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中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，但对桥粒钙粘蛋白粘附的机制研究甚少。两个因素-桥粒钙粘蛋白组成的复杂性和桥粒的不溶性-阻碍了这些结构中蛋白质-蛋白质相互作用的直接检查。然而，在理解经典钙粘蛋白粘附以及我们开发的新检测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。该应用的关键特定目的是开发一种位点特异性交联试验，能够监测桥粒中钙粘蛋白间的相互作用。这样的实验对于澄清关于桥粒形成机制的许多悬而未决的问题是必要的。其中包括：(i)桥粒钙粘蛋白是否能够产生链二聚体；（ii）这些二聚体是异二聚体、同二聚体，还是两者兼而有之；（iii）这种二聚化过程能否在体外重现，还是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；(v)桥粒是否具有特定的桥粒钙粘蛋白横向排列。我们之前对经典钙粘蛋白的实验为此类研究提供了清晰的蓝图，表明该项目是可行的。本应用程序概述了一个解决细胞-细胞粘附的一个非常难以研究但关键的方面-桥粒中蛋白质-蛋白质粘附相互作用的项目。桥粒是表皮中主要的粘附结构，它将角质形成细胞粘合在一起。尽管桥粒具有重要意义，但目前尚不清楚桥粒是如何在表皮细胞之间形成紧密、有序和动态的粘附联系的。了解这个问题是必要的，阐明严重的人类水泡皮肤病，包括天疱疮叶状和寻常性天疱疮的机制。此外，这些知识将指定化合物的目标，减少表皮细胞之间的凝聚力。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递，因此操纵它们的粘附相互作用可能为调节细胞内信号提供了一条新的途径，从而纠正过度增生、异常分化和其他皮肤异常。

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.