title: Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells.
abstract: 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.
authors.affiliation: Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20157 Milan, Italy.
authors.forename: Maddalena
authors.initials: M
authors.lastname: Fratelli
authors.forename: Hans
authors.initials: H
authors.lastname: Demol
authors.forename: Magda
authors.initials: M
authors.lastname: Puype
authors.forename: Simona
authors.initials: S
authors.lastname: Casagrande
authors.forename: Pia
authors.initials: P
authors.lastname: Villa
authors.forename: Ivano
authors.initials: I
authors.lastname: Eberini
authors.forename: Joel
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Gianazza
authors.forename: Pietro
authors.initials: P
authors.lastname: Ghezzi
mesh_terms: 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
chemical_list: D004220:Disulfides; D013438:Sulfhydryl Compounds; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen