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Multiple Factors Affecting Cellular Redox Status and Energy Metabolism Modulate Hypoxia-Inducible Factor Prolyl Hydroxylase Activity In Vivo and In Vitro

Yi Pan,^1,2, Kyle D. Mansfield,^2,, Cara C. Bertozzi,² Viktoriya Rudenko,² Denise A. Chan,³ Amato J. Giaccia,³ and M. Celeste Simon^1,2*

Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104,¹ Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104,² Center for Clinical Science Research, Department of Radiation Oncology, Stanford University, Stanford, California 94305³

Received 6 July 2006/ Returned for modification 4 August 2006/ Accepted 2 November 2006

Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O₂)-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O₂ deprivation (hypoxia). Using a fusion protein containing the human HIF-1 O₂-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-1 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-1 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 O₂ depletion.

Published ahead of print on 13 November 2006.

Y.P. and K.D.M. contributed equally to this study.

Present address: Molecular Genetics and Microbiology, Duke University, Durham, NC 27710.

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