Structure and assembly of membrane proteins at tight junctions

紧密连接处膜蛋白的结构和组装

• 批准号: 10459311
• 负责人: Alex J. Vecchio
• 金额: $ 36.22万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10459311
• 关键词: Alzheimer' s Disease; Architecture; Bacterial Toxins; Biochemical; Bioinformatics; Biophysics; Blood - brain barrier anatomy; Cell Adhesion; Cell membrane; Cells; Charge; Complex; Development; Disease; Ear Diseases; Endothelial Cells; Endothelium; Environment; Epidermis; Epithelial; Epithelial Cells; Extracellular Space; Eye diseases; Family; Food Poisoning; Functional disorder; Gland; Goals; Hepatitis; Human; Huntington Disease; Individual; Inflammatory Bowel Diseases; Integral Membrane Protein; Ions; Kidney; Knowledge; Laboratories; Limb structure; Link; Malignant Neoplasms; Membrane; Membrane Proteins; Modification; Molecular; Molecular Conformation; Molecular Disease; Molecular Structure; Organ; Organism; Parkinson Disease; Pathology; Permeability; Protein Family; Proteins; Research; Shapes; Side; Stroke; Structure; Tight Junctions; Tissues; Transport Process; Vertebrates; base; design; experimental study; insight; interdisciplinary approach; macromolecular assembly; membrane assembly; novel therapeutics; programs; reconstruction; scaffold; skin disorder; small molecule; structural biology; wasting

Project Summary Tight junctions (TJs) at the boundaries of endothelial and epithelial cells are critical in the development and function of vertebrates because they enable these tissues to separate, protect, and shape external epidermis and limbs and internal organs and glands. TJs regulate molecular transport through the spaces between individual cells (paracellular) while adhering cellular sheets. TJs perform two vital functions in tissues: 1) form barriers to restrict paracellular flux of small molecules, protecting organisms from the external environment and separating internal body compartments; and 2) creating size- and charge-selective pores, allowing permeability of ions that maintain electrochemical gradients. Numerous proteins amass at TJs to form the macromolecular assemblies necessary for barrier and pore function. But two families of membrane proteins—claudins and TAMPs (TJ-associated Marvel proteins)—predominate TJ assembly, architecture, and function. As these TJ integral membrane proteins (TJIMPs) are the sole components to span intracellular, intramembraneous, and extracellular space, they act as cytoskeletal scaffolds and assemble side-by-side within a membrane (cis) and with TJIMPs from adjacent cell membranes (trans) to form barriers and pores. The molecular structure of TJs is dynamic. Changes in protein composition, interaction, conformation, or modification—useful for assembling TJs to precisely tune paracellular transport under normal conditions—can also be mis-assembled, resulting in pathologies such as cancer, Alzheimer’s, Parkinson’s, Huntington’s, ALS, stroke, food poisoning and inflammatory bowel disease, renal wasting, hepatitis, and diseases of the skin, eyes, and ears. Molecular level insights into TJ structure and dynamics; the mechanisms of assembly that govern barrier and pore function; and how disabling these mechanisms leads to pathologies, remain unresolved matters in our fundamental understanding of TJs. We propose here a comprehensive research program that uses highly interdisciplinary approaches to determine structure–interaction–function relationships between TJIMPs at dynamic TJ microenvironments. These approaches integrate structural biology of TJIMPs and their complexes with information obtained by traditional and state-of-the-art bioinformatics, biochemical, biophysical, and functional experiments. The research program intends to resolve the underlying molecular principles of TJ assembly and disassembly by confronting technical challenges and, in the near-term, by answering specific questions on TJIMP interaction networks, the basis of gut barrier breakdown by a bacterial toxin, and the mechanisms of TJIMP form and function at the blood-brain barrier. The long-term goal of our laboratory is to elucidate the molecular bases for construction, destruction, and reconstruction of TJs, occurring both naturally or via disease-causing mechanisms, and to use the achieved insights to advance design and development of novel therapeutics to remedy TJ-related ailments.

项目摘要 内皮细胞和上皮细胞边界处的紧密连接（TJ）在发育中至关重要 脊椎动物的功能是因为它们可以使这些时间分离，保护和塑造外部表皮 以及四肢，内脏和腺体。 TJS调节通过空间之间的分子传输 在粘附细胞片的同时，单个细胞（细胞细胞）。 TJ在组织中执行两个重要功能：1）形式 限制小分子细胞细胞通量的障碍，保护生物免受外部环境和 分离内部体内室； 2）创建尺寸和电荷选择性孔，允许渗透性 维持电化学梯度的离子。 TJ在TJ中积聚的许多蛋白质形成大分子 屏障和孔功能所需的组件。但是两个膜蛋白的家族 - 丙他蛋白和 TAMP（与TJ相关的漫威蛋白） - 预先构成TJ组装，体系结构和功能。如这些TJ 整体膜蛋白（TJIMP）是跨细胞内，内膜和的唯一成分 细胞外空间，它们充当细胞骨架支架，并在膜（顺式）中并排组装 来自相邻细胞膜（反式）的TJIMP形成屏障和毛孔。 TJ的分子结构 是动态的。蛋白质成分，相互作用，构象或修饰的变化 - 用于组装 在正常条件下，TJ到精确调节细胞细胞运输 - 也可能会被错误组装，从而导致 癌症，阿尔茨海默氏症，帕金森氏症，亨廷顿，ALS，中风，食物中毒和等病理 炎症性肠病，肾脏浪费，肝炎以及皮肤，眼睛和耳朵的疾病。分子水平 对TJ结构和动态的见解；支配屏障和孔功能的组装机制； 以及这些机制如何导致病理学，在我们的基本方面仍未解决问题 对TJ的理解。我们在这里提出了一项全面的研究计划，该计划使用高度跨学科的研究 确定动态TJ处TJIMP之间的结构 - 互动 - 功能关系的方法 微环境。这些方法将TJIMP及其复合物的结构生物学与 通过传统和最先进的生物信息学，生化，生物物理和功能性获得的信息 实验。该研究计划旨在解决TJ组装的基本分子原理和 通过面对技术挑战，并在短期内回答有关特定问题的拆卸 TJIMP相互作用网络，细菌毒素的肠道障碍分解的基础，以及 血脑屏障的TJIMP形式和功能。我们实验室的长期目标是阐明 TJ的构建，破坏和重建的分子碱基，自然或通过 引起疾病的机制，并利用实现的见解来推进新颖的设计和开发 记住与TJ相关的疾病的治疗剂。