INTERCADHERIN INTERACTIONS IN DESMOSOMES

描述(申请人提供)：经典和桥粒钙粘附素是上皮细胞普遍存在的跨膜黏附受体。虽然在理解经典钙粘附素方面已经取得了重大进展，但桥粒钙粘附素的粘附性机制研究还很少。两个因素--桥粒钙粘附素组成的复杂性和桥粒的不溶性--阻碍了对这些结构中蛋白质-蛋白质相互作用的直接研究。然而，在理解经典钙粘附素粘附性方面的重大进展以及我们开发的新的分析方法使我们能够在更复杂的水平上解决桥粒中钙粘附素-钙粘附素相互作用的问题。这一应用的关键的具体目标是开发一种能够监测桥粒中钙粘附素相互作用的位点特异性交联试验。这样的分析对于澄清许多关于桥粒形成机制的公开问题是必不可少的。其中包括：(I)桥粒钙粘附素是否能够产生链二聚体；(Ii)这些二聚体是异源二聚体、同源二聚体还是两者兼而有之；(Iii)这种二聚化过程是否可以在体外复制，或者如果它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黏附相互作用；以及(V)桥粒是否具有桥粒钙粘附素的特定侧向排列。我们之前对经典钙粘附素的实验为这样的研究提供了一个明确的蓝图，并表明这个项目是可行的。这项申请概述了一个项目，以解决细胞-细胞黏附的一个非常难研究但关键的方面--桥粒中的蛋白质-蛋白质黏附相互作用。桥粒是表皮中粘连角质形成细胞的主要粘连结构。尽管桥粒具有重要意义，但它如何在表皮细胞之间形成紧密、有序和动态的粘连尚不清楚。了解这个问题对于阐明严重的人类水疱性皮肤病的机制是必要的，包括叶天疱疮和寻常型天疱疮。此外，这些知识将为降低表皮细胞之间凝聚力的化合物指明目标。这些化合物可能在伤口愈合和促进其他药物或疫苗在皮肤上的快速传递方面有应用。由于桥粒参与细胞内信号传递，因此对其黏附相互作用的调控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一条新的途径。

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.