TJIMPs are implicated in several human diseases, such as hepatitis, and cancer, as well as renal wasting disorders, ocular disease, and deafness. Tight junction barrier function may provide targets for manipulating drug transport. But the function of TJIMPs remains poorly understood. We hypothesize that select domains of TJIMPs dictate "barrier" and "fence" function, and that tight junction diversity in various epithelia is governed by the TJIMPs that constitute them. This proposal aims to understand TJIMP structure and function in molecular-level detail by: Aim 1: determining the crystal structures of one or more selected TJIMPs and, Aim 2: examining the physiological function(s) of TJIMPs. Bioinformatics will be coupled to high-throughput cloning, expression, and protein analysis technologies to select one or more targets with the best probability for successful X-ray crystallographic structure determination. Functional analysis will employ selective mutagenesis, cell adhesion assays, lipid-labeling strategies, protein localization, freeze-fracture electron microscopy, and electrophysiological means to assess "barrier" and "fence" function in situ. This research has widespread significance for understanding diseases related to the disruption of TJIMPs and provide targets for therapeutics, as well as promoting understanding of drug transport.