Research ArticlePosttranslational Modifications

Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation

See allHide authors and affiliations

Science Signaling  07 Jul 2015:
Vol. 8, Issue 384, pp. ra68
DOI: 10.1126/scisignal.aaa4312
  • Fig. 1 Proteomic identification of protein S-nitrosocysteine residues and GO analysis of modified proteins in wild-type (WT), eNOS−/−, and nNOS−/− mouse brain.

    (A) Identification of S-nitrosylated proteins and their corresponding sites of modification in mouse brain (n = 6 mice per genotype). (B) GO-based functional clustering of S-nitrosylated proteins in each genotype. The ratio of enrichment (R) score is calculated as the ratio of the observed number (O) of S-nitrosylated proteins with a specific GO annotation in the WebGestalt database to the number of annotated proteins expected (E) to be in the S-nitrosocysteine proteome. Only the top five pathways in the WT mouse with the lowest P values were considered (P < 0.01). NT, neurotransmitter. (C) Schematic of glutamate metabolism. Proteins are denoted by their short protein names, as assigned by UniProt: GLT1 (excitatory amino acid transporter 2), GDH (glutamate dehydrogenase), mAspAT (mitochondrial aspartate aminotransferase), and GS (glutamine synthetase). S-nitrosylated proteins are italicized, with the associated sites of modification indicated. Gln, glutamine; Glu, Glutamate; Asp, aspartate; Ala, alanine; α-KG, α-ketoglutarate; Cys, cysteine. (D) NOS-based dependence of S-nitrosylation (SNO) of glutamate/glutamine cycle effectors. Modified proteins (denoted as the “SNO-Fraction”) within the aforementioned pathway were confirmed by Western blot against specific targets in all genotypes. The total abundance of the indicated protein targets in 30 μg of total homogenate did not obviously differ between genotypes (n = 2 mice per genotype). (E) Quantification of the relative S-nitrosylated fraction for each glutamate/glutamine cycle protein in WT mouse brain. Bars represent means ± SEM (n = 3 mice).

  • Fig. 2 Analysis of the glutamate/glutamine cycle in WT, eNOS−/−, and nNOS−/− mouse brain.

    (A) Glutamate-associated metabolite quantification. Total metabolite concentrations were determined by HPLC and normalized to protein content (n = 3 mice per genotype). (B) Intracellular glutamine/glutamate ratios. **P < 0.01 as determined by one-way analysis of variance (ANOVA) with Tukey post hoc analysis (n = 3 mice per genotype). (C) Genotypic differences in steady-state glutamine/glutamate metabolism. 15N enrichment of glutamine/glutamate and associated metabolites was determined using GC-MS as in (41), and presented as 15N molar percent excess (MPE) of M+1 isotopomer. *P < 0.05 compared to WT by one-way ANOVA with Tukey post hoc analysis between genotypes (n = 3 mice per genotype). (D) Enzymatic activity of glutamate/glutamine effectors. Bars represent mean ± SEM (n = 3 mice per genotype) and indicate enzymatic activity relative to that measured in untreated extracts from WT mice. Activities in untreated WT extracts: GS = 1259 ± 94 nmol/mg per hour, GDH = 120 ± 15 nmol/mg per minute, mAspAT = 238 ± 12 nmol/mg per minute. WT + UV denotes extracts exposed to UV trans-illumination before assessing enzymatic activity. WT + inhibitor denotes extracts pretreated with specific enzymatic inhibitors (GS = 0.5 mM MSO, GDH = 20 μM GTP, mAspAT = 1 mM AOAA). N.D., not detectable. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to WT by one-way ANOVA with Tukey post hoc analysis.

  • Fig. 3 Regulation of GLT1 function by S-nitrosylation.

    (A to I) All results are summarized from three mice per genotype (A) or three independent experiments in cells (B to I), and plotted as means ± SEM. Cells were treated with l-cysteine (Cys) or S-nitrosocysteine (CysNO). (A) The fraction of total Na+-dependent glutamate uptake that was DHK-sensitive was greater in synaptosomes from nNOS−/− mice or synaptosomes pretreated with copper and ascorbate (WT + Cu/Asc) than in untreated WT synaptosomes. Na+-dependent uptake in untreated synaptosomes of the indicated genotype: WT = 0.24 ± 0.03 nmol/mg per minute, nNOS−/− = 0.26 ± 0.06 nmol/mg per minute, eNOS−/− = 0.25 ± 0.04 nmol/mg per minute, and WT + Cu/Asc = 0.29 ± 0.03 nmol/mg per minute. *P < 0.05 after one-way ANOVA followed by Dunnett’s post hoc analysis. (B and C) S-nitrosylation of WT GLT1 in HEK-293T cells after CysNO treatment (B) correlated with decreased glutamate uptake (C). Cys-treated WT GLT1 = 0.27 ± 0.03 nmol/mg per minute, Cys-treated C373S/C562S GLT1 = 0.26 ± 0.04 nmol/mg per minute. ****P < 0.0001 after two-way ANOVA followed by Bonferroni post hoc analysis. (D and E) Representative blots from cell surface biotinylation assays of cells expressing WT GLT1 (D) and C373S/C562S GLT1 (E). L, lysate; I, intracellular fraction; C, cell surface fraction. Five micrograms of total lysate and equivalent dilutions of the other fractions were used. T, D, and M refer to trimeric, dimeric, and monomeric GLT1, respectively. The intracellular fraction of both forms of GLT1 was increased after CysNO treatment. (F) Quantification of total GLT1 abundance in cell lysate. (G) Quantification of plasma membrane abundance of GLT1 after Cys/CysNO treatment. *P < 0.05, **P < 0.01 as determined by ANOVA with Bonferroni post hoc analysis. (H and I) S-nitrosylation of WT GLT1 was reversible (H) and correlated with a recovery in glutamate uptake (I). WT GLT1 cells were exposed to Cys (CTRL) or CysNO, then to fresh medium. Glutamate uptake and S-nitrosylation of GLT1 were assessed after CysNO exposure. *P < 0.05 after one-way ANOVA followed by Bonferroni post hoc analysis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/384/ra68/DC1

    Fig. S1. Reduction in the enrichment of S-nitrosylated proteins in eNOS−/− and nNOS−/− mice.

    Fig. S2. Additional biological processes and cellular functions identified for S-nitrosylated proteins in the mouse brain.

    Fig. S3. Identification of 15N isotopic label in glutamate-associated metabolites.

    Fig. S4. Characterization of GLT1-independent uptake in synaptosomes and GLT1-dependent uptake in cells.

    Fig. S5. S-nitrosylation augments electrostatic potential.

    Table S1. S-nitrosocysteine sites identified in wild-type, eNOS−/−, and nNOS−/− mouse brain.

  • Supplementary Materials for:

    Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation

    Karthik Raju, Paschalis-Thomas Doulias, Perry Evans, Elizabeth N. Krizman, Joshua G. Jackson, Oksana Horyn, Yevgeny Daikhin, Ilana Nissim, Marc Yudkoff, Itzhak Nissim, Kim A. Sharp, Michael B. Robinson, Harry Ischiropoulos*

    *Corresponding author. E-mail: ischirop{at}mail.med.upenn.edu

    This PDF file includes:

    • Fig. S1. Reduction in the enrichment of S-nitrosylated proteins in eNOS−/− and nNOS−/− mice.
    • Fig. S2. Additional biological processes and cellular functions identified for S-nitrosylated proteins in the mouse brain.
    • Fig. S3. Identification of 15N isotopic label in glutamate-associated metabolites.
    • Fig. S4. Characterization of GLT1-independent uptake in synaptosomes and GLT1-dependent uptake in cells.
    • Fig. S5. S-nitrosylation augments electrostatic potential.
    • Table S1. S-nitrosocysteine sites identified in wild-type, eNOS−/−, and nNOS−/− mouse brain.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.20 MB


    Citation: K. Raju, P.-T. Doulias, P. Evans, E. N. Krizman, J. G. Jackson, O. Horyn, Y. Daikhin, I. Nissim, M. Yudkoff, I. Nissim, K. A. Sharp, M. B. Robinson, H. Ischiropoulos, Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation. Sci. Signal. 8, ra68 (2015).

    © 2015 American Association for the Advancement of Science

Navigate This Article