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

Andrey I. Osipyants, Andrey A. Poloznikov, Natalya A. Smirnova, Dmitry M. Hushpulian, Anna Yu Khristichenko, Tatiana A. Chubar, Arpenik A. Zakhariants, Manuj Ahuja, Irina N. Gaisina, Bobby Thomas, Abe M. Brown, Irina G. Gazaryan, Vladimir I. Tishkov

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)46-54
Number of pages9
JournalBiochimie
Volume147
DOIs
StatePublished - Apr 2018

Keywords

  • Adaptaquin
  • Catalytic cycle
  • HIF PHD inhibitor
  • HIF1 ODD-Luciferase reporter assay
  • Jumonji demethylase
  • TET enzyme

ASJC Scopus subject areas

  • Biochemistry

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    Osipyants, A. I., Poloznikov, A. A., Smirnova, N. A., Hushpulian, D. M., Khristichenko, A. Y., Chubar, T. A., Zakhariants, A. A., Ahuja, M., Gaisina, I. N., Thomas, B., Brown, A. M., Gazaryan, I. G., & Tishkov, V. I. (2018). L-ascorbic acid: A true substrate for HIF prolyl hydroxylase? Biochimie, 147, 46-54. https://doi.org/10.1016/j.biochi.2017.12.011