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. 2002 Mar 19;99(6):3505-10.
doi: 10.1073/pnas.052592699.

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

Maddalena Fratelli  1 , 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
Affiliations

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

Maddalena Fratelli et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 1
Protein stain and glutathionylation pattern after two-dimensional electrophoresis of extracts from T cell blasts under basal conditions (control) or oxidatively stressed with 1 mM diamide or 1 mM H2O2. 35S labeling of the intracellular GSH pool and electrophoresis were performed as described in Materials and Methods. Proteins were separated based on pl (4-10 nonlinear gradient, left to right) and molecular weight (12% SDS/PAGE, top to bottom). Coomassie blue-stained maps are displayed (Upper) in comparison with fluorography (Lower). The experiment shown is representative of three and corresponds to experiment 3 as reported in Table 1.
Figure 2
Figure 2
Two-dimensional protein map of T cell blasts. The position of the spots corresponding to the proteins identified by MS fingerprinting in experiment 3, and listed in Table 1, is marked on the map of the diamide-treated sample. Sequential numbers are either above the relevant spot (black) or below (white).
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