The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging.
Sirtuins are NAD+-dependent protein deacetylases and mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 21,2. Mice lacking both SIRT3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins3. We report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. Livers from mice lacking SIRT3 show higher levels of fatty acid oxidation intermediate products and triglycerides during fasting associated with decreased levels of fatty acid oxidation when compared to wild-type mice. Mass spectrometry analysis of mitochondrial proteins shows that long-chain acyl CoA dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo, and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty acid oxidation disorders during fasting including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty acid utilization during fasting.
The mitochondrial sirtuin SIRT3 regulates metabolic homeostasis during fasting and calorie restriction. We identified mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) as an acetylated protein and a possible target of SIRT3 in a proteomics survey in hepatic mitochondria from Sirt3−/− (SIRT3KO) mice. HMGCS2 is the rate-limiting step in β-hydroxybutyrate synthesis and is hyperacetylated at lysines 310, 447, and 473 in the absence of SIRT3. HMGCS2 is deacetylated by SIRT3 in response to fasting in wild-type mice, but not in SIRT3KO mice. HMGCS2 is deacetylated in vitro when incubated with SIRT3 and in vivo by overexpression of SIRT3. Deacetylation of HMGCS2 lysines 310, 447, and 473 by incubation with wild-type SIRT3 or by mutation to arginine enhances its enzymatic activity. Molecular dynamics simulations show that in silico deacetylation of these three lysines causes conformational changes of HMGCS2 near the active site. Mice lacking SIRT3 show decreased β-hydroxybutyrate levels during fasting. Our findings show SIRT3 regulates ketone body production during fasting and provide molecular insight into how protein acetylation can regulate enzymatic activity.
Acetylation has recently emerged as an important mechanism for controlling a broad array of proteins mediating cellular adaptation to metabolic fuels. Acetylation is governed, in part, by SIRTs (sirtuins), class III NAD+-dependent deacetylases that regulate lipid and glucose metabolism in liver during fasting and aging. However, the role of acetylation or SIRTs in pathogenic hepatic fuel metabolism under nutrient excess is unknown. In the present study, we isolated acetylated proteins from total liver proteome and observed 193 preferentially acetylated proteins in mice fed on an HFD (high-fat diet) compared with controls, including 11 proteins not previously identified in acetylation studies. Exposure to the HFD led to hyperacetylation of proteins involved in gluconeogenesis, mitochondrial oxidative metabolism, methionine metabolism, liver injury and the ER (endoplasmic reticulum) stress response. Livers of mice fed on the HFD had reduced SIRT3 activity, a 3-fold decrease in hepatic NAD+ levels and increased mitochondrial protein oxidation. In contrast, neither SIRT1 nor histone acetyltransferase activities were altered, implicating SIRT3 as a dominant factor contributing to the observed phenotype. In Sirt3−/− mice, exposure to the HFD further increased the acetylation status of liver proteins and reduced the activity of respiratory complexes III and IV. This is the first study to identify acetylation patterns in liver proteins of HFD-fed mice. Our results suggest that SIRT3 is an integral regulator of mitochondrial function and its depletion results in hyperacetylation of critical mitochondrial proteins that protect against hepatic lipotoxicity under conditions of nutrient excess.
cellular metabolism; mitochondrial metabolism; NAD; non-alcoholic fatty liver disease; obesity; proteomics; sirtuin
The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae1,2. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype3. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein3, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder4,5. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome4,5. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.
In mammals, Sirt1, a member of the sirtuin family of proteins, functions as a nicotinamide adenine dinucleotide-dependent protein deactylase, and has important physiological roles, including the regulation of glucose metabolism, cell survival, and mitochondrial respiration. The initial investigations of Sirt1 deficient mice have revealed a phenotype that includes a reduced lifespan, small size, and an increased frequency of abnormal sperm. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (−/−) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Numbers of mature sperm and spermatogenic precursors, as early as d15.5 of development, are significantly reduced (∼2-10-fold less; P≤0.004) in numbers in Sirt1−/− mice, whereas Sirt1 deficiency did not effect the efficiency oocyte production following superovulation of female mice. Furthermore, the proportion of mature sperm with elevated DNA damage (∼7.5% of total epididymal sperm; P = 0.02) was significantly increased in adult Sirt1−/− males. Analysis of global gene expression by microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and viable offspring in in vitro fertilization (IVF) experiments using gametes from Sirt1−/− and sibling Sirt1+/− mice. While viable animals were derived in both Sirt1−/− X wild type and Sirt1−/− X Sirt1−/− crosses, the efficiency of producing both 2-cell zygotes and viable offspring was diminished when IVF was performed with Sirt1−/− sperm and/or oocytes. Together, these data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function.
A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3 is identified as one of major mitochondrial deacetylase 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 knock-out mice. Several acetylated Lys residues were mapped by tandem mass spectrometry and we determined the role of acetylation on Complex II activity in SIRT3 knock-out mice. In agreement with SIRT3 dependent activation of Complex I, we observed that deacetylation of 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 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 SdhA crystal structure from porcine and chicken suggest that acetylation of the hydrophilic surface of SdhA may control the substrate entry to the active site of the protein and regulate the enzyme activity. Our findings constitute the first evidence for the regulation of Complex II activity by the reversible acetylation of the SdhA subunit as a novel substrate of the NAD+- dependent deacetylase, SIRT3.
Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2Δ mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2Δ shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2Δ strains. By strong SIRT1 overexpression in sir2Δ cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) SIRT1 gene is able to complement different molecular phenotypes of the sir2Δ mutant at rDNA ii) the in vivo screening of Sirt1 activity is possible in yeast.
Sirt3 is a mitochondrial NAD+-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR−/−) and LDLR/Sirt3 double-knockout (Sirt3−/−LDLR−/−) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR−/− mice. Sirt3 deficiency does not affect atherosclerosis in LDLR−/− mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-013-0399-0) contains supplementary material, which is available to authorized users.
SIRTUIN 3; Atherosclerosis; Metabolism; Oxidative stress
Mitochondrial protein hyperacetylation is a known consequence of sustained ethanol consumption and has been proposed to play a role in the pathogenesis of alcoholic liver disease (ALD). The mechanisms underlying this altered acetylome, however, remain unknown. The mitochondrial deacetylase sirtuin 3 (SIRT3) is reported to be the major regulator of mitochondrial protein deacetylation and remains a central focus for studies on protein acetylation. To investigate the mechanisms underlying ethanol-induced mitochondrial acetylation, we employed a model for ALD in both wild-type (WT) and SIRT3 knockout (KO) mice using a proteomics and bioinformatics approach. Here, WT and SIRT3 KO groups were compared in a mouse model of chronic ethanol consumption, revealing pathways relevant to ALD, including lipid and fatty acid metabolism, antioxidant response, amino acid biosynthesis and the electron-transport chain, each displaying proteins with altered acetylation. Interestingly, protein hyperacetylation resulting from ethanol consumption and SIRT3 ablation suggests ethanol-induced hyperacetylation targets numerous biological processes within the mitochondria, the majority of which are known to be acetylated through SIRT3-dependent mechanisms. These findings reveal overall increases in 91 mitochondrial targets for protein acetylation, identifying numerous critical metabolic and antioxidant pathways associated with ALD, suggesting an important role for mitochondrial protein acetylation in the pathogenesis of ALD.
Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site-specific acetylation in wild-type murine embryonic fibroblasts to Sirt3 knockout cells. We confirm Sirt3-regulated acetylation of several mitochondrial proteins in human cells by comparing acetylation in U2OS cells overexpressing Sirt3 to U2OS cells in which Sirt3 expression was reduced by shRNA. Our data demonstrate that ablation of Sirt3 significantly increases acetylation at dozens of sites on mitochondrial proteins. Substrates of Sirt3 are implicated in various metabolic pathways, including fatty acid metabolism and the tricarboxylic acid cycle. These results imply broader regulatory roles of Sirt3 in the mitochondria by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases.
Sirtuins (SIRT1-7) are a family of NAD-dependent deacetylases and/or ADP-ribosyltransferases that are involved in metabolism, stress responses and longevity. SIRT3 is localized to mitochondria, where it deacetylates and activates a number of enzymes involved in fuel oxidation and energy production.
In this study, we performed a proteomic screen to identify SIRT3 interacting proteins and identified several subunits of complex II and V of the electron transport chain. Two subunits of complex II (also known as succinate dehydrogenase, or SDH), SDHA and SDHB, interacted specifically with SIRT3. Using mass spectrometry, we identified 13 acetylation sites on SDHA, including six novel acetylated residues. SDHA is hyperacetylated in SIRT3 KO mice and SIRT3 directly deacetylates SDHA in a NAD-dependent manner. Finally, we found that SIRT3 regulates SDH activity both in cells and in murine brown adipose tissue.
Our study identifies SDHA as a binding partner and substrate for SIRT3 deacetylase activity. SIRT3 loss results in decreased SDH enzyme activity, suggesting that SIRT3 may be an important physiological regulator of SDH activity.
FoxO transcription factors and sirtuin family deacetylases regulate diverse biological processes, including stress responses and longevity. Here we show that the Caenorhabditis elegans sirtuin SIR-2.4—homolog of mammalian SIRT6 and SIRT7 proteins—promotes DAF-16–dependent transcription and stress-induced DAF-16 nuclear localization. SIR-2.4 is required for resistance to multiple stressors: heat shock, oxidative insult, and proteotoxicity. By contrast, SIR-2.4 is largely dispensable for DAF-16 nuclear localization and function in response to reduced insulin/IGF-1-like signaling. Although acetylation is known to regulate localization and activity of mammalian FoxO proteins, this modification has not been previously described on DAF-16. We find that DAF-16 is hyperacetylated in sir-2.4 mutants. Conversely, DAF-16 is acetylated by the acetyltransferase CBP-1, and DAF-16 is hypoacetylated and constitutively nuclear in response to cbp-1 inhibition. Surprisingly, a SIR-2.4 catalytic mutant efficiently rescues the DAF-16 localization defect in sir-2.4 null animals. Acetylation of DAF-16 by CBP-1 in vitro is inhibited by either wild-type or mutant SIR-2.4, suggesting that SIR-2.4 regulates DAF-16 acetylation indirectly, by preventing CBP-1-mediated acetylation under stress conditions. Taken together, our results identify SIR-2.4 as a critical regulator of DAF-16 specifically in the context of stress responses. Furthermore, they reveal a novel role for acetylation, modulated by the antagonistic activities of CBP-1 and SIR-2.4, in modulating DAF-16 localization and function.
Sensing and responding appropriately to environmental insults is a challenge facing all organisms. In the roundworm C. elegans, the FoxO protein DAF-16 moves to the nucleus in response to stress, where it regulates gene expression and plays a key role in ensuring organismal survival. In this manuscript, we characterize SIR-2.4 as a novel factor that promotes DAF-16 function during stress. SIR-2.4 is a member of a family of proteins called sirtuins, some of which promote increased lifespan in model organisms. Worms lacking SIR-2.4 show impaired DAF-16 nuclear recruitment, DAF-16–dependent gene expression, and survival in response to a variety of stressors. SIR-2.4 regulates DAF-16 by indirectly affecting levels of a modification called acetylation on DAF-16. Overall, our work has revealed SIR-2.4 to be a key new factor in stress resistance and DAF-16 regulation in C. elegans. Future studies will address whether mammalian SIR-2.4 homologs SIRT6 and SIRT7 act similarly towards mammalian FoxO proteins.
Sirtuin 6 (Sirt6), a mammalian Sir2 (silent information regulator-2) ortholog, is an NAD+-dependent histone deacetylase that modulates chromatin structure and genomic stability. Sirt6 knockout cells demonstrate genomic instability, and a deficiency of Sirt6 in mice leads to an aging phenotype early in life. Some nuclear sirtuins, such as Sirt7, localize to the nucleolus, and others, such as Sirt1, are mainly found in the nucleoplasm, with a minor population in the nucleolus. However, Sirt6 has been reported to be a nucleoplasmic protein that is excluded from the nucleolus. Because of the importance of a protein’s localization to its interactions and functions, we evaluated Sirt6 sub-cellular localization, expression and functions throughout the cell cycle in HeLa cells. Our results showed that during interphase, Sirt6 was mostly localized to the nucleus, although it was not absent from the nucleolus. Sirt6 was enriched in the nucleolus in the G1 phase of the cell cycle, while S phase nucleoli were almost entirely free of Sirt6. During mitosis, the Sirt6 expression level was increased, and while Sirt6 was not associated with condensed chromosomes, it partially co-localized with mitotic spindles. Cells overexpressing Sirt6 had a lower proliferation rate with a lower percentage of cells in mitosis. These findings suggest roles for Sirt6 in the nucleolus and in the mitotic phase of the cell cycle.
Sirt6; Sirtuin; nucleolus; cell cycle; mitosis
Mammalian sirtuins are homologs to the yeast silent information regulator 2 (Sir2), which is an NAD-dependent deacetylase. Sirtuins are comprised of 7 proteins, and each has different target proteins. Sirtuin 1 (SIRT1) plays important roles in maintaining metabolic functions and immune responses, and SIRT3 protects cells from oxidative stress-induced cell death. Both SIRT1 and SIRT3 are regulated by metabolic status and aging. Hence, SIRT1 and SIRT3 have been researched in metabolic diseases, such as type 2 diabetes mellitus (DM), fatty liver, and heart diseases. Although these diseases have been increasing, there is little information about relation between the diseases and SIRT1 and SIRT3 in cats. Therefore we cloned SIRT1 and SIRT3 cDNA, examined mRNA expression in cat tissues, and investigated the changes in SIRT1 and SIRT3 mRNA expression in peripheral blood leukocyte of cats fed on HFD for 6 weeks.
Cat SIRT1 and SIRT3 contained a catalytic core region and showed high sequence homology with other vertebrate SIRT1 (>61.3%) and SIRT3 (>65.9%) amino acids. Real-time polymerase chain reaction analyses revealed that high expression levels were observed in the liver and skeletal muscle for SIRT1 and in the heart for SIRT3 in cats. In addition, both cat SIRT1 and SIRT3 expression levels in the pancreas were different between individuals. Cat SIRT1 mRNA expression in peripheral blood leukocytes was significantly elevated in obese cats fed on HFD (P < 0.05).
Cat SIRT1 and SIRT3 genes are highly conserved among vertebrates, and HFD feeding may be related to SIRT1 mRNA expression mechanisms in cat peripheral blood leukocytes.
Cat; Sirtuin; cDNA cloning; High-fat diet; Real-time PCR
Estrogen and insulin-like growth factor-1 (IGF-1) play important roles in mammary gland development and breast cancer. SirT1 is a highly conserved protein deacetylase that can regulate the insulin/IGF-1 signaling in lower organisms, as well as a growing number of transcription factors, including NF-κB, in mammalian cells. Whether SirT1 regulates the IGF-1 signaling for mammary gland development and function, however, is not clear. In the present study, this role of SirT1 was examined by studying SirT1-deficient mice.
SirT1-deficient (SirT1ko/ko) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1co/co) with CMV-Cre transgenic mice. Whole mount and histology analyses, immunofluorescence staining, immunohistochemistry, and western blotting were used to characterize mammary gland development in virgin and pregnant mice. The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region.
Both male and female SirT1ko/ko mice can be fertile despite the growth retardation phenotype. Virgin SirT1ko/ko mice displayed impeded ductal morphogenesis, whereas pregnant SirT1ko/ko mice manifested lactation failure due to an underdeveloped lobuloalveolar network. Estrogen implantation was sufficient to rescue ductal morphogenesis. Exogenous estrogen reversed the increased basal level of IGF-1 binding protein-1 expression in SirT1ko/ko mammary tissues, but not that of IκBα expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNFα treatment enhanced the level of the newly synthesized IκBα in SirT1ko/ko cells. SirT1 deficiency therefore affects the cellular response to multiple extrinsic signals.
SirT1 modulates the IGF-1 signaling critical for both growth regulation and mammary gland development in mice. SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis. These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells.
Sirt3 is an NAD+-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1α subunit of PDH (PDH E1α). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1α subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation.
Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3−/− livers at 3 months of age. Livers of Sirt3−/− mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Re-introduction of wild-type, but not deacetylation null Sirt3, into Sirt3−/− MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSODK122-R), increased MnSOD activity when expressed in MnSOD−/− MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3−/− MEFs with lenti-MnSODK122-R inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3−/− livers and these irradiated livers displayed significant IR-induced cell damage and micro-vacuolization in their hepatocytes.
MnSOD; Sirt3; Mitochondria; Acetylation; Carcinogenesis
Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism, and ageing1,2. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets, and physiologic functions have been unclear. Here we show that SIRT7 is an NAD+-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated ETS transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis3–6. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumourigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs, and tumour formation in vivo.
Metformin inhibits ATP production in mitochondria and this may be involved in the anti-hyperglycemic effects of the drug. Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates the function of the electron transport chain and maintains basal ATP yield. We hypothesized that metformin treatment could diminish mitochondrial ATP production through downregulation of SIRT3 expression. Glucagon and cAMP induced SIRT3 mRNA in mouse primary hepatocytes. Metformin prevented SIRT3 induction by glucagon. Moreover, metformin downregulated constitutive expression of SIRT3 in primary hepatocytes and in the liver in vivo. Estrogen related receptor alpha (ERRα) mediates regulation of Sirt3 gene by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). ERRα mRNA expression was regulated in a similar manner as SIRT3 mRNA by glucagon, cAMP and metformin. However, a higher metformin concentration was required for downregulation of ERRα than SIRT3. ERRα siRNA attenuated PGC-1α mediated induction of SIRT3, but did not affect constitutive expression. Overexpression of the constitutively active form of AMP-activated protein kinase (AMPK) induced SIRT3 mRNA, indicating that the SIRT3 downregulation by metformin is not mediated by AMPK. Metformin reduced the hepatocyte ATP level. This effect was partially counteracted by SIRT3 overexpression. Furthermore, metformin decreased mitochondrial SIRT3 protein levels and this was associated with enhanced acetylation of several mitochondrial proteins. However, metformin increased mitochondrial mass in hepatocytes. Altogether, our results indicate that metformin attenuates mitochondrial expression of SIRT3 and suggest that this mechanism is involved in regulation of energy metabolism by metformin in the liver and may contribute to the therapeutic action of metformin.
Friedreich's ataxia (FRDA) is the most common inherited human ataxia and is caused by a deficiency in the mitochondrial protein frataxin. Clinically, patients suffer from progressive spinocerebellar degeneration, diabetes and a fatal cardiomyopathy, associated with mitochondrial respiratory chain defects. Recent findings have shown that lysine acetylation regulates mitochondrial function and intermediary metabolism. However, little is known about lysine acetylation in the setting of pathologic energy stress and mitochondrial dysfunction. We tested the hypothesis that the respiratory chain defects in frataxin deficiency alter mitochondrial protein acetylation. Using two conditional mouse models of FRDA, we demonstrate marked hyperacetylation of numerous cardiac mitochondrial proteins. Importantly, this biochemical phenotype develops concurrently with cardiac hypertrophy and is caused by inhibition of the NAD+-dependent SIRT3 deacetylase. This inhibition is caused by an 85-fold decrease in mitochondrial NAD+/NADH and direct carbonyl group modification of SIRT3, and is reversed with excess SIRT3 and NAD+
in vitro. We further demonstrate that protein hyperacetylation may be a common feature of mitochondrial disorders caused by respiratory chain defects, notably, cytochrome oxidase I (COI) deficiency. These findings suggest that SIRT3 inhibition and consequent protein hyperacetylation represents a negative feedback mechanism limiting mitochondrial oxidative pathways when respiratory metabolism is compromised, and thus, may contribute to the lethal cardiomyopathy in FRDA.
SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways. However, its role in response to nutrient excess remains unknown. Thus, we investigated SIRT3 regulation of the electron transfer chain and evaluated the role of SIRT3 in hepatic lipotoxic stress. SIRT3 depleted HepG2 cells shows diffuse disruption in mitochondrial electron transfer chain functioning, a concurrent reduction in the mitochondrial membrane potential, and excess basal reactive oxygen species levels. As this phenotype may predispose to increased lipotoxic hepatic susceptibility we evaluated the expression of SIRT3 in murine liver following chronic high-fat feeding. In this nutrient-excess model SIRT3 transcript and protein levels are downregulated in parallel with increased hepatic fat storage and oxidative stress. Palmitate was used to investigate lipotoxic susceptibility in SIRT3 knockout mouse primary hepatocytes and SIRT3 siRNA depleted HepG2 cells. Under SIRT3 deficient conditions palmitate enhances reactive oxygen species and increases hepatocyte cell death. Reconstitution of SIRT3 levels and/or treatment with N-acetylcysteine ameliorates these adverse effects. In conclusion SIRT3 functions to ameliorate hepatic lipotoxicity, although paradoxically, exposure to high-fat downregulates this adaptive program in the liver. This SIRT3-dependent lipotoxic susceptibility is possibly modulated, in part, by SIRT3 mediated control of electron transfer chain flux.
SIRT3; Lipotoxicity; Electron Transfer Chain; Reactive Oxygen Species; Palmitate
Sirtuin 6 (SIRT6) is a member of the sirtuin family of NAD-dependent deacetylases. Genetic deletion of Sirt6 in mice results in a severe degenerative phenotype with impaired liver function and premature death. So far the role of SIRT6 in development and progression of hepatocellular carcinoma is unknown. We first investigated SIRT6 expression in 153 primary human liver cancers, normal and cirrhotic livers using microarray analysis. SIRT6 was significantly downregulated in both cirrhotic livers and cancer. A Sirt6 knock out (KO) gene expression signature was generated from primary hepatoctyes isolated from three week old Sirt6-deficient animals. Sirt6-deficient hepatocytes showed upregulation of established HCC-biomarkers Alpha-fetoprotein (Afp), Insulin-like growth factor 2 (Igf2), H19 and Glypican-3 (Gpc3). Furthermore decreased SIRT6 expression was observed in hepatoma cell lines that are known to be apoptosis insensitive. Re-expression of SIRT6 in HepG2 cells increased apoptosis sensitivity to CD95-stimulation or chemotherapy treatment. Loss of Sirt6 was characterized by oncogenic changes including global hypomethylation as well as metabolic changes including hypoglycemia and increased fat deposition. The hepatocyte-specific Sirt6-KO signature had prognostic impact and was enriched in patients with poorly differentiated tumors with high AFP levels as well as recurrent disease. Finally, we could demonstrate that the Sirt6-KO signature possessed a predictive value for tumors other than HCC, i.e. breast and lung cancer.
Loss of SIRT6 induces epigenetic changes which may be relevant to chronic liver diseases and HCC development. Downregulation of SIRT6 and genes dysregulated by loss of SIRT6 possess oncogenic effects in hepatocarcinogenesis. Our data demonstrate that deficiency in one epigenetic regulator predisposes a tumorigenic phenotype which ultimately has relevance for outcome of HCC and other cancer patients.
liver cancer; SIRT6; molecular pathogenesis; gene expression profile; comparative genomics
The fasting-activated longevity protein sirtuin 1 (SirT1, ref. 1) promotes gluconeogenesis in part, by increasing transcription of the key gluconeogenic genes pepck1 and g6pase2,3, through deacetylating PGC-1α and FOXO1 (ref. 4). In contrast, signal transducer and activator of transcription 3 (STAT3) inhibits glucose production by suppressing expression of these genes5,6. It is not known whether the inhibition of gluconeogenesis by STAT3 is controlled by metabolic regulation. Here we show that STAT3 phosphorylation and function in the liver were tightly regulated by the nutritional status of an animal, through SirT1-mediated deacetylation of key STAT3 lysine sites. The importance of the SirT1-STAT3 pathway in the regulation of gluconeogenesis was verified in STAT3-deficient mice in which the dynamic regulation of gluconeogenic genes by nutritional status was disrupted. Our results reveal a new nutrient sensing pathway through which SirT1 suppresses the inhibitory effect of STAT3, while activating the stimulatory effect of PGC-1α and FOXO1 on gluconeogenesis, thus ensuring maximal activation of gluconeogenic gene transcription. The connection between acetylation and phosphorylation of STAT3 implies that STAT3 may have an important role in other cellular processes that involve SirT1.
Macrophage activation and infiltration into resident tissues is known to mediate local inflammation and is a hallmark feature of metabolic syndrome. Members of the sirtuin family of proteins regulate numerous physiological processes, including those involved in nutrient regulation and the promotion of longevity. However, the important role that SIRT1, the leading sirtuin family member, plays in immune response remains unclear. In this study, we demonstrate that SIRT1 modulates the acetylation status of the RelA/p65 subunit of NF-κB and thus plays a pivotal role in regulating the inflammatory, immune, and apoptotic responses in mammals. Using a myeloid cell-specific SIRT1 knockout (Mac-SIRT1 KO) mouse model, we show that ablation of SIRT1 in macrophages renders NF-κB hyperacetylated, resulting in increased transcriptional activation of proinflammatory target genes. Consistent with increased proinflammatory gene expression, Mac-SIRT1 KO mice challenged with a high-fat diet display high levels of activated macrophages in liver and adipose tissue, predisposing the animals to development of systemic insulin resistance and metabolic derangement. In summary, we report that SIRT1, in macrophages, functions to inhibit NF-κB-mediated transcription, implying that myeloid cell-specific modulation of this sirtuin may be beneficial in the treatment of inflammation and its associated diseases.