The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.

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.

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.Conserved from bacteria to humans, the sirtuin family of NAD ϩ -dependent protein deacetylase/mono-ADP-ribosyltransferase enzymes controls a variety of cellular processes such as aging, metabolism, and gene silencing (18,24). It has been proposed that sirtuins mediate the longevity-promoting effects of calorie restriction (CR) in yeast, worms, flies, and mice (4,17,22,24). Seven mammalian sirtuins (SIRT1 to -7) are known (11,12,18,24). At least three sirtuins (SIRT3, SIRT4, and SIRT5) localize to mitochondria, suggesting the existence of sirtuin substrates in that organelle (19,26,28,(31)(32)(33). Several lines of evidence link SIRT3 to metabolism: SIRT3 is down-regulated in muscle from diabetic animals (37) and upregulated in white and brown adipose tissue in response to CR (33). Overexpression of SIRT3 in cells affects expression of genes involved in mitochondrial function (33). SIRT3 regulates the acetylation level and activity of acetyl-coenzyme A synthetase 2, a protein that may play a role in energy production in mammals under starvation conditions (20,31). SIRT4 is an ADP-ribosyltransferase that has been implicated in regulating amino acid-stimulated insulin secretion in mice via modification of glutamate dehydrogenase (GDH) (19). No functions have been reported for SIRT5.

We demonstrate a role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. In particular, transient increased expression of Sirt1 is sufficient to stimulate basal rates of autophagy. In addition, we show that Sirt1 ؊/؊ mouse embryonic fibroblasts do not fully activate autophagy under starved conditions. Reconstitution with wild-type but not a deacetylase-inactive mutant of Sirt1 restores autophagy in these cells. We further demonstrate that Sirt1 can form a molecular complex with several essential components of the autophagy machinery, including autophagy genes (Atg)5, Atg7, and Atg8. In vitro, Sirt1 can, in an NAD-dependent fashion, directly deacetylate these components. The absence of Sirt1 leads to markedly elevated acetylation of proteins known to be required for autophagy in both cultured cells and in embryonic and neonatal tissues. Finally, we show that Sirt1 ؊/؊ mice partially resemble Atg5 ؊/؊ mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. Furthermore, the in utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1 ؊/؊ pups. These results suggest that the Sirt1 deacetylase is an important in vivo regulator of autophagy and provide a link between sirtuin function and the overall cellular response to limited nutrients. mitochondria ͉ starvation ͉ acetylation ͉ aging M acroautophagy, referred to hereafter as autophagy, is an intracellular process that allows for the degradation of proteins and organelles (1-3). Morphologically, autophagy is characterized by the formation of a double-membrane structure termed the autophagosome. In yeast, the process of autophagy can be stimulated by the withdrawal of various nutrients, and evidence suggests that at least 16 separate autophagy genes (Atg) are ultimately required for the formation of the autophagosome and the subsequent induction of autophagy (1-3). Mammalian cells can also respond to nutrient withdrawal by inducing autophagy. Genetic studies have suggested that autophagy is required to maintain the animal's energetic needs during the small window of time immediately after birth but before neonates can get milk nutrients from their mothers (4). In addition, two recent studies have demonstrated that brain conditional knockouts of either Atg7 or Atg5 significantly accelerated the development of neuropathologies usually restricted to older animals (5, 6).The sirtuins are a family of NAD-dependent deacetylases that have been linked to the regulation of life span. Increased expression of Sir2 can extend the life span of model organisms, and under certain experimental conditions, it appears that the activity of Sir2 is required for the life-extending benefits of caloric restriction in organisms such as yeast and flies (7). As opposed to these relatively simple organisms, mammalian species have seven different sirtuin family members (8, 9). The closest relative of yeast Sir2 is termed Sirt1 in mammalian cells. We have demonstrated that mice subjected t...

SUMMARY Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extra-mitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.

We report the identification and characterization of a five-carbon protein post-translational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric academia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

Protein post-translational modifications (PTMs) at the lysine residue, such as lysine methylation, acetylation, and ubiquitination, are diverse, abundant, and dynamic. They play a key role in the regulation of diverse cellular physiology. Here we report discovery of a new type of lysine PTM, lysine malonylation (Kmal). Kmal was initially detected by mass spectrometry and protein sequence-database searching. The modification was comprehensively validated by Western blot, tandem MS, and high-performance liquid chromatography of synthetic peptides, isotopic labeling, and identification of multiple Kmal substrate proteins. Kmal is a dynamic and evolutionarily conserved PTM observed in mammalian cells and bacterial cells. In addition, we demonstrate that Sirt5, a member of the class III lysine deacetylases, can catalyze lysine demalonylation and lysine desuccinylation reactions both in vitro and in vivo. This result suggests the possibility of nondeacetylation activity of other class III lysine deacetylases, especially those without obvious acetylation protein substrates. Our results therefore reveal a new type of PTM pathway and identify the first enzyme that can regulate lysine malonylation and lysine succinylation status. Molecular & Cellular Proteomics 10: 10.1074/ mcp.M111.012658, 1-12, 2011.Cellular function and physiology are largely determined by the inventory of all proteins in a cell, its proteome. The collection and characterization of the proteome is critical to understanding cellular mechanisms and diseases. Proteomes in eukaryotic cells consist of over a million molecular species of proteins, easily orders of magnitude more complex than the corresponding genomes (1, 2). There are two major mechanisms for expanding the coding capacity of the human genome: mRNA splicing and protein post-translational modifications (PTMs)1 . PTMs (more than 300 types) are complex and fundamental mechanisms of cellular regulation, and have been associated with almost all known cellular pathways and disease processes (1, 2). As an example, protein phosphorylation, the most well-studied PTM, is present in more than one third of human proteins, the phosphorylation status of which can potentially be regulated by ϳ500 human protein kinases and ϳ150 phosphatases (3, 4). The modification mainly occurs at several amino acid residues: serine, threonine, tyrosine, and histidine. Protein phosphorylation makes its substrate residues more acidic, hydrophilic, and induces a charge change from ϩ1 charge to -1 (at physiological pH), which in turn modulates the structure and functions of substrate proteins.The high complexity of PTMs is also reflected by diverse modifications at -amine group of lysine residue, including methylation, acetylation, and ubiquitination. These lysine PTMs have been shown to play an important role in cellular regulations (5, 6). Recently, we identified a new type of PTM at lysine residues, lysine succinylation (7). Like phosporylation, lysine succinylation also induces a change of two negative charges in lysine re...

Aging can be defined as progressive functional decline and increasing mortality over time. Here, we review evidence linking aging to nuclear DNA lesions: DNA damage accumulates with age, and DNA repair defects can cause phenotypes resembling premature aging. We discuss how cellular DNA damage responses may contribute to manifestations of aging. We review Sir2, a factor linking genomic stability, metabolism, and aging. We conclude with a general discussion of the role of mutant mice in aging research and avenues for future investigation.