Acetylation is a small, post-translational modification that chemically modifies cellular proteins. Acetylation takes place on the ε-amino group of lysine residues on a wide range of proteins and regulates multiple protein functions, including DNA-protein interactions, transcriptional activity, subcellular localization, protein stability, and enzymatic activity . The acetylation state of a given protein results from the balanced action of histone acetyltransferases (HATs) and histone deacetylases (HDACs), enzymes that catalyze the addition and removal, respectively, of an acetyl group from a lysine residue.
Although initially discovered on histones, several non-histone proteins are lysine acetylated . An extensive proteomic survey of cellular proteins revealed that a large number of mitochondrial proteins are subject to reversible lysine acetylation . In this study, mouse liver mitochondria were purified, digested and the resulting lysate was subjected to immuno-affinity purification of lysine-acetylated peptides. Proteomic analysis of the acetylated peptides identified 277 lysine acetylation sites in 133 mitochondrial proteins, and conclusively established lysine acetylation is an abundant post-translational modification in the mitochondrion. Most lysine-acetylated proteins identified in mitochondrial fractions were metabolic enzymes. Lysine acetylation was also identified on the mitochondrial DNA encoded ATP synthase Fo subunit 8 and implies that proteins can become acetylated directly inside mitochondria.
The sirtuins are a large family of NAD+-dependent protein deacetylases that regulate the acetylation status of several proteins. They are named after the yeast silent information regulator 2 (Sir2), and regulate important biological pathways in eubacteria, archaea, and eukaryotes. Bacteria and archaea encode one or two sirtuins, but mice and humans have seven sirtuins, named SIRT1–7. The seven mammalian sirtuins occupy different subcellular compartments, such as the nucleus (SIRT1, -2, -3, -6, -7), cytoplasm (SIRT1, -2), and mitochondria (SIRT3, -4, -5) [3-8]. The sirtuins are assigned to five subclasses (I–IV and U) based on the phylogenetic conservation of a ~250 amino acid core domain [9, 10]. Among mammalian sirtuins, SIRT1, -2, and -3 are class I sirtuins, have high homology to the yeast sirtuins Sir2, Hst1, and Hst2, and exhibit robust deacetylase activity. Class II sirtuins, including mammalian SIRT4, have no detectable deacetylase activity and instead show weak ADP-ribosyltransferase activity [6, 11]. Class III sirtuins, including mammalian SIRT5, have weak deacetylase activity on histone substrates [12, 13]. Class IV sirtuins have ADP ribosyltransferase and deacetylase activity (SIRT6) or unknown activity (SIRT7) [14, 15]. Class U sirtuins are intermediate between Class I and IV and has only been observed in bacteria.
The sirtuins mediate a deacetylation reaction that uses NAD+ as a cofactor, yielding O-acetyl-ADP-ribose, the deacetylated substrate, and nicotinamide (reviewed in [16, 17]). The dependence of the sirtuins on NAD+ suggests that their enzymatic activity is directly linked to the energy status of the cell either via the cellular NAD+:NADH ratio, the absolute levels of NAD+, NADH, or nicotinamide, or a combination of these variables [18-22]. Indeed, the sirtuins have important roles in controlling metabolism in a variety of organisms (reviewed in ).
Thus, the three sirtuins located in the mitochondria (SIRT3, SIRT4, and SIRT5) are poised to mediate mitochondrial protein acetylation levels. However, deacetylase activity has not been detected by SIRT4, whereas SIRT5 only displays low levels of deacetylase activity. Initial experiments in mice lacking SIRT3 displayed high levels of mitochondrial protein acetylation. In contrast, mice lacking either SIRT4 or SIRT5 showed no obvious change in mitochondrial protein acetylation . These observations demonstrated that SIRT3 is a soluble protein in the mitochondrial matrix [25, 26], and is the major mitochondrial protein deacetylase.
Interestingly, mitochondrial acetyltransferases (MATs) have not been identified, raising the question as to how mitochondrial proteins become acetylated. Because non-enzymatic acetylation of histones with acetyl-CoA occurs in vitro on the epsilon-amino group of lysine residues under physiological conditions , high acetyl-CoA levels in the mitochondria could facilitate a similar non-enzymatic acetylation mechanism. Alternatively, MATs could mediate the acetylation reaction. Based on the unique acetylation consensus site of mitochondrial proteins, MATs could form a class of acetyltransferases different from the known nuclear and cytosolic enzymes , and could be awaiting discovery.
Together, acetylation has emerged as a highly abundant post-translational modification present in mitochondria, and deacetylation by SIRT3 is crucial for maintaining metabolic homeostasis. Because several recent studies have validated initial observations of mitochondrial protein acetylation, and identified several new acetylated mitochondrial proteins, we integrate these findings and describe below the current state of the mitochondrial protein acetylation landscape. We further describe the functional significance of acetylation on some of these mitochondrial proteins.
Mitochondrial Acetylated Protein Landscape
Overall, global mitochondrial protein acetylation is sensitive to metabolic perturbations within the cell. For example, mitochondrial protein acetylation increases in the liver during fasting. In mice, 62% of acetylated mitochondrial proteins were identified in mitochondrial fractions isolated from both fed and fasted animals, whereas 14% of acetylated mitochondrial proteins were found in fed mice, and 24% of acetylated mitochondrial proteins were in fasted fasted mice . Mice fed a calorie-restricted diet also show increases in mitochondrial protein acetylation , similar to the acetylation patterns observed during fasting. Together, nutrient deprivation is coincident with global increases in mitochondrial protein acetylation.
Paradoxically, mitochondrial protein acetylation increases in mice during high-fat diet feeding [29, 30]. Short-term high-fat diet feeding showed no changes in hepatic mitochondrial protein acetylation, but long-term high-fat diet feeding induced marked mitochondrial protein hyperacetylation . Furthermore, mitochondrial protein hyperacetylation is also observed with dietary ethanol supplementation . Thus, an altered metabolic state, such as nutrient deprivation or nutrient excess, or ethanol detoxification all lead to increases in mitochondrial protein acetylation.
To determine which proteins become acetylated under different dietary and metabolic conditions, several proteomic studies have been performed [2, 28, 32-35]. In the first study of mitochondrial protein acetylation, an estimated 20% of all mitochondrial proteins were acetylated . More recently, two studies published together identified virtually every major metabolic enzyme is acetylated, both inside and outside the mitochondria [34, 35]. Together, these studies found the metabolic proteins in glycolysis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism and glycogen metabolism were all acetylated. To gain a better understanding of the prevalence of mitochondrial protein acetylation, we integrated the acetylation sites identified in all of the major proteomic studies [2, 28, 32, 35], coupled with unpublished proteomics data . We then compared these acetylated proteins with all proteins annotated with mitochondrial subcellular localizations via gene ontology (compendium in ). Taken together, we estimate approximately 35% of all mitochondrial proteins have at least one acetylation site.
Furthermore, the majority (53%) of acetylated proteins in the mitochondria have only one or two acetylation sites (33% and 20%, respectively; Figure 2B). Remarkably, 11% of all acetylated mitochondrial proteins contained more than 10 unique acetylation sites. The top ten mitochondrial proteins with the highest levels of acetylation all had twenty or greater unique acetylation sites [Idh2: 22 sites, Hadh: 23 sites; Acat1: 24 sites, Slc25a5: 25 sites, Got2: 25 sites, Acaa2: 25 sites, Atp5a1: 26 sites, Aco2: 29 sites, Hadha: 47 sites, and Cps1: 53 sites].
To better understand the mitochondrial processes which could be regulated by mitochondrial protein acetylation, we leveraged the database for annotation, visualization, and integrated discovery (DAVID 6.7) . First, we determined which mitochondrial pathways were enriched in protein acetylation. By assigning a single, primary gene ontology (GO) term to each mitochondrial protein, we measured which mitochondrial processes contained acetylated proteins. In agreement with previously described studies, pathways involved in the generation of energy, fatty acid metabolism, sugar metabolism, and amino acid metabolism contained several acetylated proteins. We found over 50% of the proteins in these pathways were acetylated. In contrast, mitochondrial DNA maintenance, transcription, RNA processing and translation had fewer than 25% acetylated proteins in these pathways. [Summary of all annotated mitochondrial functions and their levels of acetylation; each box represents one annotated function by gene ontology (GO term); size of box indicates number of proteins labeled with common annotation; color of box indicates percent of acetylated proteins in annotated pathway (light blue ≤25%, medium blue: 26-50%, dark blue 51-75%, very dark blue: ≥76%)].
To gain further insight into the mitochondrial pathways which could be regulated by protein acetylation, we entered all acetylated mitochondrial proteins into DAVID, and identified the functional annotations of these mitochondrial acetylated protein based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, protein analysis through evolutionary relationships (PANTHER) classifications , BioCarta pathway analysis, and the enzyme commission (EC) number for chemical reactions. From this comprehensive analysis, the metabolic pathway with the highest enrichment score was oxidative phosphorylation, with 49 acetylated proteins. Indeed, protein acetylation regulates ATP production both directly by deacetylation of one subunit of Complex I (NDUFA9) , and indirectly by reducing the activity of the fatty acid oxidation pathway upstream of oxidative phosphorylation . Surprisingly, several other metabolic pathways were enriched for mitochondrial protein acetylation, for which the role of acetylation has not yet been reported; these pathways include tryptophan metabolism, arginine and proline metabolism, lysine degradation, beta-alanine metabolism, limonene and pinene degradation, ascorbate and aldarate metabolism, and histidine metabolism. In addition to metabolic pathways, pathways known to influence development of Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease were also identified by functional annotation clustering as highly enriched for mitochondrial protein acetylation. These findings support the role of mitochondrial dysfunction as a contributor to neurodegenerative disease [15, 41-43], and suggest mitochondrial protein acetylation could play an important role in these disease states.
Regulation of Mitochondrial Proteins by Reversible Acetylation
Even though a large number of acetylated proteins in the mitochondria have been identified, the effect of acetylation on most of these proteins is unknown. The first report of a role for acetylation on a mitochondrial protein described the activation of acetylated acetyl-CoA synthetase (AceCS2) by mitochondrial SIRT3-catalyzed deacetylation of a single lysine reside (K642) which lies nearby the active site [44, 45]. AceCS2 activation occurs in extra-hepatic tissues during prolonged starvation to convert acetate to acetyl-CoA for energy production, and deacetylation by SIRT3 is coincident with the response to metabolic stress.
The first identified mitochondrial acetylation event with functional consequence was a single site of AceCS2 and that site was regulated by SIRT3. In a more recent study, SIRT3 was shown to stimulate fatty acid oxidation by deacetylating one acetylation site on LCAD out of eight total acetylation sites, thereby activating the fatty acid oxidation pathway . Tissues from fasted SIRT3 knock-out mice showed reduced fat oxidation and the fasted mutant animals exhibited fatty liver disease, reduced hepatic ATP production, hypoglycemia and cold intolerance, all predicted consequences of defective fatty acid oxidation. From the eight acetylated lysine residues, K42 was identified as the major physiological site of acetylation and target of SIRT3, and was critical for the catalytic activity of LCAD. The physiological role of acetylation on lysine residues not targeted by SIRT3 is unknown. Defects in fatty acid oxidation are associated with metabolic diseases, including diabetes, cardiovascular disease, and liver steatosis. Indeed, a follow-up study identified SIRT3 as a critical regulator of mitochondrial function, and suppression by high-fat diet feeding or reduction in enzymatic activity by a point-mutation both contribute to the metabolic syndrome . LCAD hyperacetylation was induced by high-fat diet feeding and was sufficient to reduce enzymatic activity in the livers of wild-type mice, in a similar manner as SIRT3 knock-out mice. These data suggest mitochondrial protein acetylation and/or SIRT3 activity is a potential therapeutic target for the treatment of metabolic disorders.
Another study showed SIRT3 deacetylates and stimulates the catalytic activity of HMGCS2, a mitochondrial liver enzyme that catalyzes the rate-limiting step in ketone body synthesis, a critical pathway upregulated during the starvation response . Ketone bodies are an essential energy source consumed by tissues, especially brain, in place of glucose when glucose in low, and are synthesized from acetyl-CoA that has been diverted from the TCA cycle. HMGCS2 catalyzes one step in this pathway, the conversion of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. Fasted SIRT3 null mice fail to deacetylate and activate HMGCS2 and exhibit diminished levels of hepatic and serum ketone bodies. The study presents in high-resolution one mechanism by which SIRT3 deacetylation of HMGCS2 modulates activity of the enzyme. Of the eleven HMGCS2 acetylated sites identified by proteomic analysis, only three are targeted by SIRT3 (K310, K447, and K473). Acetylation of the three sites was inversely correlated with HMGCS2 activity and their deacetylation increased enzymatic activity by increasing the Vmax but not the KM for the substrates. Molecular dynamic simulations comparing unacetylated and acetylated HMGCS2 revealed acetylation induced changes in protein conformation. When unacetylated, the epsilon-amino group of K310 forms electrostatic interactions with nearby aspartate residues of the enzyme and with acetyl-CoA. These interactions are abrogated by acetylation that eliminates the positive charge on the K310 side chain, producing conformational change near K310 and these changes are propagated to critical catalytic residues distant from the site of acetylation. Thus, acetylation of some, but not all, lysine residues can change the overall structure of the enzyme leading to changes in enzymatic activity.
In addition to AceCS2, LCAD, and HMGCS2, acetylation has been shown to control the enzymatic activity of several additional mitochondrial metabolic enzymes such as malate dehydrogenase , glutamate dehydrogenase , and isocitrate dehydrogenase  in the TCA cycle, enoyl–coA hydratase/3-hydroxyacyl–coA dehydrogenase  in the fatty acid oxidation pathway, carbamoyl phosphate synthetase 1  and ornithine transcarbamoylase  in the urea cycle, and manganese superoxide dismutase [49, 50] in the antioxidant system.
Sufficient evidence now exists to conclude that reversible acetylation is critical for mitochondrial function. However, the full regulatory program of acetylation is unknown. Hundreds of acetylated mitochondrial proteins identified by mass spectrometry-based proteomics approaches that are subject to experimental artifacts have not yet been validated. Even after rigorous validation of acetylation sites on mitochondrial proteins, the presence of acetylation does not necessarily correlate with effects on protein function. For example, acetylation of lysines on HMGCS2 not targeted by SIRT3 induces no protein conformational changes , which suggests that non-physiological acetylation could occur on mitochondrial proteins. However, electrostatic surface potential will continue to drop as positively-charged epsilon-amines become acetylated, which could disrupt protein-protein interactions or substrate/cofactor binding. Thus, in order to uncover the specificity of mitochondrial protein function regulated by acetylation, additional studies describing the effects of specific acetylation/deacetylation sites are needed. Furthermore, because protein identity among mitochondria from different tissues is highly variable (only ~50% conservation) , comparison of acetylated proteins from different tissues is needed to realize possible tissue-specific differences of acetylation.
Mitochondrial protein acetylation is an emerging fundamental mechanism regulating mitochondrial proteins and overall mitochondrial function. Interestingly, the acetyl group donor for lysine acetylation, acetyl-CoA, is the end product of glucose-derived pyruvate oxidation, amino acid catabolism, and fatty acid oxidation. Therefore, mitochondrial protein acetylation could be a convergence point for carbohydrate, amino acid, and fat metabolism. Because acetyl-CoA is an indicator of the cellular energy status, acetylation could have evolved to couple metabolic enzyme activity to fluctuating levels of this key metabolite.
While more work needs to be done to fully understand this complex regulatory mechanism, mitochondrial protein acetylation is an important part of the adaptive metabolic response, where dramatic changes in energy metabolism must coordinate to ensure survival. Mitochondria are crucial for several cellular processes, including the production of more than 90% of cellular ATP, apoptosis, cell-cycle progression, proliferation, and aging; and their dysfunction has been implicated in a wide range of human metabolic and neurodegenerative diseases [52-55]. Mitochondrial protein acetylation must now be considered a highly abundant and important component of the mitochondrial metabolic regulatory network.
1. Glozak, M.A., Sengupta, N., Zhang, X., and Seto, E. (2005) Acetylation and deacetylation of non-histone proteins. Gene 363, 15-23
2. Kim, S.C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., Grishin, N.V., White, M., Yang, X.J., and Zhao, Y. (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23, 607-18
3. Blander, G. and Guarente, L. (2004) The Sir2 family of protein deacetylases. Annu Rev Biochem 73, 417-35
4. North, B.J. and Verdin, E. (2004) Sirtuins: Sir2-related NAD-dependent protein deacetylases. Genome Biol 5, 224
5. Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C., and Horikawa, I. (2005) Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol Biol Cell 16, 4623-35
6. Haigis, M.C., Mostoslavsky, R., Haigis, K.M., Fahie, K., Christodoulou, D.C., Murphy, A.J., Valenzuela, D.M., Yancopoulos, G.D., Karow, M., 6. Blander, G., Wolberger, C., Prolla, T.A., Weindruch, R., Alt, F.W., and Guarente, L. (2006) SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells. Cell 126, 941-54
7. Chen, I.Y., Lypowy, J., Pain, J., Sayed, D., Grinberg, S., Alcendor, R.R., Sadoshima, J., and Abdellatif, M. (2006) Histone H2A.z is essential for cardiac myocyte hypertrophy but opposed by silent information regulator 2alpha. J Biol Chem 281, 19369-77
8. Tanno, M., Sakamoto, J., Miura, T., Shimamoto, K., and Horio, Y. (2006) Nucleo-cytoplasmic shuttling of NAD+-dependent histone deacetylase SIRT1. J Biol Chem
9. Frye, R.A. (1999) Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity. Biochem Biophys Res Commun 260, 273-9
10. Frye, R.A. (2000) Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun 273, 793-8.
11. Ahuja, N., Schwer, B., Carobbio, S., Waltregny, D., North, B.J., Castronovo, V., Maechler, P., and Verdin, E. (2007) Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase. J Biol Chem 282, 33583-33592
12. Verdin, E., Dequiedt, F., Fischle, W., Frye, R., Marshall, B., and North, B. (2004) Measurement of mammalian histone deacetylase activity. Methods Enzymol 377, 180-96
13. Nakagawa, T., Lomb, D.J., Haigis, M.C., and Guarente, L. (2009) SIRT5 Deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle. Cell 137, 560-70
14. Kawahara, T.L.A., Michishita, E., Adler, A.S., Damian, M., Berber, E., Lin, M., McCord, R.A., Ongaigui, K.C.L., Boxer, L.D., Chang, H.Y., and Chua, K.F. (2009) SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span. Cell 136, 62-74
15. Haigis, M.C. and Sinclair, D.A. (2010) Mammalian sirtuins: biological insights and disease relevance. Annual Review of Pathology: Mechanisms of Disease 5, 253-295
16. Sauve, A.A., Wolberger, C., Schramm, V.L., and Boeke, J.D. (2006) The Biochemistry of Sirtuins. Annu Rev Biochem
17. Denu, J.M. (2005) The Sir 2 family of protein deacetylases. Curr Opin Chem Biol 9, 431-40
18. Lin, S.J., Defossez, P.A., and Guarente, L. (2000) Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126-8.
19. Lin, S.J., Kaeberlein, M., Andalis, A.A., Sturtz, L.A., Defossez, P.A., Culotta, V.C., Fink, G.R., and Guarente, L. (2002) Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418, 344-8
20. Lin, J., Wu, P.H., Tarr, P.T., Lindenberg, K.S., St-Pierre, J., Zhang, C.Y., Mootha, V.K., Jager, S., Vianna, C.R., Reznick, R.M., Cui, L., Manieri, M., Donovan, M.X., Wu, Z., Cooper, M.P., Fan, M.C., Rohas, L.M., Zavacki, A.M., Cinti, S., Shulman, G.I., Lowell, B.B., Krainc, D., and Spiegelman, B.M. (2004) Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice. Cell 119, 121-35
21. Bitterman, K.J., Anderson, R.M., Cohen, H.Y., Latorre-Esteves, M., and Sinclair, D.A. (2002) Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J Biol Chem 277, 45099-107
22. Anderson, R.M., Bitterman, K.J., Wood, J.G., Medvedik, O., and Sinclair, D.A. (2003) Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 423, 181-5
23. Finkel, T., Deng, C.X., and Mostoslavsky, R. (2009) Recent progress in the biology and physiology of sirtuins. Nature 460, 587-91
24. Lombard, D.B., Alt, F.W., Cheng, H.L., Bunkenborg, J., Streeper, R.S., Mostoslavsky, R., Kim, J., Yancopoulos, G., Valenzuela, D., Murphy, A., Yang, Y., Chen, Y., Hirschey, M.D., Bronson, R.T., Haigis, M., Guarente, L.P., Farese, R.V., Weissman, S., Verdin, E., and Schwer, B. (2007) Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation. Mol Cell Biol 27, 8807-14
25. Schwer, B., North, B.J., Frye, R.A., Ott, M., and Verdin, E. (2002) The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase. J Cell Biol 158, 647-57
26. Schwer, B., Bunkenborg, J., Verdin, R.O., Andersen, J.S., and Verdin, E. (2006) Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2. Proc Natl Acad Sci U S A
27. Paik, W.K., Pearson, D., Lee, H.W., and Kim, S. (1970) Nonenzymatic acetylation of histones with acetyl-CoA. Biochim Biophys Acta 213, 513-22
28. Schwer, B., Eckersdorff, M., Li, Y., Silva, J., Fermin, D., Kurtev, M., Giallourakis, C., Comb, M., Alt, F., and Lombard, D. (2009) Calorie Restriction Alters Mitochondrial Protein Acetylation. Aging Cell
29. Hirschey, M., Aouizerat, B., Jing, E., Shimazu, T., Grueter, C., Collins, A., Stevens, R., Lam, M., Muehlbauer, M., Schwer, B., Gao, B., Bass, N., Alt, F., Deng, C.-X., Kakar, S., Newgard, C., Farese Jr., R., Kahn, C., and Verdin, E. (2011) SIRT3 Deficiency and Mitochondrial Protein Hyperacetylation Accelerate the Development of the Metabolic Syndrome. Molecular Cell In Press,
30. Kendrick, A.A., Choudhury, M., Rahman, S.M., McCurdy, C.E., Friederich, M., Van Hove, J.L.K., Watson, P.A., Birdsey, N., Bao, J., Gius, D., Sack, M.N., Jing, E., Kahn, C.R., Friedman, J.E., and Jonscher, K.R. (2011) Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation. The Biochemical journal 433, 505-514
31. Picklo, M.J. (2008) Ethanol intoxication increases hepatic N-lysyl protein acetylation. Biochem Biophys Res Commun 376, 615-9
32. Choudhary, C., Kumar, C., Gnad, F., Nielsen, M., Rehman, M., Walther, T., Olsen, J., and Mann, M. (2009) Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions. Science 325, 834-840
33. Lombard, D. Personal Communication.
34. Wang, Q., Zhang, Y., Yang, C., Xiong, H., Lin, Y., Yao, J., Li, H., Xie, L., Zhao, W., Yao, Y., Ning, Z.-B., Zeng, R., Xiong, Y., Guan, K.-L., Zhao, S., and Zhao, G.-P. (2010) Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science 327, 1004-7
35. Zhao, S., Xu, W., Jiang, W., Yu, W., Lin, Y., Zhang, T., Yao, J., Zhou, L., Zeng, Y., Li, H., Li, Y., Shi, J., An, W., Hancock, S.M., He, F., Qin, L., Chin, J., Yang, P., Chen, X., Lei, Q., Xiong, Y., and Guan, K.-L. (2010) Regulation of cellular metabolism by protein lysine acetylation. Science 327, 1000-4
36. Pagliarini, D.J., Calvo, S.E., Chang, B., Sheth, S.A., Vafai, S.B., Ong, S.-E., Walford, G.A., Sugiana, C., Boneh, A., Chen, W.K., Hill, D.E., Vidal, M., Evans, J.G., Thorburn, D.R., Carr, S.A., and Mootha, V.K. (2008) A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112-23
37. Huang, D.W., Sherman, B.T., and Lempicki, R.A. (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 4, 44-57
38. Thomas, P.D., Kejariwal, A., Campbell, M.J., Mi, H., Diemer, K., Guo, N., Ladunga, I., Ulitsky-Lazareva, B., Muruganujan, A., Rabkin, S., Vandergriff, J.A., and Doremieux, O. (2003) PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification. Nucleic Acids Research 31, 334-341
39. Ahn, B.H., Kim, H.S., Song, S., Lee, I.H., Liu, J., Vassilopoulos, A., Deng, C.X., and Finkel, T. (2008) A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci USA 105, 14447–14452
40. Hirschey, M., Shimazu, T., Goetzman, E., Jing, E., Schwer, B., Lombard, D., Grueter, C., Harris, C., Biddinger, S., Ilkayeva, O., Stevens, R., Li, Y., Saha, A., Ruderman, N., Bain, J., Newgard, C., Farese Jr., R., Alt, F., Kahn, C., and Verdin, E. (2010) SIRT3 regulates mitochondrial fatty acid oxidation via reversible enzyme deacetylation. Nature 464, 121-125
41. Beal, M.F. (2005) Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 58, 495-505
42. Bubber, P., Haroutunian, V., Fisch, G., Blass, J.P., and Gibson, G.E. (2005) Mitochondrial abnormalities in Alzheimer brain: mechanistic implications. Annals of neurology 57, 695-703
43. Calvo, S.E. and Mootha, V.K. (2010) The mitochondrial proteome and human disease. Annual review of genomics and human genetics 11, 25-44
44. Hallows, W.C., Lee, S., and Denu, J.M. (2006) Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases. Proc Natl Acad Sci U S A 103, 10230-5
45. Schwer, B., Bunkenborg, J., Verdin, R.O., Andersen, J.S., and Verdin, E. (2006) Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2. Proc Natl Acad Sci U S A 103, 10224-9
46. Shimazu, T., Hirschey, M., Hua, L., Dittenhafer-Reed, K.E., Schwer, B., Lombard, D., Li, Y., Bunkenborg, J., Alt, F.W., Denu, J.M., Jacobson, M.P., and Verdin, E. (2010) SIRT3 Deacetylates Mitochondrial 3-Hydroxy-3-Methylglutaryl CoA Synthase 2, Increases its Enzymatic Activity and RegulatesKetone Body Production Cell Metab 12, 654-661
47. Schlicker, C., Gertz, M., Papatheodorou, P., Kachholz, B., Becker, C.F.W., and Steegborn, C. (2008) Substrates and regulation mechanisms for the human mitochondrial sirtuins Sirt3 and Sirt5. Journal of Molecular Biology 382, 790-801
48. Hallows, W.C., Yu, W., Smith, B.C., Devires, M.K., Ellinger, J.J., Someya, S., Shortreed, M.R., Prolla, T., Markley, J.L., Smith, L.M., Zhao, S., Guan, K.-L., and Denu, J.M. (2011) Sirt3 Promotes the Urea Cycle and Fatty Acid Oxidation during Dietary Restriction. Molecular cell 41, 139-149
49. Qiu, X., Brown, K., Hirschey, M.D., Verdin, E., and Chen, D. (2010) Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation. Cell Metabolism 12, 662-667
50. Tao, R., Coleman, M.C., Pennington, J.D., Ozden, O., Park, S.-H., Jiang, H., Kim, H.-S., Flynn, C.R., Hill, S., Hayes McDonald, W., Olivier, A.K., Spitz, D.R., and Gius, D. (2010) Sirt3-Mediated Deacetylation of Evolutionarily Conserved Lysine 122 Regulates MnSOD Activity in Response to Stress. Molecular cell 40, 893-904
51. Mootha, V.K., Bunkenborg, J., Olsen, J.V., Hjerrild, M., Wisniewski, J.R., Stahl, E., Bolouri, M.S., Ray, H.N., Sihag, S., Kamal, M., Patterson, N., Lander, E.S., and Mann, M. (2003) Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 115, 629-40
52. McBride, H.M., Neuspiel, M., and Wasiak, S. (2006) Mitochondria: more than just a powerhouse. Curr Biol 16, R551-60
53. Chan, D.C. (2006) Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241-52
54. Hajnoczky, G., Csordas, G., Das, S., Garcia-Perez, C., Saotome, M., Sinha Roy, S., and Yi, M. (2006) Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40, 553-60
55. Hausenloy, D.J. and Ruiz-Meana, M. Not just the powerhouse of the cell: emerging roles for mitochondria in the heart. Cardiovasc Res 88, 5-6