Characterising Novel Substrates of the Asparaginyl Hydroxylase FIH

Abstract

Factor Inhibiting the Hypoxia Inducible Factor (FIH) is an oxygen-dependent asparaginyl hydroxylase that plays a role in the cellular response to changes in oxygen concentrations. It was first found to catalyse the post translational hydroxylation of a family of transcription factors known as Hypoxia Inducible Factors (HIFs), and thus acts as a cellular sensor of hypoxia. Subsequently, other protein substrates were discovered containing ankyrin (ANK) repeat domains, although their activities were not affected by hydroxylation. FIH null mice display a metabolic phenotype which is not obviously linked to HIF regulation, supporting the existence of other important substrates of FIH. Hence the search for novel substrates of FIH was pursued, with particular interest in target proteins that upon modification by FIH would have a functional cellular outcome. Methods such as yeast two hybrid and pull down assays were previously employed to discover new substrates of FIH, but this limited searches to proteins with a relatively strong affinity for FIH. In research by a collaborator, a non-biased bioinformatic search was used to identify novel potential FIH substrates. Interestingly, the search identified a family of 5 ANK proteins present in the Orf virus (ORFV) as likely substrates of FIH. Preliminary experiments in our laboratory showed that they could interact with human FIH (hFIH), and some showed activity in CO₂ capture assays, consistent with hydroxylation. The first aim of this thesis was to determine whether the 5 ORFV ANK proteins, 008, 123, 126, 128 and 129 were substrates of FIH. Given that ORFV is an ovine virus, ovine FIH (oFIH) was cloned and purified, with recombinant protein displaying similar activity to recombinant hFIH. The ORFV ANK domains were cloned, expressed in bacteria and purified for analysis with recombinant oFIH. The hydroxylation state of these proteins was first analysed indirectly using in vitro hydroxylation assay and 008, 126 and 129 displayed high activity, consistent with being effectively hydroxylated by FIH, whereas 123 and 128 displayed low activity. Mass spectrometry (MS) of the recombinant proteins confirmed FIH-mediated hydroxylation of N40 in 008, N285 in 126, and N44 in 129, with poor ionisation preventing definitive conclusions regarding hydroxylation of 123 and 128. The ANK proteins of another closely related poxvirus, vaccinia virus (VACV), were also predicted to be substrates of FIH, but analysis of recombinant VACV ANK proteins showed no evidence of hydroxylation. These data indicate that these hydroxylation events are not conserved across the poxvirus family. The second aim was to characterise the functional role for this interaction between ORFV ANK proteins and FIH. Given the poorly characterised roles of the ORFV ANK proteins, and the relatively stable interaction between these proteins and FIH, these studies focused on the hypothesis that the ORFV ANK proteins could sequester FIH and subsequently upregulate HIF activity upon viral infection. Cell-based reporter gene assays confirmed that expression of the ORFV ANKs could sequester FIH and upregulate the HIFα transactivation domain, leading to increased HIF activity. Further supporting this hypothesis, ORFV infected cells that overexpressed or were deficient in FIH showed the induction of well characterised HIF target genes in a FIH-dependent manner. These data identified a novel mechanism of viral induced modulation of HIF activity, via the sequestration of FIH. The final aim of this thesis was to analyse OTUB1, a non-ANK and non-HIF protein discovered by our collaborators as a putative novel substrate of FIH. The hydroxylation of OTUB1 on N22 by FIH in vitro was confirmed using synthetic OTUB1 peptides in CO₂ capture assays.