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.

抽象的 致心律失常性右心室心肌病 (ARVC) 是一种基于遗传的心脏病，可导致突发性心脏病 年轻人心源性死亡。 ARVC 被称为“桥粒疾病”，因为 40% 的 ARVC 港口 桥粒成分的突变和/或丢失。然而，控制桥粒的机制 健康和疾病中的蛋白质水平和功能尚不清楚。我们确定了 CSN6，它是 COP9 信号体家族，在酵母双杂交筛选中作为新型桥粒（桥粒蛋白，DSP） 成人心脏中的相互作用蛋白。 CSN综合体的传统功能是“关闭” 通过去内基化泛素化介导蛋白质降解；然而，CSN6 在心脏中的作用是 不明确的。我们相信 CSN6 能够独特地保护心脏桥粒免遭降解和损失 加速桥粒破坏，因为 (i) CSN6 共定位于桥粒连接处，(ii) CSN6 共定位 与桥粒蛋白和 (iii) 来自新型心脏特异性 CSN6 敲除的心脏进行免疫沉淀 (CSN6-cKO) 小鼠表现出桥粒蛋白水平（及其主要靶标 connexin43）的选择性丧失。 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 的双心室形式相关，类似于我们经典的 ARVC 双心室 (DSP-cKO) 模型。 CSN6 和 DSP-cKO 心脏都选择性地表现出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 通路与桥粒连接起来。 CSN6 途径是 与人类 ARVC 相关，因为 (i) CSN6 水平在 ARVC hiPSC 衍生的心脏细胞中下调， 表现出显着的桥粒缺陷和致心律失常行为，并且 (ii) CSN6 定位从细胞中丢失 ARVC 患者心脏活检中的连接处含有桥粒 (DSP (R315C) 和 plakophilin-2 (PKP2 IVS10-1 G>C) 突变。酵母和计算机模拟分析表明，该 DSP R315C 突变是 足以废除 DSP 与 CSN6 的绑定。然而，CSN6 也与 PKP2 相关，表明 PKP2 突变也可能在这些机制中发挥作用（两次打击假设）。就CSN6而言—— 桥粒复合体，我们的数据表明其他 CSN 可能与 CSN6 分隔（“子复合体”） 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 子复合物连接到心脏桥粒，以保护桥粒不被降解 neddylation 以及该机制的失调会触发 ARVC。我们的目标是： (i) 定义 CSN6 子复合体 成员，保护桥粒免受心肌细胞降解，（ii）确定 抑制心脏特异性 CSN6 和 DSP 敲除中潜在的过度积累的蛋白质降解 小鼠和 (iii) 通过表征两种新型敲入来了解桥粒-CSN6 相互作用的作用 携带人类 ARVC 相关桥粒突变的小鼠模型，这些突变会中断与 CSN6 的结合。