Evolution of Oxygen Sensing in Animals

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

The work we are proposing to do is based on insights we have obtained into the way humans sense oxygen. Regulating the delivery of oxygen to tissues is a problem for all organisms that use it as an energy source and particularly so for large animals such as humans that are composed of many billions of cells and different types of tissues. Many human diseases such as heart attacks, strokes, cancer, and anaemia involve damage to cell and hence tissue function by low oxygen levels (hypoxia). In previous work (important components of which were supported by the BBSRC) we have identified a group of oxygenases (enzymes that catalyse the incorporation of atmospheric oxygen into their substrates) that act as cellular 'oxygen sensors',by catalysing the hydroxylation (involving addition of an oxygen atom) of specific amino acid residues in a protein called HIF (hypoxia inducible factor). Hydroxylation destroys and inactivates HIF, but since it requires oxygen, this reaction is suppressed in hypoxia, allowing HIF to become activate in hypoxic cells (hence its name). HIF is a transcription factor (a regulator of gene expression) that, when switched on, regulates many genes that are involved in altering cell metabolism, growing new blood vessels, increasing blood production and other actions that help the body to survive hypoxia. These findings have opened up a new field of research on this type of protein modification, what it does, how it is regulated by hypoxia, and how it affects the cell's responses to hypoxia. The work on HIF has also raised many questions as to whether this type of modification (hydroxylation) occurs for other types of protein within cells, and what the effects might be. Recently, we have found that the HIF system also exists in the simplest living animal, Trichoplax adhaerens, which lives in the sea. Our initial data suggest that the Trichoplax oxygen sensing system is closely related to the human system, but is much simpler, largely because Trichoplax has a much smaller genome than humans. We aim to carry out a detailed comparison between the human and Trichoplax HIF systems, to investigate whether the HIF or related oxygen sensing systems exist in other organisms. Our initial data suggests that HIF is present in all animals but not in the group of organisms from which animals are though to have evolved (choanoflagellates), leading us to propose that the HIF system evolved along with the rises in oxygen levels and multicellular animals, at or just before the famous Precambrian period in the evolution of life. This aspect of evolution has been elegantly described in the recent television series 'First Life' presented by David Attenborough. However, we have also found that enzymes related to the HIF hydroxylases also exist in bacteria even though HIF doesn't. We think that these enzymes had an oxygen sensing role via an unknown mechanism in prokaryotes, and eventually evolved into the human oxygen sensing enzymes. Our work thus ultimately aims to connect molecular and genomic analyses with evolutionary studies. However, it is our experience that the cross-species analyses can lead to deeper understanding of the underlying mechanisms of how human cells work - these are often difficult to dissect because of the complexity of human cell biology. Finally, although our work will employ the use of invertebrates, aspects of their cell biology and embryonic development appear to be well conserved with human cells, raising the possibility that they may contribute to the replacement of mammals in drug development.

Technical Summary

Following the definition of hydroxylation as regulating the transcriptional response of humans to hypoxia via the hypoxia inducible factor (HIF) system, we have focused work on the evolution of the HIF system and the possibility that post-translational hydroxylation has a wider role than previously considered. In initial work we have found that HIF itself, a HIF prolyl-hydroxylase (PHD), and the von Hippel Lindau protein which targets HIF for degradation, are functional in Trichoplax adhaerens, the simplest animal; Bioinformatic analyses suggest the HIF system occurs in all animals. We aim to investigate the extent of conservation of biochemical properties / roles of the HIF system components by a comparison of the 'key sensing' hydroxylases and their roles in T. adhaerens and other animals. The work will include detailed analyses including high-resolution structural analyses on the taPHD/HIF complexes Our second overall objective concerns the evolutionary origins of the HIF system in non-animals and the possibility that translation is regulated by 'oxygen sensing' enzymes related to the HIF hydroxylases. We have found that HIF is only present in animals. However, we have identified PHD-related enzymes in choanoflagellate animal precursors, in yeast and in prokaryotes. Cellular data suggests a role for these enzymes in hypoxic regulation in the role of gene expression. For homologues from Pseudomonas spp. we have identified a ribosomal elongation factor as a substrate opening up a new field in oxygen dependent signaling (and in prokaryotic post-translational modifications). We will thus work to define the roles of (candidate) PHD-homologous prolyl-hydroxylases from prokaryotes, yeasts, and animals in the regulation of gene expression at the level of translation. A key element of the proposed work will be investigations of the effects of hydroxylation on translational accuracy / efficiency.

Planned Impact

Impact Summary Who will benefit from the research? We intend that in the long term our research will benefit society in general by providing new insights into fundamental processes. Our results will be reported in the scientific literature. In the medium term (or shorter) we envisage that the work will be of interest to the pharmaceutical and biotechnological industries. Although not a focus of our current work we also believe that 2OG oxygenases have considerable potential as biocatalysts, as exemplified by the translation (by Kyowa-Hakko) of our work on proline-hydroxylases into a production procedure. The planned structural and biochemical analyses (in particular the high resolution crystallographic analyses) should be enabling with respect to protein engineering work on Oxygenases. Presently we believe that we are extremely fortunate to work in a field that is of both basic academic and commercial/translational interest (though this has not always been the case). If we succeed in our objectives, and our extensive preliminary data accumulated over >5 years of largely unpublished work suggests that we have a good chance of meeting most of them, the work should represent major advances in our understanding of hypoxic sensing. Although our objectives are aimed at addressing basic science questions, we believe the work is of substantial interest to the pharmaceutical industry both in terms of identifying new targets - Proteins involved in translation are of interest both as antibiotic targets and as human targets for cancer and inflammatory disease - and in terms of making more selective HIF hydroxylase inhibitors. The inhibition of non-hydroxylated over hydroxylated ribosomes is also of potential utility in targeting hypoxic tissues such as in tumours. The proposed work on the development of invertebrate animal models is also of interest from the perspective of 'ethical' drug development. Finally, the discovery of a direct role for 2OG oxygenase activity in translation will likely stimulate widespread interest in academia and industry, as occurred following the identification of the HIF hydroxylases and the JmjC histone modifying enzymes. How will they benefit? The work will provide new insights into how cells respond to limiting oxygen, a topic of basic interest to the many researchers working in signaling processes at biochemical, cellular and physiological levels. The objectives of the proposal are of interest to the pharmaceutical and biotechnological companies working on 2-oxoglutarate oxygenase inhibitors (including GSK, Merck, Amgen, Fibrogen, Astellas, Novartis, Johnson & Johnson, ReOx and others), for reasons including the following: i) The work will provide new insights into the fundamental mechanism of the role of 2OG oxygenases in hypoxia sensing in animals. ii) The work will provide high-resolution crystal structures for use in pharmaceutical design. iii) There is a reasonable likelihood that the work will lead to new pharmaceutical targets. iv) The work will help to enable the production of more selective oxygenase inhibitors. v) The work will develop and exemplify new methods for the functional assignment of genes/proteins at biochemical, cellular and physiological levels. vi) The work will help to enable protein engineering of 2OG oxygenases aimed at their cure in more efficient routes to pharmaceuticals. vii) The work will lead to the generation of new intellectual property of commercial value. viii) The work will help to retain and attract high quality researchers in / to the United Kingdom. ix) Because efficient oxygen supply is vital for all aspects of aerobic behaviour the proposed work is of interest to many sports especially high performance endurance sports, such as long distance cycling/running or sport at altitude (At the recent HypoxiaNet Conference at Davos (Jan 2011), an entire session was concerned with 'Exercise at High Altitude'.

Publications


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Hopkinson RJ (2013) Is JmjC oxygenase catalysis limited to demethylation? in Angewandte Chemie (International ed. in English)
Katz MJ (2014) Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth. in Proceedings of the National Academy of Sciences of the United States of America
 
Description Our work aimed to provide detailed insights into the evolution of hypoxia sensing in animals.
All the key objectives of our proposed work have been met. We have carried out detailed structural (including high-resolution crystallography) and kinetic analyses on the hypoxia inducible factor prolyl hydroxylase from the simplest animal, Trichoplax adhaerens, and shown that it is remarkably similar in these regards to the most important human hypoxia sensing prolyl hydroxylase (PHDs). The results have not only defined the limits of the hypoxia sensing system present in humans as being to the animal kingdom, but have identified the first prolyl hydroxylase to be identified in a prokaryote. They have also identified and characterised (including by crystal structures) a new type of prolyl hydroxylase present in organisms ranging from yeasts to humans. In yeasts the hydroxylase catalyses an unprecedented di-hydroxylation of a prolyl residue present in a key component (RP23) of the ribosome. In addition to informing in the evolution of oxygen sensing the results will aid in the pharmaceutical development of selective oxygenase inhibitors.
Exploitation Route Future work can include further studies on the regulation of translation by oxygenases and the use of the 'new' human studies (OGFOD1) in oxygenase work on the development of selective inhibition for the human hypoxia inducible factor prolyl hydroxlases.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description The work will help to enable drug discovery efforts centred on the human 2-oxoglutarate dependent oxygenases. The identification of OGFOD1 as a functionally conserved oxygenase, the active site of which is closely related to the human hypoxia inducible factory prolyl hydroxylases (PHDs) has implications for the development of selective PHD inhibitors for the treatment of anaemia and other ischaemia related disorders. We have shown that at least some PHD inhibitors in clinical trials inhibit OGFOD1. The identification of OGFOD1 as a ribosomal oxygenase also raises the possibility that modulation of its activity may be used to treat genetic diseases associated with stop codon readthrough.
Sector Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal
 
Description Chris West collaboration 
Organisation University of Oklahoma
Department Health Sciences Centre
Country United States of America 
Sector Academic/University 
PI Contribution Kinetic investigation of Toxoplasma gondii HIF hydroxylase homologue (PhyA) activity with respect to oxygen. Supply of a range of HIF hydroxylase inhibitors for screening as PhyA inhibitors. Contribution to an NIH grant application to investigate prolyl hydroxylation and oxygen sensing in Toxoplasma gondii. Academic discussion and design of future experiments.
Collaborator Contribution Provision of reagents for kinetic investigations. NIH grant application that will benefit work in our group. Academic discussion and design of future experiments.
Impact Final outcome of grant application awaited.
Start Year 2013
 
Title METHOD FOR ASSAYING OGFOD1 ACTIVITY - US patent 
Description The invention is an assay that identifies modulators of OGFOD1 activity. The assay can be used to find potential drugs against a number of genetic diseases (such as cystic fibrosis, haemophilia or muscular dystrophy) or viral conditions such as HIV. 
IP Reference US9228224 
Protection Patent granted
Year Protection Granted 2016
Licensed Commercial In Confidence
Impact OGFOD is of interest as a target and is also of use in selectivity screens for other human 2-oxoglutarate oxygenases.
 
Title OGFOD1 - European patent 
Description The invention is an assay that identifies modulators of OGFOD1 activity. The assay can be used to find potential drugs against a number of genetic diseases (such as cystic fibrosis, haemophilia or muscular dystrophy) or viral conditions such as HIB. 
IP Reference EP2675912 
Protection Patent granted
Year Protection Granted
Licensed Commercial In Confidence
Impact OGFOD is of interest as a target and is also of use in selectivity screens for other human 2-oxoglutarate oxygenases.
 
Description 'Epigenetics' Select Biosciences Symposium, Boston, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at 'Epigenetics' Select Biosciences Symposium, Boston, USA on "The role of oxygenases in epigenetics"
Year(s) Of Engagement Activity 2012
 
Description 'Redox Modulation of Health and Disease: From Inorganic Chemistry to Translational Medicine' Meeting, Erlangen, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at 'Redox Modulation of Health and Disease: From Inorganic Chemistryto Translational Medicine' Meeting, Erlangen, Germany on "The Chemistry of Oxygen Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2013
 
Description CIPSM Oktoberfest, Munich, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at CIPSM Oktoberfest, Munich, Germany on "The Chemistry of Oxygen Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2013
 
Description COST Training School, Poitiers, France 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at 'Players of the Epigenetic Symphony' COST Training School, Poitiers, France on "The role of oxygenases in epigenetic regulation"
Year(s) Of Engagement Activity 2012
 
Description Chemical Biology and Molecular Medicine (CBMM) Annual Symposium, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Talk at CBMM Annual Symposium, Cambridge on 'Chemistry of Oxygen Sensing'
Year(s) Of Engagement Activity 2013
 
Description ELRIG Drug Discovery Workshop, University of Manchester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation at ELRIG Drug Discovery Workshop, University of Manchester on "Inhibiting Epigenetic Oxygenases"
Year(s) Of Engagement Activity 2012
 
Description Frontiers in Biological Catalysis, Biochemical Society Symposium, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presenttion at Frontiers in Biological Catalysis, Biochemical Society Symposium, Cambridge on "The enzymology of oxygen sensing in animals"
Year(s) Of Engagement Activity 2012
 
Description GRC Enzyme, coenzymes and Metabolic Pathways, Waterville, USA 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation Gordon Research Conference, Waterville, USA on "The Enzymology of Oxygen Sensing in Humans and Other Organisms"
Year(s) Of Engagement Activity 2014
 
Description ISMB Symposium, Birkbeck College, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at Institute of Structural Molecular Biology (ISMB) Symposium on "Regulation of gene expression by oxygen"
Year(s) Of Engagement Activity 2012
 
Description Introduction to Epigenetic Drug Discovery SCI workshop, Great Chesterford 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at Introduction to Epigenetic Drug Discovery SCI workshop, Great Chesterford on "Is oxygen an epigenetic regulator?"
Year(s) Of Engagement Activity 2012
 
Description Lilly Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at Lilly's Chemical Biology meets Drug Discover conference on "Chemical Biology of Oxygen Sensing"
Year(s) Of Engagement Activity 2014
 
Description Mill Hill Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Mill Hill Lecture, MRC National Institute for Medical Research, London on "Chemistry of Oxygen Sensing in Humans"
Year(s) Of Engagement Activity 2013
 
Description OCISB 2012 Conference, Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation at OCISB 2012 Conference, Oxford Systems Biology: Building Networks and Bridging Scales, Oxford on "Towards a chemical understanding of hypoxic sensing in animals"
Year(s) Of Engagement Activity 2012
 
Description Oxford Cancer Research Horizons Meeting, Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk at Oxford Cancer Research Horizons Meeting, Oxford on "Chemistry of Oxygen Sensing in Humans and other Animals"
Year(s) Of Engagement Activity 2012
 
Description RSC Jeremy Knowles Lecture, St Andrews University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk at RSC Jeremy Knowles Lecture, St Andrews University, on "The chemistry of oxygen sensing in humans"
Year(s) Of Engagement Activity 2012
 
Description SEB 2014, Manchester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at The Society for Experimental Biology on "Hypoxia Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2014
 
Description Seminar at Aberdeen University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Seminar at Aberdeen University on "The Chemistry of Oxygen Sensing in Humans"
Year(s) Of Engagement Activity 2013
 
Description Seminar at Bristol 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Seminar at Bristol University on "The Chemistry of Oxygen Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2014
 
Description Seminar at CICB Paris 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at Seminar at Interdisciplinary Center Chemistry Biology-Paris (CICB-Paris) on "The Chemistry of Oxford Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2014
 
Description Seminar at Huddersfield University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Seminar at Huddersfield University on "The Chemistry of Oxygen Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2013
 
Description Seminar at Konstanz University, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Seminar at Konstanz University, Germany on "The Chemistry of Oxygen Sensing in Humans"
Year(s) Of Engagement Activity 2013
 
Description Seminar at Lillys 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation at Seminar at Lillys, Erl Wood, Windlesham on "Chemistry of Oxygen Sensing in Humans"
Year(s) Of Engagement Activity 2012
 
Description Seminar at UCL 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Seminar at University College London on "The Chemistry of Oxygen Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2014
 
Description Seminar at University of Dundee 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation at Seminar at University of Dundee on "The Chemistry of Oxygen Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2014
 
Description Seminar at University of York 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Seminar at University of York, on "Chemistry of Oxygen Sensing"
Year(s) Of Engagement Activity 2012
 
Description Seminar at the Department of Chemistry, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation at Seminar at the Department of Chemistry, Cambridge on "Chemistry of Oxygen Sensing in Animals"
Year(s) Of Engagement Activity 2012
 
Description Society of Chemical Industry's London Region Committee, New York University London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Talk at Society of Chemical Industry's London Region Committee, New York University London on "The chemistry of oxygen sensing in humans and bacteria (from antibiotics to epigenetics and back again)"
Year(s) Of Engagement Activity 2014
 
Description Talk at '125 years of Chemistry at QML', Queen Mary's London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk at '125 years of Chemistry at QML', Queen Mary's London on "The Chemistry of Oxygen Sensing in Humans"
Year(s) Of Engagement Activity 2014
 
Description Talk at Bayer, Berlin, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Talk at Bayer, Berlin, Germany on "The Chemistry of Oxygen Sensing in Humans and Other Animals"
Year(s) Of Engagement Activity 2013
 
Description Talk at Evotec 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Talk at Evotec on "The Chemistry of Oxygen Sensing in Humans and Other Animals
Year(s) Of Engagement Activity 2014
 
Description Talk at Japanese Embassy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Talk at Japanese Embassy on "The Chemistry of Oxygen Sensing in Humans"
Year(s) Of Engagement Activity 2014