Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells.
Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.
Fratelli
Maddalena
M
Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20157 Milan, Italy.
Demol
Hans
H
Puype
Magda
M
Casagrande
Simona
S
Villa
Pia
P
Eberini
Ivano
I
Vandekerckhove
Joel
J
Gianazza
Elisabetta
E
Ghezzi
Pietro
P
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds
D004220:Disulfides; D013438:Sulfhydryl Compounds; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen