TY - JOUR
T1 - L-ascorbic acid
T2 - A true substrate for HIF prolyl hydroxylase?
AU - Osipyants, Andrey I.
AU - Poloznikov, Andrey A.
AU - Smirnova, Natalya A.
AU - Hushpulian, Dmitry M.
AU - Khristichenko, Anna Yu
AU - Chubar, Tatiana A.
AU - Zakhariants, Arpenik A.
AU - Ahuja, Manuj
AU - Gaisina, Irina N.
AU - Thomas, Bobby
AU - Brown, Abe M.
AU - Gazaryan, Irina G.
AU - Tishkov, Vladimir I.
N1 - Funding Information:
The work was supported by Russian Science Foundation (grant No. 16-14-10226 ) and in part (RT-PCR experiments) by funding from Parkinson Support Group and NS101967 grant (BT).
Publisher Copyright:
© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)
PY - 2018/4
Y1 - 2018/4
N2 - 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.
AB - 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.
KW - Adaptaquin
KW - Catalytic cycle
KW - HIF PHD inhibitor
KW - HIF1 ODD-Luciferase reporter assay
KW - Jumonji demethylase
KW - TET enzyme
UR - http://www.scopus.com/inward/record.url?scp=85040356274&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85040356274&partnerID=8YFLogxK
U2 - 10.1016/j.biochi.2017.12.011
DO - 10.1016/j.biochi.2017.12.011
M3 - Article
C2 - 29289682
AN - SCOPUS:85040356274
SN - 0300-9084
VL - 147
SP - 46
EP - 54
JO - Biochimie
JF - Biochimie
ER -