Intracellular Sorting of Connexin 43 by the Ubiquitin Ligase Wwp1

通过泛素连接酶 Wwp1 对 Connexin 43 进行细胞内分选

• 批准号: 8257901
• 负责人: Lydia Eleanor Matesic
• 金额: $ 34.93万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8257901
• 关键词: Adolescence; Affect; Age; Arrhythmia; Biological Assay; Body Weight; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Childhood; Clinical; Connexin 43; Data; Development; Diagnostic; Disease; Endosomes; Event; Fibrosis; Gap Junctions; Genes; Genetic; Growth; Heart; Heart Rate; Hela Cells; Histologic; Human; Hypertrophic Cardiomyopathy; Hypertrophy; Individual; Intercalated disc; Knock-out; Knowledge; Lead; Left Ventricular Hypertrophy; Lysosomes; Mass Spectrum Analysis; Mediating; Methodology; Molecular; Mus; Muscle Cells; Mutation; Myocardium; Myopathy; Patients; Pattern; Phenotype; Play; Process; Proteasome Inhibitor; Proteins; Regulation; Risk; Role; Site; Site-Directed Mutagenesis; Sorting - Cell Movement; Specificity; Stratification; Sudden Death; Tamoxifen; Testing; Time; Transcriptional Activation; Transgenic Animals; Transgenic Mice; Ubiquitin; Ubiquitination; Ventricular Arrhythmia; Vesicle; Weight; base; emerging adult; in vivo; interstitial; mortality; mouse model; multicatalytic endopeptidase complex; novel; outcome forecast; overexpression; patient population; postnatal; public health relevance; sudden cardiac death; trafficking; ubiquitin ligase

DESCRIPTION (provided by applicant): Cardiac arrhythmias associated with hypertrophic myopathy (HCM, the most common genetic myocardial disease) are the leading cause of sudden death in athletes and young people. Grossly, HCM is characterized by left ventricular hypertrophy (LVH) in the absence of identifiable clinical causes. Histologically, the myocardium of affected individuals displays myocyte hypertrophy and disarray, disorganization of intercalated discs with altered localization of Connexin 43 (Cx43), and, usually, increased amounts of interstitial fibrosis. Although there is a correlation among prognosis, the degree of LVH, amount of fibrosis, and causative mutation, this risk stratification methodology is limited in scope. In particular, the precise mechanisms underlying ventricular arrhythmias in patients with little-to-moderate LVH and are poorly defined, especially in pediatric HCM patients, although the regulation of Cx43 likely plays a critical role. We have recently created an inducible transgenic mouse model which develops a HCM-like phenotype when the ubiquitin ligase Wwp1 is globally overexpressed. Interestingly, these mice die very suddenly at 6-12 weeks of age (equivalent to adolescence or early adulthood in humans), and this phenotype is 100% penetrant. Similar to human HCM, transgenic animals display an increase in heart weight-to-body weight ratio, moderate LVH with an accompanying transcriptional activation of hypertrophic markers, myocyte disarray, a disruption of intercalated discs, dramatically decreased Cx43 protein levels, and an irregular heart rate. Importantly, we have been able to show that WWP1 is normally expressed in vesicles within human cardiomyocytes, WWP1 is upregulated and mislocalized in HCM, and Wwp1 can ubiquitinate Cx43. Based on these observations, we posit the following central hypothesis: increased Wwp1-mediated ubiquitination of Cx43 causes lysosomal degradation of Cx43, ensuing arrhythmia, and sudden death. In order to test this hypothesis, we aim to 1) define the role of Wwp1 overexpression in cardiomyocytes and its ability to promote the HCM phenotype; 2) determine the mechanism of Wwp1-mediated intracellular trafficking of Cx43; and 3) identify the Wwp1-mediated ubiquitination site(s) on Cx43 to determine the functional significance of ubiquitination of this/these site(s). Accomplishing the Specific Aims outlined here will elucidate the molecular mechanism underlying gap junction remodeling. This information can then be applied to HCM, a frequent and devastating disease known to display mislocalized Cx43 and increased WWP1 expression, in order to identify patient populations that might be at greater risk for fatal arrhythmias. PUBLIC HEALTH RELEVANCE: Cardiac arrhythmias associated with hypertrophic cardiomyopathy are the leading cause of sudden death in athletes and young people. Currently, our understanding of why some patients are more prone to these arrhythmias is lacking, although it seems that a protein called Connexin43 is the lynchpin in this process. This application defines how a novel player, Wwp1, controls the amount of Connexin43, and as such might serve as a diagnostic to identify those hypertrophic cardiomyopathy patients most likely to suffer sudden cardiac death.

描述（由申请人提供）：与肥厚性肌病（HCM，最常见的遗传性心肌疾病）相关的心律失常是导致运动员和年轻人猝死的主要原因。大体而言，HCM的特征是左心室肥厚（LVH），没有明确的临床原因。组织学上，受影响个体的心肌表现为心肌细胞肥大和紊乱，间插椎间盘组织紊乱，连接蛋白43 （Cx43）的定位改变，通常伴有间质纤维化的增加。虽然预后、LVH程度、纤维化量和致病突变之间存在相关性，但这种风险分层方法的适用范围有限。特别是，尽管Cx43的调节可能起着关键作用，但在轻度至中度LVH患者中室性心律失常的确切机制尚不明确，特别是在儿科HCM患者中。我们最近创建了一个可诱导的转基因小鼠模型，当泛素连接酶Wwp1在全球过表达时，它会产生类似hcm的表型。有趣的是，这些小鼠在6-12周龄时突然死亡（相当于人类的青春期或成年早期），这种表型是100%渗透的。与人类HCM相似，转基因动物表现出心脏重量与体重比增加，中度LVH伴有肥厚标记物的转录激活，肌细胞紊乱，嵌入椎间盘破坏，Cx43蛋白水平显著降低，心率不规则。重要的是，我们已经能够证明WWP1在人心肌细胞的囊泡中正常表达，WWP1在HCM中上调和错定位，WWP1可以泛素化Cx43。基于这些观察结果，我们提出以下中心假设：wwp1介导的Cx43泛素化增加导致Cx43溶酶体降解，导致心律失常和猝死。为了验证这一假设，我们的目标是：1)确定Wwp1过表达在心肌细胞中的作用及其促进HCM表型的能力；2)确定wwp1介导的Cx43细胞内转运的机制；3)鉴定Cx43上wwp1介导的泛素化位点，确定该位点/这些位点泛素化的功能意义。完成本文概述的具体目标将阐明间隙连接重塑的分子机制。这些信息可以应用于HCM， HCM是一种常见的破坏性疾病，已知表现为Cx43错位和WWP1表达增加，以便确定可能具有更大致命性心律失常风险的患者群体。