Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

Abstract

The ascorbate-glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO(-)) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO(-) and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO(-). The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO(-). These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO(-) or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate-glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.

Keywords: Glutathione reductase; S-nitrosoglutathione.; S-nitrosylation; monodehydroascorbate reductase; nitration; nitric oxide; peroxynitrite; reactive nitrogen species; salinity.

Fig. 1.

Effect of nitration and S-nitrosylation on recombinant monodehydroascorbate reductase (MDAR) and glutathione reductase (GR). Effect of SIN-1 (peroxynitrite donor) on recombinant MDAR (A) and GR (B) activities. Representative immunoblot showing the grade of tyrosine nitration of MDAR (C) and chloroplastic and cytosolic GR (D), treated with different concentrations of SIN-1 and detected with an antibody against 3-nitrotyrosine (dilution 1:2500). A 5 μg aliquot of protein was used per line. Effect of S-nitrosoglutathione (GSNO) on recombinant MDAR (E) and chloroplastic GR (F). Effect of glutathione (GSH) on recombinant MDAR (G) and chloroplastic GR (H). Purified MDAR and GR proteins were incubated at different concentrations of SIN-1 at 37 ºC for 60min, GSNO at 25 ºC for 30min, or GSH at 25 ºC for 30min. The specific activity of the recombinant MDAR was 1200 nmol NADH min^–1 mg^–1 and for GR proteins it was 18 μmol NADPH min^–1 mg^–1. S-Nitrosylation of recombinant MDAR (I) and chloroplastic and cytosolic GR (J). A 5 μg aliquot of purified recombinants proteins was treated with 2mM GSH and 2mM GSNO and was subjected to the biotin switch method. Control treatments were carried out with water (lane 1) and 2mM GSH (lane 2). Additionally, recombinants proteins were S-nitrosylated with 2mM GSNO (lane 3) and reduced again with 50mM DTT (lane 4). Furthermore, GSNO-treated recombinant proteins underwent the biotin switch method without ascorbate (lane 5). Proteins were separated under non-reducing conditions by SDS–PAGE and blotted onto a PVDF membrane. Biotinylated proteins were detected using an anti-biotin antibody. Data are means ±SEM of at least three replicates. *Differences from control values were significant at P<0.05.

Fig. 2.

Comparison of the nitrated (top) and unmodified (bottom) MS/MS spectra of the peptides identified from the pea peroxisomal MDAR in the corresponding panels: (A) LFTSEIAAFYEGY*YANK, (B) TSVPDVY*AVGDV ATFPLK, and (C) SVEEYDY*LPYFYSR. Peptide fragment ions are indicated by ‘b’ if the charge is retained on the N-terminus and by ‘y’ if the charge is maintained on the C-terminus. The subscript indicates the number of amino acid residues in the fragment studied from either the N-terminus or the C-terminus. The superscript indicates the charge (1⁺ or 2⁺) of the backbone fragmentation. (This figure is available in colour at JXB online.)

Fig. 3

(A) Location of the tyrosine residues and cysteine residues susceptible to being responsible for the modulation of the enzymatic activity of pea MDAR by peroxynitrite and GSNO. (B) GSH binding site close to Cys68 located by blind docking. (This figure is available in colour at JXB online.)

Fig. 4.

Effect of SIN-1 (peroxynitrite donor) on the recombinant mutant pea MDAR I (Tyr345Phe). (A) Effect of SIN-1 on recombinant MDAR activity. (B) Representative immunoblot showing the grade of tyrosine nitration of recombinant mutant pea MDAR and detected with an antibody against 3-nitrotyrosine (dilution 1:2500). Recombinant mutant pea MDAR I (Y345F) protein was incubated at different concentrations of SIN-1 at 37 ºC for 1h. Data are means ±SEM of at least three replicates.

Fig. 5.

Protein and gene expression of MDAR and analysis of S-nitrosylated MDAR in leaves of pea plants under salinity (150mM NaCl) stress conditions. (A) Immunoblotting analysis of MDAR protein expression using an antibody against cucumber MDAR (dilution 1:3000). A 10 μg aliquot of protein was used per lane. (B) Real-time quantitative RT–PCR transcript analysis (arbitrary units) of the MDAR gene. Data are means ±SEM of at least four independent RNA samples. *Differences from control values were significant at P>0.05. (C) Detection of total S-nitrosylated proteins separated under non-reducing conditions by 12% SDS–PAGE and blotted onto a PVDF membrane. Biotinylated proteins were detected using anti-biotin antibodies as described in the Materials and methods. (D) Immunoblot of total S-nitrosylated proteins probed with an antibody against cucumber MDAR (dilution 1:3000). A 5 μg aliquot of protein was used per lane.

Fig. 6.

Regulation of the ascorbate–glutathione cycle by nitric oxide (NO). NO modulates the ascorbate–glutathione cycle throughout post-translational modifications (PTMs) as tyrosine nitration and S-nitrosylation of APX and MDAR proteins. MDAR activity is reduced after both modifications, with APX activity also being reduced by tyrosine nitration. Under nitro-oxidative stress conditions, these modifications could compromise the antioxidant capacity of the cycle. However, APX activity is enhanced by S-nitrosylation while GR activity is not significantly affected by these NO-related PTMs. This behaviour suggests that APX and GR try to detoxify hydrogen peroxide and maintain regeneration of GSH, respectively, and consequently the cellular redox state to maintain the antioxidant resistance of the ascorbate–glutathione cycle against nitro-oxidative cell conditions. (This figure is available in colour at JXB online.)