Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1
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Protein S-glutathionylation is a reversible post-translational modificationon cysteine residues forming a mixed disulfide withglutathione. S-glutathionylation, not only protects proteins fromoxidation but also regulates the functions of proteins involvedin various cellular signaling pathways. In this study, we developeda method for the detection of S-glutathionylated proteins(ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin1 (mGrx1). ProSSG was efficiently and specifically labeledwith E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection ofProSSG with semi-quantitative determination. In addition, a deglutathionylationenzyme mGrx1 specifically released E-GSHfrom ProSSG-E, which increased fluorescence allowing a sensitivedetermination of ProSSG levels. Application of the methodto the adipocyte differentiation of 3T3-L1 cells showed specificdetection of ProSSG and its increase upon differentiation induction,which was consistent with the result obtained byconventional immunoblot analysis, but with greater specificityand sensitivity.
Abstract
Protein S-glutathionylation is a reversible post-translational modificationon cysteine residues forming a mixed disulfide withglutathione. S-glutathionylation, not only protects proteins fromoxidation but also regulates the functions of proteins involvedin various cellular signaling pathways. In this study, we developeda method for the detection of S-glutathionylated proteins(ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin1 (mGrx1). ProSSG was efficiently and specifically labeledwith E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection ofProSSG with semi-quantitative determination. In addition, a deglutathionylationenzyme mGrx1 specifically released E-GSHfrom ProSSG-E, which increased fluorescence allowing a sensitivedetermination of ProSSG levels. Application of the methodto the adipocyte differentiation of 3T3-L1 cells showed specificdetection of ProSSG and its increase upon differentiation induction,which was consistent with the result obtained byconventional immunoblot analysis, but with greater specificityand sensitivity.
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