Structure and assembly of membrane proteins at tight junctions

项目摘要 内皮细胞和上皮细胞交界处的紧密连接(TJ)在发育和发育过程中起着至关重要的作用。 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。TJS通过分子间的间隙调节分子的运输 单个细胞(细胞旁)，同时粘贴细胞膜。TJS在组织中执行两个重要功能：1)形式 限制小分子细胞旁流动的屏障，保护生物体免受外部环境和 分离身体内部隔间；以及2)产生对大小和电荷有选择性的孔，允许渗透性 保持电化学梯度的离子。大量蛋白质聚集在TJ形成大分子 屏障和毛孔功能所需的组件。但有两个膜蛋白家族--Claudins和 TAMPS(TJ相关漫威蛋白)-主导TJ的组装、结构和功能。因为这些TJ 完整的膜蛋白(TJIMPs)是跨越细胞内、膜内和细胞内的唯一成分。 细胞外空间，它们充当细胞骨架支架，在膜内并排组装(顺式)和 与来自相邻细胞膜的TJIMP(反式)形成屏障和孔。TJS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--对组装很有用 TJS在正常情况下精确调节细胞旁运输-也可能被错误组装，导致 癌症、阿尔茨海默氏症、帕金森氏症、亨廷顿氏症、肌萎缩侧索硬化症、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 对TJ结构和动力学的洞察；控制屏障和孔功能的组装机制； 以及禁用这些机制如何导致病理，在我们的根本问题上仍然是悬而未决的问题 对TJ的理解。我们在这里提出了一个综合的研究计划，它使用了高度跨学科的 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。这些方法结合了TJIMP及其与TJIMP络合物的结构生物学 通过传统的和最新的生物信息学、生化、生物物理和功能 实验。该研究计划旨在解决TJ组装和TJ组装的潜在分子原理 通过面对技术挑战进行拆卸，并在短期内回答关于 TJIMP相互作用网络，细菌毒素破坏肠道屏障的基础，以及TJIMP的机制 TJIMP在血脑屏障的形态和功能。我们实验室的长期目标是阐明 TJ的构建、破坏和重建的分子基础，自然或通过 致病机制，并利用已有的见解来推进新书的设计和开发 治疗TJ相关疾病的治疗学。

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.