Characterising Novel Substrates of the Asparaginyl Hydroxylase FIH
type: CreativeWork
description: 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…
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…
author: Fabrizio, Jacqueline Alba
language: en
keywords: Ankryin proteins, HIF, FIH, Orf virus, hypoxia, hydroxylation, OTUB1
date published: 2017
date modified: 2026-07-24T00:50:44Z
provider: University of Adelaide
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Adelaide

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property ID: Handle
value: https://hdl.handle.net/2440/132758