SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion

“This bacterial enzyme is homologous to murine MCCC, with sequence identities of 47% and 66% for the a and b subunits, respectively (Sievers et al., 2011).”

“We built a phylogenetic tree from the sirtuin domain portions of all of these sequences with length greater than 40 residues using the WAG-CAT model in FastTreeMP (Price et al., 2010), version 2.1.8.”

“Each mouse subunit was superimposed over the corresponding subunits in the crystal structure template by sequence alignment and the resulting complex typed with the CHARMM forcefield (Brooks et al., 2009) and energy minimized with the smart minimizer protocol using the Generalized-Born with simple switching implicit s…”

“Originally thought to be strictly lysine deacetylases (Imai et al., 2000; Verdin et al., 2004) or ADP-ribosyltransferases (Tanny et al., 1999), more recent studies have shown that the sirtuins remove several post-translational modifications (PTMs) from lysine residues (Anderson et al., 2014).”

“The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaı́no et al., 2016) partner repository with the dataset identifier PRIDE: PXD005896.”

“Previous studies have shown that altered BCAA metabolism is associated with the progression of diabetes and cardiovascular disease (Newgard et al., 2009; Shah et al., 2010).”

“Interestingly, the structurally similar 3-methylglutaconyl-CoA (MGc-CoA; Figure 3A) is an intermediary metabolite in leucine catabolism (Wishart et al., 2013) and MG-CoA is thought to originate from the chemical or enzymatic reduction of MGc-CoA (Roe et al.”

“Conversely, ablation or inhibition of sirtuins accelerates the onset of several aging phenotypes (Houtkooper et al., 2012).”

“SIRT6 is a highly specific lysine deacetylase (Michishita et al., 2008; Mostoslavsky et al., 2006), as well as a long-chain deacylase (Jiang et al.”

“Our proteomic analysis of MCCC identified acetylation sites dispersed throughout the a subunit (Figure 4E), consistent with previous findings (Hornbeck et al., 2012).”

“SIRT5 is a lysine demalonylase (Peng et al., 2011), desuccinylase (Du et al., 2011), and deglutarylase (Tan et al., 2014).”

“Previous phylogenetic studies of sirtuins from approximately 30 (Frye, 2000) and 70 (Greiss and Gartner, 2009) sequenced species divided the human sirtuins into four classes: SIRT1–3 occupy class I, SIRT4 occupies class II, SIRT5 occupies class III, and SIRT6 and SIRT7 occupy class IV.”

“We used PROMALS3D (Pei et al., 2008) to build a 3D structure-aware alignment guided by representative structures available for human sirtuins (4IF6 A, 1J8F A, 3GLR A, 2B4Y A, 3K35 A).”

“Mitochondrial Substrate Oxidation Assays Mitochondria were isolated from liver, heart and/or skeletal muscle of SIRT4KO or WT mice, as previously described (Frezza et al., 2007).”

“We then sequenced the overexpressed SIRT4 protein by mass spectrometry and identified a putative mitochondrial target signal at residues 1–23 (Figures 1A, 1B, and S3C), which is in agreement with a previously published report (Haigis et al., 2006).”

“COBALT (Papadopoulos and Agarwala, 2007) and Uniprot were used for protein alignment.”

“SIRT6 is a highly specific lysine deacetylase (Michishita et al., 2008; Mostoslavsky et al., 2006), as well as a long-chain deacylase (Jiang et al.”

“This is consistent with abundant expression of the leucine transaminase (BCATm) but low expression of BCKDH in skeletal muscle, which is the opposite ratio of expression of these enzymes in liver (Hutson et al., 1992; Newgard, 2012; Shimomura et al., 2006; Zhou et al., 2010).”

“Furthermore, SIRT4 showed no deacylation activity against known dicarboxyl-acyl modifications (Peng et al., 2011; Tan et al., 2014) and does not efficiently remove long-chain acyl modifications (Feldman et al., 2013; Madsen et al., 2016).”

“Given the well-established role of skeletal muscle in BCAA disposal (Felig, 1975), we first collected mitochondria from wild-type mouse skeletal muscle and monitored NADH generation as described above; however, we were unable to detect NADHproduction in the presence of aKIC in isolated mitochondria prepared from skele…”

“Often, the target signal is cleaved once targeting to the mitochondria has been completed, similar to the other mitochondrial sirtuins (Nakagawa et al., 2009; Schwer et al., 2006).”

“Furthermore, SIRT4 showed no deacylation activity against known dicarboxyl-acyl modifications (Peng et al., 2011; Tan et al., 2014) and does not efficiently remove long-chain acyl modifications (Feldman et al., 2013; Madsen et al., 2016).”

“Often, the target signal is cleaved once targeting to the mitochondria has been completed, similar to the other mitochondrial sirtuins (Nakagawa et al., 2009; Schwer et al., 2006).”

“SIRT6 is a highly specific lysine deacetylase (Michishita et al., 2008; Mostoslavsky et al., 2006), as well as a long-chain deacylase (Jiang et al., 2013).”

“, 2013b), but recombinant SIRT4 does not catalyze deacetylation on a variety of substrates (Feldman et al., 2013; Verdin et al., 2004).”

“, 2009) and energy minimized with the smart minimizer protocol using the Generalized-Born with simple switching implicit solvent model (Feig et al., 2004) to an RMS convergence < 0.”

“Sirt4 ablation in mice activates glutamate dehydrogenase, leading to increased glutamine metabolism (Csibi et al., 2013; Jeong et al., 2013, 2014).”

“, 2010), and metabolic homeostasis throughout the aging process (D’Antona et al., 2010; Mansfeld et al., 2015; Martin et al., 2011).”

“Several acyl-CoA species are reactive metabolites that are the carbon source for protein modifications (Wagner et al., 2017; Wagner and Payne, 2013), but 3-methylglutaryl-CoA (MG-CoA) is not a metabolite known to be generated directly from any enzymes in humans (Wishart et al., 2013).”

“Further, leucine and BCAA metabolism are emerging as important regulators of diabetes (Newgard, 2012), cardiovascular disease (Shah et al., 2010), and metabolic homeostasis throughout the aging process (D’Antona et al.”

“Sirt4 ablation in mice activates glutamate dehydrogenase, leading to increased glutamine metabolism (Csibi et al., 2013; Jeong et al., 2013, 2014).”

“However, we observed no differences in pyruvate metabolism (Figure 5B), which is in contrast to a recent report (Mathias et al., 2014).”

“SIRT4 resides in the mitochondria and was originally thought to be an ADP-ribosyltrasferase (Ahuja et al., 2007; Haigis et al., 2006); however, this activity has been called into question as its primary enzymatic function (Du et al.”

“, 2004) or ADP-ribosyltransferases (Tanny et al., 1999), more recent studies have shown that the sirtuins remove several post-translational modifications (PTMs) from lysine residues (Anderson et al.”

“Leucine has long been known as a potent insulin secretagogue (Sener and Malaisse, 1980).”

“In particular, SIRT4 contains a homologous sirtuin deacylase domain, a conserved catalytic histidine (H161), and a Rossmann fold (Min et al., 2001) NAD+-binding motif (amino acids 62–82, 143–146, 260–262, and 286–288) (Figure 1A).”

“Additionally, several studies have shown that SIRT4 can reduce fatty acid oxidation and/or increase lipid synthesis via variousmechanisms (Ho et al., 2013; Laurent et al., 2013a, 2013b; Nasrin et al., 2010).”

“An alternative, Cell Metabolism 25, 838–855, April 4, 2017 851 but not mutually exclusive, model positions sirtuins to act as protein quality control enzymes by removing deleterious protein modifications (Wagner and Hirschey, 2014), thereby maintaining enzyme activity and metabolic flux.”

“Characterization data was in agreement with the literature (Madsen and Olsen, 2012; Wegener et al., 2003).”

“Given that SIRT5 has several solved crystal structures (Du et al., 2011; Schuetz et al., 2007), we used a structure with the substrate bound (Du et al., 2011) as a template to build a homology model of SIRT4 (Figure 2A).”

“Accordingly, the International Mouse Phenotyping Consortium reported that serum fructosamine levels, a measure of long-term glucose control, are elevated in SIRT4KO mice, further supporting our observations that SIRT4KO mice have abnormal glucose homeostasis (Koscielny et al., 2014).”

“Interestingly, our concurrent study demonstrates that reactive acyl-CoA species (RAS) have the potential to react non-enzymatically with lysine residues to generate protein modifications (Wagner et al., 2017).”

“org/ (Rost and Liu, 2003); data accessed and downloaded October 2011.”

“Importantly, activation or overexpression of sirtuins increases healthspan and lifespan in several model organisms (Hall et al., 2013).”

“In addition, it supports several more recent suggestions (Greiss and Gartner, 2009), including: the relationship between SIRT2 and SIRT3within class I, the existence of a largely fungal-specific sub-group in class I, and the great diversity of class III sirtuins.”

“The structures of SIRT5 (3RIY) and SIRT3 (3GLR) (Jin et al., 2009) also contain co-crystallized modified peptides that were utilized in the modeling studies.”

“SIRT4 resides in the mitochondria and was originally thought to be an ADP-ribosyltrasferase (Ahuja et al., 2007; Haigis et al., 2006); however, this activity has been called into question as its primary enzymatic function (Du et al., 2009).”

“Additionally, several studies have shown that SIRT4 can reduce fatty acid oxidation and/or increase lipid synthesis via variousmechanisms (Ho et al., 2013; Laurent et al., 2013a, 2013b; Nasrin et al., 2010).”

“For example, leucine allosterically activates GDH, thereby increasing aKG flux into the TCA cycle, increasing ATP production, and stimulating insulin secretion (Li et al., 2003).”

“Additionally, several studies have shown that SIRT4 can reduce fatty acid oxidation and/or increase lipid synthesis via variousmechanisms (Ho et al., 2013; Laurent et al., 2013a, 2013b; Nasrin et al., 2010).”

“Several human cancers, including those with the worst prognoses (e.g., lung, gastric, and colorectal cancer) are associated with decreased expression of SIRT4 (Jeong et al., 2013; Miyo et al., 2015).”

“Further, proper control of leucine catabolism is important for overall healthy aging (Mansfeld et al., 2015).”

“Islet Perifusion Mouse islet isolationsandperifusionswereperformedasdescribed (Campbell et al., 2016).”

“While this phenotype was mild, previous studies have shown that chronic hyperinsulinemia can lead to insulin resistance (Coleman and Hummel, 1974; Gray et al., 2010; Lee, 1981; Rajan et al., 2016).”

“This is consistent with abundant expression of the leucine transaminase (BCATm) but low expression of BCKDH in skeletal muscle, which is the opposite ratio of expression of these enzymes in liver (Hutson et al., 1992; Newgard, 2012; Shimomura et al., 2006; Zhou et al., 2010).”

“Sirt4 ablation in mice activates glutamate dehydrogenase, leading to increased glutamine metabolism (Csibi et al., 2013; Jeong et al., 2013, 2014).”

“Reduced BCAA catabolism is reported in obese, diabetic ob/ob mice and mice rendered diabetic through a high-fat diet and low-dose streptozotocin treatment (Lian et al., 2015).”

“This is consistent with abundant expression of the leucine transaminase (BCATm) but low expression of BCKDH in skeletal muscle, which is the opposite ratio of expression of these enzymes in liver (Hutson et al., 1992; Newgard, 2012; Shimomura et al., 2006; Zhou et al., 2010).”

“We then applied the GroupSim algorithm (Capra and Singh, 2008) with no window to the resulting alignment (Figure S1D) and classification of the sirtuins.”

“While the acetyl modification is not long enough to reach the a helix in SIRT3’s binding pocket (Madsen et al., 2016), medium- and long-chain uncharged acyl modifications showed favorable binding energies (Figure 2C).”

“Because sirtuins with unique enzymatic activities each occupy a distinct phylogenetic subclass, the phylogeny has been suggested to provide key information about sirtuin enzymatic activity (Hirschey, 2011).”

“To enable the identification of specific sites within SIRT4 that were likely to influence its enzymatic specificity, we subsampled the available sirtuin sequences to a set of 811 within the Pfam set of representative proteomes at 15% co-membership in UniRef50 clusters (Chen et al., 2011).”

“acyl modifications from various cellular substrates to regulate a wide range of biological pathways (Anderson et al., 2014).”

“Since chronic hyperinsulinemia can lead to the development of insulin resistance (Gray et al., 2010; Rajan et al., 2016), our data suggest that early hyperinsulinemia in SIRT4KO mice may be the primary defect driving accelerated age-induced insulin resistance.”

“To test this hypothesis, and to further validate the enzymatic activities described above (Figure 3C), we turned to another sirtuin assay based on deacylation of a fluorogenic aminomethyl-coumarin (AMC) peptide substrate (Marcotte et al., 2004).”

“, 2003), LGKsuc (Madsen and Olsen, 2012), and Kmal (Madsen and Olsen, 2012) have been previously reported.”

“Importantly, GDH is allosterically activated by leucine and its deamidated homolog aKIC (Allen et al., 2004; Fahien and Macdonald, 2011).”

“This is consistent with abundant expression of the leucine transaminase (BCATm) but low expression of BCKDH in skeletal muscle, which is the opposite ratio of expression of these enzymes in liver (Hutson et al., 1992; Newgard, 2012; Shimomura et al., 2006; Zhou et al., 2010).”

“Indeed, during leucine oxidation, three specific reactive species are generated: MGc-CoA and hydroxymethyl-CoA (HMG-CoA) are directly generated, whereas MGCoA is generated from the chemical or enzymatic (non-specific) reduction of MGc-CoA (Roe et al., 1986).”

“In wildtype mice, we observed a strong signal at 720 kDa (Figure 4L), which is the predicted molecular weight of the functionally intact dodecameric MCC complex (Huang et al., 2011).”

“Importantly, GDH is allosterically activated by leucine and its deamidated homolog aKIC (Allen et al., 2004; Fahien and Macdonald, 2011).”

“Originally thought to be strictly lysine deacetylases (Imai et al., 2000; Verdin et al., 2004) or ADP-ribosyltransferases (Tanny et al., 1999), more recent studies have shown that the sirtuins remove several post-translational modifications (PTMs) from lysine residues (Anderson et al., 2014).”

“To synthesize LGKmgc,methyl (E)-5-hydroxy-3-methylpent-2-enoate (S4) was first synthesized based on themethod described in the literature (White et al., 1982).”

“, 2010), and metabolic homeostasis throughout the aging process (D’Antona et al., 2010; Mansfeld et al., 2015; Martin et al., 2011).”

“Since chronic hyperinsulinemia can lead to the development of insulin resistance (Gray et al., 2010; Rajan et al., 2016), our data suggest that early hyperinsulinemia in SIRT4KO mice may be the primary defect driving accelerated age-induced insulin resistance.”

“(5mM) were included in all assays in an effort to clamp extramitochondrial ADP at 1mM (Clark et al., 1997).”

“We and others have found that SIRT4 binds to several enzymes of the leucine oxidation pathway (Mathias et al., 2014; Wirth et al., 2013).”

“While this phenotype was mild, previous studies have shown that chronic hyperinsulinemia can lead to insulin resistance (Coleman and Hummel, 1974; Gray et al., 2010; Lee, 1981; Rajan et al., 2016).”

“First, we turned to a well-established sirtuin deacylation assay that monitors the consumption of 32P-NAD+ (Zhu et al., 2013), based on the rationale that all sirtuins require NAD+ as a co-substrate during cata- lytic deacylation, and that the readout of this assay would be agnostic to the nature…”

“The lysine deacylase activity of SIRT4 is involved in the control of leucine metabolism and insulin secretion through regulating the acylation status of enzymes in these pathways.(225) SIRT4 also acts as a cellular lipoamidase with a preferred catalytic efficiency for lipoyl- and biotinyl-lysine modifications to its d…”

“Interestingly, NADPH is the final reductant for PRDX, and SIRT2 has been shown to deacetylate and inhibit the peroxidase activity of PRDX-1, thereby sensitizing breast cancer cells to oxidative stress-inducing agents (42).”

“Chem 273(37):23629–32 [PubMed: 9726963]”