Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric α,β-transcriptional complex that mediates the cellular response to oxygen availability in multicellular organisms, ranging from the simplest known animal Trichoplax adhaerens to humans. Dimerisation of the HIF-α and HIF-β subunits under hypoxic conditions activates the transcription of an array of genes involved in the adaptation of cells to the lowered oxygen tension. These include erythropoietin (EPO) that regulates red blood cell levels and vascular endothelial growth factor (VEGF) that regulates angiogenesis (new blood vessel growth from a pre-existing bed of vessels).
The medicinal importance of HIF regulated genes coupled to breakthroughs in our knowledge of the biochemistry of hypoxia have led to significant interest in manipulating the response for therapeutic benefit. Medicinal stimulation of the natural HIF mediated response might be used to treat ischemic illnesses such as heart disease and stroke; in contrast, inhibition of the HIF mediated hypoxic response might be used for the treatment of tumors via inhibition of angiogenesis.
The HIF-α subunits are rapidly degraded by proteasome catalysed hydrolysis (i.e. those relating to lack of oxygen) providing sufficient dioxygen is present. The rate of HIF-α degradation is regulated by the post-translational hydroxylation of conserved prolyl residues in HIF-α. The von Hippel-Lindau tumour suppressor protein (pVHL) enables binding of the prolyl hydroxylated HIF-α to a ubiquitin E3 ligase complex that catalyses ubiquitinylation of HIF-α so targeting it for hydrolysis by the ubiquitin–proteasome pathway. In collaboration with the Ratcliffe/Pugh Group in Clinical Medicine we have identified the hydroxylase enzymes that catalyse hydroxylation of HIF-α. Investigating the structures and mechanisms of the HIF prolyl hydroxylase is a current focus of our work on HIF. An Oxford University spin out company, ReOx, aims to exploit discoveries on HIF for therapeutic benefit. We have solved a crystal structure of PHD2 - one of the human prolyl hydroxylases. We are presently investigating the mechanisms and structures of the HIF prolyl hydroxylases with a view to developing a detailed chemical understanding of their role as oxygen sensors. We are exploring medicinal chemistry avenues for both tuning HIF 'on' and 'off'.
In addition to its prolyl hydroxylation HIF-α is also hydroxylated at a conserved asparaginyl residue. Hydroxylation on the β-carbon of human asparagine 803, blocks the protein-protein interaction between HIF-α and the transcriptional coactivator p300 thereby providing a second means of oxygen dependent regulation. In collaboration with the Ratcliffe/Pugh Group we have discovered that the HIF asparaginyl hydroxylase also catalyses hydroxylation of conserved motifs within a commonly occurring structural motif - the ankyrin repeat domain - present in proteins from other signaling pathways including the inflammatory response. FIH also catalyses the oxidation of aspartyl and histidinyl residues.
Techniques involved in this research include proteomics (in collaboration with Benedikt Kessler), X-ray crystallography, biological mass spectrometry, molecular biology, kinetics and organic synthesis/medicinal chemistry.
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