Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH

Andreas S Madsen,Christian Andersen,Mohammad Daoud,Kristin A Anderson,Jonas S Laursen,Saswati Chakladar,Frank K Huynh,Ana R Colaço,Donald S Backos,Peter Fristrup,Matthew D Hirschey,Christian A Olsen

doi:10.1074/jbc.m115.668699

Abstract

Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD(+)as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD(+)/NADH ratio, but it has not been formally tested for all the mammalian sirtuinsin vitro To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ϵ-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C1) to palmitoyl (C16) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, "long chain" acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1-3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD(+)-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuinsin vivo These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD(+)/NADH ratio.

Highlights

Acyllysine modifications were shown to be prone to hydrolytic cleavage by SIRT1–3 and SIRT6, supporting recent findings
We modeled the predicted interaction of SIRT3 with both the ⑀-Nacetyllysine that was contained within the original crystal structure and that of an ⑀-N-myristoyllysine and found that both acyl groups could be accommodated in the hydrophobic pocket of the enzyme (Fig. 7A)
We have prepared and evaluated chemical tool compounds containing ⑀-N-acylated lysine residues containing acyl groups ranging from formyl (C1) to palmitoyl (C16) in length as well as dicarboxyl-derived substrates

Summary

The abbreviations used are

Kac, ⑀-N-acetyllysine; aa, amino acids; AMC, 7amino-4-methylcoumarin; ADPR, adenosine diphosphate ribose; NAM, nicotinamide; HDAC, histone deacetylase. Sensitivity of Sirtuin Deacylation to NADH [19, 20, 22, 35], whereas ⑀-N-crotonyllysine has been cleaved by HDAC3 [23, 36], SIRT1 [35, 37], and SIRT2 [37] in vitro. Unlike the Zn2ϩ-dependent HDACs, the sirtuins uniquely use NADϩ as a co-substrate in their deacylation reactions [42] Because of this unique enzymatic requirement, sirtuin activity has been linked to the energetic status of the cell, and the mammalian sirtuins have been described as sensors of the NADϩ/ NADH ratio [43,44,45,46,47]. We first established a range of sirtuin-mediated lysine deacylation reactions using three different in vitro assays. The data presented here have important implications for the fundamental understanding of sirtuin activity as well as the wide scope of biology they regulate

Experimental Procedures

Results

72 Ϯ 15 76 Ϯ 7 53 Ϯ 7

Discussion

72 Ϯ 4 200 Ϯ 10