A fluorescence polarization-based interaction assay for hypoxia-inducible factor prolyl hydroxylases.
Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor 1alpha (HIF-1alpha) plays a central role in regulating transcriptional responses to hypoxia. This process requires hydroxylation of specific prolines in HIF-1alpha by HIF prolyl hydroxylase domain (PHD)-containing enzymes, leading to its specific interactions with von Hippel-Lindau protein-Elongin B-Elongin C (VBC). Here we describe a straightforward approach to apply these interactions to measure PHD activities. Employing fluorescently labeled HIF-1alpha peptides containing hydroxyproline, we developed a quantitative method based on fluorescence polarization for a systematic evaluation of binding of hydroxylated HIF-1alpha to recombinant VBC. The method was then successfully utilized for measuring the activity of the truncated, purified PHD2. The applicability of the assay was further demonstrated by examining effects of various cofactors and inhibitors for PHD2. The developed homogeneous assay would provide a convenient way of probing the biochemical properties of the HIF-1alpha-VBC interaction and PHDs, and of screening modulators for the interaction as well as the enzyme.
Cho
Hyunju
H
Life Sciences Division, Korea Institute of Science and Technology, Seoul, Republic of Korea.
Park
Hyunsung
H
Yang
Eun Gyeong
EG
D001667:Binding, Competitive; D000075065:Elongin; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D017874:Immediate-Early Proteins / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein
C000616396:ELOB protein, human; C000616395:ELOC protein, human; D000075065:Elongin; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D010455:Peptides; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human; D006909:Hydroxyproline