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).

Publications


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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
Loenarz C (2008) Expanding chemical biology of 2-oxoglutarate oxygenases. in Nature chemical biology
 
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 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 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 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