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

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 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.