Evolution of Oxygen Sensing in Animals

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.

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.

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'.