Structural and Functional Investigation of Tight Junction Membrane Proteins

紧密连接膜蛋白的结构和功能研究

基本信息

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
项目类别：
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

项目摘要

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发挥内部器官和腺体的分离、保护和塑造作用。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，但紧密连接整合膜蛋白 (TJIMP) 分为三类：occludin、claudins 和 tricellulin；被认为在其架构和功能中发挥主导作用。 TJIMP 的破坏与多种人类疾病有关，例如肝炎、癌症、肾衰竭、眼部疾病和耳聋。紧密连接屏障功能可能为操纵药物转运提供靶点。但 TJIMP 的功能仍然知之甚少。我们假设 TJIMP 的特定结构域决定了“屏障”和“栅栏”功能，并且各种上皮细胞中的紧密连接多样性由构成它们的 TJIMP 控制。该提案旨在通过以下方式详细了解 TJIMP 的结构和功能：目标 1：确定一种或多种选定的 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，以选择最有可能成功确定 X 射线晶体结构的一个或多个目标。功能性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手段来评估原位“屏障”和“栅栏”功能。这项研究对于了解与 TJIMP 破坏相关的疾病、提供治疗靶点以及促进对药物转运的了解具有广泛的意义。