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Protein S ‐glutathionylation stimulate adipogenesis by stabilizing C/EBPβ in 3T3L1 cells
Author(s) -
Watanabe Yosuke,
Watanabe Kazuhiro,
Fujioka Daisuke,
Nakamura Kazuto,
Nakamura Takamitsu,
Uematsu Manabu,
Bachschmid Markus M.,
Matsui Reiko,
Kugiyama Kiyotaka
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.201902575r
Subject(s) - glutaredoxin , adipogenesis , chemistry , mutant , microbiology and biotechnology , biochemistry , glutathione , biology , in vitro , gene , enzyme
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.