Structural and Functional Investigation of Tight Junction Membrane Proteins

紧密连接膜蛋白的结构和功能研究

• 批准号: 8565650
• 负责人: Alex J. Vecchio
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8565650
• 关键词: Algorithms; Apical; Architecture; Behavior; Binding; Biochemical; Bioinformatics; Biological; Biological Assay; Biology; Body Fluids; Cell Adhesion; Cell membrane; Cells; Characteristics; Chemicals; Chordata; Clostridium enterotoxin; Complex; Coupled; Cytoskeleton; Data; Development; Diffusion; Disease; Docking; Drug Delivery Systems; Drug Transport; Electron Microscopy; Epithelial; Epithelial Cells; Epithelium; Extracellular Domain; Extracellular Matrix; Extracellular Space; Freeze Fracturing; Gland; Goals; Hepatitis; Human body; In Situ; Individual; Integral Membrane Protein; Intercellular Junctions; Investigation; Ions; Kidney; Knowledge; Label; Link; Lipids; Malignant Neoplasms; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Conformation; Mutagenesis; Organ; Organism; Orthologous Gene; Permeability; Physiological; Physiology; Play; Probability; Property; Protein Analysis; Proteins; Research; Roentgen Rays; Role; Scaffolding Protein; Shapes; Signaling Protein; Structure; Surface; System; Technology; Tertiary Protein Structure; Testing; Tight Junctions; Tissues; Transmembrane Domain; Transport Process; Work; X-Ray Crystallography; absorption; base; computer generated; deafness; expression cloning; human disease; insight; macromolecule; mutant; novel therapeutics; occludin; protein function; protein structure; protein structure function; screening; solute; structural biology; therapeutic target; three dimensional structure; wasting

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.

描述（由申请人提供）：上皮细胞边界处的紧密连接对于多大多数细胞生物中大多数组织的发育和功能至关重要，因为它们使上皮能够在内部器官和腺体的分离，保护和塑造方面起作用。这些紧密的连接充当物理和化学的“障碍”，也充当“栅栏”，介导了调节体液组成的大分子，溶质和离子的差分运输。紧密连接由几组蛋白质组成，但三类紧密的连接整体膜蛋白（TJIMPS）：occludin，claudins和Tricellulin;人们认为在其建筑和功能中起着主导作用。 TJIMP的破坏与多种人类疾病有关，例如肝炎和癌症，以及肾脏浪费障碍，眼部疾病和耳聋。紧密的连接屏障功能可以为操纵药物运输提供目标。但是Tjimps的功能仍然很少理解。我们假设TJIMP的精选域决定了“障碍”和“围栏”功能，并且各种上皮的紧密连接多样性受构成它们的TJIMP的控制。该建议旨在通过分子级的细节来理解TJIMP结构和功能：AIM 1：确定一个或多个选定的TJIMP的晶体结构，以及AIM 2：检查TJIMPS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耦合，以选择一个或多个目标，具有成功X射线晶体学结构测定的最佳概率。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，冻结裂缝电子显微镜以及电生理手段来评估“障碍”和“围栏”功能。这项研究对于理解与TJIMP的破坏相关的疾病并提供了治疗剂的靶标以及促进对药物运输的理解，具有广泛的意义。