Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thioredoxin systems.
To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with (35)S, 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 de-glutathionylate 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.
Mario Negri Institute for Pharmacological Research, Department of Biochemistry, 20157 Milan, Italy.
Simona
S
Casagrande
Valentina
V
Bonetto
Maddalena
M
Fratelli
Elisabetta
E
Gianazza
Ivano
I
Eberini
Tania
T
Massignan
Mario
M
Salmona
Geng
G
Chang
Arne
A
Holmgren
Pietro
P
Ghezzi
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003958:Diamide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013601:T-Lymphocytes / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin
D007328:Insulin; D010446:Peptide Fragments; D003958:Diamide; D013879:Thioredoxins; D014357:Trypsin; D005978:Glutathione; D019803:Glutathione Disulfide