Functional Analysis of the Contribution of HIF-1α Proline 402 to the Interaction with VHL

Hypoxia-inducible factor 1 alpha (HIF-1α) is involved in transcription of hypoxia-inducible genes. In normoxia, it binds von Hippel-Lindau (VHL) resulting in ubiquitin-proteasome degradation. This requires hydroxylation at prolines 564 (P564) or 402 (P402) within the oxygen-dependent degradation (ODD) domain. P402 hydroxylation only occurs at higher oxygen concentrations. Its specific contribution to the HIF-1α-VHL interaction is unknown. It was hypothesized that loss of P402 in normoxia will dampen the HIF-1α-VHL interaction. HA-tagged ODD constructs (WT, P402A, P564G, and P402A/P564G) were generated. FLAG-VHL co-immunoprecipitation was lower for P402A compared to WT. In vitro ubiquitination decreased with P402 mutation when P564 was also mutated. Cycloheximide chase analysis showed that P402 loss had no significant effect on ODD half-life. These results indicate a secondary role for P402 in the HIF-1alpha-VHL interaction. Elucidating the details of P402’s role in the interaction of HIF-1alpha with VHL will enhance the understanding of mammalian oxygen sensing and ubiquitin-proteasome pathways.M.Sc