Uncovering New Functions of CSN6 in Cardiac Desmosomal Biology and Disease

摘要 致心律失常性右心室心肌病（ARVC）是一种遗传性心脏病， 年轻人的心源性死亡ARVC被称为“桥粒疾病”，因为40%的ARVC携带有 桥粒组分的突变和/或丢失。然而，控制桥粒的机制 蛋白质水平和在健康和疾病中的功能是不确定的。我们鉴定了CSN 6，它是CSN的亚基6。 COP 9信号体家族，在酵母双杂交筛选中作为新型桥粒（桥粒斑蛋白，DSP） 在成年人心脏中的相互作用蛋白。CSN综合体的传统功能是“关闭” 泛素化通过去neddylation介导蛋白质降解;然而，CSN 6在心脏中的作用是 未定义。我们相信CSN 6独特地保护心脏桥粒免于降解和丢失， 加速桥粒破坏，因为（i）CSN 6共定位于桥粒连接处，（ii）CSN 6共定位于桥粒连接处，（iii）CSN 6共定位于桥粒连接处。 与桥粒蛋白免疫沉淀和（iii）来自新的心脏特异性CSN 6敲除的心脏 （CSN 6-cKO）小鼠显示桥粒蛋白水平（及其主要靶标，连接蛋白43）的选择性丧失。 CSN 6缺失是ARVC的触发因素，因为CSN 6-cKO小鼠表现出猝死和心脏病特征 与双心室形式的ARVC相关，类似于我们的经典双心室（DSP-cKO）ARVC模型。 CSN 6和DSP-cKO心脏都选择性地显示潜在的蛋白质降解过度积累 在细胞连接处的机械，特别是连接CSN 6途径的桥粒。CSN 6通路是 （i）CSN 6水平在ARVC hiPSC衍生的心脏细胞中下调， 表现出显著的桥粒缺陷和促凋亡行为，以及（ii）CSN 6定位从细胞中丢失 来自ARVC患者的心脏活检中的连接，其含有桥粒（DSP（R315 C）和斑嗜蛋白-2 (PKP2 IVS 10 - 1G>C）突变。酵母和计算机模拟分析表明，这种DSP R315 C突变是 足以消除DSP与CSN 6的结合。然而，CSN 6也与PKP 2相关，这表明， PKP 2突变也可能在这些机制中发挥作用（二击假说）。关于CSN 6- 桥粒复合物，我们的数据表明，其他CSN可能与CSN 6区室化（“亚复合物”）， 桥粒及其定位可能依赖于CSN 6。我们假设CSN 6靶向一个 酶活性CSN亚复合物的心脏桥粒，以保护桥粒免于降解， neddylation和这种机制的失调触发ARVC。我们的目标是：（i）定义CSN 6子复合体 成员，保护桥粒在心肌细胞中不被降解，（ii）确定 在心脏特异性CSN 6和DSP敲除中抑制潜在的过度积累的蛋白质降解 小鼠和（iii）理解桥粒-CSN 6相互作用的作用，通过表征两种新的敲入， 小鼠模型，其具有中断与CSN 6结合的人ARVC相关桥粒突变。

Abstract Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic-based heart disease leading to sudden cardiac death in young people. ARVC is termed a “disease of the desmosome” as 40% of ARVC harbor mutations and/or loss of desmosomal components. However, the mechanisms that control desmosomal protein levels and function in health and disease are undefined. We identified CSN6, which is subunit 6 of the COP9 signalosome family, in a yeast-two-hybrid screen as a novel desmosomal (desmoplakin, DSP) interacting protein in the adult human heart. Traditional functions of the CSN complex are to “turn off” ubiquitination mediated protein degradation via de-neddylation; however, the role for CSN6 in the heart is undefined. We believe that CSN6 uniquely protects the cardiac desmosome from degradation and its loss accelerates desmosome destruction as (i) CSN6 co-localizes to desmosomal junctions, (ii) CSN6 co- immunoprecipitates with desmosomal proteins and (iii) hearts from novel cardiac-specific CSN6 knockout (CSN6-cKO) mice display selective loss of desmosomal protein levels (and its primary target, connexin43). CSN6 loss is a trigger for ARVC as CSN6-cKO mice exhibit sudden death and cardiac disease features associated with a biventricular form of ARVC, similar to our classic biventricular (DSP-cKO) model of ARVC. CSN6 and DSP-cKO hearts both selectively display underlying hyper-accumulation of protein degradation machinery at the cell junction, specifically linking CSN6 pathways to the desmosome. CSN6 pathways are relevant to human ARVC as (i) CSN6 levels are down-regulated in ARVC hiPSC-derived cardiac cells that exhibit striking desmosomal defects and arrhythmogenic behavior and (ii) CSN6 localization is lost from cell junctions in a cardiac biopsy from an ARVC patient harboring desmosomal (DSP (R315C) and plakophilin-2 (PKP2 IVS10-1 G>C) mutations. Yeast and in silico modeling assays reveal that this DSP R315C mutation is sufficient to abrogate DSP binding to CSN6. However, CSN6 also associates with PKP2, suggesting that the PKP2 mutation may also play a role in these mechanisms (two-hit hypothesis). In terms of the CSN6- desmosomal complex, our data suggests that other CSNs may compartmentalize (“subcomplex”) with CSN6 at the desmosome and their localization may be dependent on CSN6. We hypothesize that CSN6 targets an enzymatically active CSN subcomplex to the cardiac desmosome to protect desmosomes from degradation via neddylation, and dysregulation of this mechanism triggers ARVC. We aim to: (i) define the CSN6 subcomplex members, which protect the desmosome from degradation in cardiomyocytes, (ii) determine the effects of inhibiting the underlying hyper-accumulated protein degradation in cardiac-specific CSN6 and DSP knockout mice and (iii) understand the role of the desmosome-CSN6 interaction by characterizing two novel knockin mouse models that harbor human ARVC-associated desmosomal mutations that interrupt binding to CSN6.