title: Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro.
abstract: Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O(2) depletion.
authors.affiliation: Howard Hughes Medical Institute, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Pan
authors.forename: Kyle D
authors.initials: KD
authors.lastname: Mansfield
authors.forename: Cara C
authors.initials: CC
authors.lastname: Bertozzi
authors.forename: Viktoriya
authors.initials: V
authors.lastname: Rudenko
authors.forename: Denise A
authors.initials: DA
authors.lastname: Chan
authors.forename: Amato J
authors.initials: AJ
authors.lastname: Giaccia
authors.forename: M Celeste
authors.initials: MC
authors.lastname: Simon
mesh_terms: D000818:Animals; D002292:Carcinoma, Renal Cell / Q000473:pathology; D015687:Cell Hypoxia / Q000187:drug effects; D004579:Electron Transport / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006900:Hydroxylation / Q000187:drug effects; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D051379:Mice; D008928:Mitochondria / Q000187:drug effects; D018716:Molecular Mimicry / Q000187:drug effects; D010084:Oxidation-Reduction / Q000187:drug effects; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D011392:Proline / Q000737:chemistry; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013816:Thermodynamics; D014475:Uncoupling Agents / Q000494:pharmacology; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014475:Uncoupling Agents; D011392:Proline; D006861:Hydrogen Peroxide; D011393:Procollagen-Proline Dioxygenase; D051794:Von Hippel-Lindau Tumor Suppressor Protein