SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine

Abstract

The Sir2 family of enzymes or sirtuins are known as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases and have been implicated in the regulation of transcription, genome stability, metabolism and lifespan. However, four of the seven mammalian sirtuins have very weak deacetylase activity in vitro. Here we show that human SIRT6 efficiently removes long-chain fatty acyl groups, such as myristoyl, from lysine residues. The crystal structure of SIRT6 reveals a large hydrophobic pocket that can accommodate long-chain fatty acyl groups. We demonstrate further that SIRT6 promotes the secretion of tumour necrosis factor-α (TNF-α) by removing the fatty acyl modification on K19 and K20 of TNF-α. Protein lysine fatty acylation has been known to occur in mammalian cells, but the function and regulatory mechanisms of this modification were unknown. Our data indicate that protein lysine fatty acylation is a novel mechanism that regulates protein secretion. The discovery of SIRT6 as an enzyme that controls protein lysine fatty acylation provides new opportunities to investigate the physiological function of a protein post-translational modification that has been little studied until now.

Figure 1

Sirt6 prefers to hydrolyze long chain fatty acyl lysine in vitro. (A) HPLC traces showing Sirt6-catalyzed hydrolysis of different acyl peptides based on the H3K9 sequence. (B) H2B K12 myristoyl peptide can be hydrolyzed by Sirt6 while the corresponding acetyl peptide cannot. Reactions were carried out with 50 μM peptide, 1 μM Sirt6, 20 mM Tris pH 8.0, 0.5 mM NAD, and 1 mM DTT at 37°C for 30 min.

Figure 2

Structure basis for Sirt6 activity with long chain fatty acyl groups. (A) Overall structure of Sirt6 with myristoyl H3K9 (Myr-H3K9, green) peptide and ADP-ribose (ADPR, yellow) bound. (B) Hydrophobic residues in Sirt6 that accommodate the myristoyl (Myr) group.

Figure 3

Sirt6 regulates TNFα fatty acylation and secretion. (A) Method of using Alk14 to detect TNFα fatty acylation. (B) Sirt6 controls TNFα fatty acylation on K19 and K20. (C) H133 of Sirt6 is required for TNFα defatty-acylation. (D) Sirt6 defatty-acylates TNFα in vitro. (E) Sirt6 regulates secretion of TNFα in MEF cells. n = 6; ***P < 0.001. (F-I) Sirt6 regulates fatty acylation level and secretion of endogenous TNFα in THP-1 cells (F and G; n = 3; **P < 0.01) and bone marrow-derived macrophages (H and I; n = 5; *P < 0.02). Secretion data were expressed as mean ± sd.