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 (isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH)) may govern their 2-HG generating activity. In-vitro acetylation of these enzymes, with confirmation by western blotting, mass spectrometry, and reversibility by incubation with recombinant sirtuins, 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 sharply 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.