Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, PR China.
Department of Clinical Laboratory, Liyuan Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
J Mol Cell Cardiol. 2019 Apr;129:79-91. doi: 10.1016/j.yjmcc.2019.02.007. Epub 2019 Feb 14.
Voltage-gated sodium channel Na1.5 is critical for generation and conduction of cardiac action potentials. Mutations and expression level changes of Na1.5 are associated with cardiac arrhythmias and sudden death. The ubiquitin (Ub) conjugation machinery utilizes three enzyme activities, E1, E2, and E3, to regulate protein degradation. Previous studies from us and others showed that Nedd4-2 acts as an E3 ubiquitin-protein ligase involved in ubiquitination and degradation of Na1.5, however, more key regulators remain to be identified. In this study, we show that UBC9, a SUMO-conjugating enzyme, regulates ubiquitination and degradation of Na1.5. Overexpression of UBC9 significantly decreased Na1.5 expression and reduced sodium current densities, whereas knockdown of UBC9 expression significantly enhanced Na1.5 expression and increased sodium current densities, in both HEK293 cells and primary neonatal cardiomyocytes. Overexpression of UBC9 increased ubiquitination of Na1.5, and proteasome inhibitor MG132 blocked the effect of UBC9 overexpression on Na1.5 degradation. Co-immunoprecipitation showed that UBC9 interacts with Nedd4-2. UBC9 with mutation C93S, which suppresses SUMO-conjugating activity of UBC9, was as active as wild type UBC9 in regulating Na1.5 levels, suggesting that UBC9 regulates Na1.5 expression levels in a SUMOylation-independent manner. Our findings thus identify a key structural element of the ubiquitin-conjugation machinery for Na1.5 and provide important insights into the regulatory mechanism for ubiquitination and turnover of Na1.5.
电压门控钠离子通道 Na1.5 对于心脏动作电位的产生和传导至关重要。Na1.5 的突变和表达水平的变化与心律失常和心脏性猝死有关。泛素（Ub）缀合机制利用三种酶活性，E1、E2 和 E3，来调节蛋白质降解。我们和其他人的先前研究表明，Nedd4-2 作为一种 E3 泛素蛋白连接酶，参与 Na1.5 的泛素化和降解，然而，更多的关键调节因子仍有待确定。在这项研究中，我们表明 UBC9，一种 SUMO 缀合酶，调节 Na1.5 的泛素化和降解。UBC9 的过表达显著降低了 Na1.5 的表达并减少了钠电流密度，而 UBC9 表达的敲低则显著增强了 Na1.5 的表达并增加了钠电流密度，这在 HEK293 细胞和原代新生心肌细胞中均如此。UBC9 的过表达增加了 Na1.5 的泛素化，蛋白酶体抑制剂 MG132 阻断了 UBC9 过表达对 Na1.5 降解的影响。共免疫沉淀显示 UBC9 与 Nedd4-2 相互作用。UBC9 的 C93S 突变，抑制了 UBC9 的 SUMO 缀合活性，其调节 Na1.5 水平的活性与野生型 UBC9 相当，表明 UBC9 以 SUMOylation 非依赖性方式调节 Na1.5 的表达水平。因此，我们的发现确定了泛素缀合机制的一个关键结构元件用于 Na1.5，并为 Na1.5 的泛素化和周转的调节机制提供了重要的见解。
