Nudix hydrolases degrade protein-conjugated ADP-ribose

Casey M. Daniels, Puchong Thirawatananond, Shao En Ong, Sandra B Gabelli, Anthony Kar Lun Leung

Research output: Contribution to journalArticle

Abstract

ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR.

Original languageEnglish (US)
Article number18271
JournalScientific Reports
Volume5
DOIs
StatePublished - Dec 16 2015

Fingerprint

Adenosine Diphosphate
Proteins
Crotalus
Proteomics
Venoms
Enzymes
nudix hydrolases
NAD
Mass Spectrometry
Peptide Hydrolases

ASJC Scopus subject areas

  • General

Cite this

Nudix hydrolases degrade protein-conjugated ADP-ribose. / Daniels, Casey M.; Thirawatananond, Puchong; Ong, Shao En; Gabelli, Sandra B; Leung, Anthony Kar Lun.

In: Scientific Reports, Vol. 5, 18271, 16.12.2015.

Research output: Contribution to journalArticle

Daniels, Casey M. ; Thirawatananond, Puchong ; Ong, Shao En ; Gabelli, Sandra B ; Leung, Anthony Kar Lun. / Nudix hydrolases degrade protein-conjugated ADP-ribose. In: Scientific Reports. 2015 ; Vol. 5.
@article{87e2b2daab954ee5900bda6ca735d92d,
title = "Nudix hydrolases degrade protein-conjugated ADP-ribose",
abstract = "ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR.",
author = "Daniels, {Casey M.} and Puchong Thirawatananond and Ong, {Shao En} and Gabelli, {Sandra B} and Leung, {Anthony Kar Lun}",
year = "2015",
month = "12",
day = "16",
doi = "10.1038/srep18271",
language = "English (US)",
volume = "5",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",

}

TY - JOUR

T1 - Nudix hydrolases degrade protein-conjugated ADP-ribose

AU - Daniels, Casey M.

AU - Thirawatananond, Puchong

AU - Ong, Shao En

AU - Gabelli, Sandra B

AU - Leung, Anthony Kar Lun

PY - 2015/12/16

Y1 - 2015/12/16

N2 - ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR.

AB - ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR.

UR - http://www.scopus.com/inward/record.url?scp=84950246615&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84950246615&partnerID=8YFLogxK

U2 - 10.1038/srep18271

DO - 10.1038/srep18271

M3 - Article

C2 - 26669448

AN - SCOPUS:84950246615

VL - 5

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 18271

ER -