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

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.

Keywords: acetylation; fatty acid; histone deacetylase (HDAC); lysine myristoylation; lysine palmitoylation; nicotinamide adenine dinucleotide (NAD+); nicotinamide adenine dinucleotide (NADH); post-translational modification (PTM); protein acylation; sirtuin.

FIGURE 1.

ϵ-N-Acyllysine posttranslational modifications and synthesized sirtuin substrates. A, ϵ-N-Acyllysine posttranslational modifications from the literature. B, substrates prepared for this study. H3, core histone H3; H4, core histone H4; p53, tumor suppressor p53. Khex, ϵ-N-hexanoyllysine; Koct, ϵ-N-octanoyllysine; Kdec, ϵ-N-decanoyllysine; Klau, ϵ-N-lauroyllysine; Kpal, ϵ-N-palmitoyllysine; Ktfa, ϵ-N-trifluoroacetyllysine.

FIGURE 2.

Sirtuin deacylase activities. A, deacylation data obtained as end point readings of released fluorophore ([AMC]_final) after a 1-h incubation with each substrate at 37 °C (SIRT: 200–400 nm enzyme, 50 μm substrate, 500 μm NAD⁺; HDAC: 10 nm enzyme, 10 μm substrate), followed by development for 90 min at 25 °C (2.5 mg/ml trypsin and 2 mm nicotinamide for the sirtuins). Sirtuin deacylation data are represented as mean from two individual experiments performed in duplicate, and the values were normalized to control wells without enzyme added. B, HPLC traces (A₂₈₀) of the hydrolysis of Kac and ϵ-N-myristoyllysine (Kmyr) by SIRT1–3 (40–600 nm enzyme, 100 μm substrate (5b and 5i), 500 μm NAD⁺). *, unidentified impurity. C, [³²P]NAD⁺ consumption assay using octapeptides 4b, 4i, 4j, and 4n. Incubation with SIRT1–3 and SIRT6 in the presence of ³²P-labeled NAD⁺ followed by separation by thin layer chromatography (TLC) and visualization by autoradiography. Kfor, ϵ-N-formyllysine; Kpro, ϵ-N-propionyllysine; Kbut, ϵ-N-butyryllysine; Khex, ϵ-N-hexanoyllysine; Koct, ϵ-N-octanoyllysine; Kdec, ϵ-N-decanoyllysine; Klau, ϵ-N-lauroyllysine; Kpal, ϵ-N-palmitoyllysine; Kcr, ϵ-N-crotonyllysine; Klip, ϵ-N-lipoyllysine; Kbio, ϵ-N-biotinoyllysine; Ktfa, ϵ-N-trifluoroacetyllysine.

FIGURE 3.

HPLC-MS data of SIRT3-mediated deacylation. UV (A₂₈₀) and TIC (ES⁺) chromatograms and mass spectra at relevant time points allowing identification of acylated peptide substrates (5b and 5i) and deacylated peptide product (5n). Kmyr, ϵ-N-myristoyllysine.

FIGURE 4.

NADH inhibition of sirtuin activity. A, absorbance spectra of AMC, NAD⁺, and NADH as well as fluorescence spectra of AMC, NADH, and a combined AMC-NADH sample. The sum of the individual AMC and NADH signals is also included. B, HPLC traces (A₂₈₀) of the hydrolysis of Kac or ϵ-N-myristoyllysine (Kmyr) by SIRT1 and SIRT2 and ϵ-N-succinyllysine (Ksuc) or ϵ-N-glutaryllysine (Kglu) by SIRT5 (100 μm peptide substrate, 500 μm NAD⁺) at different NADH concentrations. C, fractional conversion for the hydrolysis of ϵ-N-acetyllysine, ϵ-N-myristoyllysine, ϵ-N-succinyllysine, and ϵ-N-glutaryllysine by SIRT1–3, SIRT5, and SIRT6 (100 μm peptide substrate, 500 μm NAD⁺), based on area under the relevant peaks.

FIGURE 5.

Concentration response of NADH, ADPR, and NAM. Residual activity of SIRT1–3 and SIRT6-mediated hydrolysis of Kac (LGKac), ϵ-N-octanoyllysine (TARKoct), and ϵ-N-myristoyllysine (TARKmyr) (300 μm peptide substrate (3b, 1f, and 1i), 1000 μm NAD⁺) as well as SIRT5-mediated hydrolysis of ϵ-N-succinyllysine (LGKsuc) and ϵ-N-glutaryllysine (LGKglu) (100 μm peptide substrate (3p and 3q), 500 μm NAD⁺) at different NADH, ADPR, and NAM concentrations. Conversions are based on relative areas under the relevant peaks in the UV chromatograms.

FIGURE 6.

Steady-state rate inhibition experiments. A, Michaelis-Menten and Lineweaver-Burk plots of NADH inhibition of SIRT1-mediated deacetylation using substrate 3b (LGKac) at varying substrate or NAD⁺ concentrations. B, Michaelis-Menten and Lineweaver-Burk plots of NADH inhibition of SIRT5-mediated desuccinylation of substrate 3p (LGKsuc) at varying substrate or NAD⁺ concentrations.

FIGURE 7.

Molecular modeling structures. A, the SIRT1 and SIRT3 active sites were analyzed for their hydrophobicity to determine the potential for binding long-chain fatty acyl groups. Structures shown are crystal structures of SIRT1 (Protein Data Bank entry 4KXQ) and model structures for active site complexes of SIRT3·Kac and SIRT3·ϵ-N-myristoyllysine (Kmyr) (modified from 3GLR). Conformations shown are the most energetically favorable. Gray areas indicate the position of a slice through the vertical z-plane. B, Conformations obtained after the production run of SIRT1 and SIRT3 in complex with ϵ-N-acetylated peptide substrate and NAD⁺ or NADH. Substrates are shown with carbon atoms in beige, and non-polar hydrogen atoms have been omitted from the peptide substrate. Isoleucine and aspartate residues forming hydrogen bonds with NAD⁺ and A, B, and C pockets for NAD⁺ binding have been labeled.