Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes

Maddalena Fratelli, Hans Demol, Magda Puype, Simona Casagrande, Ivano Eberini, Mario Salmona, Valentina Bonetto, Manuela Mengozzi, Francis Duffieux, Emeric Miclet, Angela Bachi, Joel Vandekerckhove, Elisabetta Gianazza, Pietro Ghezzi

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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 35S, 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, ubiquitinconjugating 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.

Original languageEnglish
Pages (from-to)3505-3510
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number6
DOIs
Publication statusPublished - Mar 19 2002

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Proteomics
Oxidation-Reduction
T-Lymphocytes
6-phosphogluconolactonase
Cyclophilins
Phosphopyruvate Hydratase
Proteins
Enzymes
Profilins
Actin Depolymerizing Factors
Triose-Phosphate Isomerase
Pyrophosphatases
Peptidylprolyl Isomerase
Diamide
Galectins
Phosphoglycerate Kinase
Protein Disulfide-Isomerases
Peroxiredoxins
Adenylate Kinase
Fatty Acid-Binding Proteins

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. / Fratelli, Maddalena; Demol, Hans; Puype, Magda; Casagrande, Simona; Eberini, Ivano; Salmona, Mario; Bonetto, Valentina; Mengozzi, Manuela; Duffieux, Francis; Miclet, Emeric; Bachi, Angela; Vandekerckhove, Joel; Gianazza, Elisabetta; Ghezzi, Pietro.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 6, 19.03.2002, p. 3505-3510.

Research output: Contribution to journalArticle

Fratelli, M, Demol, H, Puype, M, Casagrande, S, Eberini, I, Salmona, M, Bonetto, V, Mengozzi, M, Duffieux, F, Miclet, E, Bachi, A, Vandekerckhove, J, Gianazza, E & Ghezzi, P 2002, 'Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes', Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 6, pp. 3505-3510. https://doi.org/10.1073/pnas.052592699
Fratelli, Maddalena ; Demol, Hans ; Puype, Magda ; Casagrande, Simona ; Eberini, Ivano ; Salmona, Mario ; Bonetto, Valentina ; Mengozzi, Manuela ; Duffieux, Francis ; Miclet, Emeric ; Bachi, Angela ; Vandekerckhove, Joel ; Gianazza, Elisabetta ; Ghezzi, Pietro. / Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. In: Proceedings of the National Academy of Sciences of the United States of America. 2002 ; Vol. 99, No. 6. pp. 3505-3510.
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AU - Fratelli, Maddalena

AU - Demol, Hans

AU - Puype, Magda

AU - Casagrande, Simona

AU - Eberini, Ivano

AU - Salmona, Mario

AU - Bonetto, Valentina

AU - Mengozzi, Manuela

AU - Duffieux, Francis

AU - Miclet, Emeric

AU - Bachi, Angela

AU - Vandekerckhove, Joel

AU - Gianazza, Elisabetta

AU - Ghezzi, Pietro

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N2 - 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 35S, 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, ubiquitinconjugating 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.

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