A nonradioactive 96-well plate assay for the detection of hypoxia-inducible factor prolyl hydroxylase activity.
The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel-Lindau protein-elongin B-elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.
Oehme
Felix
F
Bayer HealthCare AG, Institute for Cardiovascular Research, Aprather Weg 18a, D-42096 Wuppertal, Germany. felix.oehme.fo@bayer-ag.de
Jonghaus
Willi
W
Narouz-Ott
Laila
L
Huetter
Joachim
J
Flamme
Ingo
I
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001681:Biological Assay / Q000379:methods; D004268:DNA-Binding Proteins / Q000032:analysis*; D049308:Dioxygenases; D000075065:Elongin; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C000616396:ELOB protein, human; C000616395:ELOC protein, human; D000075065:Elongin; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D013879:Thioredoxins; D019809:Streptavidin; D049308:Dioxygenases; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases