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

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 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 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.

Acetylation of Mitochondrial Proteins in the Heart: The Role of SIRT3

“…However, IDH2 activity was only found reduced in the liver but not in the inner ear and brain of SIRT3 KO mice ( Someya et al, 2010 ). In contrast, it was recently reported that in vitro acetylation of purified IDH2 from the porcine heart increased its activity ( Nadtochiy et al, 2017 ). These results suggest that acetylation of IDH2 influences its enzymatic activity in a tissue-dependent manner; deacetylation increases its activity in the kidney and liver but decreases its activity in the heart and has no effect in the inner ear and brain.…”

“…However, Zhao et al (2010) reported that acetylation of MDH increased its enzymatic activity in Chang liver (HeLa derivative) and HEK293 cells. Interestingly, neither acetylation nor deacetylation had any effect on the enzymatic activity of purified bovine heart MDH2 ( Nadtochiy et al, 2017 ). These results suggest that, like IDH2, the effects of acetylation on the MDH enzymatic activity are tissue-dependent.…”

“…These results suggest that, like IDH2, the effects of acetylation on the MDH enzymatic activity are tissue-dependent. In addition, differences in experimental models of acetylation, i.e., acetyl mimetics, in vitro acetylation, or SIRT3 deficiency, could account for variations in results ( Nadtochiy et al, 2017 ).…”

“…Conversely, overexpression of SIRT3 was able to reduce protein acetylation ( Karamanlidis et al, 2013 ). In contrast, SIRT3 -/- hearts have decreased mitochondrial respiration for complex I ( Koentges et al, 2015 ) and increased levels of NADH ( Nadtochiy et al, 2017 ). These results suggest a strong interdependence of OXPHOS and protein acetylation for the maintenance of mitochondrial function.…”

Cardioprotection by nicotinamide mononucleotide (NMN): Involvement of glycolysis and acidic pH

“…We recently showed that the SIRT1 inhibitor splitomicin (Sp) causes mild cellular alkalinization in primary mouse cardiomyocytes 16 . In companion experiments, we also tested the effects of the SIRT1 activator NMN, and surprisingly found that 1 mM NMN induced cellular acidification ( Figure 1A/B).…”