Biophysical and biological characterization of FIH-catalysed post-translational asparaginyl hydroxylation.

Lead Research Organisation: University of Oxford
Department Name: Clinical Medicine


The work we are proposing to do is based on recent insights we have obtained into the way cells sense oxygen. Regulating the delivery of oxygen to tissues is a problem for all organisms - particularly large animals (such as humans) that are composed of many billions of cells. Many human diseases such as heart attacks, strokes, cancer, and anaemia involve compromise of cell 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', 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 type of regulator of gene expression) and when it is switched on, it regulates a lot of genes that are involved in altering cell metabolism, growing new blood vessels, increasing blood production and other actions that help the body survive hypoxia. The work on HIF has raised a lot of 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 one of the HIF-hydroxylases enzymes also hydroxylates a very common structural domain in proteins, the ankyrin repeat domain (ARD). ARDs occur in many types of protein with many different functions, such as in transcription, cell signalling, cell structure, ion channels, chromosome integrity and aging, inflammation and differentiation. 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. It might also give fundamental clues about how the type of 'oxygen sensing' process that regulates HIF evolved. The work is goal directed and is seeking to address a problem in basic science that is potentially important for a number of different biological and biomedical fields. The two applicants are from different backgrounds (Biology and Chemistry) and are working in a partnership to combine their expertise across several biophysical, biochemical, and biological approaches to find out how common this type of protein modification is, how is affects the physical properties of the protein, and how this alters cell function.

Technical Summary

Technical summary Following definition of the central role of post-translational hydroxylation of hypoxia inducible factor (HIF) in signaling cellular hypoxia, the applicants have identified numerous and diverse ankyrin repeat domain (ARD) containing proteins including IkB and Notch family memebers as catalytic targets of the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH). The programme of work, co-directed by PJR and CJS, will take a combined biophysical, biochemical, physiological and genetic approach to define the extent, determinants and function of post-translational asparaginyl hydroxylation in ARD proteins, and the role of this process in the biology of hypoxia. Primary sequence and tertiary structural determinants of ARD hydroxylation will be defined together with detailed analyses of the effect of hydroxylation on ARD structure and stability. These analyses will be coupled with kinetic analyses on specific ARD hydroxylation events and MS analysis of ARD hydroxylation in vivo to gain an understanding of the factors governing ARD hydroxylation in cells, and its sensitivity to hypoxia and other cellular stress. The findings will underpin functional analyses that will encompass both detailed functional interrogation of specific biophysically chararacterized hydroxylation events, and screening of genetically modified cells (including FIH 'knock-out' cells) for hydroxylation events that have been 'tuned' for hypoxia signaling in a manner similar to HIF. The planned analyses will interrogate both the direct function of specific ARD hydroxylation events and the existence and nature of cross-competition between ARD-containing proteins and the HIF system. Finally building on this experience we will survey other potential sites of FIH-catalysed asparaginyl hydroxylation in the proteome, aiming to provide general insights into the role of FIH-catalysed protein hydroxylation in biological responses to hypoxia and other cellular processes.
Description The BBSRC funded research programme entitled "Biophysical and biological characterization of FIH-catalysed post-translational asparaginyl hydroxylation" has provided important insight into the regulation and biochemical activity of the cellular oxygen-sensing enzyme, Factor Inhibiting HIF. In total, 5 research papers and 1 review manuscript have been published as a direct result of the project grant support shared between the Ratcliffe and Schofield laboratories.

It is now well-established that the 2-oxoglutarate-dependent dioxygenase FIH, targets two different classes of substrate; FIH plays a pivotal role in the mammalian hypoxic response by catalyzing the hydroxylation of the key transcriptional regulator, hypoxia-inducible factor (HIF), thereby inhibiting the transcription of specific genes. In addition, FIH has been shown to catalyse the hydroxylation of a large subset of proteins bearing a common structural motif, termed the ankyrin repeat domain (ARD). For the ankyrin repeat substrates, the role of FIH-catalysed hydroxylation was less clear and provided a focus for much of the programme.

We and others have observed that FIH displays clear preferences for specific target residues within protein substrates. This prompted us to explore the sequence and structural determinants of ARD hydroxylation. We demonstrated that the efficiency of FIH-catalysed hydroxylation depends to a large extent on the flexibility and the overall fold of the AR (Hardy et al, J Mol Biol 2009). In addition, we made the surprising observation that FIH can catalyse the hydroxylation of aspartyl and histidinyl residues in ARD-containing proteins (Yang et al, JBC 2011, Yang et al, FEBS J 2011). These results have expanded the scope of FIH catalysed hydroxylations.

Biophysical studies of ankyrinR - a cytoskeletal ARD, revealed that hydroxylation increased the conformational stability of the ARD in vitro. This was also associated with a reduction in the interaction with band 3 - a known interacting partner of ankyrinR, demonstrating the potential for FIH-catalysed ARD hydroxylation to modulate protein-protein interactions (Yang et al, JBC 2011). It remains to be determined if this, or other, ARD hydroxylations function in a physiological signalling context.

In Singleton et al (JBC 2011), we published the first systematic study on the regulation of asparaginyl hydroxylation in cells using mass spectrometry. Our methodology enabled us to monitor the accrual, inhibition and decay of hydroxylation at different sites in ARD proteins, and to make direct comparisons between HIF and ARD substrates under defined conditions. We demonstrated that the hydroxylation of ARD proteins and HIF-1 is regulated by oxygen over a similar concentration range. An important implication of these findings is that the HIF transcriptional response could be modulated by competition with ARD substrates.

Finally, in a related study, we demonstrated that FIH is a peroxide sensing enzyme that is rapidly inhibited by exposure to physiological levels of oxidant stress (Masson et al, EMBO rep 2011). This finding has broad biological implications as high levels of oxidative stress are observed in both physiological (wound-healing) and pathophysiological settings (e.g. diabetes) and raises the possibility of a hitherto unappreciated role for FIH in these processes.
Exploitation Route In an academic setting, the mass spectrometry methodology described in Singleton et al. provides a platform for future studies wishing to monitor enzymatic hydroxylation in cells. Characterization of the HIF hydroxylase enzymes has to date relied on the generation of hydroxylation modification-specific antibodies that are typically difficult to generate (requiring extensive validation) and also context-specific. Given the emergence of a large class of related 2-oxoglutarate-dependent dioxygenase enzymes (many of which are predicted hydroxylases), we feel that our work will be of interest to others wishing to characterise this class of medicinally important enzymes.
Sectors Pharmaceuticals and Medical Biotechnology
Description FIH-dependent hydroxylation of ankyrin proteins has suggested that this process is involved in metabolic control, and that pharmacological inhibition might be medically beneficial.
First Year Of Impact 2016
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic
Title Quantitative mass spectrometry reveals dynamics of factor-inhibiting hypoxia-inducible factor-catalyzed hydroxylation. 
Description We have established a number of mass spectrometry compatible reporter cell lines that can be used to directly monitor FIH activity in cells 
Type Of Material Cell line 
Provided To Others? No  
Impact These cell lines have been used to demonstrate the sensitivity of FIH to hypoxia, co-factor availability and peroxide (ROS). 
Description Collaboration with Dr Benedikt Kessler 
Organisation University of Oxford
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This grant was a joint application with Department of Chemistry, University of Oxford that represents a long standing collaboration in the field of hypoxia biology. All mass spectrometry was performed in-house, in collaboration with CCMP, University of Oxford.
Start Year 2006
Description Dissemination and communication of research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Peter Ratcliffe and Chris Schofield have lectured widely of aspects of this work to a range of audiences. For instance, Peter Ratcliffe regularly speaks at local schools to highlight career opportunities in biomedical research. He has given a large number of keynote addresses at scientific meetings, including the keynote lecture at the 2012 Keystone Symposium on Hypoxia Biology.

no actual impacts realised to date
Year(s) Of Engagement Activity 2012