Oxygen sensing by HIF hydroxylases

Key Points

  • Hypoxia-inducible factor (HIF) is an α/β heterodimeric DNA-binding complex that directs an extensive transcriptional response to hypoxia. The activity of HIF is induced in hypoxic cells through the stabilization and activation of its α-subunit. HIFα subunits are regulated by a newly discovered signalling mechanism — that is, the oxygen-dependent enzymatic hydroxylation of specific amino-acid residues.

  • The hydroxylation of conserved prolyl residues in two independent degradation domains in the central region of HIFα promotes interactions with the von Hippel–Lindau ubiquitylation complex, which targets HIFα for degradation by the ubiquitin–proteasome pathway. Hydroxylation at a conserved asparaginyl residue in the HIFα carboxy-terminal activation domain blocks interaction with the p300 transcriptional co-activator.

  • To date, three HIF prolyl hydroxylases, known as prolyl hydroxylase domain (PHD)1–3, and one asparaginyl hydroxylase, known as factor inhibiting HIF (FIH), have been defined. These enzymes all belong to the non-haem, Fe2+-dependent, 2-oxoglutarate-dependent-oxygenase superfamily.

  • These enzymes possess a common 'jelly-roll' (double-stranded β-helix) core and coordinate the catalytic Fe2+ using a two-histidine, one-carboxylate 'facial triad'. During catalysis, the splitting of molecular oxygen occurs with one oxygen atom being incorporated into the HIF prolyl or asparaginyl residue and the other being incorporated into succinate during the oxidative decarboxylation of 2-oxoglutarate.

  • The absolute requirement of the HIF hydroxylases for molecular oxygen conveys oxygen sensitivity. Additional cofactor and co-substrate requirements for Fe2+, the citric-acid-cycle intermediate 2-oxoglutarate and ascorbate might help these enzymes generate the flexibility that is required for an oxygen-sensing function in complex multicellular animals.

Abstract

The transcription factor HIF (hypoxia-inducible factor) has a central role in oxygen homeostasis in animals ranging from nematode worms to man. Recent studies have shown that this factor is regulated by an unprecedented signalling mechanism that involves post-translational hydroxylation. This hydroxylation is catalysed by a set of non-haem, Fe2+-dependent enzymes that belong to the 2-oxoglutarate-dependent-oxygenase superfamily. The absolute requirement of these enzymes for molecular oxygen has provided new insights into the way cells sense oxygen.

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Figure 1: Direct transcriptional targets of HIF.
Figure 2: The domain structure of HIF1α and HIF1β/ARNT.
Figure 3: Dual regulation of HIFα subunits by prolyl and asparaginyl hydroxylation.
Figure 4: The coordination chemistry of 2-oxoglutarate-dependent oxygenases.
Figure 5: Structural insights into 2-oxoglutarate-dependent oxygenases.
Figure 6: Mechanism of the 2-oxoglutarate-dependent-oxygenase FIH.

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Acknowledgements

We thank all our colleagues for their dedicated contributions to the Oxford work in the area of hypoxic signalling, T. Murray-Rust for assistance with the figures, and the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, the Medical Research Council and the European Union for funding our research.

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Correspondence to Peter J. Ratcliffe.

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The authors are founding scientists of ReOx Ltd., a company that aims to exploit the HIF hydroxylases as therapeutic targets.

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DATABASES

Interpro

PAS domain

Swiss-Prot

Cul2

elonginB

elonginC

FIH

HIF1α

HIF2α

HIF3α

pVHL

Rbx

Glossary

PAS FAMILY

(period circadian protein (PER), aryl-hydrocarbon receptor (AHR), aryl-hydrocarbon-receptor nuclear translocator (ARNT) and single-minded protein (SIM)). A group of interacting and structurally related basic helix–loop–helix transcription factors.

ANGIOGENESIS

The growth and proliferation of new blood vessels from existing vasculature.

ERYTHROPOIESIS

The formation of erythrocytes (red blood cells).

UBIQUITIN–PROTEASOME PATHWAY

A system of selective, energy (ATP)-consuming protein degradation that involves the linking of ubiquitins to specific proteins and the subsequent targeting of these polyubiquitylated proteins to the 26S proteasome (a multi-catalytic protease).

CUPIN SUPERFAMILY

The cupins are a diverse family of plant proteins, all of which contain at least one double-stranded β-helix or jelly-roll strucural motif. This motif is also present in all structurally characterized 2-oxoglutarate-dependent oxygenases including the HIF hydroxylases, and is characteristic of the jumonji transcription factors.

TRIPLET-STATE MOLECULAR OXYGEN

Most 'natural' molecules exist in the singlet state — that is, they contain paired electrons. However, the most stable form of molecular oxygen (O2) is the triplet state, in which there are two unpaired electrons.

K m

The Michaelis constant. A kinetic parameter for a specific substrate in an enzyme-catalysed reaction. Providing certain conditions are met, the Km for a substrate can equate to its binding constant, and the lower the value of Km, the tighter the substrate binds.

pO2

The partial pressure that is exerted by molecular oxygen in a mixture of gases. It is also used to define the concentration of molecular oxygen in a solution or biological tissue that is at equilibrium with such a gas mixture.

SYSTEMIC HYPOXIA

A reduction in the partial pressure of oxygen (pO2) throughout the organism.

TISSUE ISCHAEMIA

Inadequate blood supply to a tissue, which causes other metabolic abnormalities in addition to hypoxia (for example, the defective delivery of substrates and removal of waste products).

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Schofield, C., Ratcliffe, P. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 5, 343–354 (2004). https://doi.org/10.1038/nrm1366

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