L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?

• Published in: Biochimie 2018-04, Vol.147, p.46-54
• Main Authors: Osipyants, Andrey I., Poloznikov, Andrey A., Smirnova, Natalya A., Hushpulian, Dmitry M., Khristichenko, Anna Yu, Chubar, Tatiana A., Zakhariants, Arpenik A., Ahuja, Manuj, Gaisina, Irina N., Thomas, Bobby, Brown, Abe M., Gazaryan, Irina G., Tishkov, Vladimir I.
• Format: Article
• Language: English
• Subjects: Adaptaquin; Ascorbic Acid - chemistry; Ascorbic Acid - metabolism; Catalytic cycle; Cell Line, Tumor; HIF PHD inhibitor; HIF1 ODD-Luciferase reporter assay; Humans; Jumonji demethylase; Prolyl Hydroxylases - metabolism; Prolyl Hydroxylases - pharmacology; Prolyl-Hydroxylase Inhibitors - pharmacology; Protein Binding; Stereoisomerism; TET enzyme
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2017.12.011

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40–100 μM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation. •L-Ascorbate, but not D-isoascorbate, or N-acetyl cysteine suppresses HIF1 ODD-luc reporter activation.•The suppression by L-ascorbate depends on the chemical nature of HIF prolyl hydroxylase inhibitor.•The suppression by L-ascorbate shows a saturation behavior, indicative of the Michaelis-Menten type interaction.•L-Ascorbate suppresses the induction of HIF target genes VEGF and HO-1 with the branched oxyquinoline inhibitor.