Functional assignments on human oxygenases

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.

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).