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1.  Mass spectrometry-based detection of protein acetylation 
Summary
Improved sample preparation techniques and increasingly sensitive mass spectrometry (MS) analysis have revolutionized the study of protein post-translational modifications (PTMs). Here, we describe a general approach for immunopurification and MS-based identification of acetylated proteins in biological samples. This approach is useful characterizing changes in the acetylome in response to biological interventions (1).
doi:10.1007/978-1-62703-637-5_6
PMCID: PMC3954751  PMID: 24014401
Post-translational modification; sirtuin; immunopurification; immunoprecipitation; immunoblotting
3.  SIRT3 Deacetylates Mitochondrial 3-Hydroxy-3-Methylglutaryl CoA Synthase 2 and Regulates Ketone Body Production 
Cell Metabolism  2010;12(6):654-661.
SUMMARY
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.
doi:10.1016/j.cmet.2010.11.003
PMCID: PMC3310379  PMID: 21109197
4.  Mitochondrial sirtuins in the regulation of mitochondrial activity and metabolic adaptation 
In eukaryotes, mitochondria carry out numerous functions central to cellular and organismal health. How mitochondrial activities are regulated in response to differing environmental conditions, such as variations in diet, remains an important unsolved question in biology. Here we review emerging evidence suggesting that reversible acetylation of mitochondrial proteins on lysine residues represents a key mechanism by which mitochondrial functions are adjusted to meet environmental demands. In mammals, three members of the sirtuin class of NAD+-dependent deacetylases – SIRT3, SIRT4, and SIRT5 – localize to mitochondria and regulate targets involved in a diverse array of biochemical pathways. The importance of this activity is highlighted by recent studies of SIRT3 indicating that this protein suppresses the emergence of diverse age-related pathologies: hearing loss, cardiac fibrosis, and malignancy. Together, these findings argue that mitochondrial protein acetylation represents a central means by which mammals regulate mitochondrial functions to maintain cellular and organismal homeostasis.
doi:10.1007/978-3-642-21631-2_8
PMCID: PMC3245626  PMID: 21879450
Sir2; acetylation; respiration; reactive oxygen species; β-oxidation; ketone body; apoptosis; metabolism; mitochondria; SIRT3; SIRT4; SIRT5; urea cycle; GDH; CPS1; electron transport; glutathione; SOD2; LCAD; IDH2; hearing loss; cardiac hypertrophy; cancer; HMGCS2; AceCS2
5.  THE HISTONE DEACETYLASE SIRT6 IS A NOVEL TUMOR SUPPRESSOR THAT CONTROLS CANCER METABOLISM 
Cell  2012;151(6):1185-1199.
Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, while transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. Using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size and aggressiveness of tumors. SIRT6 also functions as a novel regulator of ribosome metabolism by co-repressing MYC transcriptional activity. Lastly, SIRT6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.
doi:10.1016/j.cell.2012.10.047
PMCID: PMC3526953  PMID: 23217706
6.  SIRT3 regulates fatty acid oxidation via reversible enzyme deacetylation 
Nature  2010;464(7285):121-125.
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.
doi:10.1038/nature08778
PMCID: PMC2841477  PMID: 20203611
7.  Calorie Restriction Alters Mitochondrial Protein Acetylation 
Aging cell  2009;8(5):604-606.
Summary
Calorie restriction (CR) increases lifespan in organisms ranging from budding yeast through mammals. Mitochondrial adaptation represents a key component of the response to CR. Molecular mechanisms underlying this adaptation are largely unknown. Here we show that lysine acetylation of mitochondrial proteins is altered during CR in a tissue-specific fashion. Via large-scale mass spectrometry screening, we identify 72 candidate proteins involved in a variety of metabolic pathways with altered acetylation during CR. Mitochondrial acetylation changes may play an important role in the pro-longevity CR response.
doi:10.1111/j.1474-9726.2009.00503.x
PMCID: PMC2752488  PMID: 19594485
calorie restriction; longevity; sirtuins; mitochondria; metabolism; mass spectrometry
9.  SIRT6 IN DNA REPAIR, METABOLISM, AND AGEING 
Journal of internal medicine  2008;263(2):128-141.
Ageing, or increased mortality with time, coupled with physiologic decline, is a nearly universal yet poorly understood biological phenomenon. Studies in model organisms suggest that two conserved pathways modulate longevity: DNA damage repair and insulin/Igf1-like signaling. In addition, homologs of yeast Sir2 – the sirtuins – regulate lifespan in diverse organisms. Here, we focus on one particular sirtuin, SIRT6. Mice lacking SIRT6 develop a degenerative disorder that in some respects mimics models of accelerated ageing [1]. We discuss how sirtuins in general and SIRT6 specifically relate to other evolutionarily conserved pathways affecting ageing, and how SIRT6 might function to ensure organismal homeostasis and normal lifespan.
doi:10.1111/j.1365-2796.2007.01902.x
PMCID: PMC2486832  PMID: 18226091
Ageing; DNA Damage; Metabolism
10.  Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation▿ †  
Molecular and Cellular Biology  2007;27(24):8807-8814.
Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.
doi:10.1128/MCB.01636-07
PMCID: PMC2169418  PMID: 17923681
11.  Telomere Shortening Exposes Functions for the Mouse Werner and Bloom Syndrome Genes 
Molecular and Cellular Biology  2004;24(19):8437-8446.
The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.
doi:10.1128/MCB.24.19.8437-8446.2004
PMCID: PMC516757  PMID: 15367665
12.  Mutations in the WRN Gene in Mice Accelerate Mortality in a p53-Null Background 
Molecular and Cellular Biology  2000;20(9):3286-3291.
Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN−/−;p53−/− mice show an increased mortality rate relative to WRN+/−;p53−/− animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.
PMCID: PMC85622  PMID: 10757812
13.  C. elegans SIRT6/7 Homolog SIR-2.4 Promotes DAF-16 Relocalization and Function during Stress 
PLoS Genetics  2012;8(9):e1002948.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1002948
PMCID: PMC3441721  PMID: 23028355

Results 1-13 (13)