Intestinal Epithelial Tight Junction Structure-Function

描述（由申请方提供）：炎症性肠病（IBD）的特征为复发性肠道炎症和上皮通透性改变，导致液体/电解质损失和全身暴露于管腔抗原。渗透性变化归因于紧密连接（TJ）功能缺陷。TJ与关节下粘附连接密切相关，它们被统称为顶端连接复合体（AJC）。反过来，AJC与下面的桥粒非常接近。这些细胞间连接的功能协调是适当的细胞-细胞粘附和调节细胞旁通透性所必需的。该提案的主要目标是确定AJC中的相关结构要素，确定其关联并分析其细胞内贩运。具体目标1将确定和表征膜筏相关的细胞间连接蛋白的重要调节细胞间粘附和细胞旁通透性。AJC富集的膜筏用于产生识别肠上皮细胞细胞间连接中的独特表位的单克隆抗体。鉴定了三种候选单克隆抗体。我们确定了这些抗原之一的身份作为一种新的截短的钙粘蛋白变体，称为桥粒芯糖蛋白2（Dsg 2）。我们将阐明Dsg 2/tDs 2在调节肠上皮细胞间联系和AJC功能中的作用。其他两种抗原的鉴别将在未来的研究中确定。具体目标2将定义AJC蛋白所利用的细胞内运输途径。我们将进一步探索我们的初步研究，表明上皮细胞暴露于促炎细胞因子IFN γ诱导的AJC分解和屏障破坏与通过巨胞饮作用选择性内吞关键AJC蛋白有关。IFN γ诱导的AJC蛋白的细胞内运输将被进一步研究。具体目标3将探索密蛋白的胞外结构域在调节细胞旁通透性中的作用。研究将集中于使用生物素化和光敏诱饵肽模拟不同的细胞外结构域的密蛋白。肽-蛋白质复合物将被分离和分析。将检查与紧密连接蛋白胞外结构域的肽缔合对AJC结构和细胞旁通透性的功能后果。来自这些研究的信息将提供对AJC结构/功能的重要机制见解，并应提供对IBD中渗透性增加的校正的潜在治疗靶点的新见解。此外，了解AJCs的分子组成也将有助于设计可以通过细胞旁途径递送的分子。

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 been attributed to defective tight junction (TJ) function. The TJ is intimately affiliated with a subjacent adherens junction and they have been collectively referred to as the Apical Junctional Complex (AJC). The AJC, in turn, is in close proximity to underlying desmosomes. Functional orchestration of these intercellular junctions is required for appropriate cell-cell adhesion and regulation of paracellular permeability. The major objectives of this proposal are to identify relevant structural elements in the AJC, define their associations and analyze their intracellular trafficking. Specific Aim 1 will identify and characterize membrane raft associated intercellular junction proteins important in regulating cell-cell adhesion and paracellular permeability. AJC enriched membrane rafts were used to generate monoclonal antibodies that recognize unique epitopes in intercellular junctions of intestinal epithelial cells. Three candidate monoclonal antibodies were identified. We established identitity of one of these antigens as a novel truncated variant of a cadherin, referred to as desmoglein 2 (Dsg2). We will elucidate the role of Dsg2/tDs2 in regulating intestinal epithelial intercellular association and AJC function. Identity of the other two antigens will be established in future studies. Specific Aim 2 will define intracellular trafficking pathways utilized by AJC proteins. We will further explore our initial studies suggesting that AJC disassembly & barrier disruption induced by exposure of epithelial cells to the proinflammatory cytokine, IFNgamma is associated with select endocytosis of key AJC proteins by macropinocytosis. IFNgamma induced intracellular trafficking of AJC proteins will be further explored. Specific Aim 3 will explore the role of extracellular domains of claudin(s) in regulating paracellular permeability. Studies will focus on using biotinylated and photoactive bait peptides emulating distinct extracellular domains of claudin(s). Peptide- Protein complexes will be isolated and analyzed. Functional consequences of peptide association with claudin(s) extracellular domains on AJC structure and paracellular permeability will be examined. Information from these studies will provide important mechanistic insight into AJC structure/function and should provide novel insights into potential therapeutic targets for correction of increased permeability in IBD. Additionally, understanding the molecular composition of AJCs will also facilitate in designing molecules that can be delivered via the paracellular route.