TY - JOUR
T1 - Glutathionylation of human thioredoxin
T2 - A possible crosstalk between the glutathione and thioredoxin systems
AU - Casagrande, Simona
AU - Bonetto, Valentina
AU - Fratelli, Maddalena
AU - Gianazza, Elisabetta
AU - Eberini, Ivano
AU - Massignan, Tania
AU - Salmona, Mario
AU - Chang, Geng
AU - Holmgren, Arne
AU - Ghezzi, Pietro
PY - 2002/7/23
Y1 - 2002/7/23
N2 - To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with 35S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to deglutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.
AB - To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with 35S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to deglutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.
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U2 - 10.1073/pnas.152168599
DO - 10.1073/pnas.152168599
M3 - Article
C2 - 12119401
AN - SCOPUS:18444369324
VL - 99
SP - 9745
EP - 9749
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 15
ER -