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: 10028808
• 负责人: Alex J. Vecchio
• 金额: $ 34.94万
• 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 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10028808
• 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; Epithelium; 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; Proteins; Research; Shapes; Side; Stroke; Structure; Tight Junctions; Tissues; Transport Process; Vertebrates; base; design; experimental study; family structure; 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.

项目概要 内皮细胞和上皮细胞边界处的紧密连接（TJ）对于细胞的发育和发育至关重要。 脊椎动物的功能，因为它们使这些组织能够分离、保护和塑造外表皮 以及四肢、内脏和腺体。 TJs 调节分子之间空间的运输 单个细胞（细胞旁），同时粘附细胞片。 TJ 在组织中执行两项重要功能：1) 形成 限制小分子旁细胞通量的屏障，保护生物体免受外部环境的影响， 分隔内部身体隔室； 2) 形成尺寸和电荷选择性孔，从而实现渗透性 维持电化学梯度的离子。许多蛋白质在 TJ 处聚集形成大分子 屏障和孔隙功能所需的组件。但是膜蛋白的两个家族——密蛋白和 TAMP（TJ 相关 Marvel 蛋白）——主导 TJ 组装、结构和功能。正如这些TJ 整合膜蛋白 (TJIMP) 是跨越细胞内、膜内和细胞膜的唯一成分。 在细胞外空间，它们充当细胞骨架支架并在膜内并排组装（顺式）和 与邻近细胞膜（反式）的 TJIMP 形成屏障和孔。 TJ的分子结构 是动态的。蛋白质组成、相互作用、构象或修饰的变化——对于组装有用 在正常条件下精确调节旁细胞运输的 TJ 也可能被错误组装，从而导致 癌症、阿尔茨海默病、帕金森病、亨廷顿舞蹈病、ALS、中风、食物中毒等病症 炎症性肠病、肾衰竭、肝炎以及皮肤、眼睛和耳朵疾病。分子水平 对 TJ 结构和动态的见解；控制屏障和孔隙功能的组装机制； 以及如何禁用这些机制会导致病态，这在我们的根本问题中仍然是悬而未决的问题 对TJ的理解。我们在此提出一个综合研究计划，该计划利用高度跨学科的方法 确定动态 TJ 下 TJIMP 之间的结构-相互作用-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、生物化学、生物物理和功能学获得的信息 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具体问题来进行拆卸 TJIMP 相互作用网络、细菌毒素破坏肠道屏障的基础及其机制 TJIMP 在血脑屏障中形成并发挥作用。我们实验室的长期目标是阐明 TJ 的构建、破坏和重建的分子基础，可以自然发生或通过 致病机制，并利用所获得的见解来推进新型药物的设计和开发 治疗 TJ 相关疾病的疗法。