School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong 999077, China.
Zhujiang Hospital, Southern Medical University, 253 Industrial Road, Guangzhou 51000, Guangdong Province, China.
Int J Biol Sci. 2022 Jul 11;18(11):4595-4609. doi: 10.7150/ijbs.74407. eCollection 2022.
Myocardial infarction triggers oxidative DNA damage, apoptosis and adverse cardiac remodeling in the heart. Small ubiquitin-like modifier (SUMO) proteins mediate post-translational SUMOylation of the cardiac proteins in response to oxidative stress signals. Upregulation of isoform SUMO2 could attenuate myocardial injury via increasing protein SUMOylation. The present study aimed to discover the identity and cardioprotective activities of SUMOylated proteins. A plasmid vector for expressing N-Strep-SUMO2 protein was generated and introduced into H9c2 rat cardiomyocytes. The SUMOylated proteins were isolated with Strep-Tactin agarose beads and identified by MALDI-TOF-MS technology. As a result, γ-actin was identified from a predominant protein band of ~42 kDa and verified by Western blotting. The roles of SUMO2 and γ-actin SUMOylation were subsequently determined in a mouse model of myocardial infarction induced by ligating left anterior descending coronary artery and H9c2 cells challenged by hypoxia-reoxygenation. lentiviral-mediated SUMO2 expression in H9c2 cells were used to explore the role of SUMOylation of γ-actin. SUMOylation of γ-actin by SUMO2 was proven to be a new cardioprotective mechanism from the following aspects: 1) SUMO2 overexpression reduced the number of TUNEL positive cells, the levels of 8-OHdG and p-γ-H2ax while promoted the nuclear deposition of γ-actin in mouse model and H9c2 cell model of myocardial infarction; 2) SUMO-2 silencing decreased the levels of nuclear γ-actin and SUMOylation while exacerbated DNA damage; 3) Mutated γ-actin (KR/KR) void of SUMOylation sites failed to protect cardiomyocytes against hypoxia-reoxygenation challenge. The present study suggested that SUMO2 upregulation promoted DNA damage repair and attenuated myocardial injury via increasing SUMOylation of γ-actin in the cell nucleus.
心肌梗死会在心脏中引发氧化 DNA 损伤、细胞凋亡和不良的心脏重构。小泛素样修饰物（SUMO）蛋白可介导心脏蛋白的翻译后 SUMO 化，以响应氧化应激信号。SUMO2 同工型的上调可通过增加蛋白 SUMO 化来减轻心肌损伤。本研究旨在发现 SUMO 化蛋白的特性和心脏保护活性。生成了表达 N-Strep-SUMO2 蛋白的质粒载体，并将其引入 H9c2 大鼠心肌细胞。用 Strep-Tactin 琼脂糖珠分离 SUMO 化蛋白，并通过 MALDI-TOF-MS 技术鉴定。结果，从约 42 kDa 的主要蛋白带中鉴定出 γ-肌动蛋白，并通过 Western blot 验证。随后在结扎左前降支冠状动脉诱导的心肌梗死小鼠模型和缺氧-复氧处理的 H9c2 细胞中确定了 SUMO2 和 γ-肌动蛋白 SUMO 化的作用。使用慢病毒介导的 SUMO2 在 H9c2 细胞中的表达来探讨 γ-肌动蛋白 SUMO 化的作用。SUMO2 对 γ-肌动蛋白的 SUMO 化是通过以下几个方面证明的一种新的心脏保护机制：1）SUMO2 过表达减少 TUNEL 阳性细胞的数量、8-OHdG 和 p-γ-H2ax 的水平，同时促进心肌梗死小鼠模型和 H9c2 细胞模型中核内 γ-肌动蛋白的沉积；2）SUMO-2 沉默降低核内 γ-肌动蛋白和 SUMO 化水平，同时加剧 DNA 损伤；3）缺乏 SUMO 化位点的突变 γ-肌动蛋白（KR/KR）不能保护心肌细胞免受缺氧-复氧的挑战。本研究表明，SUMO2 的上调通过增加核内 γ-肌动蛋白的 SUMO 化来促进 DNA 损伤修复并减轻心肌损伤。
