Studies on the Molecular Mechanism of the Hypoxic Response

With Dr Emily Flashman (Dorothy Hodgkin Research Fellow) and Dr Christoph Loenarz (William R Miller Junior Research Fellow)

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.

For reviews see:

Loenarz C, Schofield CJ: Expanding chemical biology of 2-oxoglutarate oxygenases. Nat Chem Biol 2008, 4: 152-156.
Schofield CJ, Ratcliffe PJ: Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 2004, 5: 343-354.
Loenarz C, Chowdhury R, Schofield CJ, et al. Oxygenases for oxygen sensing. Pure and Applied Chemistry 2008, 80: 1837.
Schofield CJ, Ratcliffe PJ: Signalling hypoxia by HIF hydroxylases. Biochemical and Biophysical Research Communications 2005, 338: 617-626.
Hewitson KS, Schofield CJ: The HIF pathway as a therapeutic target. Drug Discovery Today 2004, 9: 704-711.
Nagel S, Talbot NP, Mecinović J, Smith TG, Buchan AM, Schofield CJ: Therapeutic Manipulation of the HIF Hydroxylases. Antioxidants & Redox Signaling 2011, 12: 481-501.

For representative publications see:

Schmierer B, Novak B, Schofield C: Hypoxia-dependent sequestration of an oxygen sensor by a widespread structural motif can shape the hypoxic response - a predictive kinetic model. BMC Systems Biology 2010, 4: 139.
Illingworth CJR, Loenarz C, Schofield CJ, Domene C: Chemical Basis for the Selectivity of the von Hippel Lindau Tumor Suppressor pVHL for Prolyl-Hydroxylated HIF-1α. Biochemistry 2010, 49: 6936-6944.
Hardy AP, Prokes I, Kelly L, Campbell ID, Schofield CJ: Asparaginyl [beta]-Hydroxylation of Proteins Containing Ankyrin Repeat Domains Influences Their Stability and Function. Journal of Molecular Biology 2009, 392: 994-1006.
Flashman E, Davies SL, Yeoh KK, Schofield CJ: Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents. Biochemical Journal 2010, 427: 135-142.
Flashman E, Hoffart LM, Hamed RB, Bollinger Jr JM, Krebs C, Schofield CJ: Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. FEBS Journal 2010, 277: 4089-4099.
Cook KM, Hilton ST, Mecinović J, Motherwell WB, Figg WD, Schofield CJ: Epidithiodiketopiperazines Block the Interaction between Hypoxia-inducible Factor-1α (HIF-1α) and p300 by a Zinc Ejection Mechanism. Journal of Biological Chemistry 2009, 284: 26831 -26838.
Chowdhury R, McDonough MA, Mecinovic J, Loenarz C, Flashman E, Hewitson KS, Domene C, Schofield CJ: Structural Basis for Binding of Hypoxia-Inducible Factor to the Oxygen-Sensing Prolyl Hydroxylases. Structure 2009, 17: 981-989.
Loenarz C, Mecinović J, Chowdhury R, McNeill LA, Flashman E, Schofield CJ: Evidence for a Stereoelectronic Effect in Human Oxygen Sensing. Angewandte Chemie International Edition 2009, 48: 1784-1787.
Kelly L, McDonough MA, Coleman ML, Ratcliffe PJ, Schofield CJ: Asparagine beta-hydroxylation stabilizes the ankyrin repeat domain fold. Mol. BioSyst. 2009, 5: 52-58.
Coleman ML, McDonough MA, Hewitson KS, Coles C, Mecinović J, Edelmann M, Cook KM, Cockman ME, Lancaster DE, Kessler BM, et al.: Asparaginyl Hydroxylation of the Notch Ankyrin Repeat Domain by Factor Inhibiting Hypoxia-inducible Factor. Journal of Biological Chemistry 2007, 282: 24027 -24038.
Cockman ME, Lancaster DE, Stolze IP, Hewitson KS, McDonough MA, Coleman ML, Coles CH, Yu X, Hay RT, Ley SC, et al.: Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proceedings of the National Academy of Sciences 2006, 103: 14767 -14772.
Hon W-C, Wilson MI, Harlos K, Claridge TDW, Schofield CJ, Pugh CW, Maxwell PH, Ratcliffe PJ, Stuart DI, Jones EY: Structural basis for the recognition of hydroxyproline in HIF-1[alpha] by pVHL. Nature 2002, 417: 975-978.
Jaakkola P, Mole DR, Tian Y-M, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim A von, Hebestreit HF, Mukherji M, Schofield CJ, et al.: Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation Complex by O2-Regulated Prolyl Hydroxylation. Science 2001, 292: 468 -472.
Epstein ACR, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, et al.: C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation. Cell 2001, 107: 43-54.
McDonough MA, Li V, Flashman E, Chowdhury R, Mohr C, Liénard BMR, Zondlo J, Oldham NJ, Clifton IJ, Lewis J, et al.: Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proceedings of the National Academy of Sciences 2006, 103: 9814 -9819.
Hewitson KS, McNeill LA, Riordan MV, Tian Y-M, Bullock AN, Welford RW, Elkins JM, Oldham NJ, Bhattacharya S, Gleadle JM, et al.: Hypoxia-inducible Factor (HIF) Asparagine Hydroxylase Is Identical to Factor Inhibiting HIF (FIH) and Is Related to the Cupin Structural Family. Journal of Biological Chemistry 2002, 277: 26351 -26355.
Loenarz C, Coleman ML, Boleininger A, Schierwater B, Holland PWH, Ratcliffe PJ, Schofield CJ: The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens. EMBO Rep 2011, 12: 63-70.