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. 2014 Aug 1;21(4):551-64.
doi: 10.1089/ars.2013.5420. Epub 2014 Mar 6.

SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress

Athanassios Vassilopoulos  1 , J Daniel Pennington, Thorkell Andresson, David M Rees, Allen D Bosley, Ian M Fearnley, Amy Ham, Charles Robb Flynn, Salisha Hill, Kristie Lindsey Rose, Hyun-Seok Kim, Chu-Xia Deng, John E Walker, David Gius
Affiliations

SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress

Athanassios Vassilopoulos et al. Antioxid Redox Signal. .

Abstract

Aims: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis.

Results: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype.

Innovation: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins.

Conclusion: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.

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Figures

<b>FIG. 1.</b>
FIG. 1.
The adenosine diphosphate (ATP) synthase complex contains multiple proteins with reversible acetyl-lysines and interacts with SIRT3. (a) Left panel, two 50 μg samples of highly purified bovine F1Fo ATP synthase were separated, transferred, and blotted with an acetyl-lysine antibody (Cell Signaling, Inc.) or stained with Coomassie. Middle panel, purified bovine F1Fo ATP synthase was treated with PCAF and blotted. Right panel, purified bovine F1Fo ATP synthase treated with PCAF was purified, treated with recombinant human SIRT3, and blotted with an acetyl-lysine antibody. (b) Skeletal muscle mitochondrial lysates from Sirt3+/+ and Sirt3−/− mice were harvested, IPed with an anti-ATP synthase antibody (Abcam, Inc.), and subsequently immunoblotted with an anti-OSCP antibody (Santa Cruz Biotechnology, Inc.), anti-ATP synthase β subunit (Abcam, Inc.), or anti-SIRT3 antibody (Cell Signaling, Inc.). (c-d) SIRT3 physically interacts with oligomycin sensitivity-conferring protein (OSCP). HCT116 cells were constructed to constitutively express a Myc-tagged SIRT3 gene, and cell extracts were (c) IPed with either an anti-Myc or an OCSP antibody and immunoblotted with an anti-SIRT3 antibody, or (d) IPed with an anti-Myc antibody and immunoblotted with an OCSP antibody. (e) IHC staining with an anti-SIRT3 and anti-OSCP antibody, with the images merged in the lower right section. Representative micrographs are shown.
<b>FIG. 2.</b>
FIG. 2.
Sirt3 knockout cells have increased mitochondrial acetylated protein and decreased ATP levels, and OSCP contains a reversible acetyl-lysine. (a) Bovine purified OSCP was mixed with or without recombinant SIRT3 in the presence or absence of nicotinamide and/or NAD+. After 1 h, samples were separated by PAGE, followed by immunoblotting with an anti-acetyl antibody (Cell Signaling, Inc.). Antibody against ATP synthase subunit γ was used as a loading control. (b) OSCP contains a reversible acetyl-lysine. Livers from isogenic, 3-month-old Sirt3+/+ mice on an AL diet (−) or fasted for 36 h (+) were harvested, IPed with an anti-OSCP antibody, and immunoblotted with an anti-acetyl antibody or anti-OSCP antibody. (c) Mitochondrial ATP levels in Sirt3 wild-type and knockout cells. Sirt3+/+ and Sirt3−/− MEFs were lysed, and ATP levels were measured using a chemiluminescence assay. Data are presented as arbitrary luminescence units for ATP levels as a ratio (or compared with) of the level in Sirt3+/+ MEFs. (d, e) Re-expression of wild-type, but not a deacetylation null mutant of Sirt3, (d) deacetylates OSCP and (e) restores ATP levels in Sirt3−/− MEFs. Fifth-passage Sirt3−/− MEFs were transfected with a control lentivirus (Cont), virus expressing a wild-type Sirt3 (wt), or one expressing the deacetylation null gene (dn) in which amino acid 248 has been changed from a histidine to tyrosine (1). Forty hours after infection, Sirt3−/− MEFs were harvested and mitochondrial extracts were IPed with an anti-OSCP antibody followed by immunoblotting with an anti-acetyl or anti-OSCP antibody. These extracts were also used to measure ATP levels as described earlier in (b). ATP levels are presented as a ratio compared with the levels observed in Sirt3+/+ MEFs. The results of all the experiments in this figure were obtained from at least three independent replicates. Representative gels are shown. Error bars represent one standard deviation. *Indicates p<0.05 by t-test.
<b>FIG. 3.</b>
FIG. 3.
SIRT3 deacetylates lysine 139. (a) Tandem mass spectrum from OSCP demonstrates acetylated lysine 139 in vivo. Liver mitochondria from wild-type and Sirt3−/− mice were resolved by SDS-PAGE followed by in-gel trypsin digestion, separation by nano-scale reverse-phase chromatography on reverse-phase columns, and analysis by OrbiTrap analyzer via an electro-spray interface. The spectrum represents the fragmentation of the peptide with a mass of 1628.9501 corresponding to the sequence TVL(AcK)SFLSPNQILK. (b) OSCP acetylation in Sirt3 wild-type and knockout mice. Livers from Sirt3+/+ and Sirt3−/− mice were used to isolate mitochondrial extracts that were used to determine the acetylation levels of OSCP amino acid 139. Data are presented as OSCPK139 acetylation as compared with Sirt3+/+ livers. (c) In vitro deacetylation of synthetic peptides. Synthetic peptide, including acetylated lysine, corresponding to the sequence of ATP synthase subunit OSCP (sequence EEATLSELKTVL(AcK)SFLSQGQ), was measured by MALDI-TOF (left). The predicted m/z for acetylated peptide is 2250.57 and for deacetylated peptide, it is 2208.56 m/z. (d) Multiple species contain a potentially reversible acetyl-lysine. The OSCP protein sequence from multiple species was BLASTed based on the reversible acetyl-lysine located at amino acid 139 in humans and mice. A 17-amino-acid motif (LSELKTVLK*SFL-SQGQV) was identified, and this motif is present in multiple species. (e) Deacetylated OSCP increases mitochondrial ATP levels. HCT116 cells expressing an OSCP shRNA (Santa Cruz Biotechnology, Inc.; see inset) were infected with lenti-OSCPK139, lenti-OSCPK139-N, or lenti-OSCPK139-R, and ATP levels were determined as described earlier (Fig. 2c legend). Equal expression of these proteins was determined (lower panel) using an anti-OSCP antibody (Santa Cruz Biotechnology, Inc.). Experiments were done in triplicate, and error bars represent one standard deviation. *Indicates p<0.05 by t-test.
<b>FIG. 4.</b>
FIG. 4.
OSCP contains a physiologically relevant reversible acetyl-lysine under cell stress. (a) Validation of an OSCP lysine 139 anti-acetyl antibody. A Flag-tagged OSCP expression vector was transfected into HEK 293T cells that contained Trichostatin A (TSA) (1 μM) and after 48 h, Flag-OSCP was IPed with an anti-Flag antibody (Sigma, Inc.). The samples were subsequently washed and incubated with purified SIRT3 protein without (lane 1) or with (lane 2) NAD. After 2 h, mixtures were immunoblotted with an anti-Ac-OSCPK139 antibody (Epitomics, Inc., - The Rabbit Monoclonal Antibody Company). Identical experiments were done with the 13-amino-acid lysine 139 acetylated peptide (peptide-139) or the control nonacetylated peptide (cont-peptide). (b) OSCP contains a CR-dependent, reversible acetyl-lysine. Livers from isogenic, 2-month-old age-matched Sirt3+/+ and Sirt3−/− mice that were placed on a CR diet for 12 weeks were harvested and mitochondrial extracts were isolated, separated, and subsequently blotted with antibodies to MnSOD, Ac-MnSODK122, Ac-MnSODK68, OSCP, Ac-OSCPK139, SIRT3, and COXIV. (c) Wild-type mice were placed on a high-fat “Western diet” (TD.88137; Harlan Teklad) as previously shown (30); livers were processed as described earlier, and samples were stained with antibodies to SIRT3, OSCP, Ac-OSCPK139, and COXIV. (d) Skeletal muscle from isogenic, 6- and 24-month-old age-matched Sirt3+/+ and Sirt3−/− mice were harvested, and mitochondrial extracts were isolated, separated, and blotted with antibodies to SIRT3, OSCP, Ac-OSCPK139, and COXIV.
<b>FIG. 5.</b>
FIG. 5.
Sirt3−/− mice exhibit decreased muscle endurance and OSCP is deacetylated in wild-type exercised mice. (a–c) After acclimatization, mice were run on a treadmill with increasing speed and slope to exhaustion. Time (a), distance (b), and work (c) were calculated from the individual performances. Bars represent mean values, and error bars represent standard error. (d) Several tissues were harvested from Sirt3+/+ and Sirt3−/− mice, and ATP was measured using the ATP determination kit from Molecular Probes. Data are presented as ATP levels measured in Sirt3−/− tissue as compared with the Sirt3+/+ tissue controls. (e, f) Skeletal muscle from isogenic Sirt3+/+ and Sirt3−/− mice both before exercise (Cont) and immediately after exercise (EX) were harvested and mitochondrial extracts were isolated, separated, and subsequently blotted with antibodies to (e) SIRT3, OSCP, Ac-OSCPK139, and COXIV or (f) with antibodies to Ac-MnSODK122, Ac-MnSODK68, and MnSOD. (g) The same samples used earlier were also used to immunoprecipitate the ATP synthase complex (see Fig. 1b), and these samples were immunoblotted with a pan-anti-acetyl antibody (Cell Signaling, Inc.). Representative blots are shown, and experiments were done in triplicate.
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