Intestinal Epithelial Tight Junction Structure-Function

描述（由申请人提供）：炎症性肠病（IBD）是 其特征在于复发性肠道炎症和改变的上皮细胞 渗透性导致液体/电解质损失和全身暴露于 管腔抗原渗透率的变化反过来又归因于 紧密连接（TJ）结构/功能缺陷。上皮TJ的详细信息 组成及其与门/栅栏功能的关系仍然是初步的。 我们最近发现富集TJ蛋白在“筏”样膜 微域本提案的主要目标是从功能上界定 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：鉴定新的细胞间连接 使用免疫组织化学方法研究参与调节肠上皮通透性的蛋白质 单克隆抗体方法。我们已经利用TJ富集的膜组分 以产生四种单克隆抗体，其识别TJS中的独特表位， 上皮细胞系和天然肠上皮细胞。主要重点 将是确定这些抗体的抗原。表位调节 将确定在炎症/修复中重要的细胞因子（IFN-γ，HGF）。 它们各自的抗原在天然正常和炎症中的表达 将分析肠组织。具体目标2：定义分子 紧密连接蛋白靶点在调节肠上皮细胞中起关键作用 使用诱饵-肽方法测定渗透性。我们将捕获蛋白质成分 在TJ复合物中使用代表TJ片段的新诱饵肽 跨膜蛋白，闭合蛋白。生物素化的光敏诱饵肽具有 已产生概括：1）27个氨基酸的卷曲螺旋区，在 2）第一和第二细胞外尾区中的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。肽蛋白结合在 将探索TJ门/栅栏功能。这些研究的信息应 揭示了对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， 预防或纠正与以下疾病相关的上皮渗透性缺陷 IBD。

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.