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Activation of the Protein Deacetylase SIRT6 by Long-chain Fatty Acids and Widespread Deacylation by Mammalian Sirtuins * ♦

Activation of the Protein Deacetylase SIRT6 by Long-chain Fatty Acids and Widespread Deacylation... REPORT THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 43, pp. 31350 –31356, October 25, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. but not SIRT1. The activation mechanism is consistent with Activation of the Protein fatty acid inducing a conformation that binds acetylated H3 with Deacetylase SIRT6 by Long-chain greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of Fatty Acids and Widespread discovering endogenous, small-molecule activators of SIRT6. Deacylation by Mammalian Sirtuins SIRT6 is one of seven members of the mammalian NAD - Received for publication, August 16, 2013, and in revised form, September 17, 2013 dependent protein deacetylases that are evolutionarily ancient Published, JBC Papers in Press, September 18, 2013, DOI 10.1074/jbc.C113.511261 ‡§ ‡§ ‡§1 Jessica L. Feldman , Josue Baeza , and John M. Denu (1). Recent studies indicate that SIRT6 plays critical roles in ‡ § From the Department of Biomolecular Chemistry and the Wisconsin intermediary metabolism and genomic stability (2). SIRT6-de- Institute for Discovery, University of Wisconsin, Madison, Wisconsin 53715 ficient mice have a striking degenerative phenotype leading to shortened lifespan and are associated with hypoglycemia and Background: Sirtuins regulate metabolism, genome defects in DNA repair (2). Deletion of SIRT6 is sufficient to maintenance, and stress responses. promote tumorigenesis independent of oncogene activation Results: Long-chain free fatty acids stimulate SIRT6 (3). SIRT6 overexpression lowers LDL and triglyceride levels, deacetylase, and sirtuins display distinct but overlapping improves glucose tolerance (4), and increases lifespan of male specificity for diverse acylated peptides. mice (5). SIRT6 also plays a role in inflammatory pathways, Conclusion: SIRT6 is activated by biologically relevant exerting anti-inflammatory effects at the transcriptional level. fatty acids, and long-chain deacylation is a general feature However, other studies suggest a pro-inflammatory effect on of sirtuins. intracellular signaling (6). Although it is clear that SIRT6 func- Significance: Discovery of endogenous, small-molecule tions in metabolism, inflammation, and genome maintenance, activators of SIRT6 demonstrates the therapeutic poten- its molecular functions remain enigmatic. tial of compounds that promote SIRT6 function. SIRT6 is grouped among the mammalian sirtuins (including SIRT4, SIRT5, and SIRT7) that display extremely low deacety- Mammalian sirtuins (SIRT1 through SIRT7) are members of a lase activity in vitro (7). SIRT1–3 display robust protein highly conserved family of NAD -dependent protein deacety- deacetylase activity, and numerous studies have identified in lases that function in metabolism, genome maintenance, and vivo substrates (35, 36). The recent observation that SIRT5 pre- stress responses. Emerging evidence suggests that some sirtuins fers succinylated/malonylated substrates helps explain the display substrate specificity toward other acyl groups attached observed low activity on acetylated substrates (8). Additional to the lysine -amine. SIRT6 was recently reported to preferen- acyl-lysine modifications have been identified in histone and tially hydrolyze long-chain fatty acyl groups over acetyl groups. non-histone proteins, including propionyl, butyryl, myristoyl, Here we investigated the catalytic ability of all sirtuins to hydro- and crotonyl (8–11), suggesting the existence of a very diverse lyze 13 different acyl groups from histone H3 peptides, ranging acylation landscape in cells. in carbon length, saturation, and chemical diversity. We find These observations have led the field to postulate that some that long-chain deacylation is a general feature of mammalian sirtuins harbor unique acyl group specificities, distinct from sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, deacetylase activity (7, 12). Consistent with this idea, Jiang et al. and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic (13) demonstrated recently that SIRT6 preferentially hydro- acid. These results provide new insight into sirtuin function and lyzes long-chain fatty acyl groups, including myristoyl and a means for cellular removal of an expanding list of endogenous palmitoyl groups, from lysine residues. A crystal structure of lysine modifications. Given that SIRT6 is a poor deacetylase in SIRT6 bound to a myristoylated peptide supported this obser- vitro, but binds and prefers to hydrolyze long-chain acylated vation. However, several prior studies indicated that SIRT6 peptides, we hypothesize that binding of certain free fatty acids acted through deacetylation of histone marks H3K9Ac (14) and (FFAs) could stimulate deacetylation activity. Indeed, we dem- H3K56Ac (15), which modulated HIF1--dependent (16), onstrate that several biologically relevant FFAs (including my- NF-B-dependent (17), and c-Myc-dependent (3) pathways. In ristic, oleic, and linoleic acids) at physiological concentrations cells, the ability of SIRT6 to promote deacetylation of histones induce up to a 35-fold increase in catalytic efficiency of SIRT6 and non-histone proteins (18) seemed at odds with the low in vitro deacetylation rates (13, 19) and with the recently * This work was supported, in whole or in part, by National Institutes of Health described demyristoylation activity (13). X-ray structural anal- Grant GM065386 (to J. M. D.), a predoctoral fellowship (to J. L. F.) from the ysis of SIRT6 suggested that the low deacetylase activity stems American Heart Association (11PRE7300059) and a predoctoral fellowship from a splayed configuration between the Rossmann fold (to J. B.) from the National Science Foundation (1256259). This article was selected as a Paper of the Week. domain and the zinc binding subdomain (19). Given this unique □ S This article contains supplemental Methods. conformation, we proposed that SIRT6 could be activated if To whom correspondence should be addressed: University of Wisconsin, the two domains could be induced to the canonical active-site 330 N. Orchard St., WID 2178, Madison, WI 53715. Tel.: 608-265-1859; Fax: 608-262-5253; E-mail: [email protected]. conformation (19). 31350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 43 •OCTOBER 25, 2013 This is an Open Access article under the CC BY license. REPORT: Activation of SIRT6 Deacetylase To provide molecular understanding of the unique catalytic NAD were incubated with 15–220 M H3K9Myr peptide in functions of SIRT6 and to resolve the apparent contradictory the presence of 0–200 M myristic acid. Data were fitted to an results discussed above, we addressed two major questions: 1) Is equation for competitive inhibition using KinetAsyst (Intelli- long-chain protein deacylation a general feature of mammalian Kinetics, State College, PA). sirtuins? and 2) Are there endogenous SIRT6 activators that RESULTS AND DISCUSSION stimulate deacetylation activity? Long-chain Deacylase Activity Is an Intrinsic Activity of Most EXPERIMENTAL PROCEDURES Sirtuins—It was recently reported that SIRT6 preferentially Expression and Purification of Recombinant SIRT1-SIRT7— hydrolyzes long-chain fatty acyl (myristoyl and palmitoyl His-tagged SIRT1–7 were overexpressed in BL21(DE3) or groups), relative to deacetylation (13). Here we investigated BL21(DE3) CobB Escherichia coli strains and purified as whether such long-chain deacylation activity was specific to described (19, 20). SIRT6 or whether this is a general feature of mammalian sir- [ P]NAD TLC Assay—Assays were performed in 20 mM tuins. We synthesized a panel of acylated lysine peptides corre- HEPES, pH 7.4, at 25 °C using [ P]NAD (American Radio- sponding to amino acids 5–13 of histone H3 containing diverse labeled Chemicals). Products were resolved by TLC and acyl--lysine modifications, including acetyl, propionyl, succi- quantified using a Typhoon FLA 9500 (GE Healthcare) (12). nyl, butyryl, crotonyl, hexanoyl, octanoyl, decanoyl, dode- TLC plates were analyzed by ImageQuant TL (GE Health- canoyl, myristoyl, or palmitoyl groups on Lys-9 (see supple- care). Intensities were determined for [ P]NAD and mental Methods) (Fig. 1A). Human sirtuins 1 through 7 32 2 O-[ P]acylADPr using spot edge average as a local back- (SIRT1–7) were recombinantly expressed and purified, and ground subtraction method. their abilities to hydrolyze each acyl group were determined. HPLC Deacylation Assay—Deacylation reactions were ana- First, the activity of SIRT6 was assessed using an excess of pep- 32  32 lyzed by reversed phase HPLC on a Kinetex C18 column (100A, tide (5 mM) and limiting [ P]NAD (0.18 M). The [ P]NAD 100  4.6 mm, 2.6-m, Phenomenex) by monitoring the for- substrate and the various O-[ P]acylADPr products were sep- mation of deacylated product at 214 nm, as described (21). Sir- arated by TLC and visualized by phosphorimaging (Fig. 1B). tuin (0.2 M)or0.8 M SIRT6 was incubated with 40 M SIRT6 displayed a clear preference for longer acyl chains, H3K9Ac, H3K9Dodec, or H3K9Myr in the presence of 0.5 mM including myristoyl and palmitoyl chains, consuming 98 and NAD and 3% DMSO at 37 °C. Product and substrate peaks 81% of the [ P]NAD (Fig. 1B), respectively, consistent with a were quantified, and deacylation rates were determined. prior study (13). In addition, SIRT6 was able to deacylate the Deacetylase Activity in the Presence of Fatty Acids (FAs)— decanoyl (96%) and dodecanoyl (98%) groups from lysine resi- SIRT6 (4 M)or2 M SIRT1 were incubated with 70 M dues. To a lesser degree, SIRT6 was able to hydrolyze the hex- H3K9Ac peptide, 0.5 mM NAD , 100 M FA (dodecanoic, my- anoyl and octanoyl groups, consuming 23 and 48% of the ristic, palmitic, oleic, linoleic, -linolenic, -linolenic). Reac- [ P]NAD , respectively. Under these assay conditions, SIRT6 tions were incubated for either 1 h (SIRT6) or 15 s (SIRT1) and was unable to remove detectable amounts of the acetyl group. quenched with TFA. To examine dose dependence, FA concen- Next, we analyzed the deacylase activity of SIRT1 through trations were varied from 0 to 1 mM for myristic acid and 0–300 SIRT7 using catalytic amounts of enzyme and a more expansive M for oleic and linoleic acid. FAs were incubated with 70 M panel of acylated H3K9 peptides, including acetyl, propionyl, H3K9Ac peptide, 0.5 mM NAD , and 4 M SIRT6 at 37 °C. succinyl, butyryl, crotonyl, hexanoyl, octanoyl, decanoyl, dode- Steady-state Kinetic Analyses—Steady-state rates were mea- canoyl, myristoyl, palmitoyl, and reduced and oxidized lipoyl sured by varying H3K9Ac peptide (0–200 M) in the presence groups. No appreciable amounts of products were observed of 0.9 M SIRT6 and 2 mM NAD  400 M myristic acid. with SIRT7. As noted in the prior experiment (Fig. 1B), SIRT6 Initial velocities were determined; data in the presence of myr- displayed a strong preference for long-chain fatty acyl groups istic acid were fitted to the Michaelis-Menten equation, and (Fig. 1C). Under these conditions, octanoyl, decanoyl, dode- data in the absence of myristic acid were fitted to a modified canoyl, and myristoyl groups were preferentially hydrolyzed. version of the equation (22). The corresponding O-[ P]acylADPr products from hexanoyl Critical Micelle Concentration—-Fold increase in fluores- and palmitoyl were detectable, but the other acyl groups did not cence of 1,6-dipheynl-1,3,5-hexatriene (DPH) was monitored accumulate to quantifiable amounts. in the presence of increasing concentration of FFAs (23). Reac- Surprisingly, SIRT1, SIRT2, SIRT3, and SIRT5 also displayed tions contained 20 mM potassium phosphate (pH 7.5), 6.7% strong deacylation activity against long-chain acyl groups. Par- DMSO, 5 M DPH, and 0–2 mM myristic acid or 0–1.5 mM ticularly striking was the overall similar preference among these oleic and linoleic acid at 37 °C. sirtuins for decanoyl and dodecanoyl, which was shared with Myristic Acid Inhibition Kinetics—SIRT6 (2 M) was incu- SIRT6 (Fig. 1, C–H). As expected, SIRT1–3 efficiently hydro- bated with 70 M H3K9Myr peptide in the presence of 0.5 mM lyzed the acetyl group from lysine (75% conversion), and to a NAD and myristic acid (0–1 mM) for 1.5 min at 37 °C. To lesser extent, the propionyl and butyryl groups (Fig. 1, D–F). determine the inhibition constant, K ,2 M SIRT6 and 0.5 mM SIRT5 was most efficient at catalyzing desuccinylation (95% is conversion) (Fig. 1H), as was reported previously (8). However, SIRT5 more efficiently hydrolyzed (2–3) decanoyl and dode- The abbreviations used are: O-acylADPr, O-acyl-ADP-ribose; FA, fatty acid; canoyl peptides as compared with the corresponding acetylated PUFA, polyunsaturated fatty acid; DPH, 1,6-dipheynl-1,3,5-hexatriene; CMC, critical micelle concentration; DMSO, dimethyl sulfoxide. form, which was hydrolyzed faster than the propionyl, octanoyl, OCTOBER 25, 2013• VOLUME 288 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 31351 REPORT: Activation of SIRT6 Deacetylase 31352 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 43 •OCTOBER 25, 2013 REPORT: Activation of SIRT6 Deacetylase and myristoyl groups. At 3 the protein concentration, SIRT4 donors are intermediates in different central metabolic path- displayed low, but measureable amounts of deoctanoylase and ways, including glycolysis, the TCA cycle, -oxidation, and FA dedecanoylase activity (Fig. 1G). synthesis (7). Many of these modifications occur on metabolic Crotonyllysine was identified as a histone modification in enzymes, suggesting a regulatory link between metabolism and several eukaryotic cells types and is associated with active pro- lysine acylation. Our results suggest that sirtuins are well suited moters or enhancers (11). A product corresponding to [ P]O- to function as the demodifying enzymes of such acylations. crotonylADPr was observed when either SIRT1 or SIRT2 was SIRT6 Deacetylase Activity Is Stimulated by Long-chain reacted with NAD and the crotonylated H3K9 peptide, con- FFAs—The fact that sirtuins other than SIRT6 displayed higher suming 20% of the [ P]NAD (Fig. 1, D and E). specific activity against long-chain acyl groups was surprising, Among sirtuins displaying quantifiable activity, SIRT1 dis- especially given the prior observation of a co-crystal structure played the greatest promiscuity for diverse acyl groups, except with bound myristoylated peptide. This structure revealed a for succinylated substrate, which neither SIRT1, SIRT2, nor long acyl chain binding cleft. Comparing SIRT6 crystal struc- SIRT6 hydrolyzed. SIRT3 and SIRT4 also exhibited activity tures with bound and unbound myristoylated peptide revealed against the succinylated substrate, but to a lesser extent than conformational differences, adopting a more canonical sirtuin SIRT5 (Fig. 1, C–H). Interestingly, a specific mammalian deli- fold when myristoylated peptide is bound (13, 19). We hypoth- poylase has not been identified (24). Here, SIRT2, and to a lesser esized that this acyl group binding pocket might bind FFAs, and extent, SIRT1, SIRT3, and SIRT4 were able to hydrolyze the if such binding could induce closure of the splayed SIRT6 sub- removal of the lipoyl group from lysine (Fig. 1, D–G). Impor- domains, then SIRT6 might show stimulated deacetylation tantly, SIRT1–3 displayed more efficient conversion of many activity. To determine whether FFAs can stimulate SIRT6 long-chain acylated peptides as compared with SIRT6. Even deacetylase activity in vitro, SIRT6 was incubated with 70 M SIRT5, an enzyme reported to harbor exquisite selectivity for H3K9Ac peptide, 0.5 mM NAD , and 100 M dodecanoic, my- succinylated peptide, hydrolyzed long-chain acyl groups with ristic, palmitic, stearic, oleic (18:1, n-9), linoleic (18:2, n-6), similar efficiency to that of SIRT6. -linolenic (18:3, n-6), or -linolenic (18:3, n-3) acids. Separate To obtain true steady-state rates of deacylation and to ensure experiments were performed with SIRT1 for comparison. that the deacylase activity observed in the above experiments Reaction substrates and products were separated by HPLC and was not due to contamination by the E. coli sirtuin CobB, quantified. Specific activities of SIRT6 and SIRT1 in the pres- SIRT1, SIRT2, SIRT3, and SIRT6 were overexpressed and puri- ence of different FAs were compared. fied from a CobB knock-out BL21(DE3) strain. The purified SIRT6-dependent deacetylation of H3K9Ac peptide exhib- enzymes were subjected to steady-state kinetic analysis, and the ited 2–6 stimulation with 7 out of 8 FAs examined (Fig. 2A): rates of deacetylation, Dedodecanoylation, and demyristoyla- oleic (5.6  0.4 times) and linoleic acid (6.2  0.5), myristic tion were determined. The resulting kinetic analysis yielded (2.5  0.3), palmitic (2.8  0.2), stearic (2.1  0.4), -linolenic trends that were in excellent agreement with the qualitative (2.7  0.3), and -linolenic (2.6  0.3) acids. Dodecanoic acid TLC-based assay, indicating that the activities do not arise from was unable to stimulate SIRT6 activity, suggesting a minimum contaminating CobB (Fig. 1I). SIRT1–3 catalyzed deacetylation chain length for stimulating deacetylation. In stark contrast, more efficiently than long-chain deacylation, whereas SIRT6 SIRT1 displayed no effect with any FA on H3K9Ac deacetyla- exhibited the opposite trend. All the sirtuins displayed robust tion, suggesting that FA-stimulated deacetylation might be a long-chain deacylase activity, with SIRT3 harboring the great- unique feature of SIRT6. est activity toward H3K9Dodec and H3K9Myr peptides. SIRT3 Next, we determined the dose-dependent effects and the was 2-, 3-, and 9 more active as a dedodecanoylase than magnitude of the -fold activation by FFAs. Deacetylase activity SIRT1, SIRT2, and SIRT6, respectively. Likewise, SIRT3 was of SIRT6 was analyzed at various concentrations of myristic, 3.5-, 5-, and 6 more active as a demyristoylase. Collectively, oleic, and linoleic acids, at fixed H3K9Ac peptide (70 M) and these results indicate that in addition to SIRT6, other mamma- NAD (500 M). Myristic, oleic, and linoleic acid were all able lian sirtuins possess intrinsic deacylation activity toward long- to stimulate SIRT6 deacetylase activity in a concentration-de- chain acyl groups, although each sirtuin displays a unique sub- pendent manner (Fig. 2B). Myristic acid stimulated deacetylase strate signature among the 13 acylated versions tested here. activity by a factor of 10.8  0.2, with an EC of 246  7 M. The expanding identification of new acyl modifications and Oleic and linoleic acid stimulated SIRT6 activity to a maximum the ability of sirtuins to remove these suggest that other abun- -fold increase of 5.8  0.2 and 6.8  0.3 and yielded an EC of dant acyl-CoAs might serve as donor molecules for lysine post- 90 7 and 100 9 M, respectively. The EC values are within translational modification. The nutrient status of the cell may favor the addition of one acyl modification over another, which the range of postprandial levels of nonesterified FAs in mice might rise and fall as a function of [acyl-CoA]. Acyl-CoA (25) and humans (26). FIGURE 1. Deacylase activity of mammalian sirtuins. A, panel of acylated lysine peptides corresponding to amino acids 5–13 of histone H3 used in TLC assays. 32 32 B, TLC assay monitoring the formation of O-[ P]acylADPr products. SIRT6 (2 M) was incubated with 1 Ci of [ P]NAD (0.18 M)and5mM acylated peptide for1h. Unmod, unmodified. C–H, quantification of TLC assay monitoring O-[ P]acylADPr formation for SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6. Sirtuin (2 M) was incubated with 5 mM acylated peptide and 12.25 M NAD (1 Ci [ P]NAD ) for 1 h. *[SIRT4] 6 M. The percentage of NAD consumed was determined by 32  32 measuring the intensities of the [ P]NAD and O-[ P]acylADPr spots. Three sets of experiments (yellow, green, blue) were performed on three separate days, using multiple enzyme preparations and peptide stocks. red, reduced; ox, oxidized. I, quantitative steady-state rates of deacetylation (black), dedodecanoyl- ation (dark gray), and demyristoylation (light gray) for SIRT1–3 and SIRT6 determined by HPLC. Error bars represent S.D. of at least three replicates. OCTOBER 25, 2013• VOLUME 288 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 31353 REPORT: Activation of SIRT6 Deacetylase FIGURE 2. Sirtuin activity in the presence of free fatty acids. A, -fold change in SIRT1 and SIRT6 deacetylase activity was monitored in the presence of various FFAs and compared with a reaction without fatty acid. SIRT1 (dark gray) and SIRT6 (light gray) were incubated with 70 M H3K9Ac peptide and 0.5 mM NAD in the presence of 100 M fatty acid and analyzed by HPLC. B, -fold increase in SIRT6 deacetylase activity when 70 M H3K9ac peptide was incubated with 0.5 mM NAD and 0 –1 mM myristic (filled circles, solid line), 0 –300 M oleic (filled diamonds, long dashed lines), and linoleic acids (open circles, short dashed lines). C, critical micelle concentration determined by DPH (5 M) assay (23) in 20 mM potassium phosphate (pH 7.5)/6.7% DMSO in the presence of varied myristic (filled circle, solid line), oleic (open circle, dash dot line), and linoleic acids (filled diamonds, long dashed line). D, steady-state kinetic analyses of SIRT6 deacetylation of 0 –200 M H3K9Ac peptide in the presence of 2 mM NAD with (filled circles) and without (filled diamonds) 400 M myristic acid. E, percentage of demyristoylase activity of SIRT6 incubated with 70 M H3K9myr peptide, 0.5 mM NAD , and 0 –1 mM myristic acid. F, myristic acid inhibition of SIRT6 demyristoylase activity displayed in double-reciprocal format. Reactions contained 2 M SIRT6, 0.5 mM NAD , and varied H3K9Myr peptide in the presence of 0 (filled circles), 25 (open circles), 50 (dark gray triangles), 100 (light shaded circles), 150 (open diamonds), and 200 (inverted triangles) M myristic acid. Data were fitted (using nonlinear least squares) to the equation for competitive inhibition and yielded a K of 15  9 M. Error bars represent S.D. of at least three replicates. is To rule out the possibility that activation was due to micelle than a substantial increase in k . In the presence of myristic cat formation, the critical micelle concentration (CMC) for the acid, the K is 9 1 M, and in the absence of myristic acid, the FFAs was determined. The CMC for myristic acid is between K is estimated to be 450 M. Together, these results suggest 600 M and1mM (Fig. 2C). The CMC for oleic acid is between that FFAs stimulate the deacetylase activity of SIRT6 by 150 and 200 M, and the CMC for linoleic acid is between 200 increasing the affinity of SIRT6 for an acetylated substrate by 35 and 300 M. Thus, the CMC is beyond the concentration times in the case of myristic acid. needed for maximal activation of each FFA, suggesting that the To test the hypothesis that myristic acid binds in the same ability to saturate the stimulated deacetylation results from spe- site as the fatty acid chain of a myristoylated lysine peptide, cific binding between enzyme and FFA. SIRT6 demyristoylase activity was measured in the presence of To investigate the activation mechanism, we performed increasing myristic acid at fixed peptide substrate concentra- steady-state kinetic analyses with increasing H3K9Ac peptide tion. Myristic acid was able to inhibit the demyristoylase activ- at saturating levels of NAD in the absence or presence of 400 ity of SIRT6 with an IC of 190  10 M (Fig. 2E), a value in M myristic acid (Fig. 2D). The data were subjected to Michaelis- good agreement to the EC determined for myristic acid-stim- Menten analysis, and k /K values were compared. The ulated deacetylation (246  7 M) and consistent with myristic cat m k /K is considered the most physiologically relevant param- acid binding to a single site shared with myristoylated peptide. cat m eter because it reflects enzyme activity at low substrate levels, To provide additional evidence that myristic acid shares the mimicking conditions typically encountered in vivo (27). Strik- same binding site with the fatty acid chain of a myristoylated ingly, the k /K value for deacetylation with 400 M myristic peptide, a detailed steady-state inhibition analysis was per- cat m 1 1 acid (230  30 s M )is 35 times faster than that for formed. The concentration of H3K9Myr peptide was varied at 1 1 deacetylation in the absence of myristic acid (6.4  2s M ). several fixed concentrations of myristic acid. The data dis- Increased catalytic efficiency is due to a decrease in K rather played clear competitive behavior and were fitted to a model of 31354 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 43 •OCTOBER 25, 2013 REPORT: Activation of SIRT6 Deacetylase competitive inhibition and plotted in double-reciprocal format matic activities provide a roadmap to uncover the biological (Fig. 2F). The analysis yielded a K value of 15  9 M for functions of diverse protein acyl modifications and the role is myristic acid. The ability of myristic acid to competitively played by sirtuins. Also, we demonstrate that SIRT6 is activated inhibit demyristoylation is consistent with free myristic acid directly by biologically relevant FAs, several of which are linked sharing the same binding pocket with myristoylated peptide. to the health benefits of dietary PUFAs. Discovery of endoge- FFA binding likely induces a conformation in SIRT6, similar to nous, small-molecule activators of SIRT6 reveals the therapeu- a myristoylated peptide, allowing for efficient deacetylation. tic potential of compounds that promote SIRT6 function, The observation that FFAs stimulate deacetylation provides particularly anti-inflammation and decreased tumorigenesis. a mechanism by which the low intrinsic activity of SIRT6 might Lastly, the precedent for FA-regulated SIRT6 activity might be activated in vivo in response to elevated levels of particular apply to other sirtuins. The fact that SIRT1 through SIRT5 bind FAs, e.g. from the diet or from fasting. Fatty acids or their and efficiently act as (e.g.) dedecanoylases suggests the possibil- metabolites, through various signaling pathways, modulate ity that other sirtuins are regulated by FAs. numerous metabolic and inflammatory processes (28–30). For instance, omega-3 fatty acids, including eicosapentaenoic acid Acknowledgment—We thank Jorge Escalante for providing the (20:5, n-3) and docosahexaenoic acid (22:6, n-3), are reported to BL21(DE3) CobB E. coli strain. exert beneficial effects against cardiovascular disease and inflammation (29, 30). Similarly, SIRT6 protects the heart from REFERENCES cardiac hypertrophy by attenuating insulin-like growth factor- 1. Michan, S., and Sinclair, D. (2007) Sirtuins in mammals: insights into their Akt signaling through H3K9Ac deacetylation at the promoters biological function. Biochem. J. 404, 1–13 of insulin-like growth factor signaling-related genes (31). 2. 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Activation of the Protein Deacetylase SIRT6 by Long-chain Fatty Acids and Widespread Deacylation by Mammalian Sirtuins * ♦

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American Society for Biochemistry and Molecular Biology
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Copyright © 2013 Elsevier Inc.
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0021-9258
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10.1074/jbc.c113.511261
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REPORT THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 43, pp. 31350 –31356, October 25, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. but not SIRT1. The activation mechanism is consistent with Activation of the Protein fatty acid inducing a conformation that binds acetylated H3 with Deacetylase SIRT6 by Long-chain greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of Fatty Acids and Widespread discovering endogenous, small-molecule activators of SIRT6. Deacylation by Mammalian Sirtuins SIRT6 is one of seven members of the mammalian NAD - Received for publication, August 16, 2013, and in revised form, September 17, 2013 dependent protein deacetylases that are evolutionarily ancient Published, JBC Papers in Press, September 18, 2013, DOI 10.1074/jbc.C113.511261 ‡§ ‡§ ‡§1 Jessica L. Feldman , Josue Baeza , and John M. Denu (1). Recent studies indicate that SIRT6 plays critical roles in ‡ § From the Department of Biomolecular Chemistry and the Wisconsin intermediary metabolism and genomic stability (2). SIRT6-de- Institute for Discovery, University of Wisconsin, Madison, Wisconsin 53715 ficient mice have a striking degenerative phenotype leading to shortened lifespan and are associated with hypoglycemia and Background: Sirtuins regulate metabolism, genome defects in DNA repair (2). Deletion of SIRT6 is sufficient to maintenance, and stress responses. promote tumorigenesis independent of oncogene activation Results: Long-chain free fatty acids stimulate SIRT6 (3). SIRT6 overexpression lowers LDL and triglyceride levels, deacetylase, and sirtuins display distinct but overlapping improves glucose tolerance (4), and increases lifespan of male specificity for diverse acylated peptides. mice (5). SIRT6 also plays a role in inflammatory pathways, Conclusion: SIRT6 is activated by biologically relevant exerting anti-inflammatory effects at the transcriptional level. fatty acids, and long-chain deacylation is a general feature However, other studies suggest a pro-inflammatory effect on of sirtuins. intracellular signaling (6). Although it is clear that SIRT6 func- Significance: Discovery of endogenous, small-molecule tions in metabolism, inflammation, and genome maintenance, activators of SIRT6 demonstrates the therapeutic poten- its molecular functions remain enigmatic. tial of compounds that promote SIRT6 function. SIRT6 is grouped among the mammalian sirtuins (including SIRT4, SIRT5, and SIRT7) that display extremely low deacety- Mammalian sirtuins (SIRT1 through SIRT7) are members of a lase activity in vitro (7). SIRT1–3 display robust protein highly conserved family of NAD -dependent protein deacety- deacetylase activity, and numerous studies have identified in lases that function in metabolism, genome maintenance, and vivo substrates (35, 36). The recent observation that SIRT5 pre- stress responses. Emerging evidence suggests that some sirtuins fers succinylated/malonylated substrates helps explain the display substrate specificity toward other acyl groups attached observed low activity on acetylated substrates (8). Additional to the lysine -amine. SIRT6 was recently reported to preferen- acyl-lysine modifications have been identified in histone and tially hydrolyze long-chain fatty acyl groups over acetyl groups. non-histone proteins, including propionyl, butyryl, myristoyl, Here we investigated the catalytic ability of all sirtuins to hydro- and crotonyl (8–11), suggesting the existence of a very diverse lyze 13 different acyl groups from histone H3 peptides, ranging acylation landscape in cells. in carbon length, saturation, and chemical diversity. We find These observations have led the field to postulate that some that long-chain deacylation is a general feature of mammalian sirtuins harbor unique acyl group specificities, distinct from sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, deacetylase activity (7, 12). Consistent with this idea, Jiang et al. and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic (13) demonstrated recently that SIRT6 preferentially hydro- acid. These results provide new insight into sirtuin function and lyzes long-chain fatty acyl groups, including myristoyl and a means for cellular removal of an expanding list of endogenous palmitoyl groups, from lysine residues. A crystal structure of lysine modifications. Given that SIRT6 is a poor deacetylase in SIRT6 bound to a myristoylated peptide supported this obser- vitro, but binds and prefers to hydrolyze long-chain acylated vation. However, several prior studies indicated that SIRT6 peptides, we hypothesize that binding of certain free fatty acids acted through deacetylation of histone marks H3K9Ac (14) and (FFAs) could stimulate deacetylation activity. Indeed, we dem- H3K56Ac (15), which modulated HIF1--dependent (16), onstrate that several biologically relevant FFAs (including my- NF-B-dependent (17), and c-Myc-dependent (3) pathways. In ristic, oleic, and linoleic acids) at physiological concentrations cells, the ability of SIRT6 to promote deacetylation of histones induce up to a 35-fold increase in catalytic efficiency of SIRT6 and non-histone proteins (18) seemed at odds with the low in vitro deacetylation rates (13, 19) and with the recently * This work was supported, in whole or in part, by National Institutes of Health described demyristoylation activity (13). X-ray structural anal- Grant GM065386 (to J. M. D.), a predoctoral fellowship (to J. L. F.) from the ysis of SIRT6 suggested that the low deacetylase activity stems American Heart Association (11PRE7300059) and a predoctoral fellowship from a splayed configuration between the Rossmann fold (to J. B.) from the National Science Foundation (1256259). This article was selected as a Paper of the Week. domain and the zinc binding subdomain (19). Given this unique □ S This article contains supplemental Methods. conformation, we proposed that SIRT6 could be activated if To whom correspondence should be addressed: University of Wisconsin, the two domains could be induced to the canonical active-site 330 N. Orchard St., WID 2178, Madison, WI 53715. Tel.: 608-265-1859; Fax: 608-262-5253; E-mail: [email protected]. conformation (19). 31350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 43 •OCTOBER 25, 2013 This is an Open Access article under the CC BY license. REPORT: Activation of SIRT6 Deacetylase To provide molecular understanding of the unique catalytic NAD were incubated with 15–220 M H3K9Myr peptide in functions of SIRT6 and to resolve the apparent contradictory the presence of 0–200 M myristic acid. Data were fitted to an results discussed above, we addressed two major questions: 1) Is equation for competitive inhibition using KinetAsyst (Intelli- long-chain protein deacylation a general feature of mammalian Kinetics, State College, PA). sirtuins? and 2) Are there endogenous SIRT6 activators that RESULTS AND DISCUSSION stimulate deacetylation activity? Long-chain Deacylase Activity Is an Intrinsic Activity of Most EXPERIMENTAL PROCEDURES Sirtuins—It was recently reported that SIRT6 preferentially Expression and Purification of Recombinant SIRT1-SIRT7— hydrolyzes long-chain fatty acyl (myristoyl and palmitoyl His-tagged SIRT1–7 were overexpressed in BL21(DE3) or groups), relative to deacetylation (13). Here we investigated BL21(DE3) CobB Escherichia coli strains and purified as whether such long-chain deacylation activity was specific to described (19, 20). SIRT6 or whether this is a general feature of mammalian sir- [ P]NAD TLC Assay—Assays were performed in 20 mM tuins. We synthesized a panel of acylated lysine peptides corre- HEPES, pH 7.4, at 25 °C using [ P]NAD (American Radio- sponding to amino acids 5–13 of histone H3 containing diverse labeled Chemicals). Products were resolved by TLC and acyl--lysine modifications, including acetyl, propionyl, succi- quantified using a Typhoon FLA 9500 (GE Healthcare) (12). nyl, butyryl, crotonyl, hexanoyl, octanoyl, decanoyl, dode- TLC plates were analyzed by ImageQuant TL (GE Health- canoyl, myristoyl, or palmitoyl groups on Lys-9 (see supple- care). Intensities were determined for [ P]NAD and mental Methods) (Fig. 1A). Human sirtuins 1 through 7 32 2 O-[ P]acylADPr using spot edge average as a local back- (SIRT1–7) were recombinantly expressed and purified, and ground subtraction method. their abilities to hydrolyze each acyl group were determined. HPLC Deacylation Assay—Deacylation reactions were ana- First, the activity of SIRT6 was assessed using an excess of pep- 32  32 lyzed by reversed phase HPLC on a Kinetex C18 column (100A, tide (5 mM) and limiting [ P]NAD (0.18 M). The [ P]NAD 100  4.6 mm, 2.6-m, Phenomenex) by monitoring the for- substrate and the various O-[ P]acylADPr products were sep- mation of deacylated product at 214 nm, as described (21). Sir- arated by TLC and visualized by phosphorimaging (Fig. 1B). tuin (0.2 M)or0.8 M SIRT6 was incubated with 40 M SIRT6 displayed a clear preference for longer acyl chains, H3K9Ac, H3K9Dodec, or H3K9Myr in the presence of 0.5 mM including myristoyl and palmitoyl chains, consuming 98 and NAD and 3% DMSO at 37 °C. Product and substrate peaks 81% of the [ P]NAD (Fig. 1B), respectively, consistent with a were quantified, and deacylation rates were determined. prior study (13). In addition, SIRT6 was able to deacylate the Deacetylase Activity in the Presence of Fatty Acids (FAs)— decanoyl (96%) and dodecanoyl (98%) groups from lysine resi- SIRT6 (4 M)or2 M SIRT1 were incubated with 70 M dues. To a lesser degree, SIRT6 was able to hydrolyze the hex- H3K9Ac peptide, 0.5 mM NAD , 100 M FA (dodecanoic, my- anoyl and octanoyl groups, consuming 23 and 48% of the ristic, palmitic, oleic, linoleic, -linolenic, -linolenic). Reac- [ P]NAD , respectively. Under these assay conditions, SIRT6 tions were incubated for either 1 h (SIRT6) or 15 s (SIRT1) and was unable to remove detectable amounts of the acetyl group. quenched with TFA. To examine dose dependence, FA concen- Next, we analyzed the deacylase activity of SIRT1 through trations were varied from 0 to 1 mM for myristic acid and 0–300 SIRT7 using catalytic amounts of enzyme and a more expansive M for oleic and linoleic acid. FAs were incubated with 70 M panel of acylated H3K9 peptides, including acetyl, propionyl, H3K9Ac peptide, 0.5 mM NAD , and 4 M SIRT6 at 37 °C. succinyl, butyryl, crotonyl, hexanoyl, octanoyl, decanoyl, dode- Steady-state Kinetic Analyses—Steady-state rates were mea- canoyl, myristoyl, palmitoyl, and reduced and oxidized lipoyl sured by varying H3K9Ac peptide (0–200 M) in the presence groups. No appreciable amounts of products were observed of 0.9 M SIRT6 and 2 mM NAD  400 M myristic acid. with SIRT7. As noted in the prior experiment (Fig. 1B), SIRT6 Initial velocities were determined; data in the presence of myr- displayed a strong preference for long-chain fatty acyl groups istic acid were fitted to the Michaelis-Menten equation, and (Fig. 1C). Under these conditions, octanoyl, decanoyl, dode- data in the absence of myristic acid were fitted to a modified canoyl, and myristoyl groups were preferentially hydrolyzed. version of the equation (22). The corresponding O-[ P]acylADPr products from hexanoyl Critical Micelle Concentration—-Fold increase in fluores- and palmitoyl were detectable, but the other acyl groups did not cence of 1,6-dipheynl-1,3,5-hexatriene (DPH) was monitored accumulate to quantifiable amounts. in the presence of increasing concentration of FFAs (23). Reac- Surprisingly, SIRT1, SIRT2, SIRT3, and SIRT5 also displayed tions contained 20 mM potassium phosphate (pH 7.5), 6.7% strong deacylation activity against long-chain acyl groups. Par- DMSO, 5 M DPH, and 0–2 mM myristic acid or 0–1.5 mM ticularly striking was the overall similar preference among these oleic and linoleic acid at 37 °C. sirtuins for decanoyl and dodecanoyl, which was shared with Myristic Acid Inhibition Kinetics—SIRT6 (2 M) was incu- SIRT6 (Fig. 1, C–H). As expected, SIRT1–3 efficiently hydro- bated with 70 M H3K9Myr peptide in the presence of 0.5 mM lyzed the acetyl group from lysine (75% conversion), and to a NAD and myristic acid (0–1 mM) for 1.5 min at 37 °C. To lesser extent, the propionyl and butyryl groups (Fig. 1, D–F). determine the inhibition constant, K ,2 M SIRT6 and 0.5 mM SIRT5 was most efficient at catalyzing desuccinylation (95% is conversion) (Fig. 1H), as was reported previously (8). However, SIRT5 more efficiently hydrolyzed (2–3) decanoyl and dode- The abbreviations used are: O-acylADPr, O-acyl-ADP-ribose; FA, fatty acid; canoyl peptides as compared with the corresponding acetylated PUFA, polyunsaturated fatty acid; DPH, 1,6-dipheynl-1,3,5-hexatriene; CMC, critical micelle concentration; DMSO, dimethyl sulfoxide. form, which was hydrolyzed faster than the propionyl, octanoyl, OCTOBER 25, 2013• VOLUME 288 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 31351 REPORT: Activation of SIRT6 Deacetylase 31352 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 43 •OCTOBER 25, 2013 REPORT: Activation of SIRT6 Deacetylase and myristoyl groups. At 3 the protein concentration, SIRT4 donors are intermediates in different central metabolic path- displayed low, but measureable amounts of deoctanoylase and ways, including glycolysis, the TCA cycle, -oxidation, and FA dedecanoylase activity (Fig. 1G). synthesis (7). Many of these modifications occur on metabolic Crotonyllysine was identified as a histone modification in enzymes, suggesting a regulatory link between metabolism and several eukaryotic cells types and is associated with active pro- lysine acylation. Our results suggest that sirtuins are well suited moters or enhancers (11). A product corresponding to [ P]O- to function as the demodifying enzymes of such acylations. crotonylADPr was observed when either SIRT1 or SIRT2 was SIRT6 Deacetylase Activity Is Stimulated by Long-chain reacted with NAD and the crotonylated H3K9 peptide, con- FFAs—The fact that sirtuins other than SIRT6 displayed higher suming 20% of the [ P]NAD (Fig. 1, D and E). specific activity against long-chain acyl groups was surprising, Among sirtuins displaying quantifiable activity, SIRT1 dis- especially given the prior observation of a co-crystal structure played the greatest promiscuity for diverse acyl groups, except with bound myristoylated peptide. This structure revealed a for succinylated substrate, which neither SIRT1, SIRT2, nor long acyl chain binding cleft. Comparing SIRT6 crystal struc- SIRT6 hydrolyzed. SIRT3 and SIRT4 also exhibited activity tures with bound and unbound myristoylated peptide revealed against the succinylated substrate, but to a lesser extent than conformational differences, adopting a more canonical sirtuin SIRT5 (Fig. 1, C–H). Interestingly, a specific mammalian deli- fold when myristoylated peptide is bound (13, 19). We hypoth- poylase has not been identified (24). Here, SIRT2, and to a lesser esized that this acyl group binding pocket might bind FFAs, and extent, SIRT1, SIRT3, and SIRT4 were able to hydrolyze the if such binding could induce closure of the splayed SIRT6 sub- removal of the lipoyl group from lysine (Fig. 1, D–G). Impor- domains, then SIRT6 might show stimulated deacetylation tantly, SIRT1–3 displayed more efficient conversion of many activity. To determine whether FFAs can stimulate SIRT6 long-chain acylated peptides as compared with SIRT6. Even deacetylase activity in vitro, SIRT6 was incubated with 70 M SIRT5, an enzyme reported to harbor exquisite selectivity for H3K9Ac peptide, 0.5 mM NAD , and 100 M dodecanoic, my- succinylated peptide, hydrolyzed long-chain acyl groups with ristic, palmitic, stearic, oleic (18:1, n-9), linoleic (18:2, n-6), similar efficiency to that of SIRT6. -linolenic (18:3, n-6), or -linolenic (18:3, n-3) acids. Separate To obtain true steady-state rates of deacylation and to ensure experiments were performed with SIRT1 for comparison. that the deacylase activity observed in the above experiments Reaction substrates and products were separated by HPLC and was not due to contamination by the E. coli sirtuin CobB, quantified. Specific activities of SIRT6 and SIRT1 in the pres- SIRT1, SIRT2, SIRT3, and SIRT6 were overexpressed and puri- ence of different FAs were compared. fied from a CobB knock-out BL21(DE3) strain. The purified SIRT6-dependent deacetylation of H3K9Ac peptide exhib- enzymes were subjected to steady-state kinetic analysis, and the ited 2–6 stimulation with 7 out of 8 FAs examined (Fig. 2A): rates of deacetylation, Dedodecanoylation, and demyristoyla- oleic (5.6  0.4 times) and linoleic acid (6.2  0.5), myristic tion were determined. The resulting kinetic analysis yielded (2.5  0.3), palmitic (2.8  0.2), stearic (2.1  0.4), -linolenic trends that were in excellent agreement with the qualitative (2.7  0.3), and -linolenic (2.6  0.3) acids. Dodecanoic acid TLC-based assay, indicating that the activities do not arise from was unable to stimulate SIRT6 activity, suggesting a minimum contaminating CobB (Fig. 1I). SIRT1–3 catalyzed deacetylation chain length for stimulating deacetylation. In stark contrast, more efficiently than long-chain deacylation, whereas SIRT6 SIRT1 displayed no effect with any FA on H3K9Ac deacetyla- exhibited the opposite trend. All the sirtuins displayed robust tion, suggesting that FA-stimulated deacetylation might be a long-chain deacylase activity, with SIRT3 harboring the great- unique feature of SIRT6. est activity toward H3K9Dodec and H3K9Myr peptides. SIRT3 Next, we determined the dose-dependent effects and the was 2-, 3-, and 9 more active as a dedodecanoylase than magnitude of the -fold activation by FFAs. Deacetylase activity SIRT1, SIRT2, and SIRT6, respectively. Likewise, SIRT3 was of SIRT6 was analyzed at various concentrations of myristic, 3.5-, 5-, and 6 more active as a demyristoylase. Collectively, oleic, and linoleic acids, at fixed H3K9Ac peptide (70 M) and these results indicate that in addition to SIRT6, other mamma- NAD (500 M). Myristic, oleic, and linoleic acid were all able lian sirtuins possess intrinsic deacylation activity toward long- to stimulate SIRT6 deacetylase activity in a concentration-de- chain acyl groups, although each sirtuin displays a unique sub- pendent manner (Fig. 2B). Myristic acid stimulated deacetylase strate signature among the 13 acylated versions tested here. activity by a factor of 10.8  0.2, with an EC of 246  7 M. The expanding identification of new acyl modifications and Oleic and linoleic acid stimulated SIRT6 activity to a maximum the ability of sirtuins to remove these suggest that other abun- -fold increase of 5.8  0.2 and 6.8  0.3 and yielded an EC of dant acyl-CoAs might serve as donor molecules for lysine post- 90 7 and 100 9 M, respectively. The EC values are within translational modification. The nutrient status of the cell may favor the addition of one acyl modification over another, which the range of postprandial levels of nonesterified FAs in mice might rise and fall as a function of [acyl-CoA]. Acyl-CoA (25) and humans (26). FIGURE 1. Deacylase activity of mammalian sirtuins. A, panel of acylated lysine peptides corresponding to amino acids 5–13 of histone H3 used in TLC assays. 32 32 B, TLC assay monitoring the formation of O-[ P]acylADPr products. SIRT6 (2 M) was incubated with 1 Ci of [ P]NAD (0.18 M)and5mM acylated peptide for1h. Unmod, unmodified. C–H, quantification of TLC assay monitoring O-[ P]acylADPr formation for SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6. Sirtuin (2 M) was incubated with 5 mM acylated peptide and 12.25 M NAD (1 Ci [ P]NAD ) for 1 h. *[SIRT4] 6 M. The percentage of NAD consumed was determined by 32  32 measuring the intensities of the [ P]NAD and O-[ P]acylADPr spots. Three sets of experiments (yellow, green, blue) were performed on three separate days, using multiple enzyme preparations and peptide stocks. red, reduced; ox, oxidized. I, quantitative steady-state rates of deacetylation (black), dedodecanoyl- ation (dark gray), and demyristoylation (light gray) for SIRT1–3 and SIRT6 determined by HPLC. Error bars represent S.D. of at least three replicates. OCTOBER 25, 2013• VOLUME 288 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 31353 REPORT: Activation of SIRT6 Deacetylase FIGURE 2. Sirtuin activity in the presence of free fatty acids. A, -fold change in SIRT1 and SIRT6 deacetylase activity was monitored in the presence of various FFAs and compared with a reaction without fatty acid. SIRT1 (dark gray) and SIRT6 (light gray) were incubated with 70 M H3K9Ac peptide and 0.5 mM NAD in the presence of 100 M fatty acid and analyzed by HPLC. B, -fold increase in SIRT6 deacetylase activity when 70 M H3K9ac peptide was incubated with 0.5 mM NAD and 0 –1 mM myristic (filled circles, solid line), 0 –300 M oleic (filled diamonds, long dashed lines), and linoleic acids (open circles, short dashed lines). C, critical micelle concentration determined by DPH (5 M) assay (23) in 20 mM potassium phosphate (pH 7.5)/6.7% DMSO in the presence of varied myristic (filled circle, solid line), oleic (open circle, dash dot line), and linoleic acids (filled diamonds, long dashed line). D, steady-state kinetic analyses of SIRT6 deacetylation of 0 –200 M H3K9Ac peptide in the presence of 2 mM NAD with (filled circles) and without (filled diamonds) 400 M myristic acid. E, percentage of demyristoylase activity of SIRT6 incubated with 70 M H3K9myr peptide, 0.5 mM NAD , and 0 –1 mM myristic acid. F, myristic acid inhibition of SIRT6 demyristoylase activity displayed in double-reciprocal format. Reactions contained 2 M SIRT6, 0.5 mM NAD , and varied H3K9Myr peptide in the presence of 0 (filled circles), 25 (open circles), 50 (dark gray triangles), 100 (light shaded circles), 150 (open diamonds), and 200 (inverted triangles) M myristic acid. Data were fitted (using nonlinear least squares) to the equation for competitive inhibition and yielded a K of 15  9 M. Error bars represent S.D. of at least three replicates. is To rule out the possibility that activation was due to micelle than a substantial increase in k . In the presence of myristic cat formation, the critical micelle concentration (CMC) for the acid, the K is 9 1 M, and in the absence of myristic acid, the FFAs was determined. The CMC for myristic acid is between K is estimated to be 450 M. Together, these results suggest 600 M and1mM (Fig. 2C). The CMC for oleic acid is between that FFAs stimulate the deacetylase activity of SIRT6 by 150 and 200 M, and the CMC for linoleic acid is between 200 increasing the affinity of SIRT6 for an acetylated substrate by 35 and 300 M. Thus, the CMC is beyond the concentration times in the case of myristic acid. needed for maximal activation of each FFA, suggesting that the To test the hypothesis that myristic acid binds in the same ability to saturate the stimulated deacetylation results from spe- site as the fatty acid chain of a myristoylated lysine peptide, cific binding between enzyme and FFA. SIRT6 demyristoylase activity was measured in the presence of To investigate the activation mechanism, we performed increasing myristic acid at fixed peptide substrate concentra- steady-state kinetic analyses with increasing H3K9Ac peptide tion. Myristic acid was able to inhibit the demyristoylase activ- at saturating levels of NAD in the absence or presence of 400 ity of SIRT6 with an IC of 190  10 M (Fig. 2E), a value in M myristic acid (Fig. 2D). The data were subjected to Michaelis- good agreement to the EC determined for myristic acid-stim- Menten analysis, and k /K values were compared. The ulated deacetylation (246  7 M) and consistent with myristic cat m k /K is considered the most physiologically relevant param- acid binding to a single site shared with myristoylated peptide. cat m eter because it reflects enzyme activity at low substrate levels, To provide additional evidence that myristic acid shares the mimicking conditions typically encountered in vivo (27). Strik- same binding site with the fatty acid chain of a myristoylated ingly, the k /K value for deacetylation with 400 M myristic peptide, a detailed steady-state inhibition analysis was per- cat m 1 1 acid (230  30 s M )is 35 times faster than that for formed. The concentration of H3K9Myr peptide was varied at 1 1 deacetylation in the absence of myristic acid (6.4  2s M ). several fixed concentrations of myristic acid. The data dis- Increased catalytic efficiency is due to a decrease in K rather played clear competitive behavior and were fitted to a model of 31354 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 43 •OCTOBER 25, 2013 REPORT: Activation of SIRT6 Deacetylase competitive inhibition and plotted in double-reciprocal format matic activities provide a roadmap to uncover the biological (Fig. 2F). The analysis yielded a K value of 15  9 M for functions of diverse protein acyl modifications and the role is myristic acid. The ability of myristic acid to competitively played by sirtuins. Also, we demonstrate that SIRT6 is activated inhibit demyristoylation is consistent with free myristic acid directly by biologically relevant FAs, several of which are linked sharing the same binding pocket with myristoylated peptide. to the health benefits of dietary PUFAs. Discovery of endoge- FFA binding likely induces a conformation in SIRT6, similar to nous, small-molecule activators of SIRT6 reveals the therapeu- a myristoylated peptide, allowing for efficient deacetylation. tic potential of compounds that promote SIRT6 function, The observation that FFAs stimulate deacetylation provides particularly anti-inflammation and decreased tumorigenesis. a mechanism by which the low intrinsic activity of SIRT6 might Lastly, the precedent for FA-regulated SIRT6 activity might be activated in vivo in response to elevated levels of particular apply to other sirtuins. 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Journal

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

Published: Oct 25, 2013

Keywords: Gene Regulation; Histone Deacetylase; Metabolism; Polyunsaturated Fatty Acids; Post-translational Modification; Sirtuins; coA; Enzymology; Fatty Acids

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