Abstract
Mitochondria play a central role in oxidative energy metabolism and age-related diseases such as cancer. Accumulation of spurious oxidative damage can cause cellular dysfunction. Antioxidant pathways that rely on NADPH are needed for the reduction of glutathione and maintenance of proper redox status. The mitochondrial matrix protein isocitrate dehydrogenase 2 (IDH2) is a major source of NADPH. Previously, we demonstrated that the NAD(+)-dependent deacetylase SIRT3 was essential for the prevention of age-related hearing loss in mice fed a calorically restricted diet. Here we provide direct biochemical and biological evidence establishing an exquisite regulatory relationship between IDH2 and SIRT3 under acute and chronic caloric restriction. The regulated site of acetylation was mapped to Lys-413, an evolutionarily invariant residue. Site-specific, genetic incorporation of N(ε)-acetyllysine into position 413 of IDH2 revealed that acetylated IDH2 displays a dramatic 44-fold loss in activity. Deacetylation by SIRT3 fully restored maximum IDH2 activity. The ability of SIRT3 to protect cells from oxidative stress was dependent on IDH2, and the deacetylated mimic, IDH2(K413R) variant was able to protect Sirt3(-/-) mouse embryonic fibroblasts from oxidative stress through increased reduced glutathione levels. Together these results uncover a previously unknown mechanism by which SIRT3 regulates IDH2 under dietary restriction. Recent findings demonstrate that IDH2 activities are a major factor in cancer, and as such, these results implicate SIRT3 as a potential regulator of IDH2-dependent functions in cancer cell metabolism.
Highlights
NADϩ-dependent deacetylase SIRT3 is essential for the prevention of age-related hearing loss during caloric restriction
Mapping Functional Acetylation Sites in isocitrate dehydrogenase 2 (IDH2)—Recent proteomic studies reported that 13 different lysine residues in IDH2 were modified by acetylation [12,13,14]; it was unknown which sites played a critical role in regulating IDH2 activity
We proposed that SIRT3 deacetylates IDH2 and stimulates an increase in NADPH, which is utilized by the mitochondrion antioxidant system to combat reactive oxygen species (ROS)
Summary
NADϩ-dependent deacetylase SIRT3 is essential for the prevention of age-related hearing loss during caloric restriction. The ability of SIRT3 to protect cells from oxidative stress was dependent on IDH2, and the deacetylated mimic, IDH2K413R variant was able to protect Sirt3؊/؊ mouse embryonic fibroblasts from oxidative stress through increased reduced glutathione levels. Together these results uncover a previously unknown mechanism by which SIRT3 regulates IDH2 under dietary restriction. Sirt3Ϫ/Ϫ mice showed no response to CR, Sirt3Ϫ/Ϫ mice on a CR diet exhibited higher levels of NADPH, of reduced glutathione, and of IDH2 activity in mitochondria These observations led us to postulate that IDH2 may be a direct in vivo target of SIRT3 [2]. In addition to regulation of mitochondrial redox status, these results implicate SIRT3 as a general regulator of IDH2 functions, in cancer cell metabolism
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