Department of Internal Medicine II, University of Yamanashi, Chuo, Japan.
Department of Medicine, Vascular Biology Section, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
FASEB J. 2020 Apr;34(4):5827-5837. doi: 10.1096/fj.201902575R. Epub 2020 Mar 5.
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 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.
活性氧 (ROS) 在脂肪生成和肥胖过程中会增加。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，即 S-谷胱甘肽化，然后被谷氧还蛋白-1 (Glrx) 选择性去除。我们之前曾报道过，Glrx 敲除小鼠的蛋白质 S-谷胱甘肽化增加，并通过未知机制发展为肥胖。在这项研究中，我们证明 3T3L1 脂肪细胞分化会增加 ROS 和蛋白质 S-谷胱甘肽化。Glrx 缺失会增加 3T3L1 细胞中的蛋白质 S-谷胱甘肽化和脂质含量。Glrx 的补充会降低 Glrx KO 3T3L1 细胞的脂质含量。Glrx 缺失还会增加脂肪生成转录因子 CCAAT 增强子结合蛋白 (C/EBP) β 的蛋白表达和蛋白质 S-谷胱甘肽化。蛋白质 S-谷胱甘肽化会降低 C/EBPβ 与蛋白激活 STAT 的抑制剂 (PIAS) 1 的相互作用，PIAS1 是一种小泛素相关修饰酶 E3 连接酶，可促进 C/EBPβ 的降解。使用截断突变体 C/EBPβ 的实验表明，PIAS1 与 C/EBPβ 的富含肝脏的抑制蛋白 (LIP) 区域相互作用。此外，质谱分析鉴定了 C/EBPβ 的 LIP 区域中 Cys201 和 Cys296 的蛋白质 S-谷胱甘肽化。C201S、C296S 双突变 C/EBPβ 可防止蛋白质 S-谷胱甘肽化，并保持与 PIAS1 的相互作用。总之，Glrx 缺失通过增加 C/EBPβ 的蛋白质 S-谷胱甘肽化来刺激 3T3L1 细胞分化和脂肪生成，从而稳定和增加 C/EBPβ 蛋白水平。
