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. Colaco; Donald S. Backos; Peter Fristrup; Matthew D. Hirschey; Christian A. Olsen

doi:10.1074/jbc.m115.668699

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. Colaco, Donald S. Backos, Peter Fristrup, Matthew D. Hirschey, Christian A. Olsen

49 Citationer (Scopus)

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 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.

Originalsprog	Engelsk
Tidsskrift	Journal of Biological Chemistry
Vol/bind	291
Udgave nummer	13
Sider (fra-til)	7128-7141
Antal sider	14
ISSN	0021-9258
DOI	https://doi.org/10.1074/jbc.m115.668699
Status	Udgivet - 25 mar. 2016

Adgang til dokumentet

10.1074/jbc.m115.668699

http://www.jbc.org/content/291/13/7128.full.pdfLicens: CC BY

Citationsformater

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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 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. ",

keywords = "acetylation, fatty acid, histone deacetylase (HDAC), nicotinamide adenine dinucleotide (NADH), protein acylation, sirtuin, lysine myristoylation, lysine palmitoylation, nicotinamide adenine dinucleotide (NAD plus ), post-translational modification (PTM)",

author = "Madsen, {Andreas S.} and Christian Andersen and Mohammad Daoud and Anderson, {Kristin A.} and Laursen, {Jonas S.} and Saswati Chakladar and Huynh, {Frank K.} and Colaco, {Ana R.} and Backos, {Donald S.} and Peter Fristrup and Hirschey, {Matthew D.} and Olsen, {Christian A.}",

year = "2016",

month = mar,

day = "25",

doi = "10.1074/jbc.m115.668699",

language = "English",

volume = "291",

pages = "7128--7141",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

number = "13",

}

TY - JOUR

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

AU - Madsen, Andreas S.

AU - Andersen, Christian

AU - Daoud, Mohammad

AU - Anderson, Kristin A.

AU - Laursen, Jonas S.

AU - Chakladar, Saswati

AU - Huynh, Frank K.

AU - Colaco, Ana R.

AU - Backos, Donald S.

AU - Fristrup, Peter

AU - Hirschey, Matthew D.

AU - Olsen, Christian A.

PY - 2016/3/25

Y1 - 2016/3/25

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 - nicotinamide adenine dinucleotide (NADH)

KW - protein acylation

KW - sirtuin

KW - lysine myristoylation

KW - lysine palmitoylation

KW - nicotinamide adenine dinucleotide (NAD plus )

KW - post-translational modification (PTM)

U2 - 10.1074/jbc.m115.668699

DO - 10.1074/jbc.m115.668699

M3 - Journal article

C2 - 26861872

SN - 0021-9258

VL - 291

SP - 7128

EP - 7141

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 13

ER -