Modulation of the Intercellular Junction Cadherins

该项目的长期目标是了解如何通过调节介导细胞间连接的蛋白质相互作用来调节紧密的细胞间连接以改善药物递送。本提案的短期目标是了解E-钙粘蛋白介导的细胞间连接机制，并评估钙粘蛋白肽调节钙粘蛋白相互作用以改善细胞旁药物递送的能力。紧密的细胞间连接至少在一定程度上是由称为e -钙粘蛋白的细胞表面蛋白介导的。钙粘蛋白介导的细胞-细胞粘附是由来自一个细胞的e -钙粘蛋白分子与来自另一个细胞的e -钙粘蛋白分子相互作用产生的。我们的假设是，与钙粘蛋白-钙粘蛋白相互作用结合区域中发现的肽序列相似，可用于以平衡方式调节e -钙粘蛋白介导的细胞粘附；因此，它们可用于鉴定e -钙粘蛋白形成细胞间连接的机制。此外，来自e -钙粘蛋白序列的肽可能调节钙粘蛋白-钙粘蛋白相互作用，并为细胞旁药物递送创造通道。这些建议的研究将使我们了解肠粘膜和血脑屏障（BBB）等生物屏障的调节机制。为此，单层牛脑微血管内皮细胞（BBMECs）、结肠腺癌-2 （Caco-2）细胞和Madin-Darby犬肾（MDCK）细胞将被用作血脑屏障、肠黏膜和肾脏屏障的体外模型；这些细胞紧密的细胞间连接处有e -钙粘蛋白。因此，本研究将重点比较钙粘蛋白肽在这些不同类型细胞中调节e -钙粘蛋白介导的细胞-细胞粘附的能力和选择性。其次，E-cadherin的细胞外结构域（EC domain）蛋白会过度表达，以阐明E-cadherin的亲同性相互作用；本研究还将涉及：a)钙粘蛋白肽与ec结构域蛋白之间的平衡结合研究，b) ec结构域蛋白的结构研究，以及c)钙粘蛋白肽与ec结构域蛋白结合构象的确定。肽/蛋白质的结构将通过核磁共振、CD和分子模型来确定。最后，在体外细胞培养模型中，将优化钙粘蛋白肽，并将其用于调节细胞间连接，以改善标记分子的细胞旁递送。了解e -钙粘蛋白- e -钙粘蛋白相互作用的机制对于理解生物屏障的完整性以及e -钙粘蛋白在包括肿瘤在内的不同组织中维持细胞-细胞粘附的作用至关重要。

The long-term objective of this project is to understand how to modulate tight intercellular junctions for improving drug delivery by regulating protein interactions that mediate the intercellular junctions. The short- term objectives of this proposal are to understand the mechanisms of E- cadherin-mediated intercellular junctions and to evaluate the ability of cadherin peptides to modulate cadherin interactions for improving paracellular drug delivery. Tight intercellular junctions are mediated, at least in part, by cell surface proteins called E-cadherins. Cadherin- mediated cell-cell adhesion is produced by homophilic interactions in which E-cadherin molecules from one cell interact with E-cadherin molecules from another cell. Our hypothesis is that peptide sequences similar to those found in the binding region of cadherin-cadherin interactions can be used to modulate E-cadherin-mediated cell adhesion in an equilibrium fashion; thus, they can be used to identify the mechanisms of intercellular junction formation by E-cadherins. Furthermore, peptides derived from E-cadherin sequence may modulate cadherin-cadherin interactions and create channels for paracellular drug delivery. These proposed studies would allow us to understand the mechanisms of modulation of biological barriers such as the intestinal mucosa and the blood-brain barrier (BBB). For this purpose, monolayers of bovine-brain microvessel endothelial cells (BBMECs), colon adenocarcinoma-2 (Caco-2) cells, and Madin-Darby Canine Kidney (MDCK) cells will be used as in vitro models for the BBB, intestinal mucosa, and kidney barriers; these cells have E-cadherins in their tight intercellular junctions. Therefore, this work will be focused on comparing the ability and selectivity of cadherin peptides to regulate the E-cadherin mediated cell-cell adhesion in these different types of cells. Secondly, the extracellular domain (EC domain) proteins from E- cadherin will be overexpressed to elucidate the homophilic interaction of E-cadherins; this study will also involve: a) equilibrium binding studies between cadherin peptides and EC-domain proteins, b) structural studies of the EC-domain proteins, and c) determination of the bound conformational of cadherin peptides to EC-domain protein. The peptide/protein structures will be determined by NMR, CD, and molecular modeling. Finally, cadherin peptides will be optimized and used to modulate intercellular junctions for improving paracellular delivery of marker molecules using in vitro cell culture models. Understanding the mechanisms of E-cadherin-E-cadherin interactions may be essential for understanding the integrity of biological barriers in general and the role of E-cadherins in maintaining cell-cell adhesion in different tissues, including tumors.