Structure and assembly of membrane proteins at tight junctions

项目摘要 内皮细胞和上皮细胞边界处的紧密连接（TJ）在内皮细胞和上皮细胞的发育和分化中是至关重要的。 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 四肢内脏和腺体TJ通过间隙调节分子运输 单个细胞（细胞旁），同时粘附细胞片。TJ在组织中执行两个重要功能：1）形成 限制小分子的细胞旁通量的屏障，保护生物体免受外部环境的影响， 分离体内隔室;和2）产生大小和电荷选择性孔，允许渗透性 维持电化学梯度的离子许多蛋白质聚集在TJ处形成大分子 这些组件是屏障和孔隙功能所必需的。但是两个膜蛋白家族--claudins和 TAMPs（TJ相关的奇迹蛋白）-主导TJ组装，结构和功能。因为这些TJ 整合膜蛋白（TJIMPs）是跨越细胞内、膜内和膜外的唯一组分。 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）， 与来自相邻细胞膜（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-对组装有用 在正常条件下精确调节细胞旁转运的TJ也可能被错误组装，导致 病理学如癌症、阿尔茨海默病、帕金森病、亨廷顿病、ALS、中风、食物中毒和 炎症性肠病、肾衰竭、肝炎以及皮肤、眼睛和耳朵的疾病。分子水平 深入了解TJ结构和动力学;控制屏障和孔隙功能的组装机制; 以及如何禁用这些机制导致病理，仍然是我们的基本问题， 理解TJ。我们在这里提出了一个全面的研究计划，使用高度跨学科的 确定动态TJ下TJIMP之间结构-相互作用-功能关系的方法 微环境这些方法整合了TJIMP及其复合物的结构生物学， 通过传统和最先进的生物信息学、生物化学、生物物理学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， 通过面对技术挑战，并在近期内，通过回答有关 TJIMP相互作用网络是细菌毒素破坏肠道屏障的基础， TJIMP在血脑屏障中形成和发挥作用。我们实验室的长期目标是阐明 自然发生或通过其他途径发生的TJ的构建、破坏和重建的分子基础 致病机制，并利用所取得的见解，以推进设计和开发新的 治疗TJ相关疾病的药物

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.