Regulation of succinate dehydrogenase activity by SIRT3 in mammalian mitochondria

Abstract

A member of the sirtuin family of NAD(+)-dependent deacetylases, SIRT3, is identified as one of the major mitochondrial deacetylases located in mammalian mitochondria responsible for deacetylation of several metabolic enzymes and components of oxidative phosphorylation. Regulation of protein deacetylation by SIRT3 is important for mitochondrial metabolism, cell survival, and longevity. In this study, we identified one of the Complex II subunits, succinate dehydrogenase flavoprotein (SdhA) subunit, as a novel SIRT3 substrate in SIRT3 knockout mice. Several acetylated Lys residues were mapped by tandem mass spectrometry, and we determined the role of acetylation in Complex II activity in SIRT3 knockout mice. In agreement with SIRT3-dependent activation of Complex I, we observed that deacetylation of the SdhA subunit increased the Complex II activity in wild-type mice. In addition, we treated K562 cell lines with nicotinamide and kaempferol to inhibit deacetylase activity of SIRT3 and stimulate SIRT3 expression, respectively. Stimulation of SIRT3 expression decreased the level of acetylation of the SdhA subunit and increased Complex II activity in kaempherol-treated cells compared to control and nicotinamide-treated cells. Evaluation of acetylated residues in the SdhA crystal structure from porcine and chicken suggests that acetylation of the hydrophilic surface of SdhA may control the entry of the substrate into the active site of the protein and regulate the enzyme activity. Our findings constitute the first evidence of the regulation of Complex II activity by the reversible acetylation of the SdhA subunit as a novel substrate of the NAD(+)-dependent deacetylase, SIRT3.

Figure 1. Detection of SdhA as a novel SIRT3 substrate in SIRT3 knock-out mice liver mitochondria

Acetylated proteins and SIRT3 expression in Sirt3^+/+, Sirt3^+/−, and Sirt3^−/− mice liver mitochondria were evaluated by immunoblotting analysis using various antibodies. A) Mitochondrial lysates prepared as described in the Materials and Methods were separated on SDS-PAGE and increased acetylation of mitochondrial proteins was detected by immunoblotting probed with N-acetyl lysine (N-acetyl K) antibody. As a control for equal loading, protein blot was developed with Hsp60 antibody. B) SIRT3 protein levels in the liver or isolated liver mitochondria from wild type or SIRT3 deficient mice were detected by immunoblotting analysis using SIRT3 antibody. Mitochondrial protein MnSOD and cytoplasmic protein tubulin were also detected as controls. C) Approximately, 2mg of Sirt3^−/− mice liver mitochondrial lysate were layered on 34% sucrose cushion and fractioned into five separate layers (the top and bottom fractions of the sucrose cushion is shown by the arrow). Equal volumes of each fraction were separated on SDS-PAGE gel and acetylated proteins in each fraction were detected by immunoblotting analysis. The total Sirt3^−/− mice liver mitochondrial lysate (ML) layered on the cushion was also analyzed to locate the acetylated proteins in the fractions. Arrows show the location of SIRT3 substrates glutamate dehydrogenase (GDH) and the flavoprotein subunit of succinate dehydrogenase (SdhA). D) Approximately, 50 μl of fraction 3 from Sirt3^+/+ or Sirt3^−/− mice liver mitochonria was separated on 2D-gels and acetylated proteins were detected with N-acetyl lysine antibody. The acetylated 2D-gel spots corresponding to the Coomassie Blue stained gel spots were in-gel digested and identified by mass spectrometry. The protein identification determined by mass spectrometry was confirmed by immunoblotting using SdhA antibody.

Figure 1. Detection of SdhA as a novel SIRT3 substrate in SIRT3 knock-out mice liver mitochondria

Acetylated proteins and SIRT3 expression in Sirt3^+/+, Sirt3^+/−, and Sirt3^−/− mice liver mitochondria were evaluated by immunoblotting analysis using various antibodies. A) Mitochondrial lysates prepared as described in the Materials and Methods were separated on SDS-PAGE and increased acetylation of mitochondrial proteins was detected by immunoblotting probed with N-acetyl lysine (N-acetyl K) antibody. As a control for equal loading, protein blot was developed with Hsp60 antibody. B) SIRT3 protein levels in the liver or isolated liver mitochondria from wild type or SIRT3 deficient mice were detected by immunoblotting analysis using SIRT3 antibody. Mitochondrial protein MnSOD and cytoplasmic protein tubulin were also detected as controls. C) Approximately, 2mg of Sirt3^−/− mice liver mitochondrial lysate were layered on 34% sucrose cushion and fractioned into five separate layers (the top and bottom fractions of the sucrose cushion is shown by the arrow). Equal volumes of each fraction were separated on SDS-PAGE gel and acetylated proteins in each fraction were detected by immunoblotting analysis. The total Sirt3^−/− mice liver mitochondrial lysate (ML) layered on the cushion was also analyzed to locate the acetylated proteins in the fractions. Arrows show the location of SIRT3 substrates glutamate dehydrogenase (GDH) and the flavoprotein subunit of succinate dehydrogenase (SdhA). D) Approximately, 50 μl of fraction 3 from Sirt3^+/+ or Sirt3^−/− mice liver mitochonria was separated on 2D-gels and acetylated proteins were detected with N-acetyl lysine antibody. The acetylated 2D-gel spots corresponding to the Coomassie Blue stained gel spots were in-gel digested and identified by mass spectrometry. The protein identification determined by mass spectrometry was confirmed by immunoblotting using SdhA antibody.

Figure 2. Acetylation of SdhA at conserved K179, K485, K498, and K538 residues

A) The CID spectrum of acetylated peptide detected in LC-MS/MS analysis of 2D-gel spot of SdhA from SIRT3 knock-out mice mitochondria. B) Primary sequence alignment of acetylated peptides from mice SdhA and its homologs from different species. The human, bovine, pig, chicken, and E. coli SdhA were aligned with acetylated peptides of mouse SdhA. (*) denotes the acetylated Lys residues detected in the LC-MS/MS analysis. The alignment was created with CLUSTALW program in Biology Workbench and displayed in BOXSHADE. C) Crystal structure model of the chicken SdhA (PDB# 1YQ3) representing the all four subunits SdhA (green), SdhB (cyan), SdhC, and SdhD (yellow and pink, respectively). The conserved Lys residues found to be acetylated in mouse SdhA (shown by asterisks in B) were colored in red.

Figure 2. Acetylation of SdhA at conserved K179, K485, K498, and K538 residues

A) The CID spectrum of acetylated peptide detected in LC-MS/MS analysis of 2D-gel spot of SdhA from SIRT3 knock-out mice mitochondria. B) Primary sequence alignment of acetylated peptides from mice SdhA and its homologs from different species. The human, bovine, pig, chicken, and E. coli SdhA were aligned with acetylated peptides of mouse SdhA. (*) denotes the acetylated Lys residues detected in the LC-MS/MS analysis. The alignment was created with CLUSTALW program in Biology Workbench and displayed in BOXSHADE. C) Crystal structure model of the chicken SdhA (PDB# 1YQ3) representing the all four subunits SdhA (green), SdhB (cyan), SdhC, and SdhD (yellow and pink, respectively). The conserved Lys residues found to be acetylated in mouse SdhA (shown by asterisks in B) were colored in red.

Figure 2. Acetylation of SdhA at conserved K179, K485, K498, and K538 residues

A) The CID spectrum of acetylated peptide detected in LC-MS/MS analysis of 2D-gel spot of SdhA from SIRT3 knock-out mice mitochondria. B) Primary sequence alignment of acetylated peptides from mice SdhA and its homologs from different species. The human, bovine, pig, chicken, and E. coli SdhA were aligned with acetylated peptides of mouse SdhA. (*) denotes the acetylated Lys residues detected in the LC-MS/MS analysis. The alignment was created with CLUSTALW program in Biology Workbench and displayed in BOXSHADE. C) Crystal structure model of the chicken SdhA (PDB# 1YQ3) representing the all four subunits SdhA (green), SdhB (cyan), SdhC, and SdhD (yellow and pink, respectively). The conserved Lys residues found to be acetylated in mouse SdhA (shown by asterisks in B) were colored in red.

Figure 3. Regulation of Succinate Dehydrogenase (Complex II) activity by acetylation of SdhA

Hyper acetylation of SdhA decreases Complex II activity in SIRT3 knock-out mice. A) Equal amounts of lysates obtained from Sirt3^+/+, Sirt3^+/−, and Sirt3^−/− mice liver mitochondria were separated on 12% SDS-PAGE and probed with N-acetyl lysine (N-acetyl K), SdhA and Hsp60 antibodies. B) Complex II activity was measured as the rate of DCIP reduction, monitored at 600 nm using different amounts of mitochondrial lysates from Sirt3^+/+ and Sirt3^−/− mice liver mitochondria. Asterisks denote p<0.005.

Figure 4. Role of SIRT3 over-expression on SdhA deacetylation and Complex II activity

Over-expression of SIRT3 in HIB1B cells increases Complex II activity by deacetylation of SdhA. A) Mitochondria from control and HIB1B cells stably expressing Flag-tagged truncated (tSIRT3) and full-length (fSIRT3) were isolated and about 20 μg of the mitochondrial lysate from each cell line was separated on 12% SDS-PAGE. Immunoblotting analyses were performed with antibodies described in Fig. 3A and Flag-tag antibody. B) Immunoblotting analysis of K562 cell lysates obtained from control (Cont), nicotinamide (Nam), and kaempferol (Kaem) treated cells. Approximately, 20 μg of control and treated K562 cell lysates from each sample was loaded onto 12% SDS-PAGE and immunoblotting was performed as described above. Actin and Hsp60 blots were shown to ensure equal loading in the protein lanes. C) Equal amounts (about 2mg) of control and treated K562 cells lysates were layered on 34% sucrose cushion and fractionated into 8-1 mL aliquots after high speed centrifugation. Equal volumes of each fraction (–8) were acetone precipitated and loaded on 12% SDS-PAGE gels for immunoblotting analysis. Arrows show the location of acetylated protein overlapping with the SdhA signal. D) Complex II activity was monitored using different amounts of kaempferol and nicotinamide treated K562 cell lysates. The analysis was done in triplicate and values shown are the mean ±SD.

Figure 4. Role of SIRT3 over-expression on SdhA deacetylation and Complex II activity

Over-expression of SIRT3 in HIB1B cells increases Complex II activity by deacetylation of SdhA. A) Mitochondria from control and HIB1B cells stably expressing Flag-tagged truncated (tSIRT3) and full-length (fSIRT3) were isolated and about 20 μg of the mitochondrial lysate from each cell line was separated on 12% SDS-PAGE. Immunoblotting analyses were performed with antibodies described in Fig. 3A and Flag-tag antibody. B) Immunoblotting analysis of K562 cell lysates obtained from control (Cont), nicotinamide (Nam), and kaempferol (Kaem) treated cells. Approximately, 20 μg of control and treated K562 cell lysates from each sample was loaded onto 12% SDS-PAGE and immunoblotting was performed as described above. Actin and Hsp60 blots were shown to ensure equal loading in the protein lanes. C) Equal amounts (about 2mg) of control and treated K562 cells lysates were layered on 34% sucrose cushion and fractionated into 8-1 mL aliquots after high speed centrifugation. Equal volumes of each fraction (–8) were acetone precipitated and loaded on 12% SDS-PAGE gels for immunoblotting analysis. Arrows show the location of acetylated protein overlapping with the SdhA signal. D) Complex II activity was monitored using different amounts of kaempferol and nicotinamide treated K562 cell lysates. The analysis was done in triplicate and values shown are the mean ±SD.

Figure 4. Role of SIRT3 over-expression on SdhA deacetylation and Complex II activity

Over-expression of SIRT3 in HIB1B cells increases Complex II activity by deacetylation of SdhA. A) Mitochondria from control and HIB1B cells stably expressing Flag-tagged truncated (tSIRT3) and full-length (fSIRT3) were isolated and about 20 μg of the mitochondrial lysate from each cell line was separated on 12% SDS-PAGE. Immunoblotting analyses were performed with antibodies described in Fig. 3A and Flag-tag antibody. B) Immunoblotting analysis of K562 cell lysates obtained from control (Cont), nicotinamide (Nam), and kaempferol (Kaem) treated cells. Approximately, 20 μg of control and treated K562 cell lysates from each sample was loaded onto 12% SDS-PAGE and immunoblotting was performed as described above. Actin and Hsp60 blots were shown to ensure equal loading in the protein lanes. C) Equal amounts (about 2mg) of control and treated K562 cells lysates were layered on 34% sucrose cushion and fractionated into 8-1 mL aliquots after high speed centrifugation. Equal volumes of each fraction (–8) were acetone precipitated and loaded on 12% SDS-PAGE gels for immunoblotting analysis. Arrows show the location of acetylated protein overlapping with the SdhA signal. D) Complex II activity was monitored using different amounts of kaempferol and nicotinamide treated K562 cell lysates. The analysis was done in triplicate and values shown are the mean ±SD.