Functional assignments on human oxygenases
Lead Research Organisation:
University of Oxford
Department Name: Oxford Chemistry
Abstract
Regulating oxygen delivery to cells is a problem for all aerobic organisms ranging from bacteria to humans. In mammals, the lungs, heart and blood are all devoted to this task, and human diseases (such as strokes, heart attacks and anaemia) often involve damage to organs by low oxygen levels (hypoxia). Basic science work supported by the BBSRC has provided insights into how cells 'sense' and respond to hypoxia. The work has helped to identify a set of 'oxygenases' (a type of enzyme that catalyses the incorporation of atmospheric oxygen into their substrates) that catalyse the addition of oxygen atoms to a protein called hypoxia inducible factor (HIF), so called because its level is raised under low oxygen concentrations. HIF is important as it occurs in a very wide range of organisms including insects, worms and humans; it enables the expression of a range of genes that work to help the organism overcome the challenge of hypoxia. In humans these genes enable a response to hypoxia and include those involved in blood vessel and red blood cell formation. Addition of oxygen to HIF stops its ability to enable the expression of the genes involved in the hypoxic response. When there is sufficient oxygen the oxygenases can catalyse its addition to HIF, but when oxygen levels fall HIF is no longer modified and it is free to enable expression of the genes involved in the hypoxic response. We hope that the results of this work will result in new treatments for diseases involving the cardiovascular system. By inhibiting the HIF oxygenases with small molecule drugs, it should be possible to improve the body's natural defence against damage from low oxygen concentrations. However to do this safely without causing side effects will require more knowledge of other human oxygenases, that have been revealed by analysis of the human genome. The work on the HIF system has raised questions as to the extent of the role of oxygenases in controlling the expression of genes in other pathways and indeed biology as a whole. This proposal seeks to go some way towards addressing these questions by studying human oxygenases, some of which are known to be biomedicinally important from work at the physiological level but for which there is little or no data in terms of their actual substrates and roles at a biochemical level. There are technical problems in working with human cells compared to, for example, those from microorganisms. However, we have chosen to work with human enzymes, in part, because the worldwide efforts in genome sequencing and other large scale projects studying the proteins present in human cells have provided a resource we can utilise to help assign biochemical roles for the oxygenases, and, in part, because we hope that the work will be useful in the development of new therapies based on a better understanding of human metabolism.
Technical Summary
Work from the applicants' laboratories has led to the identification of a set of non-haem Fe(II) and 2-oxoglutarate (2OG) oxygenases that catalyse the post-translational hydroxylation of specific prolyl and asparaginyl residues in the alpha-subunits of a transcription factor termed hypoxia inducible factor (HIF). The hydroxylations regulate the activity of the HIF complex through proteolysis and co-activator recruitment. HIF itself directs an extensive transcriptional cascade, which plays a central role in oxygen homeostasis. Thus the HIF hydroxylases directly connect oxygen availability with the regulation of a major transcriptional pathway, and have provided a new focus for understanding of oxygen sensitive signal pathways. An important element in the work was the use of structural information and mechanistic data (generated in BBSRC funded work) in combination with genomic sequence data to identify the HIF hydroxylases. Crystallographic and biochemical analyses on the HIF asparaginyl hydroxylase (Factor inhibiting HIF, FIH), have led to the (re)assignment of a number of the JmjC transcription factors, some of which are already known to be of biomedicinal importance from medicinal data, as Fe(II) and 2OG oxygenases involved in transcriptional regulation. In extensive preliminary data we have identified alternative substrates for FIH and demonstrated that two other JmjC proteins, the phosphatidylserine receptor (PSR) and Mina53 (mineral dust induce gene), are 2OG oxygenases, though we have not yet identified their substrates. This proposal seeks to address the question of the extent of the involvement of post-translational hydroxylation in human signalling pathways by functional analyses on the JmjC 2-oxoglurate oxygenases. Initially we will focus on FIH, PSR and Mina53 and then will extend the work to other members of the JmjC 2OG oxygenases. From our work on the HIF hydroxylases we are well aware of the challenges and pitfalls in making functional assignments relevant at the endogenous level. Thus although the objectives of the project are ambitious we will be focusing on a specific family with which we have extensive expertise. We will apply an integrated approach employing techniques from biochemistry, cell biology and structural biology. We are requesting funding for five years for two post-doctoral assistants plus technical support, who will be responsible for the biochemical and cell biology aspects of the work. The objectives, milestones and activities of the project are clearly defined. One of the PDRAs will focus on protein production and characterisation (including in vitro analysis of substrates) whilst the other will focus on the identification of potential substrates using immunoprecipitation, yeast-two hybrid, and affinity purification methods. The PDRAs will work in a well-organised environment; their work will complement and enhance ongoing structural efforts on the human 2OG oxygenases (in collaboration with the Structural Genomics Consortium).
Organisations
Publications
Banh RS
(2016)
PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia.
in Nature cell biology
Brewitz L
(2020)
Small-molecule active pharmaceutical ingredients of approved cancer therapeutics inhibit human aspartate/asparagine-ß-hydroxylase.
in Bioorganic & medicinal chemistry
Chan M
(2016)
Pharmacological targeting of the HIF hydroxylases - A new field in medicine development
in Molecular Aspects of Medicine
Chan MC
(2016)
Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases.
in The Journal of biological chemistry
Church C
(2009)
A mouse model for the metabolic effects of the human fat mass and obesity associated FTO gene.
in PLoS genetics
Cole MA
(2016)
On the pivotal role of PPARa in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.
in FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Dickinson RS
(2017)
Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitro and in vivo.
in Wellcome open research
Ge W
(2012)
Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans.
in Nature chemical biology
Gerken T
(2007)
The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.
in Science (New York, N.Y.)
Loenarz C
(2011)
Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases.
in Trends in biochemical sciences
Loenarz C
(2008)
Expanding chemical biology of 2-oxoglutarate oxygenases.
in Nature chemical biology
Mantri M
(2011)
The 2-Oxoglutarate-Dependent Oxygenase JMJD6 Catalyses Oxidation of Lysine Residues to give 5 S -Hydroxylysine Residues
in ChemBioChem
Mantri M
(2012)
Self-hydroxylation of the splicing factor lysyl hydroxylase, JMJD6
in MedChemComm
Mantri M
(2010)
Crystal Structure of the 2-Oxoglutarate- and Fe(II)-Dependent Lysyl Hydroxylase JMJD6.
in Journal of molecular biology
Markolovic S
(2016)
Structure-function relationships of human JmjC oxygenases-demethylases versus hydroxylases.
in Current opinion in structural biology
Markolovic S
(2018)
The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.
in Nature chemical biology
Mayer M
(2018)
A Fluorescent Benzo[ g ]isoquinoline-Based HIF Prolyl Hydroxylase Inhibitor for Cellular Imaging
in ChemMedChem
McAllister TE
(2016)
Recent Progress in Histone Demethylase Inhibitors.
in Journal of medicinal chemistry
McDonough MA
(2010)
Structural studies on human 2-oxoglutarate dependent oxygenases.
in Current opinion in structural biology
Rose NR
(2011)
Inhibition of 2-oxoglutarate dependent oxygenases.
in Chemical Society reviews
Rotili D
(2011)
A photoreactive small-molecule probe for 2-oxoglutarate oxygenases.
in Chemistry & biology
Thinnes C
(2019)
Selective Inhibitors of a Human Prolyl Hydroxylase (OGFOD1) Involved in Ribosomal Decoding
in Chemistry - A European Journal
Thinnes CC
(2014)
Targeting histone lysine demethylases - progress, challenges, and the future.
in Biochimica et biophysica acta
Description | All animals must have the ability to sense and respond to changes in oxygen availability, such as when we go to high altitude. One mechanism that coordinates our response to this 'hypoxia' involves the oxygen dependent regulation of the synthesis of proteins that enable us to respond appropriately - for example in hypoxia we make more erythropoietin or EPO that in turn switches on red blood cell formation. Work from Oxford showed that EPO production is regulated by the direct reaction of oxygen with types of proteins called transcription factors, that turn on the conversion of genetic DNA into messenger RNA, which in turn is converted into the worker proteins, such as EPO. The introduction of oxygen into the transcription factors is catalysed by a family of enzymes called oxygenases. In our new work we have shown that oxygenases are not only involved in the regulation of the conversion of DNA into RNA, but also in the conversion of RNA into proteins, which is catalysed by ribosomes. The discovery of the ribosomal oxygenases, or ROX, has opened up new possibilities for the treatment of diseases such as cancer, because tumours are hypoxic. In unexpected developments the work has also shown that oxygenases are involved in RNA-cutting, or splicing. In one case we have even found that an oxygenase is linked to obesity. The biological work of our project also benefitted from chemical tools produced as part of a BBSRC funded scheme - Selective Chemical Intervention in Biological Systems. Overall the work has shown that all steps in protein production in cells interface with oxygen and have revealed new targets for the pharmaceutical industry. The results of the work have been reported in prestigious journals such as Science, have attracted interest from a wide range of research communities and, in the course of the obesity work, the general public. |
Exploitation Route | The results are of general biological interest because they have revealed that, in addition to the transcriptional machinery, the MRNA splicing and translational machineries are modified by direct reaction with atmospheric oxygen. The findings have opened up new potential pharmaceutical targets, will enhance selectivity studies in work on existing targets (e.g. transcription factor hydroxylases), and have opened the way to explore new treatments for genetic diseases caused by incorrect stop codon insertion, e.g. some forms of cystic fibrosis. |
Sectors | Chemicals Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | The project has been remarkably successful in attracting industrial interest. It has directly helped to acquire funding from the Wellcome Trust and industrial partners, including GSK and Pfizer, for a project aimed at identifying epigenetic probe compounds. Further, it has helped to enable new collaborations (funding for a PDRA) with two pharmaceutical companies on the identification of compounds that modify nucleic acid structure and function and for the treatment of obesity. We have hosted numerous industrial workers in our laboratories and have supplied both inhibitors and biological reagents to industrial laboratories. A spin-out company, OxEpi, was formed with seed-corn investment from Sofinova, with a view to developing certain types of human oxygenase inhibitors - the company has been closed but with a profit. |
First Year Of Impact | 2012 |
Sector | Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal Economic |
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 | 2nd World Epigenetics Summit, 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 | Presentation at 2nd World Epigenetics Summit, Munich, Germany on "The role of oxygenases in epigenetics" |
Year(s) Of Engagement Activity | 2011 |
Description | Bertram Dillon Steele Lecture |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | From Penicillins to Epigenetics - Adventures at the Interface of Chemistry and Biology |
Year(s) Of Engagement Activity | 2018 |
Description | Chemistry & Biology Interface workshop, Salerno, Italy |
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 Chemistry & Biology Interface workshop, Salerno, Italy on "The molecular mechanism of oxygen sensing in humans" |
Year(s) Of Engagement Activity | 2011 |
Description | Epigenetics Europe, 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 Epigenetics Europe, London on 'Histone demethylases - functions and therapeutic possibilities' |
Year(s) Of Engagement Activity | 2013 |
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 | Hypoxia Symposium, University of Nantes, Nantes, France |
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 Hypoxia Symposium, University of Nantes, Nantes, France on "Role of post-translational hydroxylation in the hypoxic response" |
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 | Medical Epigenetics and Drug Discovery FRIAS Workshop, Freiburg, Germay |
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 Medical Epigenetics and Drug Discovery FRIAS Workshop, Freiburg, Germany on "Structure based drug discovery" |
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 | Seminar at Sapienza University of Rome, Rome, Italy |
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 Seminar at Sapienza University of Rome, Rome, Italy on "The Chemical Basis of Oxygen Sensing in Humans" |
Year(s) Of Engagement Activity | 2011 |
Description | Seminar at University of Dundee |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Other audiences |
Results and Impact | From Penicillins to Epigenetics - Adventures at the Interface of Chemistry and Biology |
Year(s) Of Engagement Activity | 2018 |
Description | Seminar at Vertex, Abingdon, Oxford |
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 Vertex, Abingdon, Oxford on "Functional, Structural and Mechanistic Studies on Human Oxygenases |
Year(s) Of Engagement Activity | 2011 |
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 |