A Conserved Family of Prolyl-4-Hydroxylases That Modify HIF

We identified a conserved family of hypoxia-inducible factor prolyl hydoxylase enzymes that appear to be responsible for this posttranslational modification

Richard K. Bruick; Steven L. McKnight

2002

Scholarcy highlights

  • Mammalian cells respond to changes in oxygen availability through a conserved pathway that is regulated by the hypoxia-inducible factor
  • We identified a conserved family of HIF prolyl hydoxylase enzymes that appear to be responsible for this posttranslational modification
  • Inappropriate accumulation of HIF caused by forced expression of the HIF-1α subunit under normoxic conditions was attenuated by coexpression of HPH
  • Expression of each gene product was confirmed by Western blot analysis with an antibody specific for the COOH-terminal V5 tag. 12.5 μl of each in vitro transcription/translation reaction was incubated for 30 min at 30°C in a reaction buffer containing 20 mM tris-Cl, 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 2 mM 2-oxoglutarate, 2 mM ascorbate, and 250 μM FeSO4 in the presence of 30 μl of ImmunoPure Immobilized Streptavidin beads that had previously been incubated with 1 μg of peptide for 30 min at room temperature and washed three times to remove excess peptide
  • Cells were incubated under normoxic or hypoxic conditions for an additional 15 hours, and total RNA was prepared with RNA STAT-60
  • The beads were washed three times with cold NTEN buffer, and bound-VHL was measured by scintillation counting.-labeled human VHL was synthesized from the human VHL cDNA cloned into the pcDNA3.1/V5-HIS vector with the TNT Coupled Reticulocyte Lysate System and-l-Met and desalted with a PD-10 column

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