title: Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor.
abstract: The 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.
authors.affiliation: School of Molecular and Biomedical Science and The ARC Special Research Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, Australia.
authors.forename: Sarah
authors.initials: S
authors.lastname: Linke
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Hampton-Smith
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Peet
mesh_terms: D000818:Animals; D017089:Ankyrin Repeat; D002245:Carbon Dioxide / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D002245:Carbon Dioxide; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human