Protein S-glutathionylation stimulate adipogenesis by stabilizing C/EBPβ in 3T3L1 cells.
Protein S-glutathionylation stimulate adipogenesis by stabilizing C/EBPβ in 3T3L1 cells.
复制标题
DOI：
10.1096/fj.201902575r
复制
发表时间：
2020-04
期刊：
影响因子：
--
通讯作者：
Kugiyama K
中科院分区：
文献类型：
--
作者：
Watanabe Y;Watanabe K;Fujioka D;Nakamura K;Nakamura T;Uematsu M;Bachschmid MM;Matsui R;Kugiyama K
活性氧（ROS）在脂肪形成和肥胖过程中增加。氧化剂与蛋白质的半胱氨酸残基反应形成谷胱甘肽（GSH）加合物（S-谷胱甘肽化），并被谷氧还蛋白-1（Glrx）选择性去除。我们以前曾报道过Glycoprotein基因敲除小鼠的蛋白S-谷胱甘肽化增加，并通过一种未知的机制发展为肥胖。在这项研究中，我们证明了3 T3 L1脂肪细胞分化增加ROS和蛋白质S-谷胱甘肽化。在3 T3 L1细胞中，Glycoprotein消融升高蛋白S-谷胱甘肽化和脂质含量。补充Glycine可降低Glycine KO 3 T3 L1细胞的脂质含量。Glycoprotein KO还增加脂肪形成转录因子CCAAT增强子结合蛋白（C/EBP）β的蛋白表达和蛋白S-谷胱甘肽化。蛋白S-谷胱甘肽化降低了C/EBPβ与活化STAT（皮亚斯）1的蛋白抑制剂的相互作用，STAT 1是一种促进C/EBPβ降解的小泛素相关修饰剂（SUMO）E3连接酶。用截短的突变体C/EBPβ进行的实验表明，PIAS 1与C/EBPβ的肝脏富集抑制蛋白（LIP）区域相互作用。此外，质谱分析鉴定了C/EBPβ的LIP区域中Cys 201和Cys 296的蛋白S-谷胱甘肽化。C201 S、C296 S双突变体C/EBPβ阻止了蛋白S-谷胱甘肽化并保留了与PIAS 1的相互作用。总之，Glycine消融通过增加C/EBPβ的蛋白S-谷胱甘肽化，稳定和增加C/EBPβ蛋白水平，刺激3 T3 L1细胞分化和脂肪形成。
Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) β. Protein S-glutathionylation decreased the interaction of C/EBPβ and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier (SUMO) E3 ligase that facilitates C/EBPβ degradation. Experiments with truncated mutant C/EBPβ demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPβ. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPβ. The C201S, C296S double mutant C/EBPβ prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPβ, stabilizing and increasing C/EBPβ protein levels.