Cytosolic Isocitrate Dehydrogenase from Arabidopsis thaliana Is Regulated by Glutathionylation

Abstract

NADP-dependent (Nicotinamide Adénine Dinucléotide Phosphate-dependent) isocitrate dehydrogenases (NADP-ICDH) are metabolic enzymes involved in 2-oxoglutarate biosynthesis, but they also supply cells with NADPH. Different NADP-ICDH genes are found in Arabidopsis among which a single gene encodes for a cytosolic ICDH (cICDH) isoform. Here, we show that cICDH is susceptible to oxidation and that several cysteine (Cys) residues are prone to S-nitrosylation upon nitrosoglutathione (GSNO) treatment. Moreover, we identified a single S-glutathionylated cysteine Cys363 by mass-spectrometry analyses. Modeling analyses suggest that Cys363 is not located in the close proximity of the cICDH active site. In addition, mutation of Cys363 consistently does not modify the activity of cICDH. However, it does affect the sensitivity of the enzyme to GSNO, indicating that S-glutathionylation of Cys363 is involved in the inhibition of cICDH activity upon GSNO treatments. We also show that glutaredoxin are able to rescue the GSNO-dependent inhibition of cICDH activity, suggesting that they act as a deglutathionylation system in vitro. The glutaredoxin system, conversely to the thioredoxin system, is able to remove S-nitrosothiol adducts from cICDH. Finally, NADP-ICDH activities were decreased both in a catalase2 mutant and in mutants affected in thiol reduction systems, suggesting a role of the thiol reduction systems to protect NADP-ICDH activities in planta. In line with our observations in Arabidopsis, we found that the human recombinant NADP-ICDH activity is also sensitive to oxidation in vitro, suggesting that this redox mechanism might be shared by other ICDH isoforms.

Keywords: Arabidopsis thaliana; Isocitrate dehydrogenase; glutaredoxin; glutathionylation; nitrosylation.

Figure 1

Cytosolic ICDH1 activity. ICDH (Isocitrate dehydrogenase) activity was measured for 800 ng of ICDH by monitoring the change in absorbance at 340 nm due to the isocitrate-dependent rate of NADP⁺ reduction at 25 °C in 1 mL reaction medium containing 5 mM MgCl2, 250 μM of NADP⁺ and (A) different concentrations (0–5 mM) of DL-isocitric acid or (B) different concentrations (0–1 mM) of NADP in 100 mM phosphate buffer (KOH, pH 7.5). (C) 2.5 mM DL-isocitric acid in 100 mM phosphate buffer at different pH (6–8.5). Error bars represent SE (Standard Error) (n = 3).

Figure 2

Cytosolic ICDH activity is sensitive to oxidation. 4.38 µM (200 ng/µL) ICDH were incubated with different concentrations of (A) H₂O₂/GSH, (B) nitrosoglutathione (GSNO) or (C) oxidized glutathione (GSSG) for 15 min at 25 °C. This reaction mixture was diluted 250 times in reaction buffer during the assay. ICDH activity was measured for 800 ng of ICDH at 25 °C in 1 mL reaction medium containing 100 mM phosphate buffer (KOH, pH 7.5), 5 mM MgCl2, 250 µM of NADP⁺ and 2.5 mM DL-isocitric acid by monitoring the change in absorbance at 340 nm due to the isocitrate-dependent rate of NADP⁺ reduction. Error bars represent SE (n = 3). * p < 0.05, ** p < 0.01 for statistical differences compared to non-treated samples (Student’s t test).

Figure 3

Glutathionylation of cICDH. (A) cICDH was treated or not with 1 mM GSNO for 30 min at 25 °C. Samples were trypsin digested and analyzed by nanoLC-MSMS. The panels show fragmentation spectra matching peptides with either unmodified (top) or with glutathionylated C363 (bottom). The same glutathionylated residue was identified in three biological repetitions. (B) Modeled cICDH (residues 4–408) homodimer (grey and green chains) based on human cytosolic ICDH. Conserved amino acids (R111, R134, Y141, T214, D252, D279, R314, H315) with the most plant and mammalian NADP-ICDH proteins, in the active site pocket are shown in different colors depending on the nature of the residue: R, blue; Y, purple; T, orange; D, red; H, grey. The conserved cysteine C363 in each chain is represented in yellow (arrowheads). In the right panel, the cICDH homodimer was rotated 180°.

Figure 4

cICDH is S-nitrosylated and is denitrosylated by the GRX system. (A) 2.19 µM of recombinant wild-type cICDH or Cys-mutated versions of cICDH (cICDH-C363S) were treated with or without 1 mM GSNO for 30 min at 25 °C, and subjected to the biotin-switch assay in presence or absence of sodium ascorbate. (B) After treatment with GSNO (1 mM), the protein was treated with GRXC1 (5 µM) alone (lane 2), GRXC1, GSH (0.8 mM) and GR (0.45 µM) (Lane 3), NTRA (3 µM) alone (lane 4) and NTRA+TRXh3 (4.59 µM) (lane 5) for 30 min at 25 °C and subjected to the biotin-switch assay in the presence of sodium ascorbate. (C) The same experimental design in the presence of GSH (0.8 mM) (lane 2), GR (5 µM) (lane 3), GrxC1 (5 µM) (lane 4) and GRXC1+GR+GSH (lane 5). Afterwards, the proteins were separated by reducing SDS-PAGE and transferred onto nitrocellulose membrane. Total ICDH (bottom panel) or S-nitrosylated ICDH (top panel) was detected using an anti-His antibody.

Figure 5

Cysteine-dependent regulation of cICDH activity. (A) ICDH activity was measured for 800 ng of ICDH and ICDH-C363S by monitoring the change in absorbance of NADPH at 340 nm due to the isocitrate-dependent rate of NADP⁺ reduction at 25 °C in 1 mL reaction medium containing 5 mM MgCl₂, 250 μM of NADP⁺ and different (0–5 mM) concentrations of DL-isocitric acid. (B) ICDH were incubated with 1 mM GSNO and measured in the same conditions than described previously. (C) 800 ng of cICDH were incubated with 0.5 mM GSNO, 0.75 mM GSSG for 15 min at 25 °C. The samples were diluted 2-fold and then incubated with 1 mM NADPH in the presence of NTRA (3 µM) and TRXh3 or TRXh5 (4.59 µM) (D) Same experimental design as in (C), but in presence of GR (0.45 µM), GSH (0.8 mM) and GRXC1 or GRXC2 (5 µM). Error bars represent SE (n = 3). * p < 0.01, ** p < 0.001 for statistical differences compared to non-treated samples (Student’s t test).

Figure 6

ICDH activity in planta. (A) ICDH activity was measured for 40 µg of protein extracts at 25 °C by monitoring the change in absorbance at 340 nm due to the isocitrate-dependent rate of NADP+ reduction. Wild-type (Col-0) or mutant A. thaliana plants were grown in soil in a controlled growth chamber (180 µE m⁻² s⁻¹, 16 h day/8 h night, 22 °C 55% RH day, 20 °C 60% RH night) for 2 weeks. (B) Same design as in (A). In Col-O+GSNO and Col-0+SNP, protein extracts of wild-type plants were treated with 1 mM GSNO or SNP for 30 min before performing activity tests. Error bars represent SE (n = 3). * p < 0.01, ** p < 0.001 for statistical differences compared to non-treated samples (Student’s t test).