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The asparaginyl hydroxylase FIH [factor inhibiting HIF (hypoxia-inducible factor)] was first identified as a protein that inhibits transcriptional activation by HIF, through hydroxylation of an asparagine residue in the CAD (C-terminal activation domain). More recently, several ARD [AR (ankyrin repeat) domain]-containing proteins were identified as FIH substrates using FIH interaction assays. Although the function(s) of these ARD hydroxylations is unclear, expression of the ARD protein Notch1 was shown to compete efficiently with HIF CAD for asparagine hydroxylation and thus to enhance HIF activity. The ARD is a common protein domain with over 300 examples in the human proteome. However, the extent of hydroxylation among ARD proteins, and the ability of other members to compete with HIF–CAD for FIH, is not known. In the present study we assay for asparagine hydroxylation in a bioinformatically predicted FIH substrate, the targeting subunit of myosin phosphatase, MYPT1. Our results confirm hydroxylation both in cultured cells and in endogenous protein purified from animal tissue. We show that the extent of hydroxylation at three sites is dependent on FIH expression level and that hydroxylation is incomplete under basal conditions even in the animal tissue. We also show that expression of MYPT1 enhances HIF–CAD activity in a manner consistent with competition for FIH and that this property extends to other ARD proteins. These results extend the range of FIH substrates and suggest that cross-competition between ARDs and HIF–CAD, and between ARDs themselves, may be extensive and have important effects on hypoxia signalling.

Low cellular oxygenation (hypoxia) represents a significant threat to the viability of affected tissues. Multicellular organisms have evolved a highly conserved signalling pathway that directs many of the changes in gene expression that underpin physiological oxygen homoeostasis. Oxygen-sensing enzymes in this pathway control the activity of the HIF (hypoxia-inducible factor) transcription factor by the direct incorporation of molecular oxygen into the post-translational hydroxylation of specific residues. This represents the canonical hypoxia signalling pathway which regulates a plethora of genes involved in adaptation to hypoxia. The HIF hydroxylases have been identified in other biological contexts, consistent with the possibility that they have other substrates. Furthermore, several intracellular proteins have been demonstrated, directly or indirectly, to be hydroxylated, although the protein hydroxylases responsible have yet to be identified. This chapter will summarize what is currently known about the canonical HIF hydroxylase signalling pathway and will speculate on the existence of other oxygen-sensing enzymes and the role they may play in signalling hypoxia through other pathways.

The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent Km (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent Km (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1α. The present results, albeit obtained under non-physiological conditions, imply that the apparent Km (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.

Recent studies have indicated that regulatory mechanisms underlying the oxygen-dependent expression of the haematopoietic growth factor erythropoietin are widely operative in non-erythropoietin-producing cells and are involved in the regulation of other genes. An important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals such as cobaltous ions, and by iron chelation. We have investigated the extent of operation of this system in the regulation of a range of genes concerned with energy metabolism. The effects of hypoxia (1% oxygen), cobaltous ions and desferrioxamine on gene expression in tissue-culture cells was studied using RNase protection assays. Hypoxia induced the expression of glucose transporters in an isoform-specific manner; GLUT-1 and GLUT-3 were induced by hypoxia, whereas expression of GLUT-2 was decreased. Isoenzyme-specific regulation by hypoxia was also observed for genes encoding phosphofructokinase, aldolase and lactate dehydrogenase. For all of these genes, responses to cobaltous ions and desferrioxamine correlated in both direction and magnitude with the response to hypoxia. In contrast, a reduction in mitochondrial transcripts was observed in hypoxia, but these changes were not mimicked by either cobaltous ions or desferrioxamine. These findings indicate that similarities with erythropoietin regulation extend to the oxygen-dependent regulation of genes encoding glucose transporters and glycolytic enzymes but not to the regulation of mitochondrial transcripts, and they show that in glucose metabolism regulation by this system is isoenzyme- or isoform-specific.

The inability to account for large systematic variations in total purine nucleotide content of perfused rat hearts led to the demonstration that the soluble adenine nucleotides are in rapid equilibrium with a highly phosphorylated hetero-oligomeric derivative whose structure appears to be 3-phospho[glyceroyl-gamma-triphospho-5′-adenosine-3′-3-phosp ho]4glyceroyl- gamma-triphospho-5′-adenosine [Hutchinson, Morris & Mowbray (1986) Biochem. J. 234, 623-627]. Analogous techniques to those used with hearts for specifically labelling tissue purine nucleotides followed by extration and purification of nucleotides from the trichloroacetic acid-precipitable fraction show the existence of a corresponding rapid equilibrium between ATP and an oligomeric tetraphosphoadenosine derivative in perfused kidneys.