Structural and Functional Investigation of Tight Junction Membrane Proteins

描述（由申请人提供）：上皮细胞边界的紧密连接对多细胞生物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、保护和形成中发挥作用。这些紧密连接充当物理和化学“屏障”，也充当“栅栏”，介导调节体液成分的大分子、溶质和离子的差异运输。紧密连接由几组蛋白质组成，但有三类紧密连接积分膜蛋白（TJIMPs）： occludin、claudin和tricellulin；被认为在它们的结构和功能中起着主导作用。TJIMPs的破坏与几种人类疾病有关，如肝炎和癌症，以及肾耗损疾病、眼病和耳聋。紧密结屏障功能可能为操纵药物转运提供靶点。但是人们对TJIMPs的功能仍然知之甚少。我们假设TJIMPs的选择结构域决定了“屏障”和“栅栏”功能，并且各种上皮中的紧密连接多样性由构成它们的TJIMPs控制。本研究旨在通过以下方法在分子水平上详细了解TJIMP的结构和功能：目标1：确定一个或多个选定的TJIMP的晶体结构；目标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，选择一个或多个具有最佳概率的目标，以成功确定x射线晶体结构。功能

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.