2-Oxoglutarate (2OG) and Fe(II) dependent oxygenases have a broad range of substrates, extending from histones to fatty acids. Several 2OG oxygenases have nucleic acid substrates, with members of the AlkB subfamily being responsible for nucleic acid modification and repair. The AlkB protein itself is part of the Escherichia coli adaptive response, protecting the DNA from methylation damage. Methyl lesions are repaired by a direct removal mechanism via a hydroxylated intermediate, with release of formaldehyde. Homologues of AlkB have been identified throughout the vertebrates, with nine known human homologues: AlkB homologue 1-8 (ABH1-8) and Fat, mass and obesity associated protein (FTO). ABH2, ABH3 and FTO catalyse similar reactions to AlkB, whereas ABH8 methylates then hydroxylates modified wobble-position uridines in tRNA. The remaining homologues are of unknown function. The FTO gene is associated with obesity in humans, a link confirmed by mouse models; mice lacking FTO are thinner than wildtype individuals, whereas overexpression of FTO leads to increased mass. Investigation of recombinant FTO identified a novel C terminal helical domain which appears to mediate protein dimerisation in vitro. A loss of function mutation in this C terminal domain produces a lean phenotype in mice, emphasising the importance of this domain for the protein’s function in vivo. The FTO protein was further studied in cells, and localisation of several protein variant constructs were studied by immunofluorescence. Cell lysis and immunoprecipitation techniques were developed that enable proteomic analyses of proteins with which FTO may interact in cells. No protein interactors were confidently identified, suggesting that FTO may not interact with specific proteins in cells, and instead may preferentially interact with nucleic acids. Studies were initiated on two further members of the ABH family, ABH1 and ABH7. Recombinant proteins were prepared and characterised as 2OG oxygenases, however initial attempts to identify potential histone or nucleic acid substrates were not successful. Both proteins were found to be localised in the mitochondria, however proteomic analysis was unable to identify proteins interacting with either protein in cells. Selective inhibitors are required for in vivo inhibition of the ABH proteins. AlkB and ABH2 proteins were purified and characterised, and a formaldehyde dehydrogenase-coupled assay was developed to follow activity of these DNA demethylases. A dynamic combinatorial mass spectrometry method was employed to identify novel inhibitor scaffolds for AlkB, leading to the successful discovery of the first series of potent and selective inhibitors for this class of enzymes. Crystal structures of AlkB in complex with the most potent compounds were obtained, rationalising the inhibition observed. This work therefore suggests that therapeutic inhibition of this family of 2OG oxygenases is likely to be tractable.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:559730 |
| Date | January 2011 |
| Creators | Bagg, Eleanor Amy Louise |
| Contributors | Schofield, Christopher J. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:eaaf3427-3001-464f-8e7b-68a4a3f8fbcf |
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