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细胞间紧密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的机制，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部分由称为E-钙粘蛋白的细胞表面蛋白介导。钙粘蛋白介导的细胞间粘附是由嗜同性相互作用产生的，其中来自一个细胞的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。我们的假设是，在钙粘蛋白-钙粘蛋白相互作用的结合区中发现的肽序列相似，可用于调节E-钙粘蛋白介导的细胞粘附的平衡方式，因此，它们可用于确定由E-钙粘蛋白的细胞间连接形成的机制。此外，来源于E-钙粘蛋白序列的肽可以调节钙粘蛋白-钙粘蛋白相互作用并产生用于细胞旁药物递送的通道。这些拟议的研究将使我们能够了解生物屏障，如肠粘膜和血脑屏障（BBB）的调制机制。为此，将牛脑微血管内皮细胞（BBMEC）、结肠腺癌-2（Caco-2）细胞和Madin-Darby犬肾（MDCK）细胞单层用作BBB、肠粘膜和肾屏障的体外模型;这些细胞在其紧密细胞间连接中具有E-钙粘蛋白。因此，这项工作将集中在比较钙粘蛋白肽的能力和选择性，以调节E-钙粘蛋白介导的细胞-细胞粘附在这些不同类型的细胞。其次，将过量表达E-钙粘蛋白的胞外结构域（EC结构域）蛋白，以阐明E-钙粘蛋白的嗜同性相互作用;本研究还将涉及：a）钙粘蛋白肽和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，和c）钙粘蛋白肽与EC结构域蛋白的结合构象的测定。肽/蛋白质结构将通过NMR、CD和分子建模确定。最后，钙粘蛋白肽将被优化并用于调节细胞间连接，以改善使用体外细胞培养模型的标记分子的细胞旁递送。了解E-cadherin-E-cadherin相互作用的机制对于理解一般生物屏障的完整性以及E-cadherins在维持不同组织（包括肿瘤）中细胞-细胞粘附中的作用可能是至关重要的。

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.