Glutathionylation of Pea Chloroplast 2-Cys Prx and Mitochondrial Prx IIF Affects Their Structure and Peroxidase Activity and Sulfiredoxin Deglutathionylates Only the 2-Cys Prx

Abstract

Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys¹⁷⁴), but not the peroxidatic equivalent (Cys⁵²), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys⁵⁹) and resolving (Cys⁸⁴) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins.

Keywords: 2-Cys peroxiredoxin; glutathione redox state; glutathionylation; peroxiredoxin IIF; post-translational modification; reactive nitrogen species; reactive oxygen species; sulfiredoxin.

FIGURE 1

Elution profile after size exclusion chromatography (A) through Superdex-200 HR 10/30 column of native (untreated) recombinant pea 2-Cys Prx and after its treatment with 10 mM DTT (+DTT) and 10 mM DTT + 5 mM GSSG (+DTT+GSSG). Asterisks indicate the samples subsequently analyzed by MALDI TOF/TOF. Pattern of oligomerization (B) analyzed by native PAGE and Coomasie staining of pre-reduced protein treated with GSSG or DTT, the non-treated protein (-DTT, -GSSG) being used as control.

FIGURE 2

Elution profile after size exclusion chromatography through Superdex-200 HR 10/30 column of pre-reduced pea 2-Cys Prx (+DTT) and after its treatment with 5 mM GSNO for different incubation times. Asterisks indicate the samples subsequently analyzed by MALDI TOF/TOF.

FIGURE 3

Elution profile after size exclusion chromatography through Superdex-200 HR 10/30 column of DTT-reduced pea 2-Cys Prx and after its treatment with different S-nitrosylating and glutathionylating agents: 750 μM SNP, 750 μM SNP + 5 mM GSH and 5 mM GSNO.

FIGURE 4

Elution profile after size exclusion chromatography through Superdex-200 HR 10/30 column of native (untreated form) recombinant pea Prx IIF and after its treatment with 10 mM DTT and 5 mM GSSG and GSNO. Asterisks indicate the samples subsequently analyzed by MALDI TOF/TOF.

FIGURE 5

Glutathionylation of recombinant 2-Cys Prx and Prx IIF by different concentrations of GSH/GSSG. DTT-reduced Prx proteins were incubated with the different concentrations for 5 min at 37°C and the excess of glutathionylating agents was removed. A DTT-treated sample was used as negative control. Samples (15 μg protein) were immediately analyzed by western-blot using a specific monoclonal glutathione antibody, and a representative example is shown. Numbers show the mean of the densitometric analysis of at least 4 independent experiments, relative to the first band in each of the forms of the proteins. Pounceau S stained membranes were used as loading controls. Dec: decameric and Dim: dimeric forms of the proteins.

FIGURE 6

Peroxidase activities of recombinant 2-Cys Prx and Prx IIF after the treatment with 5 mM GSSG. Peroxidase activity was measured in previously DTT-reduced proteins (control) and in GSSG-treated proteins after incubation with H₂O₂ for 10 min at 37°C, using trichloroacetic acid to stop the reactions, as described in material and methods. H₂O₂ was then quantified using the eFOX method.

FIGURE 7

Deglutathionylation of recombinant 2-Cys Prx (A) and Prx IIF (B) proteins by pea recombinant Srx. Prx proteins were treated with 5 mM GSSG and then incubated for different times with recombinant DTT-treated Srx. The samples (3 μg of protein) were analyzed by western-blot using a monoclonal glutathione antibody. The loading was checked using specific polyclonal 2-Cys Prx and Prx IIF antibodies.

FIGURE 8

Proposed mechanism of glutathionylation of the decameric reduced form of 2-Cys Prx after treatment with 5 mM GSSG, 5 mM GSNO or 750 μM SNP. Glutathionylation of the 2-Cys Prx can be caused directly by GSSG or GSNO or indirectly by GSH after nitrosylation of the protein by SNP. The glutathionylation of the decameric 2-Cys Prx induces dimerization of the protein.