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

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.

Paracrine Induction of HIF by Glutamate in Breast Cancer: EglN1 Senses Cysteine

“…We found that self-inactivation of EglN1 is linked to oxidation of cysteine residues 208, 266, 302, and either 323 or 326 (or both). Of note, it was recently reported that EglN1 cysteine residues 302 and 326 mediate EglN1 dimer formation when EglN1 is oxidatively inactivated (Lee et al, 2016). Clearly, additional studies will be required to determine how, mechanistically, oxidation of the intramolecular cysteine residues identified here regulates EglN1 activity and to understand why this enzyme evolved to sense both oxygen and cysteine.…”

Mitochondria control acute and chronic responses to hypoxia

“…Multiple lines of evidence have indicated that the PHDs may be oxidized by reactive oxygen and nitrogen species leading to inhibition in vitro. Candidate cysteine residues required for the PHD oxidation have been identified (Lee 2016). A new proposed mechanism indicates that oxidation of PHD2 leads to the formation of inactive homodimers through disulfide bridge formation (Chowdhury 2011, Lee 2016).…”

The role of mitochondria in cardiac development and protection

“…In hypoxia, ROS release is required as a signal to stabilize HIF-1α, resulting in metabolic reprogramming with a generalized hypoxia response, including increased expression of HIF-1α itself [29] (Figure 1). Elevated cytoplasmic ROS levels have been reported to also activate Keap1, the binding partner of nuclear factor erythroid-derived-like 2 (NFE2l2 or Nrf2) [30].…”