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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1

  • Hwang, Sungwon (Department of Medical Biotechnology and Research Institute of Cell Culture, Yeungnam University) ;
  • Iram, Sana (Department of Medical Biotechnology and Research Institute of Cell Culture, Yeungnam University) ;
  • Jin, Juno (Department of Medical Biotechnology and Research Institute of Cell Culture, Yeungnam University) ;
  • Choi, Inho (Department of Medical Biotechnology and Research Institute of Cell Culture, Yeungnam University) ;
  • Kim, Jihoe (Department of Medical Biotechnology and Research Institute of Cell Culture, Yeungnam University)
  • Received : 2021.09.29
  • Accepted : 2021.10.26
  • Published : 2022.03.31
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Abstract

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity.

Keywords

Acknowledgement

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A 03044512 and 2020R1I1A3060716).

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