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Contenuto archiviato il 2024-05-27

Molecular Mechanism of Oxygen Sensing by Enzymes

Final Report Summary - MOOSE (Molecular Mechanism of Oxygen Sensing by Enzymes)

MOOSE - SUMMARY
Christopher Schofield & Peter Ratcliffe
Prior to the onset of the project work from the applicants’ laboratories had shown that oxygen dependent regulation of the hypoxia inducible factor (HIF) involves the post-translational hydroxylation of specific prolyl- and asparaginyl-residues as catalysed by a set of four human non-haem Fe(II) and 2-oxoglutarate dependent dioxygenases – the action of these enzymes provides a direct link between oxygen availability and the regulation of human gene expression. The overall objective of our ERC project – Molecular Mechanism of Oxygen Sensing by Enzymes (MOOSE) was to capitalize on opportunities arising from the discovery of the prolyl-hydroxylase domain (PHD)- and asparaginyl-hydroxylase (FIH) enzymes as oxygen sensors, by undertaking an interdisciplinary programme of work into the chemistry, physiology, and therapeutics of human hypoxia signalling pathways. Our objectives were to further our basic understanding of the hypoxic response and to enable pharmaceutical efforts in its manipulation. The project has succeeded in all its overall objectives of providing detailed structural and chemical characterisations of human hydroxylase enzymes that underpin their physiological role as oxygen sensors, scoping the extent and role(s) of an unprecedented biological signalling mode, and developing novel small-molecule/inhibitors that enable specific manipulation of hypoxia signalling pathways.
In the first Workpackage (Understanding the HIF hydroxylases as oxygen sensors – from high resolution structures to physiological analysis) we carried out extensive studies on molecular aspects of the oxygen sensing enzymes. The work employed detailed structural analyses on isolated HIF hydroxylases in complex with their substrates and in cell studies using mass spectrometry and specific antibodies. An important outcome of the work was the identification of biochemical/biophysical differences between HIF hydroxylases (especially the PHDs) and related enzymes not involved in hypoxia sensing. Crystallographic studies on the HIF hydroxylases enabled inhibition work by ourselves and others and provided insights into the substrate selectivities of the PHDs (selective) and FIH (very promiscuous).
The second Workpackage (Extent and role of intracellular protein hydroxylation) was exceptionally productive including in unexpected ways. Initially we focused on the promiscuous nature of FIH catalysis – we found FIH not only accepts any ankyrin repeat domain proteins, in a manner that enables FIH to act as a tuneable hypoxia sensor, but that it can accept not only asparagine- (as it does in HIF hydroxylation), but also aspartate- and histidine-residues as substrates in humans. Further, with recombinant proteins FIH even accepts serine- and leucine-residues. These observations are of interest not because they extend the range of known post-translational modifications, but because they suggest that the family of enzymes (2-oxoglutarate oxygenases) to which the HIF hydroxylases belong could be used as flexible catalysts for protein modification. Finally, the work on FIH heralded our discovery of other oxygenases catalysing novel protein modifications, notably the ribosomal oxygenases, some of which, like FIH with some substrates, also catalyse histidine-hydroylation.
In the final Workpackage, we developed inhibitors of individual human HIF hydroxylases. The work involved use of the methods / structural information generated in the other 2 Workpackages. In addition to classical methods we applied combinatorial chemistry coupled to mass spectrometric analyses. An important output of the work was development of probe compounds which are available to the community without patent protection.
The project resulted in multiple publications, patent applications, training of young researchers who have gone onto academia and industry, multiple widely used reagents, and has enabled pharmaceutical efforts with several companies carrying out clinical trials on PHD inhibitors.