Oxidative Dimerization of PHD2 is Responsible for its Inactivation and Contributes to Metabolic Reprogramming via HIF-1α Activation.

Gibok Lee,Yoon-Mi Lee,Dong-Kug Choi,Young Jun Kim,Jeong-Hwa Jang,Taek-In Oh,Hyung-Sik Won,Jong-Wan Park,Pere Puigserver,Beong-Ou Lim,Ji-Hong Lim,Jae-Wan Choi

doi:10.1038/srep18928

Abstract

Prolyl hydroxylase domain protein 2 (PHD2) belongs to an evolutionarily conserved superfamily of 2-oxoglutarate and Fe(II)-dependent dioxygenases that mediates homeostatic responses to oxygen deprivation by mediating hypoxia-inducible factor-1α (HIF-1α) hydroxylation and degradation. Although oxidative stress contributes to the inactivation of PHD2, the precise molecular mechanism of PHD2 inactivation independent of the levels of co-factors is not understood. Here, we identified disulfide bond-mediated PHD2 homo-dimer formation in response to oxidative stress caused by oxidizing agents and oncogenic H-rasV12 signalling. Cysteine residues in the double-stranded β-helix fold that constitutes the catalytic site of PHD isoforms appeared responsible for the oxidative dimerization. Furthermore, we demonstrated that disulfide bond-mediated PHD2 dimerization is associated with the stabilization and activation of HIF-1α under oxidative stress. Oncogenic H-rasV12 signalling facilitates the accumulation of HIF-1α in the nucleus and promotes aerobic glycolysis and lactate production. Moreover, oncogenic H-rasV12 does not trigger aerobic glycolysis in antioxidant-treated or PHD2 knocked-down cells, suggesting the participation of the ROS-mediated PHD2 inactivation in the oncogenic H-rasV12-mediated metabolic reprogramming. We provide here a better understanding of the mechanism by which disulfide bond-mediated PHD2 dimerization and inactivation result in the activation of HIF-1α and aerobic glycolysis in response to oxidative stress.

Highlights

Several reports have suggested that elevated reactive oxygen species (ROS) levels lead to PHD2 inactivation and subsequently to HIF-α stabilization by regulating the levels of ascorbate, ferrous iron, or Krebs cycle intermediates[16,17,18,19]
Several reports have shown that cytosolic proteins, including pyruvate kinase M2 (PKM2), phosphatase and tensin homolog (PTEN), NEMO, and protein tyrosine phosphatases (PTPs), are directly regulated by intracellular ROS through inter or intramolecular disulfide bond formation[36,37,38,39]
Several reports have shown that oxidizing ferrous iron inactivates PHD2 under oxidative stress[16,17], it seems unlikely that this is the sole mechanism of PHD2 regulation

Summary

Introduction

Several reports have suggested that elevated ROS levels lead to PHD2 inactivation and subsequently to HIF-α stabilization by regulating the levels of ascorbate, ferrous iron, or Krebs cycle intermediates[16,17,18,19]. Proliferating cancer cells compared to normal differentiated cells exhibit different metabolic pathways to support their biomass synthesis such as amino acids, lipids, and nucleic acids. They show increased glucose uptake and lactate production[21], known as Warburg effect, to support their high rates of proliferation. Several oncogenic pathways such as HIF-1α , c-Myc, PI-3K, and Ras play important roles in enhancing aerobic glycolysis and suppressing oxidative phosphorylation, thereby suggesting their involvement in the metabolic reprogramming of cancer cells[22]. We show that PHD2 dimerization and its inactivation caused by oncogenic H-rasV12-associated oxidative stress result in HIF-1α activation and consequent increased glucose flux and lactate production

Methods

Results

Conclusion