A comprehensive screening system for damaged nucleotide-binding proteins

Daisuke Tsuchimoto, Teruaki Iyama, Mari Nonaka, Nona Abolhassani, Eiko Ohta, Sakumi Kunihiko, Yusaku Nakabeppu

Research output: Contribution to journalReview article

8 Citations (Scopus)

Abstract

To identify novel nucleotide pool sanitizing enzymes, we have established a comprehensive screening system for damaged nucleotide-binding proteins based on proteomics technology. In the screening system, affinity chromatography with resins carrying various damaged nucleotides is used for the purification of binding proteins, and the purified proteins are identified by mass-spectrometry. Inosine triphosphate (ITP) is a deleterious damaged nucleotide, and can be generated by nitrosative deamination of ATP or phosphorylation of inosine monophosphate (IMP). Using the above system, we performed screens for ITP-binding proteins from mouse and human cell extracts, and identified several ITP-binding enzymes. We identified both mouse inosine triphosphatase (ITPA) and human ITPA, well-known ITP hydrolyzing enzymes, as ITP-binding proteins. These results support the validity of this screening system. In addition to ITPA, we identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an ITP-binding protein. Biochemical analysis revealed that NUDT16 selectively hydrolyzes deoxyinosine diphosphate (dIDP) and IDP to deoxyinosine monophosphate (dIMP) and IMP, respectively. dITP and ITP are also hydrolyzed by NUDT16 to a lesser extent. The knockdown of NUDT16 in HeLa MR cells suppressed cell proliferation, and was accompanied by a significantly increased accumulation of strand breaks in nuclear DNA, suggesting that NUDT16 has an essential role in the maintenance of genome stability. RS21-C6, another ITP-binding protein identified in our screen, binds not only to ITP, but also to ATP. RS21-C6 hydrolyzes dCTP and 5-halo-dCTP, but does not hydrolyze ITP or ATP. It is likely that RS21-C6 may control dCTP levels or eliminate 5-halo-dCTP in the nucleotide pools. In conclusion, the results of these studies show that our screening system is applicable in studying the health effects of damaged nucleotides and cellular sanitizing systems for nucleotide pools.

Original languageEnglish
Pages (from-to)37-42
Number of pages6
JournalMutation Research - Genetic Toxicology and Environmental Mutagenesis
Volume703
Issue number1
DOIs
Publication statusPublished - Nov 28 2010

Fingerprint

Inosine Triphosphate
Carrier Proteins
Nucleotides
Inosine Monophosphate
Adenosine Triphosphate
Enzymes
Inosine Diphosphate
Deamination
Diphosphates
Genomic Instability
Cell Extracts
Affinity Chromatography
HeLa Cells
Nucleosides
Reproducibility of Results
Proteomics
Mass Spectrometry
Proteins
Maintenance
Phosphorylation

All Science Journal Classification (ASJC) codes

  • Genetics
  • Health, Toxicology and Mutagenesis

Cite this

A comprehensive screening system for damaged nucleotide-binding proteins. / Tsuchimoto, Daisuke; Iyama, Teruaki; Nonaka, Mari; Abolhassani, Nona; Ohta, Eiko; Kunihiko, Sakumi; Nakabeppu, Yusaku.

In: Mutation Research - Genetic Toxicology and Environmental Mutagenesis, Vol. 703, No. 1, 28.11.2010, p. 37-42.

Research output: Contribution to journalReview article

@article{652505135fbf4860ad5191d6f11f3e95,
title = "A comprehensive screening system for damaged nucleotide-binding proteins",
abstract = "To identify novel nucleotide pool sanitizing enzymes, we have established a comprehensive screening system for damaged nucleotide-binding proteins based on proteomics technology. In the screening system, affinity chromatography with resins carrying various damaged nucleotides is used for the purification of binding proteins, and the purified proteins are identified by mass-spectrometry. Inosine triphosphate (ITP) is a deleterious damaged nucleotide, and can be generated by nitrosative deamination of ATP or phosphorylation of inosine monophosphate (IMP). Using the above system, we performed screens for ITP-binding proteins from mouse and human cell extracts, and identified several ITP-binding enzymes. We identified both mouse inosine triphosphatase (ITPA) and human ITPA, well-known ITP hydrolyzing enzymes, as ITP-binding proteins. These results support the validity of this screening system. In addition to ITPA, we identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an ITP-binding protein. Biochemical analysis revealed that NUDT16 selectively hydrolyzes deoxyinosine diphosphate (dIDP) and IDP to deoxyinosine monophosphate (dIMP) and IMP, respectively. dITP and ITP are also hydrolyzed by NUDT16 to a lesser extent. The knockdown of NUDT16 in HeLa MR cells suppressed cell proliferation, and was accompanied by a significantly increased accumulation of strand breaks in nuclear DNA, suggesting that NUDT16 has an essential role in the maintenance of genome stability. RS21-C6, another ITP-binding protein identified in our screen, binds not only to ITP, but also to ATP. RS21-C6 hydrolyzes dCTP and 5-halo-dCTP, but does not hydrolyze ITP or ATP. It is likely that RS21-C6 may control dCTP levels or eliminate 5-halo-dCTP in the nucleotide pools. In conclusion, the results of these studies show that our screening system is applicable in studying the health effects of damaged nucleotides and cellular sanitizing systems for nucleotide pools.",
author = "Daisuke Tsuchimoto and Teruaki Iyama and Mari Nonaka and Nona Abolhassani and Eiko Ohta and Sakumi Kunihiko and Yusaku Nakabeppu",
year = "2010",
month = "11",
day = "28",
doi = "10.1016/j.mrgentox.2010.06.005",
language = "English",
volume = "703",
pages = "37--42",
journal = "Mutation Research - Genetic Toxicology and Environmental Mutagenesis",
issn = "1383-5718",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - A comprehensive screening system for damaged nucleotide-binding proteins

AU - Tsuchimoto, Daisuke

AU - Iyama, Teruaki

AU - Nonaka, Mari

AU - Abolhassani, Nona

AU - Ohta, Eiko

AU - Kunihiko, Sakumi

AU - Nakabeppu, Yusaku

PY - 2010/11/28

Y1 - 2010/11/28

N2 - To identify novel nucleotide pool sanitizing enzymes, we have established a comprehensive screening system for damaged nucleotide-binding proteins based on proteomics technology. In the screening system, affinity chromatography with resins carrying various damaged nucleotides is used for the purification of binding proteins, and the purified proteins are identified by mass-spectrometry. Inosine triphosphate (ITP) is a deleterious damaged nucleotide, and can be generated by nitrosative deamination of ATP or phosphorylation of inosine monophosphate (IMP). Using the above system, we performed screens for ITP-binding proteins from mouse and human cell extracts, and identified several ITP-binding enzymes. We identified both mouse inosine triphosphatase (ITPA) and human ITPA, well-known ITP hydrolyzing enzymes, as ITP-binding proteins. These results support the validity of this screening system. In addition to ITPA, we identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an ITP-binding protein. Biochemical analysis revealed that NUDT16 selectively hydrolyzes deoxyinosine diphosphate (dIDP) and IDP to deoxyinosine monophosphate (dIMP) and IMP, respectively. dITP and ITP are also hydrolyzed by NUDT16 to a lesser extent. The knockdown of NUDT16 in HeLa MR cells suppressed cell proliferation, and was accompanied by a significantly increased accumulation of strand breaks in nuclear DNA, suggesting that NUDT16 has an essential role in the maintenance of genome stability. RS21-C6, another ITP-binding protein identified in our screen, binds not only to ITP, but also to ATP. RS21-C6 hydrolyzes dCTP and 5-halo-dCTP, but does not hydrolyze ITP or ATP. It is likely that RS21-C6 may control dCTP levels or eliminate 5-halo-dCTP in the nucleotide pools. In conclusion, the results of these studies show that our screening system is applicable in studying the health effects of damaged nucleotides and cellular sanitizing systems for nucleotide pools.

AB - To identify novel nucleotide pool sanitizing enzymes, we have established a comprehensive screening system for damaged nucleotide-binding proteins based on proteomics technology. In the screening system, affinity chromatography with resins carrying various damaged nucleotides is used for the purification of binding proteins, and the purified proteins are identified by mass-spectrometry. Inosine triphosphate (ITP) is a deleterious damaged nucleotide, and can be generated by nitrosative deamination of ATP or phosphorylation of inosine monophosphate (IMP). Using the above system, we performed screens for ITP-binding proteins from mouse and human cell extracts, and identified several ITP-binding enzymes. We identified both mouse inosine triphosphatase (ITPA) and human ITPA, well-known ITP hydrolyzing enzymes, as ITP-binding proteins. These results support the validity of this screening system. In addition to ITPA, we identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an ITP-binding protein. Biochemical analysis revealed that NUDT16 selectively hydrolyzes deoxyinosine diphosphate (dIDP) and IDP to deoxyinosine monophosphate (dIMP) and IMP, respectively. dITP and ITP are also hydrolyzed by NUDT16 to a lesser extent. The knockdown of NUDT16 in HeLa MR cells suppressed cell proliferation, and was accompanied by a significantly increased accumulation of strand breaks in nuclear DNA, suggesting that NUDT16 has an essential role in the maintenance of genome stability. RS21-C6, another ITP-binding protein identified in our screen, binds not only to ITP, but also to ATP. RS21-C6 hydrolyzes dCTP and 5-halo-dCTP, but does not hydrolyze ITP or ATP. It is likely that RS21-C6 may control dCTP levels or eliminate 5-halo-dCTP in the nucleotide pools. In conclusion, the results of these studies show that our screening system is applicable in studying the health effects of damaged nucleotides and cellular sanitizing systems for nucleotide pools.

UR - http://www.scopus.com/inward/record.url?scp=78549286641&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78549286641&partnerID=8YFLogxK

U2 - 10.1016/j.mrgentox.2010.06.005

DO - 10.1016/j.mrgentox.2010.06.005

M3 - Review article

C2 - 20542141

AN - SCOPUS:78549286641

VL - 703

SP - 37

EP - 42

JO - Mutation Research - Genetic Toxicology and Environmental Mutagenesis

JF - Mutation Research - Genetic Toxicology and Environmental Mutagenesis

SN - 1383-5718

IS - 1

ER -