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

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.

Vitamin C effects on 5-hydroxymethylcytosine and gene expression in osteoblasts and chondrocytes: Potential involvement of PHD2

“…AA is a cofactor for prolyl hydroxylase domain-containing (PHD) proteins, a family of dioxygenases which depend upon the presence of 2-oxoglutarate, Fe 2+ , and oxygen to function. [32] PHDs can hydroxylate proline residues in the hypoxia-inducible factors (HIFs), causing the HIFs to undergo ubiquitination followed by proteasomal degradation. [52] Indeed, we have demonstrated that PHD2-mediated proteasomal degradation of HIF1α is a mechanism of AA’s action in bone.…”

“…Interestingly, the PHDs are part of the same enzyme family as the TETs, and a recent study has provided evidence that, in addition to its role as a reducing agent, L-ascorbate acts as a true co-substrate for PHD2 in the same way it does for the TETs. [32] Since AA can induce DNA demethylation, and since PHD2 mediates AA effects in bone and cartilage, we hypothesized that some of the AA effect in osteoblasts and chondrocytes may be due to TET- and/or PHD2-mediated increases in 5-hmC in the promoter regions of genes important for osteoblast and chondrocyte function. To test this hypothesis, we first examined the effects of AA on bone and cartilage at a phenotypic and gene expression level.…”

Kinetic parameters of human aspartate/asparagine–β-hydroxylase suggest that it has a possible function in oxygen sensing

“…The peptides and their hydroxylation products were analyzed by SPE-MS monitoring substrate depletion and product formation (+16 Da mass shift). Initially, the assay conditions were optimized (Supporting Figure S2) The highest AspH activity was observed in 50 mM HEPES buffer (pH 7.5) without additional salts in the presence of 2OG, ferrous ammonium sulphate (FAS), and L-ascorbic acid (LAA, which enhances the activity of many isolated 2OG oxygenases) ( Figure 1c) (46)(47)(48). Compared to our previously reported MALDI-MS assay conditions (36), the AspH concentration was reduced 100 fold to 0.1 μM in the SPE-MS based assay, significantly reducing the enzyme required, thus potentially more accurately reflecting physiological conditions.…”

“…LAA is commonly added to assay buffers to enhance the activity of isolated 2OG oxygenases (e.g. for the procollagen and HIF-α prolyl hydroxylases) (46)(47)(48). In some cases LAA might act as a co-substrate effectively replacing 2OG (e.g.…”

Antitumor and antiviral activities of 4-substituted 1,2,3-triazolyl-2,3-dibenzyl-L-ascorbic acid derivatives

“…It was shown that L-ASA inhibits the growth of KRAS and BRAF mutant colorectal cancer cells by causing oxidative stress and accumulation of reactive oxygen species (ROS), the latter leading to inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an energy crisis and consequently cell death [6]. Lascorbic acid is an established activator of non-heme iron a-ketoglutarate (aKG) dioxygenases, such as hypoxia inducible factor prolyl hydroxylases (HIF PHD), and epigenetic ten-eleven translocation (TET) enzymes, which are directly relevant to cancer susceptibility and progression [7,8]. HIF-1 is a critical mediator of the cellular response to hypoxia, making it an attractive molecular target for anticancer therapy.…”