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Mitochondrial Protein Acylation and Intermediary Metabolism: Regulation by Sirtuins and Implications for Metabolic Disease *

Mitochondrial Protein Acylation and Intermediary Metabolism: Regulation by Sirtuins and... MINIREVIEW THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 51, pp. 42436 –42443, December 14, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Three Mitochondrial Sirtuins Mitochondrial Protein Acylation Mammals contain seven sirtuins (SIRT1–7) that are charac- and Intermediary Metabolism: terized by an evolutionarily conserved sirtuin core domain homologous to Sir2, a yeast protein that increases life span (4, Regulation by Sirtuins and 5). SIRT1–7 are localized in distinct subcellular compartments. Implications for Metabolic SIRT1, SIRT6, and SIRT7 are found in the nucleus; SIRT2 is primarily cytosolic; and SIRT3–5 are found in mitochondria. Disease Sirtuins have different levels of NAD -dependent protein Published, JBC Papers in Press, October 18, 2012, DOI 10.1074/jbc.R112.404863 ‡§ ‡ ‡¶1 John C. Newman , Wenjuan He , and Eric Verdin deacetylase activity. This reaction couples lysine deacetylation to NAD hydrolysis to yield O-acetyl-ADP-ribose, the deacety- From the Gladstone Institute of Virology and Immunology, San Francisco, § ¶ California 94158 and the Division of Geriatrics and Department of lated substrate, and nicotinamide (reviewed in Refs. 6 and 7). Medicine, University of California, San Francisco, California 94143 SIRT1–3 exhibit robust protein deacetylase activity, whereas the others have only weak and highly selective (SIRT5–7) or The sirtuins are a family of NAD -dependent protein undetectable (SIRT4) protein deacetylase activity. So far, only deacetylases that regulate cell survival, metabolism, and longev- weak ADP-ribosyltransferase activity has been described for ity. Three sirtuins, SIRT3–5, localize to mitochondria. Expres- SIRT4 (8, 9). The dependence of sirtuins on NAD suggests sion of SIRT3 is selectively activated during fasting and calorie that their enzymatic activity is directly linked to the energy restriction. SIRT3 regulates the acetylation level and enzymatic status of the cell via the cellular NAD :NADH ratio; the abso- activity of key metabolic enzymes, such as acetyl-CoA synthe- lute levels of NAD , NADH, or nicotinamide; or a combination tase, long-chain acyl-CoA dehydrogenase, and 3-hydroxy-3- of these variables (10–14). methylglutaryl-CoA synthase 2, and enhances fat metabolism during fasting. SIRT5 exhibits demalonylase/desuccinylase Mitochondrial Protein Acetylation activity, and lysine succinylation and malonylation are abun- Reversible protein acetylation occurs primarily at the -a- dant mitochondrial protein modifications. No convincing enzy- mino group of lysine residues (for a recent review of mechanis- matic activity has been reported for SIRT4. Here, we review the tically distinct N-terminal acetylation, see Ref. 15). Like other emerging role of mitochondrial sirtuins as metabolic sensors post-translational modifications, lysine acetylation regulates that respond to changes in the energy status of the cell and mod- diverse protein properties, including DNA-protein interac- ulate the activities of key metabolic enzymes via protein tions, subcellular localization, protein stability, protein-protein deacylation. interaction, and enzymatic activity (16). Mitochondrial proteins are subject to extensive lysine acety- lation (17, 18). Acetylated mitochondrial proteins include those Proper mitochondrial function is required for metabolic involved in energy metabolism, such as in the TCA cycle, oxi- homeostasis and involves careful regulation of the activity of dative phosphorylation, -oxidation of lipids, amino acid multiple metabolic enzymes. Changes in mitochondrial num- metabolism, carbohydrate metabolism, nucleotide metabo- ber and activity are implicated in aging, cancer, and the patho- lism, and the urea cycle (2, 3). Interestingly, 44% of mitochon- genesis of the metabolic syndrome, a group of metabolic abnor- drial dehydrogenases are acetylated. Among them, 14 use malities characterized by central obesity, dyslipidemia, high NAD as the electron acceptor to catalyze biochemical reac- blood pressure, and increased fasting glucose levels (1). tions in oxidative catabolic routes. The importance of acetyla- Protein acetylation is increasingly recognized as an impor- tion is further supported by the high degree of conservation of tant post-translational modification for a number of key meta- many sites from Drosophila to humans (19). bolic pathways (2, 3). Lysine malonylation and succinylation were recently identified in several mitochondrial proteins, and SIRT3 Is the Major Mitochondrial Protein Deacetylase the mitochondrial sirtuin SIRT5 was found to have demalony- Endogenous SIRT3 is a soluble protein in the mitochondrial lase/desuccinylase activity. Here, we review the emerging role matrix (20, 21). Interestingly, SIRT3 is translated in the cyto- of protein acylation and its regulation by sirtuins in mitochon- plasm as a longer, enzymatically inactive precursor and drial biology and metabolic regulation. imported into the mitochondrion. After import, the first 100 amino acids of SIRT3 are proteolytically cleaved, leading to a final enzymatically active SIRT3 of 28 kDa. A small fraction of * This work was supported, in whole or in part, by National Institutes of Health Grants P30 DK026743 (to the UCSF Liver Center) and R24 DK085610 from SIRT3 resides in the nucleus as well (22). The initial controversy NIDDK. This work was also supported by a Senior Scholarship in Aging from regarding the mitochondrial localization of mouse SIRT3 was the Ellison Medical Foundation and institutional support from the J. David resolved by cloning of additional mouse SIRT3 cDNAs that Gladstone Institutes. Eric Verdin is a member of the Scientific Advisory Board of Sirtris/GSK, a company involved in the commercialization of sir- encode a protein that is imported to the mitochondrial matrix, tuin-related discoveries. This is the third article in the Thematic Minireview like human SIRT3 (23, 24). Series on Sirtuins: From Biochemistry to Health and Disease. SIRT3 appears to be the major mitochondrial deacetylase To whom correspondence should be addressed. E-mail: everdin@ gladstone.ucsf.edu. because mice lacking SIRT3, but not mice lacking SIRT4 or This is an Open Access article under the CC BY license. 42436 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 51 •DECEMBER 14, 2012 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation SIRT5, show a striking hyperacetylation of mitochondrial pro- tissue. SIRT3 accelerates amino acid catabolism and nitrogen teins (25). SIRT3 expression is highest in the most metabolically waste disposal by deacetylating and activating GLUD1 (gluta- active tissues, including liver, kidney, and heart (26, 27), and is mate dehydrogenase 1), a major cataplerotic enzyme (38). increased in glucose-poor fasting states, including calorie Catabolism of most amino acids requires transfer of the -a- restriction in liver and kidney (28–32). Expression in skeletal mino moiety to -ketoglutarate by an aminotransferase, form- muscle also increases under calorie restriction (31, 33) but has ing glutamate. GLUD1 regenerates -ketoglutarate from gluta- been reported to both increase and decrease with fasting (26, mate and releases nitrogen to the urea cycle as ammonia (39). 29, 33). Interestingly, SIRT3 expression initially increases on a SIRT3 accelerates the urea cycle by deacetylating and activating high-fat diet (HFD) in liver and skeletal muscle, but chronic ornithine transcarbamylase, the key mitochondrial enzyme in high-fat feeding leads to decreased SIRT3 expression (26, 31, the urea cycle. Mice lacking SIRT3 exhibit a metabolic profile 33–35). SIRT3 expression also decreases in mouse models of similar to that in human disorders of the urea cycle, including type 2 diabetes mellitus (26, 33). increased serum ornithine and reduced citrulline levels (the substrate and product, respectively, of ornithine transcarbam- SIRT3 Regulates Intermediary Metabolism oylase) (30). SIRT3 targets many enzymes that together help mediate the Carbohydrate Metabolism—By promoting fat oxidation, switch to fasting metabolism, as tissues move away from glu- SIRT3 indirectly suppresses carbohydrate utilization. In con- cose as a source of energy and metabolic intermediates to trast, cancer cells favor glucose as a source of energy, a process instead utilize lipids and amino acids. referred to as the Warburg effect (40). SIRT3 down-regulation Lipid Metabolism—SIRT3 promotes the efficient utilization is frequently observed in tumors and enhances glucose utiliza- of lipids as a primary source of acetyl-CoA during fasting by tion by allowing an increase in reactive oxygen species (ROS) deacetylating and activating long-chain acyl-CoA dehydrogen- that stimulate hypoxia-inducible factor 1, a transcription fac- ase, a key enzyme in the -oxidation of fatty acids (28). Mice tor that drives the expression of glycolytic genes (41–43). lacking SIRT3 accumulate -oxidation precursors and inter- SIRT3 also regulates the acetylation of the peptidyl-prolyl mediates, including triglycerides and long-chain fatty acids. isomerase cyclophilin D. In the absence of SIRT3, this leads to These mice also share other characteristics of human disorders activation of hexokinase II on the outer mitochondrial mem- of fatty acid oxidation, including cold intolerance and reduced brane, facilitating the rapid production of glucose 6-phosphate basal ATP levels (28). SIRT3 also regulates ketone body produc- (41, 44). tion by deacetylating and activating 3-hydroxy-3-methylglu- Reactive Oxygen Species—SIRT3 also regulates the produc- taryl-CoA synthase 2, the rate-limiting enzyme in ketone body tion of ROS generated as a by-product of oxidative phosphory- biosynthesis. Accordingly, mice lacking SIRT3 show reduced lation. First, SIRT3 deacetylates and activates isocitrate dehy- fasting serum levels of ketone bodies (36). SIRT3 also deacety- drogenase 2, an enzyme in the TCA cycle that helps to replenish lates and activates acetyl-CoA synthetase 2, an enzyme in extra- the mitochondrial pool of NADPH (45). NADPH is used by hepatic tissues that activates acetate into acetyl-CoA (21, 37). glutathione reductase to maintain glutathione in its reduced Acetate itself is produced in the liver from acetyl-CoA and can antioxidant form. Second, SIRT3 deacetylates and activates the be distributed to extrahepatic tissues as a form of energy (36). ROS-scavenging enzyme manganese superoxide dismutase, SIRT3 therefore facilitates the catabolism of fatty acids in the thereby reducing oxidative damage in the liver (46–48). Mice liver and the peripheral use of lipid-derived acetate and ketone lacking SIRT3 therefore show increased oxidative stress (46), bodies during fasting. particularly on a HFD (34), and lose the reduction of ROS levels Nitrogen Metabolism—Oxidation of acetyl-CoA to CO by normally observed under calorie restriction (45). the TCA cycle is a central pathway in energy metabolism. How- Oxidative Phosphorylation—Mice lacking SIRT3 consume ever, the TCA cycle also functions in biosynthetic pathways in 10% less O and produce up to 50% less ATP than wild-type which intermediates leave the cycle to be converted primarily mice, suggesting that SIRT3 regulates the activity of the respi- to glucose, fatty acids, or nonessential amino acids. Equilibrium ratory chain (27, 33). SIRT3 deacetylates and activates mito- of the substrates of the TCA cycle is maintained by two pro- chondrial respiratory chain complexes, including NDUFA9 cesses called anaplerosis and cataplerosis. Anaplerosis refers to (complex I) (27) and SDHA (complex II) (43, 49). Accordingly, the replenishment of critical anions. Pyruvate carboxylase, mice lacking SIRT3 have lower complex I and II activities than which generates oxalacetate directly in the mitochondria, is the wild-type mice (43, 49). SIRT3 also regulates ATP synthase major anaplerotic enzyme. Conversely, 4- and 5-carbon inter- (35). mediates that enter the TCA cycle during the catabolism of Accelerated Metabolic Syndrome in the Absence amino acids cannot be fully oxidized and therefore must be of SIRT3 removed by cataplerosis. Cataplerosis may in turn be linked to biosynthetic processes, such as hepatic gluconeogenesis, fatty The metabolic syndrome is defined by central obesity, insulin acid synthesis in the liver, and glyceroneogenesis in adipose resistance, hyperlipidemia, hyperglycemia, and hypertension (50). Physical inactivity, diet, and several genes and their prod- ucts (including leptin,  -adrenergic receptor, hormone-sensi- The abbreviations used are: HFD, high-fat diet; ROS, reactive oxygen species; tive lipase, lipoprotein lipase, insulin receptor substrate 1, PC-1, PGC-1, peroxisome proliferator-activated receptor- coactivator 1; and skeletal muscle glycogen synthase) are implicated in the CrAT, carnitine acetyltransferase; ACC, acetyl-CoA carboxylase; MCD, mal- onyl-CoA decarboxylase. pathogenesis of the metabolic syndrome (51–54). Other meta- DECEMBER 14, 2012• VOLUME 287 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42437 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation FIGURE 1. Mitochondrial acetyl-CoA, malonyl-CoA, and succinyl-CoA metabolism. Metabolic pathways resulting from the oxidation of glucose, fatty acids, and amino acids and leading to the synthesis of acetyl-CoA, malonyl-CoA, and succinyl-CoA are shown. Also shown are the two mechanisms leading to export of acetyl-CoA from mitochondria: ATP citrate lyase (CrAT) and carnitine/acylcarnitine translocase (CACT). BCAA, branched-chain amino acid; PK, pyruvate kinase; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PCC, propionyl-CoA carboxylase; LCAD, long-chain acyl-CoA dehydrogenase; BCAT, branched-chain aminotransferase; BCKD, branched-chain -keto acid dehydrogenase; MCM, methylmalonyl-CoA mutase; CPT, carnitine palmitoyltransferase. bolic abnormalities, such as aberrant lipogenesis (55, 56), ulation characterized by fatty liver disease (The NASH Clinical increased inflammation (57, 58), reduced fatty acid oxidation Research Network), patients meeting the criteria for metabolic (59, 60), and increased oxidative stress, have also been impli- syndrome were more likely to carry the SIRT3 rs11246020 “A” cated. Sustained weight loss and exercise are protective, as minor allele. In a follow-up study of8000 Finnish men focus- might be increased activation of fatty acid oxidation (61). ing specifically on rs11246020, the frequency of this allele and a Lack of SIRT3 and the resulting mitochondrial protein metabolic syndrome diagnosis were significantly correlated hyperacetylation are associated with accelerated development (34). However, this association was relatively weak (odds ratio of the metabolic syndrome (34). Wild-type mice fed a HFD of 1.3) and was not observed with all definitions of the meta- develop obesity, hyperlipidemia, type 2 diabetes mellitus, insu- bolic syndrome. Remarkably, the SIRT3 rs11246020 polymor- lin resistance, and non-alcoholic steatohepatitis (62–65). We phism induces a mutation within the catalytic domain of SIRT3 reported that the development of each of these consequences of (V208I). Mutation of Val-208 to isoleucine reduces SIRT3 HFD feeding is significantly accelerated in mice lacking SIRT3 enzyme efficiency by increasing the K for NAD and reducing (34). In addition, mice lacking SIRT3 show dramatically the V , consistent with the model that reduction of SIRT3 max enhanced levels of proinflammatory cytokines, including IL-6 enzymatic activity increases susceptibility to the metabolic and TNF-, another frequent manifestation of the metabolic syndrome. syndrome. Finally, we found that 90% of SIRT3 knock-out SIRT3, Acetylation, and Metabolic Inflexibility mice develop hepatocellular carcinoma, a cancer associated with the metabolic syndrome in humans (66), when placed on a We hypothesize that high mitochondrial acetyl-CoA levels HFD. and mitochondrial protein hyperacetylation cause metabolic Interestingly, prolonged exposure (13 weeks) to HFD feed- inflexibility. Acetyl-CoA, malonyl-CoA, and succinyl-CoA are ing in wild-type mice results in a reduction of hepatic SIRT3 important intracellular metabolites. They are present in mito- expression (34, 35), whereas acute HFD feeding leads to a tem- chondria and the cytosol and are variously derived from the porary increase in SIRT3 protein expression (34). A HFD sup- catabolism of carbohydrates, fatty acids, or proteins (Fig. 1). presses SIRT3 expression via suppression of peroxisome prolif- Intramitochondrial concentrations of acetyl-CoA and succi- erator-activated receptor- coactivator 1 (PGC-1) (67, 68), a nyl-CoA are in the millimolar range (70), a level that can initiate major regulator of SIRT3 expression (69). Reintroducing exog- non-enzymatic acetylation reactions (71). Importantly, global enous PGC-1 rescues the loss of SIRT3 in HFD-fed mice (34). protein acetylation in mitochondria correlates with elevated Preliminary evidence also supports a role of SIRT3 in the production of acetyl-CoA in such varied states as fasting, calo- pathogenesis of the metabolic syndrome in humans. In a pop- rie restriction, HFD, and ethanol intoxication (28, 34, 72–74). Acetyl-CoA is produced during the aerobic catabolism of carbohydrates from pyruvate, during -oxidation of long-chain M. D. Hirschey and E. Verdin, unpublished data. J. Y. Huang and E. Verdin, unpublished data. fatty acids, and from the catabolism of some amino acids or 42438 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 51 •DECEMBER 14, 2012 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation decarboxylation of malonyl-CoA (Fig. 1) (39). There is growing in the nucleus. Identifying the true enzymatic activity of SIRT4 evidence that levels of acetyl-CoA regulate fuel utilization and will undoubtedly shed light on its function. that dysregulated acetyl-CoA levels have a role in the pathogen- SIRT5, a Protein Demalonylase and Desuccinylase esis of insulin resistance and the metabolic syndrome. During fasting, acetyl-CoA can either feed into the TCA cycle for SIRT5 possesses unique potent demalonylase and desucciny- energy production or be used for ketogenesis or acetate produc- lase activities (79, 81). Malonyllysine and succinyllysine modi- tion (primarily in the liver) (39). During feeding, acetyl-CoA is fications occur in a variety of organisms from yeast to human exported from the mitochondria to the cytoplasm via citrate by (81, 82). Malonylation and succinylation are detected in meta- the activity of ATP citrate lyase. Excess acetyl-CoA can also be bolic enzymes, including isocitrate dehydrogenase 2, serine exported from the mitochondria via the activity of the enzyme hydroxymethyltransferase, glyceraldehyde-3-phosphate dehy- carnitine acetyltransferase (CrAT) (75). This enzyme is present drogenase, GLUD1, malate dehydrogenase 2, citrate synthase, in the mitochondrial matrix and combines acetyl-CoA and carbamoyl phosphate synthetase 1, 3-hydroxy-3-methylglu- carnitine into acetylcarnitine (76), which can be directly taryl-CoA synthase 2, thiosulfate sulfurtransferase, and aspar- exchanged across the mitochondrial membrane by carnitine/ tate aminotransferase (79, 81, 82). Mice lacking SIRT5 show acylcarnitine translocase (Fig. 1). global protein hypermalonylation and hypersuccinylation, sug- Increased mitochondrial levels of acetyl-CoA induced by gesting that it is the major protein demalonylase and desucci- fatty acid oxidation allosterically inhibit the activity of pyruvate nylase (81). The biological significance of lysine malonylation dehydrogenase, a mitochondrial enzyme complex that converts and succinylation and how lysine malonylation and succinyla- pyruvate into acetyl-CoA and thereby couples glycolysis and tion regulate enzymatic activity are currently unknown. In glucose oxidation. This acetyl-CoA-mediated inhibition repre- addition to these novel enzymatic activities, SIRT5 may also sents part of the glucose-fatty acid cycle originally proposed by function as a protein deacetylase on a restricted number of sub- Randle to explain the lipid-induced suppression of muscle glu- strates, such as the urea cycle enzyme carbamoyl phosphate cose disposal, a hallmark of obesity-associated insulin resist- synthetase 1 (83). ance (77). A pivotal role of intramitochondrial acetyl-CoA con- Succinyl-CoA and Malonyl-CoA Are Critical Metabolic centrations in metabolic control is further supported by recent Intermediates studies of CrAT (75). Mice with a muscle-specific deletion of CrAT exhibit compromised glucose tolerance and decreased As we discussed above, hyperacetylation of mitochondrial metabolic flexibility. This latter phenomenon was recently proteins associated with loss of SIRT3 disrupts the normal met- identified in obese humans as an inability to switch from fatty abolic switch toward fatty acid utilization that occurs during acid to glucose oxidation during the transition from fasting to prolonged fasting (28, 34). Because many metabolic enzymes feeding and may be a key manifestation of the metabolic syn- are also malonylated or succinylated (81, 82), SIRT5-mediated drome (78). Muoio et al. (75) proposed that CrAT promotes demalonylation or desuccinylation of metabolic enzymes may metabolic flexibility and increases insulin action by enhancing modulate metabolic pathways in a similar fashion under condi- mitochondrial export of excess acetyl residues. On the basis of tions of high malonyl-CoA or succinyl-CoA levels. SIRT5 is our observations of conditions associated with high acetyl-CoA therefore likely to emerge in the future as an important regula- levels, we predict that CrAT deletion leads to mitochondrial tor of intermediary metabolism. protein hyperacetylation and that dysregulated mitochondrial Succinyl-CoA is an intermediate in the TCA cycle and also a protein acetylation might represent the molecular mechanism precursor for porphyrin synthesis (39). Catabolism of odd- of metabolic inflexibility. In this context, the ability of SIRT3 to chain fatty acids and of some amino acids (e.g. branched-chain remove excess mitochondrial protein acetylation could there- amino acids, such as leucine, isoleucine, and valine) generates fore lead to increased metabolic flexibility and increased insulin propionyl-CoA, which is first carboxylated to methylmalonyl- sensitivity. CoA and then converted to succinyl-CoA (Fig. 1) (39). Branched-chain amino acids are the most abundant essential SIRT4, an Enzyme without a Substrate amino acids (84). Muscle represents 40% of the total mass of Unlike the well defined role of SIRT3 in acetylation, the pre- mammals and is therefore the largest metabolic organ. Muscle cise enzymatic function of SIRT4 is unclear. It may possess acts as a critical fuel reserve site in starvation or other glucose- weak ADP-ribosyltransferase activity (8, 9); however, this activ- poor states (85, 86) and accounts for50% of the capacity of the ity is 1000-fold slower than that of a bacterial ADP-ribosyl- tissues to catabolize branched-chain amino acids (87). We transferase, raising doubt about its physiological significance therefore expect that succinyl-CoA production will rise during (79). SIRT4 regulates insulin secretion (8, 9). Intriguingly and fasting. We do not know yet whether succinyl-CoA levels and unlike SIRT3, SIRT4 expression is reduced during calorie global protein succinylation correlate, as is observed between restriction and is increased in mouse models of diabetes (72, acetyl-CoA levels and mitochondrial protein acetylation. 80). SIRT4 negatively regulates fatty acid oxidation in liver and Malonyl-CoA pools in mitochondria and the cytosol are also muscle: knockdown of SIRT4 expression enhances fatty acid tightly regulated. Cytosolic malonyl-CoA is synthesized by the oxidation and mitochondrial respiration (80). This may be carboxylation of acetyl-CoA by acetyl-CoA carboxylase (ACC), mediated by increased SIRT1, PGC-1, and CPT1 expression and the decarboxylation of malonyl-CoA by malonyl-CoA in the absence of SIRT4 (80). Nevertheless, it is not clear how decarboxylase (MCD) regenerates acetyl-CoA (88). However, lack of SIRT4 in the mitochondrion affects gene transcription the mitochondrial pool of malonyl-CoA is generated by the DECEMBER 14, 2012• VOLUME 287 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42439 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation activity of propionyl-CoA carboxylase on acetyl-CoA, with the acetylation/succinylation and decreased metabolic flexibility. reverse reaction again catalyzed by MCD (88). ACC and MCD We propose that SIRT3 and SIRT5, in cooperation with other are tightly regulated by a variety of factors, including levels of enzymes in this pathway, such as CrAT, deacetylate and desuc- glucose, insulin, and AMP-activated protein kinase (88). cinylate mitochondrial proteins and thereby promote maximal Whole-cell malonyl-CoA levels decrease during fasting and metabolic flexibility. We further propose that a failure of this diabetic conditions and rapidly double after feeding (88). Mal- protective mechanism underlies the pathogenesis of the meta- onyl-CoA is the precursor for de novo fatty acid synthesis but is bolic syndrome and metabolic inflexibility. also a critical inhibitor of fatty acid oxidation. It binds to and Much work remains to be done to test this mechanism link- inhibits CPT1 on the mitochondrial outer membrane, thereby ing mitochondrial protein acylation and metabolic disease. Of inhibiting the transport of fatty acids into mitochondria for particular importance is the identification of the acyl moiety -oxidation (88), a regulatory process referred to as the reverse targeted by SIRT4. There are many other acyl-CoAs beside the Randle cycle. Drug inhibition or genetic disruption of MCD three discussed in this minireview, all with the potential of activity leads to increased intracellular malonyl-CoA levels, inducing the same type of modifications on mitochondrial pro- decreased fatty acid oxidation, and increased glucose oxidation teins. Their possible roles in intermediary metabolism and met- (89, 90). Mammals encode two isoforms of ACC: ACC1 is abolic disease regulation should represent fertile grounds for enriched in lipogenic tissues, where it produces cytosolic mal- future investigations. Mechanistic links to other obesity-related onyl-CoA as a precursor for lipogenesis, and ACC2 is preferen- diseases, such as diabetic nephropathy, are tempting but tially expressed in oxidative tissues, where it negatively regu- remain unknown. Finally, although the ubiquity of protein lates fatty acid oxidation (91). ACC2 knock-out mice show acetylation argues for a non-enzymatic mechanism, this does increased -oxidation in both liver and muscle. They are lean, not exclude the existence of a specific mitochondrial acetyl- hyperphagic, and resistant to obesity and diet-induced diabetes transferase. Future research effort should address this impor- (92, 93). Malonyl-CoA also regulates, directly or indirectly, tant remaining question. physiological or pathological conditions, such as muscle con- traction, cardiac ischemia, -cell secretion of insulin, and the Acknowledgments—We thank John Carroll for figure preparation hypothalamic control of appetite (88). These findings illustrate and Gary Howard for editorial review. the emerging but still partial understanding of the role of mal- onyl-CoA. The discovery of lysine malonylation as a post-trans- REFERENCES lational modification and its regulation by SIRT5 suggests the intriguing possibility that protein malonylation represents one 1. Wallace, D. C. (2005) A mitochondrial paradigm of metabolic and degen- erative diseases, aging, and cancer: a dawn for evolutionary medicine. of the mechanisms by which malonyl-CoA levels regulate inter- Annu. Rev. Genet. 39, 359–407 mediary metabolism. 2. 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(2006) Ab- 100, 10207–10212 DECEMBER 14, 2012• VOLUME 287 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42443 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Mitochondrial Protein Acylation and Intermediary Metabolism: Regulation by Sirtuins and Implications for Metabolic Disease *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2012 Elsevier Inc.
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0021-9258
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DOI
10.1074/jbc.r112.404863
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Abstract

MINIREVIEW THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 51, pp. 42436 –42443, December 14, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Three Mitochondrial Sirtuins Mitochondrial Protein Acylation Mammals contain seven sirtuins (SIRT1–7) that are charac- and Intermediary Metabolism: terized by an evolutionarily conserved sirtuin core domain homologous to Sir2, a yeast protein that increases life span (4, Regulation by Sirtuins and 5). SIRT1–7 are localized in distinct subcellular compartments. Implications for Metabolic SIRT1, SIRT6, and SIRT7 are found in the nucleus; SIRT2 is primarily cytosolic; and SIRT3–5 are found in mitochondria. Disease Sirtuins have different levels of NAD -dependent protein Published, JBC Papers in Press, October 18, 2012, DOI 10.1074/jbc.R112.404863 ‡§ ‡ ‡¶1 John C. Newman , Wenjuan He , and Eric Verdin deacetylase activity. This reaction couples lysine deacetylation to NAD hydrolysis to yield O-acetyl-ADP-ribose, the deacety- From the Gladstone Institute of Virology and Immunology, San Francisco, § ¶ California 94158 and the Division of Geriatrics and Department of lated substrate, and nicotinamide (reviewed in Refs. 6 and 7). Medicine, University of California, San Francisco, California 94143 SIRT1–3 exhibit robust protein deacetylase activity, whereas the others have only weak and highly selective (SIRT5–7) or The sirtuins are a family of NAD -dependent protein undetectable (SIRT4) protein deacetylase activity. So far, only deacetylases that regulate cell survival, metabolism, and longev- weak ADP-ribosyltransferase activity has been described for ity. Three sirtuins, SIRT3–5, localize to mitochondria. Expres- SIRT4 (8, 9). The dependence of sirtuins on NAD suggests sion of SIRT3 is selectively activated during fasting and calorie that their enzymatic activity is directly linked to the energy restriction. SIRT3 regulates the acetylation level and enzymatic status of the cell via the cellular NAD :NADH ratio; the abso- activity of key metabolic enzymes, such as acetyl-CoA synthe- lute levels of NAD , NADH, or nicotinamide; or a combination tase, long-chain acyl-CoA dehydrogenase, and 3-hydroxy-3- of these variables (10–14). methylglutaryl-CoA synthase 2, and enhances fat metabolism during fasting. SIRT5 exhibits demalonylase/desuccinylase Mitochondrial Protein Acetylation activity, and lysine succinylation and malonylation are abun- Reversible protein acetylation occurs primarily at the -a- dant mitochondrial protein modifications. No convincing enzy- mino group of lysine residues (for a recent review of mechanis- matic activity has been reported for SIRT4. Here, we review the tically distinct N-terminal acetylation, see Ref. 15). Like other emerging role of mitochondrial sirtuins as metabolic sensors post-translational modifications, lysine acetylation regulates that respond to changes in the energy status of the cell and mod- diverse protein properties, including DNA-protein interac- ulate the activities of key metabolic enzymes via protein tions, subcellular localization, protein stability, protein-protein deacylation. interaction, and enzymatic activity (16). Mitochondrial proteins are subject to extensive lysine acety- lation (17, 18). Acetylated mitochondrial proteins include those Proper mitochondrial function is required for metabolic involved in energy metabolism, such as in the TCA cycle, oxi- homeostasis and involves careful regulation of the activity of dative phosphorylation, -oxidation of lipids, amino acid multiple metabolic enzymes. Changes in mitochondrial num- metabolism, carbohydrate metabolism, nucleotide metabo- ber and activity are implicated in aging, cancer, and the patho- lism, and the urea cycle (2, 3). Interestingly, 44% of mitochon- genesis of the metabolic syndrome, a group of metabolic abnor- drial dehydrogenases are acetylated. Among them, 14 use malities characterized by central obesity, dyslipidemia, high NAD as the electron acceptor to catalyze biochemical reac- blood pressure, and increased fasting glucose levels (1). tions in oxidative catabolic routes. The importance of acetyla- Protein acetylation is increasingly recognized as an impor- tion is further supported by the high degree of conservation of tant post-translational modification for a number of key meta- many sites from Drosophila to humans (19). bolic pathways (2, 3). Lysine malonylation and succinylation were recently identified in several mitochondrial proteins, and SIRT3 Is the Major Mitochondrial Protein Deacetylase the mitochondrial sirtuin SIRT5 was found to have demalony- Endogenous SIRT3 is a soluble protein in the mitochondrial lase/desuccinylase activity. Here, we review the emerging role matrix (20, 21). Interestingly, SIRT3 is translated in the cyto- of protein acylation and its regulation by sirtuins in mitochon- plasm as a longer, enzymatically inactive precursor and drial biology and metabolic regulation. imported into the mitochondrion. After import, the first 100 amino acids of SIRT3 are proteolytically cleaved, leading to a final enzymatically active SIRT3 of 28 kDa. A small fraction of * This work was supported, in whole or in part, by National Institutes of Health Grants P30 DK026743 (to the UCSF Liver Center) and R24 DK085610 from SIRT3 resides in the nucleus as well (22). The initial controversy NIDDK. This work was also supported by a Senior Scholarship in Aging from regarding the mitochondrial localization of mouse SIRT3 was the Ellison Medical Foundation and institutional support from the J. David resolved by cloning of additional mouse SIRT3 cDNAs that Gladstone Institutes. Eric Verdin is a member of the Scientific Advisory Board of Sirtris/GSK, a company involved in the commercialization of sir- encode a protein that is imported to the mitochondrial matrix, tuin-related discoveries. This is the third article in the Thematic Minireview like human SIRT3 (23, 24). Series on Sirtuins: From Biochemistry to Health and Disease. SIRT3 appears to be the major mitochondrial deacetylase To whom correspondence should be addressed. E-mail: everdin@ gladstone.ucsf.edu. because mice lacking SIRT3, but not mice lacking SIRT4 or This is an Open Access article under the CC BY license. 42436 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 51 •DECEMBER 14, 2012 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation SIRT5, show a striking hyperacetylation of mitochondrial pro- tissue. SIRT3 accelerates amino acid catabolism and nitrogen teins (25). SIRT3 expression is highest in the most metabolically waste disposal by deacetylating and activating GLUD1 (gluta- active tissues, including liver, kidney, and heart (26, 27), and is mate dehydrogenase 1), a major cataplerotic enzyme (38). increased in glucose-poor fasting states, including calorie Catabolism of most amino acids requires transfer of the -a- restriction in liver and kidney (28–32). Expression in skeletal mino moiety to -ketoglutarate by an aminotransferase, form- muscle also increases under calorie restriction (31, 33) but has ing glutamate. GLUD1 regenerates -ketoglutarate from gluta- been reported to both increase and decrease with fasting (26, mate and releases nitrogen to the urea cycle as ammonia (39). 29, 33). Interestingly, SIRT3 expression initially increases on a SIRT3 accelerates the urea cycle by deacetylating and activating high-fat diet (HFD) in liver and skeletal muscle, but chronic ornithine transcarbamylase, the key mitochondrial enzyme in high-fat feeding leads to decreased SIRT3 expression (26, 31, the urea cycle. Mice lacking SIRT3 exhibit a metabolic profile 33–35). SIRT3 expression also decreases in mouse models of similar to that in human disorders of the urea cycle, including type 2 diabetes mellitus (26, 33). increased serum ornithine and reduced citrulline levels (the substrate and product, respectively, of ornithine transcarbam- SIRT3 Regulates Intermediary Metabolism oylase) (30). SIRT3 targets many enzymes that together help mediate the Carbohydrate Metabolism—By promoting fat oxidation, switch to fasting metabolism, as tissues move away from glu- SIRT3 indirectly suppresses carbohydrate utilization. In con- cose as a source of energy and metabolic intermediates to trast, cancer cells favor glucose as a source of energy, a process instead utilize lipids and amino acids. referred to as the Warburg effect (40). SIRT3 down-regulation Lipid Metabolism—SIRT3 promotes the efficient utilization is frequently observed in tumors and enhances glucose utiliza- of lipids as a primary source of acetyl-CoA during fasting by tion by allowing an increase in reactive oxygen species (ROS) deacetylating and activating long-chain acyl-CoA dehydrogen- that stimulate hypoxia-inducible factor 1, a transcription fac- ase, a key enzyme in the -oxidation of fatty acids (28). Mice tor that drives the expression of glycolytic genes (41–43). lacking SIRT3 accumulate -oxidation precursors and inter- SIRT3 also regulates the acetylation of the peptidyl-prolyl mediates, including triglycerides and long-chain fatty acids. isomerase cyclophilin D. In the absence of SIRT3, this leads to These mice also share other characteristics of human disorders activation of hexokinase II on the outer mitochondrial mem- of fatty acid oxidation, including cold intolerance and reduced brane, facilitating the rapid production of glucose 6-phosphate basal ATP levels (28). SIRT3 also regulates ketone body produc- (41, 44). tion by deacetylating and activating 3-hydroxy-3-methylglu- Reactive Oxygen Species—SIRT3 also regulates the produc- taryl-CoA synthase 2, the rate-limiting enzyme in ketone body tion of ROS generated as a by-product of oxidative phosphory- biosynthesis. Accordingly, mice lacking SIRT3 show reduced lation. First, SIRT3 deacetylates and activates isocitrate dehy- fasting serum levels of ketone bodies (36). SIRT3 also deacety- drogenase 2, an enzyme in the TCA cycle that helps to replenish lates and activates acetyl-CoA synthetase 2, an enzyme in extra- the mitochondrial pool of NADPH (45). NADPH is used by hepatic tissues that activates acetate into acetyl-CoA (21, 37). glutathione reductase to maintain glutathione in its reduced Acetate itself is produced in the liver from acetyl-CoA and can antioxidant form. Second, SIRT3 deacetylates and activates the be distributed to extrahepatic tissues as a form of energy (36). ROS-scavenging enzyme manganese superoxide dismutase, SIRT3 therefore facilitates the catabolism of fatty acids in the thereby reducing oxidative damage in the liver (46–48). Mice liver and the peripheral use of lipid-derived acetate and ketone lacking SIRT3 therefore show increased oxidative stress (46), bodies during fasting. particularly on a HFD (34), and lose the reduction of ROS levels Nitrogen Metabolism—Oxidation of acetyl-CoA to CO by normally observed under calorie restriction (45). the TCA cycle is a central pathway in energy metabolism. How- Oxidative Phosphorylation—Mice lacking SIRT3 consume ever, the TCA cycle also functions in biosynthetic pathways in 10% less O and produce up to 50% less ATP than wild-type which intermediates leave the cycle to be converted primarily mice, suggesting that SIRT3 regulates the activity of the respi- to glucose, fatty acids, or nonessential amino acids. Equilibrium ratory chain (27, 33). SIRT3 deacetylates and activates mito- of the substrates of the TCA cycle is maintained by two pro- chondrial respiratory chain complexes, including NDUFA9 cesses called anaplerosis and cataplerosis. Anaplerosis refers to (complex I) (27) and SDHA (complex II) (43, 49). Accordingly, the replenishment of critical anions. Pyruvate carboxylase, mice lacking SIRT3 have lower complex I and II activities than which generates oxalacetate directly in the mitochondria, is the wild-type mice (43, 49). SIRT3 also regulates ATP synthase major anaplerotic enzyme. Conversely, 4- and 5-carbon inter- (35). mediates that enter the TCA cycle during the catabolism of Accelerated Metabolic Syndrome in the Absence amino acids cannot be fully oxidized and therefore must be of SIRT3 removed by cataplerosis. Cataplerosis may in turn be linked to biosynthetic processes, such as hepatic gluconeogenesis, fatty The metabolic syndrome is defined by central obesity, insulin acid synthesis in the liver, and glyceroneogenesis in adipose resistance, hyperlipidemia, hyperglycemia, and hypertension (50). Physical inactivity, diet, and several genes and their prod- ucts (including leptin,  -adrenergic receptor, hormone-sensi- The abbreviations used are: HFD, high-fat diet; ROS, reactive oxygen species; tive lipase, lipoprotein lipase, insulin receptor substrate 1, PC-1, PGC-1, peroxisome proliferator-activated receptor- coactivator 1; and skeletal muscle glycogen synthase) are implicated in the CrAT, carnitine acetyltransferase; ACC, acetyl-CoA carboxylase; MCD, mal- onyl-CoA decarboxylase. pathogenesis of the metabolic syndrome (51–54). Other meta- DECEMBER 14, 2012• VOLUME 287 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42437 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation FIGURE 1. Mitochondrial acetyl-CoA, malonyl-CoA, and succinyl-CoA metabolism. Metabolic pathways resulting from the oxidation of glucose, fatty acids, and amino acids and leading to the synthesis of acetyl-CoA, malonyl-CoA, and succinyl-CoA are shown. Also shown are the two mechanisms leading to export of acetyl-CoA from mitochondria: ATP citrate lyase (CrAT) and carnitine/acylcarnitine translocase (CACT). BCAA, branched-chain amino acid; PK, pyruvate kinase; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PCC, propionyl-CoA carboxylase; LCAD, long-chain acyl-CoA dehydrogenase; BCAT, branched-chain aminotransferase; BCKD, branched-chain -keto acid dehydrogenase; MCM, methylmalonyl-CoA mutase; CPT, carnitine palmitoyltransferase. bolic abnormalities, such as aberrant lipogenesis (55, 56), ulation characterized by fatty liver disease (The NASH Clinical increased inflammation (57, 58), reduced fatty acid oxidation Research Network), patients meeting the criteria for metabolic (59, 60), and increased oxidative stress, have also been impli- syndrome were more likely to carry the SIRT3 rs11246020 “A” cated. Sustained weight loss and exercise are protective, as minor allele. In a follow-up study of8000 Finnish men focus- might be increased activation of fatty acid oxidation (61). ing specifically on rs11246020, the frequency of this allele and a Lack of SIRT3 and the resulting mitochondrial protein metabolic syndrome diagnosis were significantly correlated hyperacetylation are associated with accelerated development (34). However, this association was relatively weak (odds ratio of the metabolic syndrome (34). Wild-type mice fed a HFD of 1.3) and was not observed with all definitions of the meta- develop obesity, hyperlipidemia, type 2 diabetes mellitus, insu- bolic syndrome. Remarkably, the SIRT3 rs11246020 polymor- lin resistance, and non-alcoholic steatohepatitis (62–65). We phism induces a mutation within the catalytic domain of SIRT3 reported that the development of each of these consequences of (V208I). Mutation of Val-208 to isoleucine reduces SIRT3 HFD feeding is significantly accelerated in mice lacking SIRT3 enzyme efficiency by increasing the K for NAD and reducing (34). In addition, mice lacking SIRT3 show dramatically the V , consistent with the model that reduction of SIRT3 max enhanced levels of proinflammatory cytokines, including IL-6 enzymatic activity increases susceptibility to the metabolic and TNF-, another frequent manifestation of the metabolic syndrome. syndrome. Finally, we found that 90% of SIRT3 knock-out SIRT3, Acetylation, and Metabolic Inflexibility mice develop hepatocellular carcinoma, a cancer associated with the metabolic syndrome in humans (66), when placed on a We hypothesize that high mitochondrial acetyl-CoA levels HFD. and mitochondrial protein hyperacetylation cause metabolic Interestingly, prolonged exposure (13 weeks) to HFD feed- inflexibility. Acetyl-CoA, malonyl-CoA, and succinyl-CoA are ing in wild-type mice results in a reduction of hepatic SIRT3 important intracellular metabolites. They are present in mito- expression (34, 35), whereas acute HFD feeding leads to a tem- chondria and the cytosol and are variously derived from the porary increase in SIRT3 protein expression (34). A HFD sup- catabolism of carbohydrates, fatty acids, or proteins (Fig. 1). presses SIRT3 expression via suppression of peroxisome prolif- Intramitochondrial concentrations of acetyl-CoA and succi- erator-activated receptor- coactivator 1 (PGC-1) (67, 68), a nyl-CoA are in the millimolar range (70), a level that can initiate major regulator of SIRT3 expression (69). Reintroducing exog- non-enzymatic acetylation reactions (71). Importantly, global enous PGC-1 rescues the loss of SIRT3 in HFD-fed mice (34). protein acetylation in mitochondria correlates with elevated Preliminary evidence also supports a role of SIRT3 in the production of acetyl-CoA in such varied states as fasting, calo- pathogenesis of the metabolic syndrome in humans. In a pop- rie restriction, HFD, and ethanol intoxication (28, 34, 72–74). Acetyl-CoA is produced during the aerobic catabolism of carbohydrates from pyruvate, during -oxidation of long-chain M. D. Hirschey and E. Verdin, unpublished data. J. Y. Huang and E. Verdin, unpublished data. fatty acids, and from the catabolism of some amino acids or 42438 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 51 •DECEMBER 14, 2012 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation decarboxylation of malonyl-CoA (Fig. 1) (39). There is growing in the nucleus. Identifying the true enzymatic activity of SIRT4 evidence that levels of acetyl-CoA regulate fuel utilization and will undoubtedly shed light on its function. that dysregulated acetyl-CoA levels have a role in the pathogen- SIRT5, a Protein Demalonylase and Desuccinylase esis of insulin resistance and the metabolic syndrome. During fasting, acetyl-CoA can either feed into the TCA cycle for SIRT5 possesses unique potent demalonylase and desucciny- energy production or be used for ketogenesis or acetate produc- lase activities (79, 81). Malonyllysine and succinyllysine modi- tion (primarily in the liver) (39). During feeding, acetyl-CoA is fications occur in a variety of organisms from yeast to human exported from the mitochondria to the cytoplasm via citrate by (81, 82). Malonylation and succinylation are detected in meta- the activity of ATP citrate lyase. Excess acetyl-CoA can also be bolic enzymes, including isocitrate dehydrogenase 2, serine exported from the mitochondria via the activity of the enzyme hydroxymethyltransferase, glyceraldehyde-3-phosphate dehy- carnitine acetyltransferase (CrAT) (75). This enzyme is present drogenase, GLUD1, malate dehydrogenase 2, citrate synthase, in the mitochondrial matrix and combines acetyl-CoA and carbamoyl phosphate synthetase 1, 3-hydroxy-3-methylglu- carnitine into acetylcarnitine (76), which can be directly taryl-CoA synthase 2, thiosulfate sulfurtransferase, and aspar- exchanged across the mitochondrial membrane by carnitine/ tate aminotransferase (79, 81, 82). Mice lacking SIRT5 show acylcarnitine translocase (Fig. 1). global protein hypermalonylation and hypersuccinylation, sug- Increased mitochondrial levels of acetyl-CoA induced by gesting that it is the major protein demalonylase and desucci- fatty acid oxidation allosterically inhibit the activity of pyruvate nylase (81). The biological significance of lysine malonylation dehydrogenase, a mitochondrial enzyme complex that converts and succinylation and how lysine malonylation and succinyla- pyruvate into acetyl-CoA and thereby couples glycolysis and tion regulate enzymatic activity are currently unknown. In glucose oxidation. This acetyl-CoA-mediated inhibition repre- addition to these novel enzymatic activities, SIRT5 may also sents part of the glucose-fatty acid cycle originally proposed by function as a protein deacetylase on a restricted number of sub- Randle to explain the lipid-induced suppression of muscle glu- strates, such as the urea cycle enzyme carbamoyl phosphate cose disposal, a hallmark of obesity-associated insulin resist- synthetase 1 (83). ance (77). A pivotal role of intramitochondrial acetyl-CoA con- Succinyl-CoA and Malonyl-CoA Are Critical Metabolic centrations in metabolic control is further supported by recent Intermediates studies of CrAT (75). Mice with a muscle-specific deletion of CrAT exhibit compromised glucose tolerance and decreased As we discussed above, hyperacetylation of mitochondrial metabolic flexibility. This latter phenomenon was recently proteins associated with loss of SIRT3 disrupts the normal met- identified in obese humans as an inability to switch from fatty abolic switch toward fatty acid utilization that occurs during acid to glucose oxidation during the transition from fasting to prolonged fasting (28, 34). Because many metabolic enzymes feeding and may be a key manifestation of the metabolic syn- are also malonylated or succinylated (81, 82), SIRT5-mediated drome (78). Muoio et al. (75) proposed that CrAT promotes demalonylation or desuccinylation of metabolic enzymes may metabolic flexibility and increases insulin action by enhancing modulate metabolic pathways in a similar fashion under condi- mitochondrial export of excess acetyl residues. On the basis of tions of high malonyl-CoA or succinyl-CoA levels. SIRT5 is our observations of conditions associated with high acetyl-CoA therefore likely to emerge in the future as an important regula- levels, we predict that CrAT deletion leads to mitochondrial tor of intermediary metabolism. protein hyperacetylation and that dysregulated mitochondrial Succinyl-CoA is an intermediate in the TCA cycle and also a protein acetylation might represent the molecular mechanism precursor for porphyrin synthesis (39). Catabolism of odd- of metabolic inflexibility. In this context, the ability of SIRT3 to chain fatty acids and of some amino acids (e.g. branched-chain remove excess mitochondrial protein acetylation could there- amino acids, such as leucine, isoleucine, and valine) generates fore lead to increased metabolic flexibility and increased insulin propionyl-CoA, which is first carboxylated to methylmalonyl- sensitivity. CoA and then converted to succinyl-CoA (Fig. 1) (39). Branched-chain amino acids are the most abundant essential SIRT4, an Enzyme without a Substrate amino acids (84). Muscle represents 40% of the total mass of Unlike the well defined role of SIRT3 in acetylation, the pre- mammals and is therefore the largest metabolic organ. Muscle cise enzymatic function of SIRT4 is unclear. It may possess acts as a critical fuel reserve site in starvation or other glucose- weak ADP-ribosyltransferase activity (8, 9); however, this activ- poor states (85, 86) and accounts for50% of the capacity of the ity is 1000-fold slower than that of a bacterial ADP-ribosyl- tissues to catabolize branched-chain amino acids (87). We transferase, raising doubt about its physiological significance therefore expect that succinyl-CoA production will rise during (79). SIRT4 regulates insulin secretion (8, 9). Intriguingly and fasting. We do not know yet whether succinyl-CoA levels and unlike SIRT3, SIRT4 expression is reduced during calorie global protein succinylation correlate, as is observed between restriction and is increased in mouse models of diabetes (72, acetyl-CoA levels and mitochondrial protein acetylation. 80). SIRT4 negatively regulates fatty acid oxidation in liver and Malonyl-CoA pools in mitochondria and the cytosol are also muscle: knockdown of SIRT4 expression enhances fatty acid tightly regulated. Cytosolic malonyl-CoA is synthesized by the oxidation and mitochondrial respiration (80). This may be carboxylation of acetyl-CoA by acetyl-CoA carboxylase (ACC), mediated by increased SIRT1, PGC-1, and CPT1 expression and the decarboxylation of malonyl-CoA by malonyl-CoA in the absence of SIRT4 (80). Nevertheless, it is not clear how decarboxylase (MCD) regenerates acetyl-CoA (88). However, lack of SIRT4 in the mitochondrion affects gene transcription the mitochondrial pool of malonyl-CoA is generated by the DECEMBER 14, 2012• VOLUME 287 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42439 MINIREVIEW: Sirtuins and Mitochondrial Protein Acylation activity of propionyl-CoA carboxylase on acetyl-CoA, with the acetylation/succinylation and decreased metabolic flexibility. reverse reaction again catalyzed by MCD (88). ACC and MCD We propose that SIRT3 and SIRT5, in cooperation with other are tightly regulated by a variety of factors, including levels of enzymes in this pathway, such as CrAT, deacetylate and desuc- glucose, insulin, and AMP-activated protein kinase (88). cinylate mitochondrial proteins and thereby promote maximal Whole-cell malonyl-CoA levels decrease during fasting and metabolic flexibility. We further propose that a failure of this diabetic conditions and rapidly double after feeding (88). Mal- protective mechanism underlies the pathogenesis of the meta- onyl-CoA is the precursor for de novo fatty acid synthesis but is bolic syndrome and metabolic inflexibility. also a critical inhibitor of fatty acid oxidation. It binds to and Much work remains to be done to test this mechanism link- inhibits CPT1 on the mitochondrial outer membrane, thereby ing mitochondrial protein acylation and metabolic disease. Of inhibiting the transport of fatty acids into mitochondria for particular importance is the identification of the acyl moiety -oxidation (88), a regulatory process referred to as the reverse targeted by SIRT4. There are many other acyl-CoAs beside the Randle cycle. Drug inhibition or genetic disruption of MCD three discussed in this minireview, all with the potential of activity leads to increased intracellular malonyl-CoA levels, inducing the same type of modifications on mitochondrial pro- decreased fatty acid oxidation, and increased glucose oxidation teins. Their possible roles in intermediary metabolism and met- (89, 90). Mammals encode two isoforms of ACC: ACC1 is abolic disease regulation should represent fertile grounds for enriched in lipogenic tissues, where it produces cytosolic mal- future investigations. Mechanistic links to other obesity-related onyl-CoA as a precursor for lipogenesis, and ACC2 is preferen- diseases, such as diabetic nephropathy, are tempting but tially expressed in oxidative tissues, where it negatively regu- remain unknown. Finally, although the ubiquity of protein lates fatty acid oxidation (91). ACC2 knock-out mice show acetylation argues for a non-enzymatic mechanism, this does increased -oxidation in both liver and muscle. They are lean, not exclude the existence of a specific mitochondrial acetyl- hyperphagic, and resistant to obesity and diet-induced diabetes transferase. Future research effort should address this impor- (92, 93). 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(2006) Ab- 100, 10207–10212 DECEMBER 14, 2012• VOLUME 287 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42443

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Dec 14, 2012

Keywords: Metabolic Diseases; Metabolism; Mitochondria; Protein Acylation; Sirtuins

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