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

Abstract

Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD(+)-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ε-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6.

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

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. B, TLC assay monitoring the formation of O-[³²P]acylADPr products. SIRT6 (2 μm) was incubated with 1 μCi of [³²P]NAD⁺ (0.18 μm) and 5 mm acylated peptide for 1 h. Unmod, unmodified. C–H, quantification of TLC assay monitoring O-[³²P]acylADPr formation for SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6. Sirtuin (2 μm) was incubated with 5 mm acylated peptide and 12.25 μm NAD⁺ (1 μCi [³²P]NAD⁺) for 1 h. *[SIRT4] = 6 μm. The percentage of NAD⁺ consumed was determined by measuring the intensities of the [³²P]NAD⁺ and O-[³²P]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), dedodecanoylation (dark gray), and demyristoylation (light gray) for SIRT1–3 and SIRT6 determined by HPLC. Error bars represent S.D. of at least three replicates.

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 μm H3K9Ac peptide and 0.5 mm NAD⁺ in the presence of 100 μm fatty acid and analyzed by HPLC. B, -fold increase in SIRT6 deacetylase activity when 70 μm H3K9ac peptide was incubated with 0.5 mm NAD⁺ and 0–1 mm myristic (filled circles, solid line), 0–300 μm oleic (filled diamonds, long dashed lines), and linoleic acids (open circles, short dashed lines). C, critical micelle concentration determined by DPH (5 μm) 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 μm H3K9Ac peptide in the presence of 2 mm NAD⁺ with (filled circles) and without (filled diamonds) 400 μm myristic acid. E, percentage of demyristoylase activity of SIRT6 incubated with 70 μm 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 μm 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) μm myristic acid. Data were fitted (using nonlinear least squares) to the equation for competitive inhibition and yielded a K_is of 15 ± 9 μm. Error bars represent S.D. of at least three replicates.