Inhibition of a viral prolyl hydroxylase

The hydroxylation of prolyl-residues in eukaryotes is important in collagen biosynthesis and in hypoxic signalling. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are drug targets for the treatment of anaemia, while the procollagen prolyl hydroxylases and other 2-oxoglutarate dependent oxygenases are potential therapeutic targets for treatment of cancer, fibrotic disease, and infection. We describe assay development and inhibition studies for a procollagen prolyl hydroxylase from Paramecium bursaria chlorella virus 1 (vCPH). The results reveal HIF PHD inhibitors in clinical trials also inhibit vCPH. Implications for the targeting of the human PHDs and microbial prolyl hydroxylases are discussed.     

Siah proteins, HIF prolyl hydroxylases, and the physiological response to hypoxia

New evidence suggests that at least two members of the family of hypoxia-inducible factor (HIF) prolyl hydroxylases that regulate HIF stability in response to oxygen (O2) availability are also targeted for proteosome-dependent degradation by the E3 ubiquitin ligases Siah1a and Siah2. This preview examines cellular responses to O2 deprivation (hypoxia) and the complexity of the regulation of the HIF O2 sensing pathway in mammals.     

The HIF prolyl hydroxylase PHD3 is a potential substrate of the TRiC chaperonin

Hypoxia-inducible factor-1 (HIF) is regulated by oxygen-dependent prolyl hydroxylation. Of the three HIF prolyl hydroxylases (PHD1, 2 and 3) identified, PHD3 exhibits restricted substrate specificity in vitro and is induced in different cell types by diverse stimuli. PHD3 may therefore provide an interface between oxygen sensing and other signalling pathways. We have used co-purification and mass spectrometry to identify proteins that interact with PHD3. The cytosolic chaperonin TRiC was found to copurify with PHD3 in extracts from several cell types. Our results indicate that PHD3 is a TRiC substrate, providing another step at which PHD3 activity may be regulated.     

Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay

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 K(m) (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent K(m) (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-1alpha. The present results, albeit obtained under non-physiological conditions, imply that the apparent K(m) (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.