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

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

Research output: Contribution to journalJournal articleResearchpeer-review

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 NADas 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 sirtuins in 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 sirtuins in vivo. These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD+/NADH ratio.
Original languageEnglish
JournalJournal of Biological Chemistry
Volume291
Issue number13
Pages (from-to)7128-7141
Number of pages14
ISSN0021-9258
DOIs
Publication statusPublished - 2016

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

Cite this

Madsen, A. S., Andersen, C., Daoud, M. M., Anderson, K. A., Laursen, J. S., Chakladar, S., ... Olsen, C. A. (2016). Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH. Journal of Biological Chemistry, 291(13), 7128-7141. https://doi.org/10.1074/jbc.M115.668699
Madsen, Andreas Stahl ; Andersen, Christian ; Daoud, Mohammad Mahdi ; Anderson, Kristin A. ; Laursen, Jonas S. ; Chakladar, Saswati ; Huynh, Frank K. ; Colaço, Ana Rita Freitas ; Backos, Donald S ; Fristrup, Peter ; Hirschey, Matthew D. ; Olsen, Christian Adam. / Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 13. pp. 7128-7141.
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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",
author = "Madsen, {Andreas Stahl} and Christian Andersen and Daoud, {Mohammad Mahdi} and Anderson, {Kristin A.} and Laursen, {Jonas S.} and Saswati Chakladar and Huynh, {Frank K.} and Cola{\cc}o, {Ana Rita Freitas} and Backos, {Donald S} and Peter Fristrup and Hirschey, {Matthew D.} and Olsen, {Christian Adam}",
year = "2016",
doi = "10.1074/jbc.M115.668699",
language = "English",
volume = "291",
pages = "7128--7141",
journal = "Journal of Biological Chemistry",
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publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
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Madsen, AS, Andersen, C, Daoud, MM, Anderson, KA, Laursen, JS, Chakladar, S, Huynh, FK, Colaço, ARF, Backos, DS, Fristrup, P, Hirschey, MD & Olsen, CA 2016, 'Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH', Journal of Biological Chemistry, vol. 291, no. 13, pp. 7128-7141. https://doi.org/10.1074/jbc.M115.668699

Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH. / Madsen, Andreas Stahl; Andersen, Christian; Daoud, Mohammad Mahdi; Anderson, Kristin A.; Laursen, Jonas S.; Chakladar, Saswati; Huynh, Frank K.; Colaço, Ana Rita Freitas; Backos, Donald S; Fristrup, Peter; Hirschey, Matthew D.; Olsen, Christian Adam.

In: Journal of Biological Chemistry, Vol. 291, No. 13, 2016, p. 7128-7141.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH

AU - Madsen, Andreas Stahl

AU - Andersen, Christian

AU - Daoud, Mohammad Mahdi

AU - Anderson, Kristin A.

AU - Laursen, Jonas S.

AU - Chakladar, Saswati

AU - Huynh, Frank K.

AU - Colaço, Ana Rita Freitas

AU - Backos, Donald S

AU - Fristrup, Peter

AU - Hirschey, Matthew D.

AU - Olsen, Christian Adam

PY - 2016

Y1 - 2016

N2 - 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 sirtuins in 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 sirtuins in vivo. These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD+/NADH ratio.

AB - 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 sirtuins in 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 sirtuins in vivo. These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD+/NADH ratio.

KW - Acetylation

KW - Fatty acid

KW - Histone deacetylase (HDAC)

KW - Lysine myristoylation

KW - Lysine palmitoylation

KW - Nicotinamide adenine dinucleotide (NAD+)

KW - Nicotinamide adenine dinucleotide (NADH)

KW - Post-translational modification (PTM)

KW - Protein acylation

KW - Sirtuin

U2 - 10.1074/jbc.M115.668699

DO - 10.1074/jbc.M115.668699

M3 - Journal article

VL - 291

SP - 7128

EP - 7141

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 13

ER -