Uncovering New Functions of CSN6 in Cardiac Desmosomal Biology and Disease

揭示 CSN6 在心脏桥粒生物学和疾病中的新功能

• 批准号: 9754240
• 负责人: Farah Sheikh
• 金额: $ 39.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754240
• 关键词: Address; Adult; Arrhythmogenic Right Ventricular Dysplasia; Automobile Driving; Behavior; Binding; Biochemical; Biological Assay; Biology; Biopsy; COPS5 gene; Cardiac; Cardiac Myocytes; Cells; Complex; Computer Simulation; Connexin 43; Data; Defect; Desmosomes; Disease; Electrophysiology (science); Exhibits; Family; Genetic; Health; Heart; Heart Diseases; Human; In Vitro; Intercellular Junctions; Interruption; Intervention; Knock-in Mouse; Knock-out; Knockout Mice; Left; Link; Mediating; Modeling; Molecular; Molecular Analysis; Mus; Mutation; Myocardium; Pathway interactions; Patients; Pattern; Pharmacology; Phenotype; Physiological; Play; Proteins; Proteomics; Role; Structure; Sudden Death; Testing; Therapeutic; Tissues; Ubiquitin; Ubiquitination; Yeasts; base; cDNA Library; desmoplakin; effective therapy; in vivo; induced pluripotent stem cell; inherited cardiomyopathy; inhibitor/antagonist; insight; member; mouse model; novel; plakophilin 2; protein degradation; small hairpin RNA; sudden cardiac death; yeast two hybrid system

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.

摘要 致心律失常性右室心肌病(ARVC)是一种遗传性心脏病，可导致突发性 年轻人的心源性死亡。ARVC被称为“桥粒疾病”，因为40%的ARVC 桥粒成分的突变和/或丢失。然而，控制桥粒的机制 蛋白质在健康和疾病中的水平和功能尚未确定。我们鉴定了CSN6，这是该基因的第6亚基 COP9信号小体家族，在酵母双杂交筛选中作为一个新的桥粒(桥粒蛋白，DSP) 成人心脏中相互作用的蛋白质。CSN综合体的传统功能是“关闭” 泛素化通过脱氢酶介导蛋白质降解；然而，CSN6在心脏中的作用是 未定义。我们认为CSN6是唯一保护心脏桥粒不被降解和丢失的基因 加速桥粒破坏，因为(I)CSN6共定位于桥粒连接，(Ii)CSN6共定位于桥粒连接 新的心脏特异性CSN6基因敲除的心脏与桥粒蛋白的免疫共沉淀物 (CSN6-CKO)小鼠表现出桥粒蛋白水平的选择性丢失(及其主要靶标连接蛋白43)。 CSN6缺失是ARVC的触发因素，因为CSN6-CKO小鼠表现出猝死和心脏病特征 与ARVC的双室形式相关，类似于我们的经典ARVC双室(DSP-CKO)模型。 CSN6和DSP-CKO心脏都选择性地显示潜在的蛋白质降解过度积累 细胞连接处的机械，特别是将CSN6通路连接到桥粒。CSN6途径包括 与人类ARVC相关：(1)在ARVC来源的心肌细胞中，CSN6水平下调， 表现出显著的桥粒缺陷和致心律失常行为，以及(Ii)CSN6从细胞中定位丢失 1例伴有桥粒(DSPR315C)和血小板亲和素-2的ARVC患者心脏活检中的连接 (PKP2 IVS10-1G&gt；C)突变。酵母菌和计算机模拟分析表明，该DSPR315C突变是 足以废除数字信号处理器与CSN6的绑定。然而，CSN6也与PKP2相关，这表明 PKP2突变也可能在这些机制中发挥作用(两次打击假说)。在CSN6方面- 桥粒复合体，我们的数据表明，其他CSN可能与CSN6在 桥粒及其定位可能依赖于CSN6。我们假设CSN6的目标是 具有酶活性的CSN亚复合体与心脏桥粒结合，保护桥粒免受降解 糖尿病，这一机制的失调会触发ARVC。我们的目标是：(I)定义CSN6亚复合体 保护桥粒在心肌细胞中不被降解的成员，(Ii)决定 抑制心脏特异性CSN6和DSP基因敲除中潜在的超积累蛋白降解 小鼠和(Iii)通过描述两个新的敲打信号来了解桥粒-CSN6相互作用的作用 携带人类ARVC相关桥粒突变的小鼠模型，该突变中断与CSN6的结合。