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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.

Original publication

DOI

10.1128/MCB.01223-06

Type

Journal article

Journal

Mol Cell Biol

Publication Date

02/2007

Volume

27

Pages

912 - 925

Keywords

Animals, Carcinoma, Renal Cell, Cell Hypoxia, Electron Transport, Electrophoresis, Polyacrylamide Gel, Energy Metabolism, Gene Expression Regulation, Humans, Hydrogen Peroxide, Hydroxylation, Hypoxia-Inducible Factor 1, alpha Subunit, Mice, Mitochondria, Molecular Mimicry, Oxidation-Reduction, Procollagen-Proline Dioxygenase, Proline, Protein Transport, RNA, Messenger, Recombinant Fusion Proteins, Thermodynamics, Uncoupling Agents, Von Hippel-Lindau Tumor Suppressor Protein