title: Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes.
abstract: Formation of mixed disulfides between glutathione and the cysteines of some proteins (glutathionylation) has been suggested as a mechanism through which protein functions can be regulated by the redox status. The aim of this study was to identify the proteins of T cell blasts that undergo glutathionylation under oxidative stress. To this purpose, we radiolabeled cellular glutathione with (35)S, exposed T cells to oxidants (diamide or hydrogen peroxide), and performed nonreducing, two-dimensional electrophoresis followed by detection of labeled proteins by phosphorimaging and their identification by mass spectrometry techniques. We detected several proteins previously not recognized to be glutathionylated, including cytoskeletal proteins (vimentin, myosin, tropomyosin, cofilin, profilin, and the already known actin), enzymes (enolase, aldolase, 6-phosphogluconolactonase, adenylate kinase, ubiquitin-conjugating enzyme, phosphoglycerate kinase, triosephosphate isomerase, and pyrophosphatase), redox enzymes (peroxiredoxin 1, protein disulfide isomerase, and cytochrome c oxidase), cyclophilin, stress proteins (HSP70 and HSP60), nucleophosmin, transgelin, galectin, and fatty acid binding protein. Based on the presence of several protein isoforms in control cells, we suggest that enolase and cyclophilin are heavily glutathionylated under basal conditions. We studied the effect of glutathionylation on some of the enzymes identified in the present study and found that some of them (enolase and 6-phosphogluconolactonase) are inhibited by glutathionylation, whereas the enzymatic activity of cyclophilin (peptidylprolyl isomerase) is not. These findings suggest that protein glutathionylation might be a common mechanism for the global regulation of protein functions.
authors.affiliation: Mario Negri Institute for Pharmacological Research, 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: Ivano
authors.initials: I
authors.lastname: Eberini
authors.forename: Mario
authors.initials: M
authors.lastname: Salmona
authors.forename: Valentina
authors.initials: V
authors.lastname: Bonetto
authors.forename: Manuela
authors.initials: M
authors.lastname: Mengozzi
authors.forename: Francis
authors.initials: F
authors.lastname: Duffieux
authors.forename: Emeric
authors.initials: E
authors.lastname: Miclet
authors.forename: Angela
authors.initials: A
authors.lastname: Bachi
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: D002478:Cells, Cultured; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D013058:Mass Spectrometry; D008970:Molecular Weight; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D020543:Proteome / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012394:Rosaniline Dyes; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
chemical_list: D004220:Disulfides; D016877:Oxidants; D020543:Proteome; D012394:Rosaniline Dyes; D003958:Diamide; C048139:Coomassie blue; D006861:Hydrogen Peroxide; D005978:Glutathione