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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的心律失常右心肌病（ARVC）是一种基于遗传的心脏病，导致突然年轻人心脏死亡。 ARVC被称为“脱糖体的疾病”，为40％的ARVC港口突变和/或脱骨成分的损失。但是，控制脱发的机制蛋白质水平和健康和疾病的功能是不确定的。我们确定了CSN6，这是一个亚基6 COP9信号体系列，在酵母2杂交屏幕中，作为一种新型的脱发（Desmoplakin，DSP）在成年人心脏中相互作用的蛋白质。 CSN综合体的传统功能是“关闭” 泛素化介导的蛋白质降解通过脱发；但是，CSN6在心脏中的作用是不明确的。我们认为，CSN6独特地保护心脏脱骨体免受降解及其损失（i）csn6加速脱骨破坏，将脱斑量化为脱骨连接，（ii）CSN6共同存在新颖心脏特异性CSN6基因敲除的脱发蛋白和（iii）心脏的免疫沉淀（CSN6-CKO）小鼠表现出脱骨蛋白水平的选择性损失（及其主要靶标，Connexin43）。 CSN6损失是ARVC的触发因素，因为CSN6-CKO小鼠暴露了猝死和心脏病特征与ARVC的双脑室形式相关，类似于我们经典的双室（DSP-CKO）ARVC模型。 CSN6和DSP-CKO心脏都有选择性地显示蛋白质降解的基础超积累细胞连接处的机械，专门将CSN6途径连接到朝底。 CSN6途径是与人ARVC相关的AS（i）CSN6水平在ARVC HIPSC衍生的心脏细胞中被下调表现出醒目的脱骨缺陷和心律失常行为，（ii）CSN6定位从细胞中丢失来自ARVC患者的心脏活检连接点，该患者携带脱染色体（DSP（R315C）和Plakophilin-2）（PKP2 IVS10-1 g> c）突变。酵母和计算机建模测定法显示了DSP R315C突变是足以消除DSP与CSN6结合。但是，CSN6也与PKP2相关联，这表明 PKP2突变也可能在这些机制中起作用（两次打击假设）。根据CSN6- 我们的数据表明，其他CSN可能会与csn6划分（“子复合”）向突变及其本地化可能取决于CSN6。我们假设CSN6针对酶促活性的CSN子复合到心脏脱骨体，以保护脱糖体免受降解的降解这种机制的Neddylation和失调触发ARVC。我们的目标是：（i）定义CSN6子复合构件，保护脱骨体免受心肌细胞降解的降解，（ii）确定抑制心脏特异性CSN6和DSP基因敲除中的潜在超耗尽的蛋白质降解小鼠和（iii）通过表征两个新型敲击素来了解脱骨体-CSN6相互作用的作用具有与人类ARVC相关的脱发突变的小鼠模型中断与CSN6结合的小鼠模型。