Catechol type polyphenol is a potential modifier of protein sulfhydryls: Development and application of a new probe for understanding the dietary polyphenol actions

Takeshi Ishii, Miki Ishikawa, Noriyuki Miyoshi, Mayuko Yasunaga, Mitsugu Akagawa, Koji Uchida, Yoshimasa Nakamura

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The oxidation of dietary polyphenols with a catechol structure leads to the formation of an o-quinone structure, which rapidly reacts with sulfhydryls such as glutathione and protein cysteine residues. This modification may be important for understanding the redox regulation of cell functions by polyphenols. In this study, to investigate the catechol modification of protein sulfhydryls, we used 3,4-dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect protein modification by catechol type polyphenols using a biotinylated DPA (Bio-DPA). The oxidation-dependent electrophilic reactivity of DPA with peptide sulfhydryls was confirmed by both mass spectrometry and nuclear magnetic resonance spectroscopy. When RL34 cells were treated with Bio-DPA, the significant incorporation of Bio-DPA into a 40 kDa protein was observed by Western blot analysis. The band was identified by mass spectrometry as the cytoskeletal protein, β-actin. This identification was confirmed by the pull-down assay with anti-β-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous β-actin, RL34 cells were coexposed to Bio-DPA and the flavonoids quercetin, (-)-epicatechin, and (-)-epicatechin gallate. Upon exposure of the cells to Bio-DPA in the presence of the flavonoids, we observed a significant decrease in the DPA-modified β-actin. These results indicate that β-actin is one of the major targets of protein modification by catechol type polyphenols and that Bio-DPA is an useful probe for understanding the redox regulation by dietary polyphenols. Furthermore, Keap1, a scaffold protein to the actin cytoskeleton controlling cytoprotective enzyme genes, was also identified as another plausible target of the catechol type polyphenols by oxidative modification of the intracellular sulfhydryls. These results provide an alternative approach to understand that catechol type polyphenol is a potential modifier of redox-dependent cellular events through sulfhydryl modification.

Original languageEnglish
Pages (from-to)1689-1698
Number of pages10
JournalChemical Research in Toxicology
Volume22
Issue number10
DOIs
Publication statusPublished - Oct 19 2009

Fingerprint

Polyphenols
Actins
Acetic Acid
Proteins
Flavonoids
Oxidation-Reduction
Mass spectrometry
Mass Spectrometry
Oxidation
Cytoskeletal Proteins
catechol
Catechin
Quercetin
Actin Cytoskeleton
Scaffolds
Nuclear magnetic resonance spectroscopy
Glutathione
Cysteine
Anti-Idiotypic Antibodies
Assays

ASJC Scopus subject areas

  • Toxicology

Cite this

Catechol type polyphenol is a potential modifier of protein sulfhydryls : Development and application of a new probe for understanding the dietary polyphenol actions. / Ishii, Takeshi; Ishikawa, Miki; Miyoshi, Noriyuki; Yasunaga, Mayuko; Akagawa, Mitsugu; Uchida, Koji; Nakamura, Yoshimasa.

In: Chemical Research in Toxicology, Vol. 22, No. 10, 19.10.2009, p. 1689-1698.

Research output: Contribution to journalArticle

Ishii, Takeshi ; Ishikawa, Miki ; Miyoshi, Noriyuki ; Yasunaga, Mayuko ; Akagawa, Mitsugu ; Uchida, Koji ; Nakamura, Yoshimasa. / Catechol type polyphenol is a potential modifier of protein sulfhydryls : Development and application of a new probe for understanding the dietary polyphenol actions. In: Chemical Research in Toxicology. 2009 ; Vol. 22, No. 10. pp. 1689-1698.
@article{a7832604c8184dce8c336f9cd9f37bd7,
title = "Catechol type polyphenol is a potential modifier of protein sulfhydryls: Development and application of a new probe for understanding the dietary polyphenol actions",
abstract = "The oxidation of dietary polyphenols with a catechol structure leads to the formation of an o-quinone structure, which rapidly reacts with sulfhydryls such as glutathione and protein cysteine residues. This modification may be important for understanding the redox regulation of cell functions by polyphenols. In this study, to investigate the catechol modification of protein sulfhydryls, we used 3,4-dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect protein modification by catechol type polyphenols using a biotinylated DPA (Bio-DPA). The oxidation-dependent electrophilic reactivity of DPA with peptide sulfhydryls was confirmed by both mass spectrometry and nuclear magnetic resonance spectroscopy. When RL34 cells were treated with Bio-DPA, the significant incorporation of Bio-DPA into a 40 kDa protein was observed by Western blot analysis. The band was identified by mass spectrometry as the cytoskeletal protein, β-actin. This identification was confirmed by the pull-down assay with anti-β-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous β-actin, RL34 cells were coexposed to Bio-DPA and the flavonoids quercetin, (-)-epicatechin, and (-)-epicatechin gallate. Upon exposure of the cells to Bio-DPA in the presence of the flavonoids, we observed a significant decrease in the DPA-modified β-actin. These results indicate that β-actin is one of the major targets of protein modification by catechol type polyphenols and that Bio-DPA is an useful probe for understanding the redox regulation by dietary polyphenols. Furthermore, Keap1, a scaffold protein to the actin cytoskeleton controlling cytoprotective enzyme genes, was also identified as another plausible target of the catechol type polyphenols by oxidative modification of the intracellular sulfhydryls. These results provide an alternative approach to understand that catechol type polyphenol is a potential modifier of redox-dependent cellular events through sulfhydryl modification.",
author = "Takeshi Ishii and Miki Ishikawa and Noriyuki Miyoshi and Mayuko Yasunaga and Mitsugu Akagawa and Koji Uchida and Yoshimasa Nakamura",
year = "2009",
month = "10",
day = "19",
doi = "10.1021/tx900148k",
language = "English",
volume = "22",
pages = "1689--1698",
journal = "Chemical Research in Toxicology",
issn = "0893-228X",
publisher = "American Chemical Society",
number = "10",

}

TY - JOUR

T1 - Catechol type polyphenol is a potential modifier of protein sulfhydryls

T2 - Development and application of a new probe for understanding the dietary polyphenol actions

AU - Ishii, Takeshi

AU - Ishikawa, Miki

AU - Miyoshi, Noriyuki

AU - Yasunaga, Mayuko

AU - Akagawa, Mitsugu

AU - Uchida, Koji

AU - Nakamura, Yoshimasa

PY - 2009/10/19

Y1 - 2009/10/19

N2 - The oxidation of dietary polyphenols with a catechol structure leads to the formation of an o-quinone structure, which rapidly reacts with sulfhydryls such as glutathione and protein cysteine residues. This modification may be important for understanding the redox regulation of cell functions by polyphenols. In this study, to investigate the catechol modification of protein sulfhydryls, we used 3,4-dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect protein modification by catechol type polyphenols using a biotinylated DPA (Bio-DPA). The oxidation-dependent electrophilic reactivity of DPA with peptide sulfhydryls was confirmed by both mass spectrometry and nuclear magnetic resonance spectroscopy. When RL34 cells were treated with Bio-DPA, the significant incorporation of Bio-DPA into a 40 kDa protein was observed by Western blot analysis. The band was identified by mass spectrometry as the cytoskeletal protein, β-actin. This identification was confirmed by the pull-down assay with anti-β-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous β-actin, RL34 cells were coexposed to Bio-DPA and the flavonoids quercetin, (-)-epicatechin, and (-)-epicatechin gallate. Upon exposure of the cells to Bio-DPA in the presence of the flavonoids, we observed a significant decrease in the DPA-modified β-actin. These results indicate that β-actin is one of the major targets of protein modification by catechol type polyphenols and that Bio-DPA is an useful probe for understanding the redox regulation by dietary polyphenols. Furthermore, Keap1, a scaffold protein to the actin cytoskeleton controlling cytoprotective enzyme genes, was also identified as another plausible target of the catechol type polyphenols by oxidative modification of the intracellular sulfhydryls. These results provide an alternative approach to understand that catechol type polyphenol is a potential modifier of redox-dependent cellular events through sulfhydryl modification.

AB - The oxidation of dietary polyphenols with a catechol structure leads to the formation of an o-quinone structure, which rapidly reacts with sulfhydryls such as glutathione and protein cysteine residues. This modification may be important for understanding the redox regulation of cell functions by polyphenols. In this study, to investigate the catechol modification of protein sulfhydryls, we used 3,4-dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect protein modification by catechol type polyphenols using a biotinylated DPA (Bio-DPA). The oxidation-dependent electrophilic reactivity of DPA with peptide sulfhydryls was confirmed by both mass spectrometry and nuclear magnetic resonance spectroscopy. When RL34 cells were treated with Bio-DPA, the significant incorporation of Bio-DPA into a 40 kDa protein was observed by Western blot analysis. The band was identified by mass spectrometry as the cytoskeletal protein, β-actin. This identification was confirmed by the pull-down assay with anti-β-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous β-actin, RL34 cells were coexposed to Bio-DPA and the flavonoids quercetin, (-)-epicatechin, and (-)-epicatechin gallate. Upon exposure of the cells to Bio-DPA in the presence of the flavonoids, we observed a significant decrease in the DPA-modified β-actin. These results indicate that β-actin is one of the major targets of protein modification by catechol type polyphenols and that Bio-DPA is an useful probe for understanding the redox regulation by dietary polyphenols. Furthermore, Keap1, a scaffold protein to the actin cytoskeleton controlling cytoprotective enzyme genes, was also identified as another plausible target of the catechol type polyphenols by oxidative modification of the intracellular sulfhydryls. These results provide an alternative approach to understand that catechol type polyphenol is a potential modifier of redox-dependent cellular events through sulfhydryl modification.

UR - http://www.scopus.com/inward/record.url?scp=70350237026&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70350237026&partnerID=8YFLogxK

U2 - 10.1021/tx900148k

DO - 10.1021/tx900148k

M3 - Article

C2 - 19743802

AN - SCOPUS:70350237026

VL - 22

SP - 1689

EP - 1698

JO - Chemical Research in Toxicology

JF - Chemical Research in Toxicology

SN - 0893-228X

IS - 10

ER -