A fluorescence polarization-based interaction assay for hypoxia-inducible factor prolyl hydroxylases

Abstract

Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor lot (HIF-1 alpha) plays a central role in regulating transcriptional responses to hypoxia. This process requires hydroxylation of specific prolines in HIF-1 alpha 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-1 alpha(peptides containing hydroxyproline, we developed a quantitative method based on fluorescence polarization for a systematic evaluation of binding of hydroxylated HIF-1 alpha 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-1 alpha VBC interaction and PHDs, and of screening modulators for the interaction as well as the enzyme. (c) 2005 Elsevier Inc. All rights reserved.