Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor

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dc.contributor.authorKarttunen, S.
dc.contributor.authorHampton-Smith, R.
dc.contributor.authorPeet, D.
dc.contributor.editorSies, H.
dc.contributor.editorBrune, B.
dc.contributor.organisationCentre for the Molecular Genetics of Development
dc.date.issued2007
dc.descriptionAvailable online 12 November 2007.
dc.description.abstractThe hypoxia-inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen-sensitive hydroxylases-three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF-alpha (alpha) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia. HIF-alpha asparaginyl hydroxylation is catalyzed by factor-inhibiting HIF-1 (FIH-1) and directly regulates the transcription activity of the HIF-alpha proteins. Recent work has demonstrated that, in addition to hydroxylating HIF-alpha, FIH-1 can also hydroxylate the ankyrin domains of a wide range of proteins. This paper presents in vitro and cell-based techniques for the preliminary characterization of ankyrin domain-containing proteins as FIH-1 substrates and interacting proteins. Strategies are presented for the expression and purification of FIH-1 from mammalian or bacterial cells. Similar to the HIF-alpha proteins, the ankyrin-containing substrates are examined as purified proteins expressed in bacteria and overexpressed in mammalian cells or in the form of synthetic peptides. Specific conditions for the efficient expression of ankyrin-containing proteins compared with the HIF-alpha substrates in Escherichia coli are detailed. Hydroxylation is rapidly inferred, utilizing the described in vitro CO(2) capture assay. Finally, substrate and non-substrate interactions are examined using in vitro affinity pull-down assays and mammalian cell-based co-immunoprecipitation assays. Together, these methods are rapid and well suited to the preliminary characterization of potential substrates of the therapeutically relevant oxygen-sensing enzyme FIH-1.
dc.description.statementofresponsibilitySarah Linke, Rachel J. Hampton‐Smith and Daniel J. Peet
dc.description.urihttp://www.sciencedirect.com/science/bookseries/00766879
dc.identifier.citationMethods in Enzymology, 2007; 435:61-85
dc.identifier.doi10.1016/S0076-6879(07)35004-0
dc.identifier.issn0076-6879
dc.identifier.issn1557-7988
dc.identifier.orcidPeet, D. [0000-0002-6085-8936]
dc.identifier.urihttp://hdl.handle.net/2440/47857
dc.language.isoen
dc.publisherElsevier Academic Press Inc
dc.rightsCopyright © 2007 Elsevier Inc. All rights reserved.
dc.source.urihttps://doi.org/10.1016/s0076-6879(07)35004-0
dc.subjectAnimals
dc.subjectHumans
dc.subjectEscherichia coli
dc.subjectCarbon Dioxide
dc.subjectMixed Function Oxygenases
dc.subjectProteins
dc.subjectRecombinant Proteins
dc.subjectTranscription Factors
dc.subjectRepressor Proteins
dc.subjectImmunoprecipitation
dc.subjectAnkyrin Repeat
dc.subjectSubstrate Specificity
dc.titleCharacterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor
dc.typeJournal article
pubs.publication-statusPublished

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