Dataset Information

Intermediary metabolite precursor dimethyl-2-ketoglutarate stabilizes hypoxia-inducible factor-1? by inhibiting prolyl-4-hydroxylase PHD2.

ABSTRACT: Hypoxia-inducible factor 1? (HIF-1?), a major mediator of tumor physiology, is activated during tumor progression, and its abundance is correlated with therapeutic resistance in a broad range of solid tumors. The accumulation of HIF-1? is mainly caused by hypoxia or through the mutated succinate dehydrogenase A (SDHA) or fumarate hydratase (FH) expression to inhibit its degradation. However, its activation under normoxic conditions, termed pseudohypoxia, in cells without mutated SDHA or FH is not well documented. Here, we show that dimethyl-2-ketoglutarate (DKG), a cell membrane-permeable precursor of a key metabolic intermediate, ?-ketoglutarate (?-KG), known for its ability to rescue glutamine deficiency, transiently stabilized HIF-1? by inhibiting activity of the HIF prolyl hydroxylase domain-containing protein, PHD2. Consequently, prolonged DKG-treatment under normoxia elevated HIF-1? abundance and up-regulated the expression of its downstream target genes, thereby inducing a pseudohypoxic condition. This HIF-1? stabilization phenotype is similar to that from treatment of cells with desferrioxamine (DFO), an iron chelator, or dimethyloxalyglycine (DMOG), an established PHD inhibitor, but was not recapitulated with other ?-KG analogues, such as Octyl-2KG, MPTOM001 and MPTOM002. Our study is the first example of an ?-KG precursor to increase HIF-1? abundance and activity. We propose that DKG acts as a potent HIF-1? activator, highlighting the potential use of DKG to investigate the contribution of PHD2-HIF-1? pathway to tumor biology.

SUBMITTER: Hou P

PROVIDER: S-EPMC4242664 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Intermediary metabolite precursor dimethyl-2-ketoglutarate stabilizes hypoxia-inducible factor-1α by inhibiting prolyl-4-hydroxylase PHD2.

Hou Peifeng P Kuo Ching-Ying CY Cheng Chun-Ting CT Liou Jing-Ping JP Ann David K DK Chen Qiang Q

PloS one 20141124 11

Hypoxia-inducible factor 1α (HIF-1α), a major mediator of tumor physiology, is activated during tumor progression, and its abundance is correlated with therapeutic resistance in a broad range of solid tumors. The accumulation of HIF-1α is mainly caused by hypoxia or through the mutated succinate dehydrogenase A (SDHA) or fumarate hydratase (FH) expression to inhibit its degradation. However, its activation under normoxic conditions, termed pseudohypoxia, in cells without mutated SDHA or FH is no ...[more]

PMID: 25420025

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Project description:Prolyl hydroxylase domain 2 (PHD2) is deemed a primary oxygen sensor in humans, yet many details of its underlying mechanism are still not fully understood. (Fe(2+) + ?KG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying three coordination sites, a bidentate ?-ketoglutarate occupying two sites, and an aquo ligand in the final site. Turnover is thought to be initiated upon release of the aquo ligand, creating a site for O(2) to bind at the iron. Herein we show that steady-state turnover is faster under acidic conditions, with k(cat) exhibiting a kinetic pK(a) = 7.22. A variety of spectroscopic probes were employed to identify the active-site acid, through comparison of (Fe(2+) + ?KG)PHD2 at pH 6.50 with pH 8.50. The near-UV circular dichroism spectrum was virtually unchanged at elevated pH, indicating that the secondary structure did not change as a function of pH. UV-visible and Fe X-ray absorption spectroscopy indicated that the primary coordination sphere of Fe(2+) changed upon increasing the pH; extended X-ray absorption fine structure analysis found a short Fe-(O/N) bond length of 1.96 Å at pH 8.50, strongly suggesting that the aquo ligand was deprotonated at this pH. Solvent isotope effects were measured during steady-sate turnover over a wide pH-range, with an inverse solvent isotope effect (SIE) of k(cat) observed ((D(2)O)k(cat) = 0.91 ± 0.03) for the acid form; a similar SIE was observed for the basic form of the enzyme ((D(2)O)k(cat) = 0.9 ± 0.1), with an acid equilibrium offset of ?pK(a) = 0.67 ± 0.04. The inverse SIE indicated that aquo release from the active site Fe(2+) immediately precedes a rate-limiting step, suggesting that turnover in this enzyme may be partially limited by the rate of O(2) binding or activation, and suggesting that aquo release is relatively slow. The unusual kinetic pK(a) further suggested that PHD2 might function physiologically to sense both intracellular pO(2) as well as pH, which could provide for feedback between anaerobic metabolism and hypoxia sensing.

Dataset Information

Intermediary metabolite precursor dimethyl-2-ketoglutarate stabilizes hypoxia-inducible factor-1? by inhibiting prolyl-4-hydroxylase PHD2.

Publications

Intermediary metabolite precursor dimethyl-2-ketoglutarate stabilizes hypoxia-inducible factor-1α by inhibiting prolyl-4-hydroxylase PHD2.

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