title: 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.
authors: From the Center for Biopharmaceuticals, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark, the Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark, andreas.madsen@sund.ku.dk.
authors: Andreas S
authors: AS
authors: Madsen
authors: the Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
authors: Christian
authors: C
authors: Andersen
authors: the Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
authors: Mohammad
authors: M
authors: Daoud
authors: the Duke Molecular Physiology Institute, Duke University Medical Center, Durham, North Carolina 27701, and.
authors: Kristin A
authors: KA
authors: Anderson
authors: From the Center for Biopharmaceuticals, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors: Jonas S
authors: JS
authors: Laursen
authors: From the Center for Biopharmaceuticals, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors: Saswati
authors: S
authors: Chakladar
authors: the Duke Molecular Physiology Institute, Duke University Medical Center, Durham, North Carolina 27701, and.
authors: Frank K
authors: FK
authors: Huynh
authors: the Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
authors: Ana R
authors: AR
authors: Colaço
authors: the Computational Chemistry and Biology Core Facility, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors: Donald S
authors: DS
authors: Backos
authors: the Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
authors: Peter
authors: P
authors: Fristrup
authors: the Duke Molecular Physiology Institute, Duke University Medical Center, Durham, North Carolina 27701, and matthew.hirschey@duke.edu.
authors: Matthew D
authors: MD
authors: Hirschey
authors: From the Center for Biopharmaceuticals, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark, the Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark, cao@sund.ku.dk.
authors: Christian A
authors: CA
authors: Olsen
mesh_terms: D000215:Acylation; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006655:Histone Deacetylases / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives*; D013058:Mass Spectrometry; D056004:Molecular Dynamics Simulation; D009243:NAD / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry; D037761:Sirtuins / Q000737:chemistry*; D012996:Solutions
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D007527:Isoenzymes; D009842:Oligopeptides; D011994:Recombinant Proteins; D012996:Solutions; D009243:NAD; D037761:Sirtuins; D006655:Histone Deacetylases; D008239:Lysine
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