Intestinal Epithelial Tight Junction Structure-Function

描述(申请人提供)：炎症性肠病(IBD) 以复发性肠炎和上皮细胞改变为特征 渗透性导致液体/电解质流失和全身暴露于 管腔抗原。渗透率的变化反过来又被归因于 紧密连接(TJ)结构/功能有缺陷。上皮性TJ的细节 其组成及其与闸门/栅栏功能的关系仍处于初级阶段。 我们最近发现TJ蛋白富含在“筏”状的膜上。 微域。这项提案的主要目标是在功能上定义 上皮细胞TJ中的相关结构元素 明确的目标。具体目标1：确定新的细胞间连接 参与调节肠上皮通透性的蛋白质 单抗方法。我们已经利用了富含TJ的膜馏分 产生四种可识别鸡TJ独特表位的单抗 上皮细胞系和天然肠上皮细胞。主要关注点 将致力于定义这些抗体的抗原。表位调控方式 将确定在炎症/修复中起重要作用的细胞因子(干扰素-γ、肝细胞生长因子)。 它们各自的抗原在正常和炎症性疾病中的表达 将对肠道组织进行分析。具体目标2：定义分子 紧密连接蛋白在肠道上皮细胞调控中的作用靶点 使用诱饵-多肽方法的渗透性。我们将捕获蛋白质组分 在TJ复合体中使用代表TJ片段的新型诱饵肽 跨膜蛋白，封闭蛋白。生物素化的光活性诱饵多肽 简而言之：1)一个27AA的盘绕线圈区域 胞质尾部；2)第一和第二胞外21个氨基酸区域 循环。上皮细胞中的多肽蛋白质复合体将使用 亲和素的固体骨架。多肽蛋白结合的功能后果 将探索TJ门/栅栏的功能。来自这些研究的信息应该 揭示对TJ和TJ的结构/功能的重要机械见解 应该为治疗的潜在靶点提供新的见解 预防或纠正与以下相关的上皮通透性缺陷 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.