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Title: Post-translational modifications of HIF and related enzymes.
Author: Murray-Rust, Thomas Ayrton
ISNI:       0000 0001 3436 6519
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2006
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The ability to adapt to a large range of changes in oxygen tension is crucial for both the longand short-tenn survival of all aerobic organisms. A key protein involved in the response to low oxygen levels (hypoxia) is hypoxia inducible factor (HIF), an a,p-transcription factor that upregulates many genes. Some of the genes upregulated by HIP are involved in processes that counteract hypoxia, including those involved in vasculogenesis, erythropoiesis and energy metabolism. In order to maintain tight control over the hypoxic response, HIP-a is regulated by several oxygen-dependent post-translational modifications. Hydroxylations of HIP-a at specific prolyl and asparaginyl residues regulate both the stability and transcriptional activity of HIP. In addition, regulation of HIP stability and activity by phosphorylation, ubiquitination and acetylation have all recently been reported. As a consequence of the reported acetylation of LysS32 of HIF-la by the acetyltransferase human analogue of arrest defective 1 (hARDl), this project sought to investigate the effect acetylation had on the known hydroxylation of ProS64 of HIF-la. These studies, along with reports from other laboratories published during the course of this work, strongly suggest that, at least during in vitro acetyltransferase assays, hARDl does not acetylate HIF-la. However, during the investigation a slow self-acetylation reaction of hARD1 was observed. This was subsequently continned to be a self-mediated process, suggesting residual catalytic hARDl activity consistent with its main known function, as the catalytic subunit of a heterodimeric complex with Na-terminal acetyltransferase activity. The project also investigated the analogous oxygen sensing system in the fruit fly Drosophila melanogaster. The protein Similar (Sima) has been identified as a HIF homologue and several in vivo studies in flies have shown it to be crucial in the regulation of D. melanogaster development. A homologue of the human HIF prolyl hydroxylases, Fatiga, has also been identified, and one of the aims of this work was to produce this protein and confmn its ability to hydroxylate Sima in vivo. Although production of soluble Fatiga protein was not achieved in this project, Sima protein was produced and shown to be a substrate of a truncated fonn of human PHD2, thereby providing further evidence for the conserved nature of oxygen sensing pathways in higher eukaryotes. Finally, the Skp1 protein in the slime mould Dictyostelium is another protein known to undergo post-translational hydroxylation. The prolyl hydroxylase responsible for this bears similarity to the human prolyl hydroxylase, PHD2, and so the relationship between human Skpland human PHD2 was investigated, specifically whether a truncated fonn of PHD2 could hydroxylate Skpl. The results described here show that human Skpl protein, at least in vitro, does not function as a PHD2 substrate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available