Research Guides

Department of Chemistry University of Oxford

Dr Emily Flashman

Mechanisms of Oxygen Sensing by the HIF Hydroxylases

All animals can respond to conditions of low oxygen (hypoxia) via a transcription factor protein, the hypoxia-inducible factor (HIF). In hypoxia, HIF upregulates genes which allow the body to adapt to decreased oxygen availability (the hypoxic response). A hypoxic response is seen in many cancer cells, either because of hypoxic conditions in tumours, or because cellular metabolic changes affect the normal regulation of HIF levels. The HIF hydroxylases are enzymes which downregulate HIF in the presence of oxygen. Our work focuses on understanding how these enzymes react with oxygen at the molecular level to enable them to be efficient oxygen sensors. Much of this work is carried out in collaboration with Prof. Chris Schofield's group.

Mechanism of Oxygen Sensing

The HIF hydroxylases are part of a broad family of enzymes, the Fe(II)/2-oxoglutarate dependent oxygenases, which have a variety of functions in biology. Using stopped-flow transient kinetic techniques, and in collaboration with Prof.’s Carsten Krebs and J. Marty Bollinger at Penn State University, we have found that one of the HIF hydroxylases, PHD2, reacts particularly slowly with oxygen. This is most likely to be related to its specific role as an oxygen sensor. We are interested in understanding what makes PHD2 unusual amongst the Fe(II)/2-oxoglutarate oxygenases in its reaction with oxygen, and are currently investigating this by looking at how variants of PHD2 function under different oxygen conditions. We are also interested in looking at other Fe(II)/2-oxoglutarate dependent oxygenases to understand which are able to act as oxygen sensors and what molecular features define the oxygen sensors.

Influence of Reducing Agents

HIF levels are affected by oxidising and reducing agents such as ascorbate, especially in cancer cells. These effects are primarily mediated by the HIF hydroxylases, although the precise mechanism is unknown. We are investigating how reducing agents increase the activity of the HIF hydroxylases, and interestingly, why different HIF hydroxylase reactions seem to be differentially affected by different reducing agents.

Looking Into Cells

The contents of a test tube are very different to the contents of a cell, and it is therefore important to be able to translate in vitro findings to a cellular context. To this end, we are trying to find ways of characterising the mechanistic properties of the HIF hydroxylases in a cellular context. We are currently investigating whether in-cell EPR can be used to visualise the active site of these enzymes under different cellular stress conditions.

 

Tarhonskaya H, Hardy AP, Howe EA, Loik ND, Kramer HB, McCullagh JS, Schofield CJ, Flashman E (2015). Kinetic investigations of the role of factor inhibiting hypoxia-inducible factor (FIH) as an oxygen sensor. J Biol Chem. 290: 19726-19742.

Hancock RL, Dunne K, Walport LJ, Flashman E, Kawamura A (2015). Epigenetic regulation by histone demethylases in hypoxia. Epigenomics. 7: 791-811.

Tarhonskaya H, Chowdhury R, Leung IK, Loik ND, McCullagh JS, Claridge TD, Schofield CJ and Flashman E (2014). Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen. Biochem J. 463: 363-372.

Tarhonskaya H, Szöllössi A, Leung IK, Bush JT, Henry L, Chowdhury R, Iqbal A, Claridge TD, Schofield CJ and Flashman E (2014). Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases. Biochemistry. 53: 2483-2493.         

Tarhonskaya H, Rydzik AM, Leung IK, Loik ND, Chan MC, Kawamura A, McCullagh JS, Claridge TD, Flashman E and Schofield CJ (2014). Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases. Nat Commun. 5: 3423.

Sanchez-Fernandez EM, Tarhonskaya H, Al-Qahtani K, Hopkinson RJ, McCullagh JS, Schofield CJ, Flashman E (2013). Investigations on the oxygen dependence of a 2-oxoglutarate histone demethylase. Biochem J. 449: 491-496.

Flashman E, Hoffart LM, Hamed RB, Bollinger Jr JM, Krebs C, Schofield CJ (2010). Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. FEBS J. 277: 4089-4099

Flashman E, Davies SL, Yeoh KK, Schofield CJ (2010). Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents. Biochem J. 427: 135-142.