A nonradioactive 96-well plate assay for the detection of hypoxia-inducible factor prolyl hydroxylase activity

Anal Biochem. 2004 Jul 1;330(1):74-80. doi: 10.1016/j.ab.2004.03.066.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / chemistry
  • Biological Assay / methods
  • DNA-Binding Proteins / analysis*
  • Dioxygenases
  • Elongin
  • Humans
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Hypoxia-Inducible Factor-Proline Dioxygenases
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / genetics
  • Nuclear Proteins / analysis*
  • Peptides / analysis*
  • Peptides / chemistry
  • Procollagen-Proline Dioxygenase / chemistry*
  • Procollagen-Proline Dioxygenase / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Sensitivity and Specificity
  • Streptavidin / chemistry
  • Thioredoxins / chemistry
  • Thioredoxins / genetics
  • Transcription Factors / analysis*
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics

Substances

  • Antibodies, Monoclonal
  • DNA-Binding Proteins
  • ELOB protein, human
  • ELOC protein, human
  • Elongin
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Multiprotein Complexes
  • Nuclear Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Thioredoxins
  • Streptavidin
  • Dioxygenases
  • Procollagen-Proline Dioxygenase
  • EGLN3 protein, human
  • Hypoxia-Inducible Factor-Proline Dioxygenases