Tier 4 — mechanistic

Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins

Jessica L. Feldman, Josue Baeza, John M. Denu
Journal of Biological Chemistry 2013 Volume 288, issue 43, pages 31350–31356

Bibliography

PubMed
PMID 24052263
PubMed Central
PMC3829447
DOI
10.1074/jbc.C113.511261
Funding
National Institutes of Health Grant GM065386 (to J.M.D.); American Heart Association predoctoral fellowship 11PRE7300059 (to J.L.F.); National Science Foundation predoctoral fellowship 1256259 (to J.B.).
Competing interests
Not stated in the manuscript text

Study snapshot

DesignIn vitro deacylation profiling of recombinant human sirtuins (SIRT1 through SIRT7) against a synthetic panel of H3K9 peptides bearing 13 different acyl groups; quantitative HPLC steady-state kinetics; determination of fatty acid EC50 and inhibition constants; critical micelle concentration control experiments.
ModelRecombinant human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7 purified from BL21(DE3) or BL21(DE3) CobB knockout E. coli strains.
SampleThree or more replicates per condition for all reported specific activities; kinetic parameters derived from full concentration-response curves.
InterventionH3K9 peptides bearing acetyl, propionyl, succinyl, butyryl, crotonyl, hexanoyl, octanoyl, decanoyl, dodecanoyl, myristoyl, palmitoyl, or reduced/oxidised lipoyl groups; free fatty acids dodecanoic, myristic, palmitic, stearic, oleic (18:1 n-9), linoleic (18:2 n-6), γ-linolenic (18:3 n-6), α-linolenic (18:3 n-3) at 0–1 mM.
DurationIndividual reactions 15 s to 1 h depending on assay.
EndpointsDeacylation rates for each sirtuin against each of 13 acyl substrates; fold-activation of SIRT6 deacetylation by each fatty acid; EC50, IC50, Kis, and Michaelis-Menten parameters (kcat, Km, kcat/Km) for SIRT6 in the presence and absence of fatty acids.

What the study showed, in plain terms

This paper made two connected discoveries. First, most mammalian Sirtuin enzymes (SIRT1 through SIRT5) can remove long-chain fatty acid groups from lysine residues, not just acetyl groups. Second, and more consequentially for SIRT6 biology, free fatty acids can directly activate SIRT6 as a histone deacetylase.

The authors built a panel of 13 differently-modified histone H3 peptides — acetyl, succinyl, crotonyl, lipoyl, and a series of fatty acid chains from six to sixteen carbons long — and tested each sirtuin for its ability to remove each modification. SIRT6 preferred long-chain fatty acid groups over acetyl, as previously reported. But SIRT1, SIRT2, and SIRT3, which are known as strong deacetylases, could also remove long-chain fatty acids efficiently, and SIRT1 and SIRT2 also removed crotonyl groups. SIRT1 through SIRT4 could also remove lipoyl modifications, activity that had not been characterised in mammals before.

Testing free fatty acids on SIRT6 revealed dose-dependent activation. Oleic acid and linoleic acid — abundant dietary fatty acids that circulate at high concentrations after a meal — activated SIRT6 five- to seven-fold with EC50 values of approximately 90 and 100 μM, within the physiological range. Myristic acid activated more strongly (up to about 11-fold) but required higher concentrations. Detailed kinetics showed the fatty acids improved SIRT6's efficiency approximately 35-fold, principally by tightening its grip on the acetylated substrate. Free fatty acids and myristoylated peptide competed for the same binding site, consistent with the activation working through the acyl-binding channel identified in earlier crystal structures.

The authors propose that dietary or fasting-induced changes in circulating fatty acid levels could act as endogenous activation signals for SIRT6 — a plausible molecular link between nutrient status and the metabolic and inflammatory programmes SIRT6 regulates.

Key findings

  • Long-chain fatty acid deacylation is a general property of mammalian sirtuins: SIRT1, SIRT2, SIRT3, and SIRT5 all removed decanoyl and dodecanoyl groups from H3K9 peptides at rates comparable to or greater than SIRT6.
  • SIRT3 was the most active dedodecanoylase and demyristoylase of the tested sirtuins, exceeding SIRT6's activity on H3K9myr by roughly six-fold in quantitative HPLC kinetics.
  • SIRT1 and SIRT2 acted as decrotonylases, and SIRT1 through SIRT4 could remove lipoic acid groups from lysine — activities not previously characterised in mammalian sirtuins.
  • Seven of eight tested free fatty acids stimulated SIRT6 deacetylase activity 2- to 6-fold: myristic, palmitic, stearic, oleic, linoleic, γ-linolenic, and α-linolenic acids; dodecanoic acid did not activate, indicating a minimum chain-length requirement.
  • Oleic and linoleic acids activated SIRT6 with EC50 values of approximately 90 μM and 100 μM respectively — concentrations within the postprandial physiological range of circulating non-esterified fatty acids in humans and mice.
  • Myristic acid stimulated SIRT6 up to 10.8-fold at 246 μM EC50; critical micelle concentration measurements confirmed activation was due to specific binding rather than micelle formation.
  • Steady-state kinetics showed myristic acid increased the kcat/Km for H3K9Ac deacetylation approximately 35-fold, driven primarily by a decrease in Km (from around 450 μM to 9 μM) rather than an increase in kcat.
  • Myristic acid inhibited SIRT6 demyristoylation competitively with respect to myristoylated peptide (Kis approximately 15 μM), confirming that free fatty acids and long-chain acyl substrates share the same binding site.
  • None of the tested free fatty acids activated SIRT1 deacetylation of H3K9Ac, suggesting fatty acid activation may be a unique feature of SIRT6 among the mammalian sirtuins.

What this study can and cannot tell us

All experiments were performed on recombinant sirtuins with synthetic H3K9 peptide substrates. Physiological substrates (nucleosomes, full-length histones, or non-histone acetylated proteins) were not tested. Whether fatty acid activation operates on nucleosomal SIRT6 in intact chromatin remains untested.

Cellular and in vivo relevance is inferred, not demonstrated. No cell or animal experiments were performed to show that dietary or fasting-induced changes in circulating fatty acid levels actually activate SIRT6-dependent histone deacetylation in tissues.

The activating fatty acid concentrations (EC50 approximately 90–246 μM) approach the critical micelle concentration for the same lipids. The authors performed appropriate micelle controls, but the effective concentration of monomeric fatty acid at maximum activation is uncertain in complex biological media containing serum albumin, membranes, and lipoproteins.

The finding that SIRT1, SIRT2, and SIRT3 also efficiently remove long-chain fatty acyl groups from H3K9 peptides in vitro raises questions about substrate specificity in cells that this paper does not resolve. Whether these activities operate on real cellular substrates or which sirtuin is the dominant defatty-acylase for any given lysine site was not addressed.

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