Ratcliffe / Pugh Group
Maintenance of oxygen homeostasis is a fundamental physiological challenge and hypoxia (inadequate oxygen availability) is a common feature of many medical conditions including cancer and ischaemic vascular diseases. The laboratory, led by Professor Peter Ratcliffe (FRS) and Professor Chris Pugh has defined novel pathways that signal hypoxia though the post-translational hydroxylation of specific amino acids in target proteins including hypoxia inducible factor (HIF), and is exploring the biochemistry, physiology, patho-physiology and therapeutic manipulation of these pathways.
HIF is the central regulator of transcriptional responses to hypoxia and is itself regulated by oxygen availability through dual degradation and inactivation pathways involving prolyl and asparaginyl hydroxylation. Prolyl hydroxylation at specific residues in a central oxygen-dependent destruction domain promotes interaction with the von Hippel-Lindau ubiquitin ligase and hence proteolytic destruction via the ubiquitin-proteasome pathway; asparaginyl hydroxylation within the C-terminal transactivation domain blocks co-activator recruitment to the complex. These reactions are catalysed via a series of non-haem Fe(II) and 2-oxoglutarate-dependent dioxygenases that act as cellular oxygen sensors. In hypoxia these hydroxylations are impaired allowing HIF to escape destruction and activate transcription.
Current programmes of research:
Cellular Biochemistry of Oxygen Sensing
The aim of this programme is to understand the biochemical basis of the oxygen-sensitive signal generated by the HIF hydroxyalses. In addition to the absolute requirement for dioxygen as substrate, the HIF prolyl and asparaginyl hydroxylases require Fe(II), 2-oxoglutarate and ascorbate as co-factors/co-substrates. The aim is to understand how these biochemical requirements transduce the hydroxylation signals that regulate the HIF pathway. Current data indicates that in addition to the direct sensing of dioxygen availability, metabolic and redox signals are integrated at this point. In collaboration with Professor Christopher Schofield, Department of Chemistry structural, kinetic, mass spectrometric, genetic, and cell biological methods are being applied to better understand these processes.
Wider functions for oxygen sensitive intracellular protein hydroxylation
The central role of post-translational prolyl and asparaginyl hydroxylation in the regulation of HIF has raised the question as to whether this mode of signalling operates more widely in biology. Hitherto, post-translational hydroxylation has been thought to be largely restricted to structural functions in extracellular proteins, for instance in the stabilization of collagens. Recently the laboratory has identified a range of ankyrin repeat domain (ARD) proteins including IκB family members, and Notch receptors as targets of the HIF asparaginyl hydroxylase (FIH). This indicates that intracellular protein hydroxylation is more widespread than has been assumed and suggests a wider function for the process. A range of cell biological, mass spectrometric, structural, bioinformatic and evolutionary methodologies are being used in the laboratory to investigate these possibilities in collaboration with Professor Schofield’s group.
Integrated physiology of HIF prolyl hydroxylase pathways
In higher animals oxygen delivery though the pulmonary, cardiac and vascular systems must be precisely co-ordinated with organ growth and metabolic control to achieve oxygen homeostasis. The group is analysing the role of the HIF prolyl hydroxylase system in these key physiological functions, and assessing the potential for therapeutic manipulation, by transgenic and knock-out genetic technology in collaboration with Professor Peter Carmeliet (University of Leuven) and Professor Patrick Maxwell (University College, London). Studies of human integrative physiology, particularly cardio-pulmonary responses to hypoxia are in progress in collaboration with Professor Peter Robbins (Department of Physiology, Anatomy and Genetics).
Hypoxia pathways in cancer
The HIF pathway is commonly up-regulated in cancer by genetic and environmental stimuli including tumour hypoxia, and dysregulation of metabolic and redox pathways. In particular inactivation of the von Hippel-Lindau tumour suppressor is observed in the majority of renal cancers and causes constitutive upregulation of the HIF pathway by disabling HIF proteolysis. Upregulation of HIF target genes including growth factors, chemokines and matrix remodelling proteins likely contribute to the highly invasive and angiogenic phenotype of these tumours. The group is interested in defining the tumour promoting hypoxia pathways in greater detail and using the analysis for clinical treatment stratification in collaboration with Professor Adrian Harris, CRUK Oncology Unit.
Translational programme - hypoxia therapeutics in ischaemic vascular disease
Inhibition of HIF hydroxylation by 2-oxoglutarate analogues up-regulates the HIF pathway, potentially inducing erythropoietic, cytoprotective, reparative, regenerative and pro-angiogenic responses that may be of use in a range of human diseases. The group is particularly interested in developing effective anti-ischaemia strategies and has access to appropriate pharmacological probes through collaborations with Professor Schofield’s group and with industrial partners including the University spin-out company ReOx Ltd.