Mass-spectrometry analysis and live cell/zebrafish imaging to discover the function of novel post-translational modifications

To survive, multicellular organism need to sense and respond to changes in oxygen level. Episodes of low oxygen (hypoxia) occur during many physiological processes and also in pathological circumstances such as stroke, ageing and cancer. Hypoxia inducible factor (HIF) is the main transcription factor involved in the adaptation to hypoxia and it is therefore crucial to understand how HIF is regulated by both the available oxygen levels and other signalling cues experienced by the cells. We have initiated an un-biased mass spectrometry (MS)-based proteomic analysis of HIF family member post-translational modifications (PTMs), identifying numerous novel PTMs. We now seek to define their function, by studying site conservation and the functional effect of the PTM on HIF activity. Remarkably, one novel phosphorylation site in human HIF-1a is absent in fish, which constitutes an ancient evolutionary mutation in species constantly exposed to low oxygen environments.

The aim of the project is to establish the role and function of the newly discovered HIF PTMs in cells and in vivo using model organisms (zebrafish). This project builds on recent discoveries of HIF PTMs by the labs of C Eyers and V See. These PTMs are predicted to regulate HIF stability or activity. Understanding the function of these modifications is timely as it could contribute to the quest for therapeutic regulators of HIF function, a current focus of pharmaceutical industry.

Experimental approach: A combination of peptide and protein-based Mass Spectrometry analysis will be used to understand how these novel HIF PTMs occur in combination (at the level of the intact protein). Native ion mobility-MS, together with other biochemical strategies will be used to assess the PTMs' impact on protein conformation, stability, and protein/DNA binding interactions. The consequence of the PTMs on HIF subcellular localisation, diffusion, binding interaction and transcription activity will be investigated by live cell imaging, using mutants of HIF fused to fluorescent tags. To fully characterise the function of some of the HIF mutants in vivo, zebrafish lines will be generated (collaboration with the University of Sheffield). The student will therefore be working both at the forefront of protein modifications analysis and live cell imaging, using cell lines and zebrafish models.