Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents
- PMID: 20055761
- DOI: 10.1042/BJ20091609
Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents
Abstract
The HIF (hypoxia-inducible factor) hydroxylases [PHDs or EGLNs (prolyl hydroxylases), which in humans are PHD isoforms 1-3, and FIH (factor inhibiting HIF)] regulate HIF levels and activity. These enzymes are Fe(II)/2-oxoglutarate-dependent oxygenases, many of which are stimulated by ascorbate. We have investigated the ascorbate dependence of PHD2-catalysed hydroxylation of two prolyl hydroxylation sites in human HIF-1alpha, and of FIH-catalysed hydroxylation of asparaginyl hydroxylation sites in HIF-1alpha and in a consensus ankyrin repeat domain peptide. The initial rate and extent of hydroxylation was increased in the presence of ascorbate for each of these reactions. When ascorbate was replaced with structural analogues, the results revealed that the ascorbate side chain was not important in its contribution to HIF hydroxylase catalysis, whereas modifications to the ene-diol portion of the molecule negated the ability to promote hydroxylation. We investigated whether alternative reducing agents (glutathione and dithiothreitol) could be used to promote HIF hydroxylase activity, and found partial stimulation of hydroxylation in an apparently enzyme- and substrate-specific manner. The results raise the possibility of developing reducing agents targeted to specific HIF hydroxylase-catalysed reactions.
Similar articles
-
Catalytic properties of the asparaginyl hydroxylase (FIH) in the oxygen sensing pathway are distinct from those of its prolyl 4-hydroxylases.J Biol Chem. 2004 Mar 12;279(11):9899-904. doi: 10.1074/jbc.M312254200. Epub 2003 Dec 29. J Biol Chem. 2004. PMID: 14701857
-
Src activates HIF-1α not through direct phosphorylation of HIF-1α specific prolyl-4 hydroxylase 2 but through activation of the NADPH oxidase/Rac pathway.Carcinogenesis. 2011 May;32(5):703-12. doi: 10.1093/carcin/bgr034. Epub 2011 Feb 18. Carcinogenesis. 2011. PMID: 21335603
-
Protein Hydroxylation by Hypoxia-Inducible Factor (HIF) Hydroxylases: Unique or Ubiquitous?Cells. 2019 Apr 26;8(5):384. doi: 10.3390/cells8050384. Cells. 2019. PMID: 31035491 Free PMC article. Review.
-
Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites.Biochem J. 2011 Jun 1;436(2):363-9. doi: 10.1042/BJ20101201. Biochem J. 2011. PMID: 21410436
-
Hydroxylation of hypoxia-inducible transcription factors and chemical compounds targeting the HIF-alpha hydroxylases.Curr Med Chem. 2007;14(17):1853-62. doi: 10.2174/092986707781058850. Curr Med Chem. 2007. PMID: 17627521 Review.
Cited by
-
Nuclear and Cytoplasmic Functions of Vitamin C.Chem Res Toxicol. 2020 Oct 19;33(10):2515-2526. doi: 10.1021/acs.chemrestox.0c00348. Epub 2020 Oct 1. Chem Res Toxicol. 2020. PMID: 33001635 Free PMC article. Review.
-
Gulonolactone Addition to Human Hepatocellular Carcinoma Cells with Gene Transfer of Gulonolactone Oxidase Restores Ascorbate Biosynthesis and Reduces Hypoxia Inducible Factor 1.Biomedicines. 2014 Mar 5;2(1):98-109. doi: 10.3390/biomedicines2010098. Biomedicines. 2014. PMID: 28548062 Free PMC article.
-
The Role of Oxygen Homeostasis and the HIF-1 Factor in the Development of Neurodegeneration.Int J Mol Sci. 2024 Apr 23;25(9):4581. doi: 10.3390/ijms25094581. Int J Mol Sci. 2024. PMID: 38731800 Free PMC article. Review.
-
Myths, artifacts, and fatal flaws: identifying limitations and opportunities in vitamin C research.Nutrients. 2013 Dec 16;5(12):5161-92. doi: 10.3390/nu5125161. Nutrients. 2013. PMID: 24352093 Free PMC article. Review.
-
The Loss of HIF1α Leads to Increased Susceptibility to Cadmium-Chloride-Induced Toxicity in Mouse Embryonic Fibroblasts.J Toxicol. 2011;2011:391074. doi: 10.1155/2011/391074. Epub 2011 Jul 17. J Toxicol. 2011. PMID: 21811500 Free PMC article.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
