INTERCADHERIN INTERACTIONS IN DESMOSOMES

描述（由申请人提供）：经典钙粘蛋白和桥粒钙粘蛋白是上皮细胞普遍存在的跨膜粘附受体。虽然在理解经典钙粘蛋白粘附方面取得了重大进展，但桥粒钙粘蛋白的粘附机制研究甚少。两个因素-桥粒钙粘蛋白组成的复杂性和桥粒的不溶性-阻碍了直接检查这些结构中的蛋白质-蛋白质相互作用。然而，在理解经典的钙粘蛋白粘附以及我们开发的新检测方法方面取得了重大进展，使我们能够在更复杂的水平上解决桥粒中钙粘蛋白-钙粘蛋白相互作用的问题。本申请的关键具体目的是开发一种能够监测桥粒中钙粘蛋白间相互作用的位点特异性交联测定。这样的分析是必不可少的澄清许多悬而未决的问题桥粒形成的机制。其中包括：（i）桥粒钙粘蛋白是否能够产生链二聚体;（ii）这些二聚体是否是异二聚体、同二聚体或两者;（iii）这种二聚化过程是否可以在体外再现，或者它是否只能在桥粒内的活细胞中进行;（iv）细胞内桥粒蛋白是否调节细胞外粘附相互作用;以及（v）桥粒是否具有桥粒钙粘蛋白的特异性横向排列。我们以前的经典钙粘蛋白实验为这样的研究提供了一个清晰的蓝图，并表明这个项目是可行的。这个应用程序概述了一个项目，以解决一个非常困难的研究，但细胞-细胞粘附的关键方面-蛋白质-蛋白质粘附相互作用的桥粒。桥粒是表皮中将角质形成细胞粘合在一起的主要粘附结构。尽管桥粒具有重要意义，但尚不清楚桥粒如何在表皮细胞之间形成紧密、有序和动态的粘附连接。了解这个问题对于阐明严重的人类起泡性皮肤病（包括落叶型天疱疮和寻常型天疱疮）的机制是必要的。此外，这些知识将为降低表皮细胞之间凝聚力的化合物指定目标。这些化合物可用于伤口愈合和促进其他药物或疫苗在整个皮肤中的快速递送。由于桥粒参与细胞内信号传导，因此操纵它们的粘附相互作用可能为调节细胞内信号以纠正过度增殖、异常分化和其他皮肤异常提供新的途径。

DESCRIPTION (provided by applicant): Classic and desmosomal cadherins are ubiquitous transmembrane adhesion receptors of epithelial cells. While significant progress has been made toward understanding classic cadherin adhesion, the mechanisms of adhesion by desmosomal cadherins have been poorly studied. Two factors - the complexity of desmosome cadherin composition and the insolubility of desmosomes - have hampered direct examination of protein-protein interactions in these structures. However, significant progress in understanding classic cadherin adhesion as well new assays that we developed have enabled us to address the question of cadherin-cadherin interactions in desmosomes on a more sophisticated level. The key Specific Aim of this application is the development of a site-specific cross-linking assay that is able to monitor intercadherin interactions in desmosomes. Such an assay is essential for clarifying many open questions about the mechanisms of desmosome formation. Among them are the following: (i) whether desmosomal cadherins are able to produce strand dimers; (ii) whether these dimers are heterodimers, homodimers, or both; (iii) whether this dimerization process can be reproduced in vitro or if it can proceed only in living cells within desmosomes; (iv) whether intracellular desmosomal proteins regulate extracellular adhesive interactions; and (v) whether desmosomes have a specific lateral alignment of desmosomal cadherins. Our previous experiments with classic cadherins provide a clear blueprint for such a study and indicate that this project is feasible. This application outlines a project to resolve a very difficult-to-study but critical aspect of cell-cell adhesion - the protein-protein adhesive interactions in desmosomes. Desmosomes are major adhesion structures in the epidermis that glue together keratinocytes. Despite their significance, it is not known how desmosomes form the tight, ordered and dynamic adhesive links between epidermal cells. Understanding this question is necessary for elucidating the mechanisms of severe human blistering skin diseases, including pemphigus foliaceus and pemphigus vulgaris. Furthermore, such knowledge will specify targets for compounds that decrease cohesion between epidermal cells. These compounds may have applications in wound healing and in promoting fast delivery of other drugs or vaccines throughout the skin. Since desmosomes are involved in intracellular signaling, the manipulation of their adhesive interactions may present a new avenue for modulating intracellular signals in order to correct hyperproliferation, abnormal differentiation and other skin abnormalities.