Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yongin 446-701, Korea.
J Biol Chem. 2012 Jul 6;287(28):24043-52. doi: 10.1074/jbc.M112.364117. Epub 2012 May 10.
The N-end rule pathway is a proteolytic system in which destabilizing N-terminal amino acids of short lived proteins are recognized by recognition components (N-recognins) as an essential element of degrons, called N-degrons. In eukaryotes, the major way to generate N-degrons is through arginylation by ATE1 arginyl-tRNA-protein transferases, which transfer Arg from aminoacyl-tRNA to N-terminal Asp and Glu (and Cys as well in mammals). We have shown previously that ATE1-deficient mice die during embryogenesis with defects in cardiac and vascular development. Here, we characterized the arginylation-dependent N-end rule pathway in cardiomyocytes. Our results suggest that the cardiac and vascular defects in ATE1-deficient embryos are independent from each other and cell-autonomous. ATE1-deficient myocardium and cardiomyocytes therein, but not non-cardiomyocytes, showed reduced DNA synthesis and mitotic activity ~24 h before the onset of cardiac and vascular defects at embryonic day 12.5 associated with the impairment in the phospholipase C/PKC-MEK1-ERK axis of Gα(q)-mediated cardiac signaling pathways. Cardiac overexpression of Gα(q) rescued ATE1-deficient embryos from thin myocardium and ventricular septal defect but not from vascular defects, genetically dissecting vascular defects from cardiac defects. The misregulation in cardiovascular signaling can be attributed in part to the failure in hypoxia-sensitive degradation of RGS4, a GTPase-activating protein for Gα(q). This study is the first to characterize the N-end rule pathway in cardiomyocytes and reveals the role of its arginylation branch in Gα(q)-mediated signaling of cardiomyocytes in part through N-degron-based, oxygen-sensitive proteolysis of G-protein regulators.
N 端规则途径是一种蛋白水解系统，其中不稳定的短寿命蛋白的 N 端氨基酸被识别元件（N 识别物）识别为降解物（称为 N 降解物）的必需元件。在真核生物中，生成 N 降解物的主要方法是通过 ATE1 精氨酰-tRNA 蛋白转移酶的精氨酰化，该酶将 Arg 从氨酰-tRNA 转移到 N 端的 Asp 和 Glu（哺乳动物中也有 Cys）。我们之前已经表明，ATE1 缺陷小鼠在胚胎发生过程中因心脏和血管发育缺陷而死亡。在这里，我们描述了心肌细胞中依赖于精氨酰化的 N 端规则途径。我们的结果表明，ATE1 缺陷胚胎中的心脏和血管缺陷彼此独立且为细胞自主性的。ATE1 缺陷的心肌及其内部的心肌细胞，但不是非心肌细胞，在胚胎第 12.5 天心脏和血管缺陷发生前约 24 小时显示出 DNA 合成和有丝分裂活性降低，与 Gα(q) 介导的心脏信号通路中 PLC/PKC-MEK1-ERK 轴的损伤有关。心脏过表达 Gα(q) 可挽救 ATE1 缺陷胚胎免于出现薄心肌和室间隔缺损，但不能挽救血管缺陷，从遗传上区分了血管缺陷和心脏缺陷。心血管信号的失调部分归因于缺氧敏感的 RGS4（Gα(q) 的 GTP 酶激活蛋白）降解失败。这项研究首次描述了心肌细胞中的 N 端规则途径，并揭示了其精氨酰化分支在 Gα(q) 介导的心肌细胞信号中的作用，部分是通过基于 N 降解物的、氧敏感的 G 蛋白调节剂的蛋白水解。
