Structural studies of tight junction proteins

紧密连接蛋白的结构研究

• 批准号: 8439261
• 负责人: John M Flanagan
• 金额: $ 44.7万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8439261
• 关键词: Alzheimer' s Disease; Area; Asthma; Binding; Biochemical; Biological; Biological Assay; Biology; Blood; Blood-Retinal Barrier; Brain; Brain Neoplasms; Cell physiology; Cells; Cellular Structures; Coiled-Coil Domain; Collaborations; Complex; Cytoplasmic Tail; Data; Disease; Drug Delivery Systems; Endothelial Cells; Epithelial; Epithelial Cells; Extracellular Fluid; Family; Functional disorder; Head; Health; Herpes zoster disease; Human; Hybrids; Individual; Infection; Ions; Irritable Bowel Syndrome; Kidney Diseases; Knowledge; Lead; Length; Lung; Malignant Neoplasms; Medicine; Membrane; Membrane Proteins; Methods; Michigan; Modeling; Molecular; N-terminal; Pennsylvania; Permeability; Pharmaceutical Preparations; Phosphorylation; Physiological; Physiology; Property; Protein Binding; Proteins; Protocols documentation; Publishing; Regulation; Regulatory Element; Research; Resolution; Retina; Retinal Diseases; Role; Serine; Site; Stroke; Structure; Surface; Techniques; Testing; Therapeutic; Therapeutic Agents; Tight Junctions; Tissues; Tyrosine; Universities; Vascular Dementia; Work; X-Ray Crystallography; apical membrane; base; basolateral membrane; college; disorder prevention; fluid flow; gastrointestinal epithelium; human disease; improved; in vivo; insight; novel; novel strategies; novel therapeutics; occludin; pathogen; public health relevance; receptor; research study; response; solute; tool; treatment strategy; uptake

DESCRIPTION (provided by applicant): Tight junctions (TJs) are essential cellular structures that form the selective paracellular barriers in endothelial and epithelial cells, including the blood-brain, and blood-retinal barriers, lung and gut epithelium. The barrier properties of TJs regulate metabolite flux, fluid flow and drug uptake into these tissues. Dysfunction in TJ barrier properties is observed in a large number of human diseases including cancer (e.g. brain tumors), stroke, retinopathies, kidney disorders, irritable bowel syndrome, Alzheimer's disease and vascular dementia, and asthma. Moreover, TJ components are used as cellular receptors by a range of pathogens. Thus, an improved understanding of the basic structural and functional biology of TJs will have wide ranging impact for human health. Specifically, this information may lead to improved drug delivery methods, novel protocols for regulating TJ permeability in disease and prevention of some infections. Currently, little is known about the molecular basis for TJ barrier properties and how they are regulated. In this proposal, we describe a combined structural, biochemical and cellular and molecular approach to illuminate the function of occludin (Occ), a transmembrane component of TJs, which has been implicated in the regulation of TJ barrier properties. These studies build upon our previously published and unpublished data implicating Occ, and its binding partner zona occludens 1 (ZO-1) protein, in regulating barrier properties conferred by TJs. The planned studies are organized into three specific aims. In Aim1, experiments are described, using a cell biological approach to determine the molecular mechanism(s) for the effects of phosphorylation of Serine 471 in Occ (S471) on TJ cellular structure function. In Aim2, we describe experiments to determine the structural details of the ZO-1/Occ protein-binding core, which includes S471, and with their functional binding complexes In Aim 3, we extend these approaches to determine the contribution of S471 phosphorylation of Occ to its interaction with ZO-1 using full length Occ and dimeric ZO-1 constructs. Together, the results of these three aims will provide a structural and biochemical basis for understanding Occ function and starting point for developing novel strategies for modulating TJ barrier properties that target the ZO-1/Occ complex.

描述（由申请人提供）：紧密连接（TJ）是重要的细胞结构，在内皮和上皮细胞中形成选择性细胞旁屏障，包括血脑、血视网膜屏障、肺和肠上皮。 TJ 的屏障特性可调节这些组织的代谢通量、液体流动和药物摄取。在许多人类疾病中都观察到 TJ 屏障特性功能障碍，包括癌症（例如脑肿瘤）、中风、视网膜病、肾脏疾病、肠易激综合征、阿尔茨海默病、血管性痴呆和哮喘。此外，TJ 成分被多种病原体用作细胞受体。因此，更好地了解 TJ 的基本结构和功能生物学将对人类健康产生广泛的影响。具体来说，这些信息可能会导致药物输送方法的改进、调节疾病中 TJ 通透性和预防某些感染的新方案。目前，人们对 TJ 屏障特性的分子基础及其调控方式知之甚少。在本提案中，我们描述了一种结合结构、生化、细胞和分子的方法来阐明 Occludin (Occ) 的功能，occludin (Occ) 是 TJ 的跨膜成分，与 TJ 屏障特性的调节有关。这些研究建立在我们之前发表和未发表的数据基础上，这些数据涉及 Occ 及其结合伴侣闭塞带 1 (ZO-1) 蛋白在调节 TJ 赋予的屏障特性方面的作用。计划中的研究分为三个具体目标。在 Aim1 中，描述了使用细胞生物学方法确定 Occ (S471) 中丝氨酸 471 磷酸化对 TJ 细胞结构功能影响的分子机制的实验。在 Aim2 中，我们描述了确定 ZO-1/Occ 蛋白结合核心（包括 S471 及其功能性结合复合物）结构细节的实验。在 Aim 3 中，我们扩展这些方法，使用全长 Occ 和二聚 ZO-1 构建体来确定 Occ S471 磷酸化对其与 ZO-1 相互作用的贡献。这三个目标的结果将为理解 Occ 功能提供结构和生化基础，并为开发针对 ZO-1/Occ 复合物的调节 TJ 屏障特性的新策略提供起点。