A Novel Desmosomal COP9 Signalosome Complex in Epidermal Differentiation

 描述（由申请人提供）：桥粒是细胞间的粘附连接，是经历机械应力的组织（如表皮）的完整性所必需的。它们的机械功能是通过连接的中间丝细胞骨架跨膜桥粒钙粘蛋白，通过一个复杂的细胞质蛋白质在犰狳和plakin家庭。已知通过基因突变、细菌毒素或自身抗体破坏桥粒结构和功能会导致严重的起泡性疾病和角化病，这些疾病和角化病的治疗选择有限。然而，在何种程度上损害的粘附与新出现的信号转导功能的桥粒有助于疾病的发病机制是未知的。最近显示，桥粒钙粘蛋白桥粒芯糖蛋白1（Dsg 1）的胞质结构域（其首先在细胞致力于分化时表达）通过抑制EGFR/MAPK信号传导促进表皮分化。为了阐明Dsg 1细胞质尾区在分化中的信号传导作用，进行了酵母双杂交筛选。在通过筛选鉴定的推定结合配偶体中是COP 9信号体亚基3，Cops 3。COP 9是一种蛋白质复合体，由8个亚基（Cops 1-Cops 8）组成。该复合物从cullin-RING连接酶中去除Nedd 8稳定蛋白修饰，以调节cullin-RING连接酶的活化循环（其促进蛋白质的泛素化和降解）。Nedd 8蛋白还显示出修饰膜受体（例如EGFR），这可以促进受体稳定。已知Dsg 1的异位表达通过抑制EGFR信号传导促进表皮分化，但在Cops 3缺陷细胞中不能这样做。这一观察结果表明，Dsg 1需要Cops 3来抑制EGFR/MAPK并促进表皮分化。此外，生化分析显示Cops 3与Dsg 1和桥粒斑蛋白Desmoplakin（Dp）两者相关联，并且cullin家族成员（Cul 3）与Dp相关联。这些数据表明，桥粒有不止一个界面来支撑COP 9信号体发挥功能。基于这些观察，假设桥粒充当C 0 P9信号体的支架以使cullin和EGFR去neddylate，并通过抑制EGFR信号传导促进表皮分化。Aim 1将定义COP 9信号体和桥粒之间的关联，以及桥粒分子影响COP 9依赖性去neddylation活性的程度。Aim 2将决定Cops 3如何通过抑制表皮角质形成细胞中的EGFR/MAPK信号传导来协助Dsg 1依赖性分化。该项目将深入了解一种新型桥粒-COP 9信号复合物的功能及其在表皮分化中的作用。了解桥粒如何整合机械和细胞内信号传导将有助于确定新的生物学途径作为治疗与桥粒损伤相关的严重皮肤病的潜在靶点。

 DESCRIPTION (provided by applicant): Desmosomes are intercellular adhesive junctions, which are required for integrity of tissues that experience mechanical stress, such as the epidermis. Their mechanical functions are facilitated by the linkage of the intermediate filament cytoskeleton to transmembrane desmosomal cadherins through a complex of cytoplasmic proteins in the armadillo and plakin families. It is known that disrupting desmosome structure and function through gene mutations, bacterial toxins or autoantibodies results in severe blistering disorders and keratodermas that have limited treatment options. However, the extent to which impairment of adhesion versus newly emerging signaling functions of desmosomes contributes to disease pathogenesis is unknown. It was recently shown that the cytoplasmic domain of the desmosomal cadherin Desmoglein 1 (Dsg1), which is first expressed as cells commit to differentiate, promotes epidermal differentiation by inhibiting EGFR/MAPK signaling. Towards elucidating the signaling role of the Dsg1 cytoplasmic tail in differentiation, a yeast two hybrid screen was performed. Among the putative binding partners identified by the screen was the COP9 signalosome subunit 3, Cops3. COP9 is complex of proteins, comprising eight subunits (Cops1-Cops8). This complex removes Nedd8 stabilizing protein modifications from cullin-RING ligases, to regulate activation cycles of cullin-RING ligases (which promote ubiquitination and degradation of proteins). The Nedd8 protein has also been shown to modify membrane receptors (e.g. EGFR), which can promote receptor stabilization. Ectopic expression of Dsg1 is known to promote epidermal differentiation through the suppression of EGFR signaling, but it is unable to do so in Cops3-deficient cells. This observation suggests that Dsg1 requires Cops3 to inhibit EGFR/MAPK and promote epidermal differentiation. Further, biochemical analyses revealed that Cops3 associates with both Dsg1 and the desmosomal plakin protein Desmoplakin (Dp), and that cullin family members (Cul3) associate with Dp. These data suggest that there is more than one interface for the desmosome to scaffold the COP9 signalosome for functioning. Based on these observations, it is hypothesized that the desmosome acts as a scaffold for the COP9 signalosome to de-neddylate cullins and EGFR, and promotes epidermal differentiation by inhibition of EGFR signaling. Aim1 will define the association between the COP9 signalosome and the desmosome, and the extent to which desmosome molecules affect COP9-dependent de-neddylation activity. Aim2 will determine how Cops3 assists in Dsg1- dependent differentiation by suppressing EGFR/MAPK signaling in epidermal keratinocytes. This project will provide insight into the functions of a novel desmosome-COP9 signaling complex and its role in epidermal differentiation. Understanding how desmosomes integrate mechanical and intracellular signaling will help identify new biological pathways as potential targets for therapeutics to treat the severe skin disorders associated with desmosomal impairment.