Potential mechanisms linking SIRT activity and hypoxic 2-hydroxyglutarate generation: no role for direct enzyme (de)acetylation

Biochem J. 2017 Aug 10;474(16):2829-2839. doi: 10.1042/BCJ20170389.

Abstract

2-Hydroxyglutarate (2-HG) is a hypoxic metabolite with potentially important epigenetic signaling roles. The mechanisms underlying 2-HG generation are poorly understood, but evidence suggests a potential regulatory role for the sirtuin family of lysine deacetylases. Thus, we hypothesized that the acetylation status of the major 2-HG-generating enzymes [lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH)] may govern their 2-HG-generating activity. In vitro acetylation of these enzymes, with confirmation by western blotting, mass spectrometry, reversibility by recombinant sirtuins and an assay for global lysine occupancy, yielded no effect on 2-HG-generating activity. In addition, while elevated 2-HG in hypoxia is associated with the activation of lysine deacetylases, we found that mice lacking mitochondrial SIRT3 exhibited hyperacetylation and elevated 2-HG. These data suggest that there is no direct link between enzyme acetylation and 2-HG production. Furthermore, our observed effects of in vitro acetylation on the canonical activities of IDH, MDH and LDH appeared to contrast with previous findings wherein acetyl-mimetic lysine mutations resulted in the inhibition of these enzymes. Overall, these data suggest that a causal relationship should not be assumed between acetylation of metabolic enzymes and their activities, canonical or otherwise.

Keywords: acetylation; epigenetics; hypoxia; ischemia; sirtuins.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylation
  • Animals
  • Cell Hypoxia
  • Enzyme Assays
  • Glutarates / metabolism*
  • HEK293 Cells
  • Humans
  • Isocitrate Dehydrogenase / genetics
  • Isocitrate Dehydrogenase / metabolism
  • Kinetics
  • L-Lactate Dehydrogenase / genetics
  • L-Lactate Dehydrogenase / metabolism
  • Lysine / metabolism*
  • Malate Dehydrogenase / genetics
  • Malate Dehydrogenase / metabolism
  • Male
  • Mice
  • Mice, Knockout
  • Mitochondria, Heart / enzymology*
  • Mitochondrial Proteins / genetics*
  • Mitochondrial Proteins / metabolism
  • Protein Processing, Post-Translational*
  • Signal Transduction
  • Sirtuin 3 / deficiency
  • Sirtuin 3 / genetics*

Substances

  • Glutarates
  • Mitochondrial Proteins
  • Sirt3 protein, mouse
  • alpha-hydroxyglutarate
  • L-Lactate Dehydrogenase
  • Malate Dehydrogenase
  • Isocitrate Dehydrogenase
  • Sirtuin 3
  • Lysine