Daly, Leonard 
ORCID: 0000-0001-9712-9676
(2020)
Identification and characterisation of the post-translational modifications that regulate the Hypoxia Inducible Factors, HIF-1α and HIF-2α.
PhD thesis, University of Liverpool.
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Abstract
Eukaryotic organisms, including human, require molecular oxygen (O2) to survive. During periods of low O2 availability (hypoxia), a family of protein transcription factors become stabilised (Hypoxia Inducible Factors, HIF) and allow the adaption to hypoxia by regulating gene expression. Hypoxic adaption is required for cellular survival and is considered a hallmark of cancer. HIF is a heterodimeric protein consisting of a stable beta subunit (HIF1β) and an O2 labile alpha subunits (HIF-1α or HIF-2α). The two HIFα isoforms share ~50% sequence homology, yet have different target genes, O2 sensitivity and sub-nuclear localisation. The O2-dependent stability of HIFα subunits is due to an O2 dependent, proline hydroxylation post translational modification (PTM) resulting in degradation. The current understanding of post-translational regulation beyond the O2 dependent hydroxylation are poorly understood. Previous published studies aiming to identify HIF PTMs did it using a targeted/biased approach and failed to compare isoforms. Therefore, the primary aim of this work was to expand the understanding of the regulatory network of HIFα proteins, by obtaining an unbiased identification of PTMs and binding partners using a proteomics approach. We have performed an in-depth analysis of PTMs across ~90% of the total protein sequence for HIF-1α and HIF-2α in response to hypoxia, with a specific focus on phosphorylation. In total, ~50 different PTMs were confidently identified (~25 of which were phosphorylation) for each HIFα proteins, with the majority of these PTM sites being novel. Identified PTM sites were investigated through a combination of hypoxia regulation, evolutionary analysis, domain localisation and crystal structure modelling to identify potentially interesting sites to prioritise functional characterisation. This led to the discovery of HIF-1α Serine 31 phosphorylation, a previously superficially investigated site, as a potentially important mechanism to fully abolish HIF-1α-mediated transcription by preventing its binding to DNA. In addition, HIFα binding partners in response to hypoxia were identified. We demonstrated that many more proteins interact with HIFα proteins than currently known. The binding partner profiles were hypoxia-dependent, especially for HIF-2α which had >10 fold more binding partners confidently identified in hypoxia than normoxia. Combined with Gene ontology (GO) analysis, the binding partners identified strongly suggest a role for HIFα with mitochondria. Whilst we have discovered many novel data, this project has opened many avenues for further investigation. Overall, it is clear that the current understanding of HIF mediated hypoxia signalling is incomplete, and that the signalling pathways at play are orders of magnitude more complex than the current understanding
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health and Life Sciences > Faculty of Health and Life Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 04 Feb 2020 16:53 |
| Last Modified: | 19 Jan 2023 00:08 |
| DOI: | 10.17638/03071020 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3071020 |
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