SIRT3 protein deacetylates isocitrate dehydrogenase 2 (IDH2) and regulates mitochondrial redox status

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.

FIGURE 1.

A, maintenance of unacetylated Lys-413 is necessary for IDH2 activity. Wild-type IDH2 and K413R, K413Q, K272Q, K256Q, and K263Q mutants were expressed in HEK293 cells. Proteins were purified by immunoprecipitation (IP), IDH2 levels were normalized for protein, and activity assays were performed. Wild-type IDH2 activity was set as 100%. Bars and error bars represent mean and S.D. of triplicate assays. B and C, kinetic comparison of wild-type IDH2 and variants. Lys-413 was mutated to glutamine or arginine and bacterially expressed, recombinant proteins were purified, and steady-state kinetic analyses were performed. Comparison of IDH2 (open circles) and variants K413R (open squares) and K413Q (open diamonds) shows that the Lys-413 is important in catalysis.

FIGURE 2.

A, in vivo site-specific incorporation of acetylated Lys-413 in IDH2. IDH2 and IDH2^K413Ac were recombinantly expressed and purified and analyzed by 12% SDS-PAGE or detected in total lysates by Western blot with an anti-His₆ antibody or anti-pan acetyllysine antibody. NAM, nicotinamide; AcK, acetyllysine. B, relative IDH2 activity assays using purified IDH2 and IDH2^K413Ac. C and D, SIRT3 specifically deacetylates IDH2^K413Ac and rescues activity. C, recombinant IDH2^K413Ac from A was incubated with purified recombinant SIRT3 with or without NAD⁺ at 37 °C for 1 h. Acetylation status was assessed by Western blotting with anti-acetyllysine antibody. An anti-His Western blot shows that equivalent IDH2 protein levels were used. D, IDH2 activity assays were performed using the corresponding IDH2^K413Ac or unacetylated IDH2 from C. E and F, steady-state kinetic analysis of wild-type IDH2 and IDH2^K413Ac. Recombinant proteins were purified by nickel affinity chromatography, and kinetic assays were performed as described under “Experimental Procedures.” Comparison of wild-type IDH2 (open circles) and IDH2^K413Ac (open squares) reveals that acetylation greatly affects V_max and V_max/K_m. Data are means ± S.E.

FIGURE 3.

A and B, NADPH concentrations in mitochondria decrease when endogenous IDH2 is knocked down in HEK293 cells. A, Western blotting (WB) with anti-IDH2 antibody confirms IDH2 expression. B, measurements with errors are shown for two different stable cell populations from each type of transfection (SiRNA control, SiRNA of Idh2) (n = 3). p values indicate values significantly different from control (p < 0.05). C and D, IDH2 is critical to mediate the SIRT3-dependent protection in HEK293 cells treated with oxidant stressors hydrogen peroxide (H₂O₂) (C) or menadione (D). The three different stable cell populations (vector (VEC), SIRT3 stable expression, and IDH2 knockdown in stable SIRT3 cells, supplemental Fig. 2A) were transiently exposed to either 1 mm H₂O₂ or 25 μm menadione (n = 16). Data are means ± S.E.

FIGURE 4.

A, in cells, IDH2 is acetylated on lysine 413. A specific acetyllysine 413 antibody was generated using the peptide SGAMT(AC)KDLAGC. HEK293 cells were co-transfected by pcDNA3-IDH2-FLAG and pcDNA3-IDH2K413R-FLAG. IDH2 proteins were immunoprecipitated (IP) using FLAG beads. IDH2 Lys-413 acetylation levels were detected using the site-specific antibody. WB, Western blotting; NAM, nicotinamide; AcK, acetyllysine. B, low glucose decreases acetylation level of IDH2 Lys-413 and increases IDH2 activity. IDH2-FLAG was overexpressed in HEK293 cells following treatment with varying glucose levels (5, 3, 1, and 0 g/liter) for 6 h. Cell lysates were resolved by SDS-PAGE and detected by Western blotting with anti-SIRT3 antibody. IDH2-FLAG was immunoprecipitated with anti-FLAG beads, and IDH2 activity was measured and normalized to IDH2 protein levels; quantifications of the amounts of IDH2 were performed by anti-FLAG antibody and anti-acetylated Lys-413 antibody from A. Error bars represent standard error measurement (S.E.) (n = 3), p < 0.05. C, Bottom and middle, Western blot analysis of SIRT3 (bottom) and levels of acetylated lysine 413 (middle) in liver from 5-month-old WT or Sirt3^−/− mice fed either control diet (CD) or calorie-restricted diet (CR). Top, endogenous acetylated IDH2 was isolated by immunoprecipitation with anti-IDH2 antibody followed by Western blotting with anti-acetyllysine 413 IDH2 antibody (middle).

FIGURE 5.

A and B, GSH:GSSG ratios are significantly increased when IDH2^K413R was stably overexpressed in Sirt3^−/− MEFs. Measurements with errors are shown for the five different stable cell populations from each type of transfection (vector (VEC), IDH2^K413Q, and IDH2^K413R in Sirt3^−/− MEFs) (n = 3), (p < 0.01) in cells treated with oxidants hydrogen peroxide (H₂O₂) (A) and menadione (B). C and D, IDH2^K413R overexpression is sufficient to protect Sirt3^−/− MEFs from hydrogen peroxide (H₂O₂) (C) and menadione (D). Cells were transiently exposed to either 1 mm H₂O₂ or 25 μm menadione (n = 16) (p < 0.05). Data are means ± S.E.