Intestinal Epithelial Tight Junction Structure-Function

肠上皮紧密连接结构-功能

• 批准号: 6524462
• 负责人: ASMA NUSRAT
• 金额: $ 27.78万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524462
• 关键词: avidin; cell line; gastrointestinal epithelium; hepatocyte growth factor; human tissue; inflammatory bowel diseases; interferon gamma; laboratory mouse; membrane permeability; membrane proteins; membrane structure; monoclonal antibody; protein binding; protein structure function; tight junctions

DESCRIPTION (provided by applicant): Inflammatory bowel disease (IBD) is characterized by relapsing intestinal inflammation and altered epithelial permeability resulting in fluid/electrolyte loss and systemic exposure to luminal antigens. Permeability changes have, in turn, been attributed to defective tight junction (TJ) structure/function. Details of epithelial TJ composition and its relationship to gate/fence function are still rudimentary. We have recently shown enrichment TJ proteins in "raft" like membrane microdomains. The major objective of this proposal is to define functionally relevant structural elements in TJs of epithelial cells with the following two specific aims. Specific Aim 1: To identify novel intercellular junction proteins involved in regulation of intestinal epithelial permeability using a monoclonal antibody approach. We have utilized TJ-enriched membrane fractions to generate four monoclonal antibodies that recognize unique epitopes in TJs of epithelial cell lines and native intestinal epithelial cells. The primary focus will be on defining the antigens for these antibodies. Epitope modulation by cytokines (IFN-gamma, HGF) important in inflammation/repair will be determined. Expression of their respective antigens in native normal and inflamed intestinal tissues will be analyzed. Specific Aim 2: To define molecular targets for tight junction proteins key in regulating intestinal epithelial permeability using a bait-peptide approach. We will capture protein components in the TJ complex using novel bait peptides representing segments of the TJ transmembrane protein, occludin. Biotinylated, photoactive bait peptides have been generated to recapitulate: 1) a 27 aa coiled-coil region in the cytoplasmic tail; and 2) 21 aa regions in the first and second extracellular loops. Peptide protein complexes in epithelial cells will be captured using a solid matrix of avidin. Functional consequences of peptide protein binding on TJ gate/fence function will be explored. Information from these studies should reveal important mechanistic insights into the structure/function of TJs and should provide novel insights into potential therapeutic targets for the prevention or correction of epithelial permeability defects associated with IBD.

描述（由申请人提供）： 炎症性肠病（IBD）是 其特征是复发性肠道炎症和上皮细胞改变 渗透性导致液体/电解质损失和全身暴露 管腔抗原。渗透率的变化又归因于 紧密连接 (TJ) 结构/功能有缺陷。上皮TJ详细信息 其组成及其与门/栅栏功能的关系仍处于初级阶段。 我们最近展示了在“筏”状膜中富集 TJ 蛋白 微域。该提案的主要目标是在功能上定义 上皮细胞 TJ 中的相关结构元件具有以下两个 具体目标。具体目标 1：识别新的细胞间连接 参与调节肠上皮通透性的蛋白质 单克隆抗体方法。我们利用了富含 TJ 的膜组分 生成四种单克隆抗体，可识别 TJ 中的独特表位 上皮细胞系和天然肠上皮细胞。主要焦点 将定义这些抗体的抗原。表位调节 将确定对炎症/修复重要的细胞因子（IFN-γ、HGF）。 其各自抗原在天然正常和炎症中的表达 将分析肠道组织。具体目标 2：定义分子 紧密连接蛋白的靶点是调节肠上皮的关键 使用诱饵肽方法测定渗透性。我们将捕获蛋白质成分 在 TJ 复合体中使用代表 TJ 片段的新型诱饵肽 跨膜蛋白，occludin。生物素化的光活性诱饵肽具有 已生成概括：1）27个aa卷曲线圈区域 细胞质尾； 2) 第一和第二细胞外膜中的 21 个氨基酸区域 循环。上皮细胞中的肽蛋白复合物将使用 抗生物素蛋白固体基质。肽蛋白结合的功能后果 将探讨 TJ 门/栅栏功能。来自这些研究的信息应该 揭示对 TJ 结构/功能的重要机制见解 应该为潜在的治疗靶点提供新的见解 预防或纠正与相关的上皮通透性缺陷 炎症性肠病。