Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues. Hydroxylation of specific prolyl residues by 2-oxoglutarate (2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-␣. Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-␣ abrogates interaction with p300, preventing transcriptional activation. Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-␣. Since FIH contains certain motifs present in iron-and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase. Assays using recombinant FIH and HIF-␣ fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen. Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.Hypoxia in animals activates a broad range of homeostatic responses via induction of a transcriptional complex termed hypoxia-inducible factor (HIF) 1 (1, 2). HIF is a heterodimer of ␣-and -subunits with regulation by dioxygen availability being mediated by post-translational modification of the ␣-subunits (1, 2). In mammalian cells, two HIF-␣ subunit isoforms (HIF-1␣ and HIF-2␣) are regulated by dioxygen levels. Each HIF-␣ protein contains an internal oxygen-dependent degradation domain possessing targeting motifs for proteolytic regulation and a C-terminal transactivation domain (CAD) independently regulated by dioxygen, irrespective of changes in protein abundance, through interaction with the CH1 domain of the co-activator p300 (for review, see Ref.3).The oxygen-dependent degradation of HIF-␣ by proteolysis is regulated by the hydroxylation of specific prolyl residues (Pro-402 and Pro-564 in human HIF-1␣) that mediate recognition of HIF-␣ by the von Hippel-Lindau (VHL) ubiquitinylation complex and consequent proteasomal destruction (4 -7). Combined structural analysis and genetic approaches led to the identification of three isoforms of human HIF prolyl hydroxylase (PHD1-3, prolyl hydroxylase domain) together with homologues in a range of organisms (8,9). In vitro analyses together with sequence and mutational analyses identified these as belonging to a subfamily of the Fe(II)-and 2-oxoglutarate (2-OG)-dependent oxygenases (4,5,8,9). Limiting oxygen availability in hypoxia, or direct inhibition of the PHD enzymes by cobaltous ions and iron chelators, allows HIF-␣ to escape hydroxylation and recognition by pVHL, providing insights into the mechanism by which these stimuli suppress HIF-␣ degradation and activate the transcriptional casca...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.