钙网蛋白乙酰转移酶活性促进肌动蛋白聚合减轻心肌缺血再灌注损伤

Myocardial ischemia-reperfusion injury is a common clinical emergency. Acetylation PTM regulator has potential therapeutic effect. In-depth exploration of the targets and mechanism is of great significance for CHD prevention and treatment. Actin cytoskeleton is one of the key factors to control cardiomyocyte survival and remodeling, and its polymerization and depolymerization is regulated by Nε-lysine acetylation. Calreticulin (CRT) is a multifunctional protein, which has acetyltransferase activity, mainly residents in ER lumin and stresses induce its intracellular translocation. We previously found that non-ER CRT promotes actin polymerization, stabilizes cytoskeleton and reduces cell injury, but the exact mechanism has not been clarified. In this project, we intend to use the mouse I/R and the neonatal rat cardiomyocyte H/R model to investigate the effect of CRT on α-actin acetylation, polymerization and depolymerization and myocardial injury. In vivo Co-IP and in vitro GST pull down are used to confirm the interaction between CRT and α-actin. We also measure the acetyltransferase enzyme activity constant, determine the acetylation site by LC-MS/MS, and use site-specific mutagenesis to verify, in order to confirm that CRT catalyzes α-actin Nε-lysine acetylation, promoting its polymerization, stabilizing cytoskeletal structures, reducing myocardial ischemia/reperfusion injury, and expecting to be a new drug target for CVD.

心肌缺血再灌注损伤是临床常见的心血管急症，调节蛋白乙酰化修饰的药物对其具有潜在治疗效果，深入探讨作用机制对冠心病防治具有重要意义。细胞骨架蛋白α-actin是调控心肌损伤修复的关键分子，其聚合解聚受Nε-赖氨酸乙酰化调节；Ca2+结合分子伴侣——钙网蛋白(CRT)是具有乙酰转移酶活性的多功能蛋白，主要定位于内质网，应激可诱导其胞内转位；课题组前期发现非内质网CRT促进actin聚合，稳定微丝骨架减轻细胞损伤。本项目拟在整体I/R模型和细胞H/R模型上研究CRT对α-actin乙酰化、微丝聚合及心肌损伤的影响；采用in vivo免疫共沉淀和in vitro GST pull down验证CRT与α-actin的相互作用、测定酶活性常数、确定乙酰化位点并进行定点突变验证，旨在证实CRT催化α-actin Nε-乙酰化，促进微丝聚合，稳定细胞骨架，减轻心肌缺血再灌注损伤，有望开发新的药物靶点。

心肌缺血再灌注损伤是临床常见的心血管急症，调节蛋白乙酰化修饰的药物对其具有潜在治疗效果，深入探讨作用机制对冠心病防治具有重要意义。细胞骨架蛋白α-actin是调控心肌损伤修复的关键分子，其聚合解聚受Nε-赖氨酸乙酰化调节；Ca2+结合分子伴侣——钙网蛋白(CRT)是具有乙酰转移酶活性的多功能蛋白，主要定位于内质网，应激可诱导其胞内转位；课题组前期发现非内质网CRT促进actin聚合，稳定微丝骨架减轻细胞损伤。本项目在整体I/R模型和细胞H/R模型上证实钙网蛋白预处理可使α-actin乙酰化增加，促进微丝聚合，稳定细胞骨架，减轻心肌缺血再灌注损伤；采用in vivo免疫共沉淀和in vitro GST pull down证实CRT与α-actin存在直接相互作用；采用LC-MS/MS质谱分析钙网蛋白自身乙酰化位点及其催化α-actin乙酰化位点，发现钙网蛋白N结构域Lys-64和P结构域Lys-209可发生自身乙酰化，可催化actin第61位和第68位赖氨酸残基发生乙酰化；分别构建Lys-64和Lys-209点突变的重组CRT，证实CRT自身乙酰化可催化α-actin Nε-乙酰化，促进微丝聚合，稳定细胞骨架，减轻心肌缺血再灌注损伤，有望开发新的药物靶点。