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, beta-actin. This identification was confirmed by the pull-down assay with anti-beta-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous beta-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 beta-actin. These results indicate that beta-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.
authors.affiliation: Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ishii
authors.forename: Miki
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Miyoshi
authors.forename: Mayuko
authors.initials: M
authors.lastname: Yasunaga
authors.forename: Mitsugu
authors.initials: M
authors.lastname: Akagawa
authors.forename: Koji
authors.initials: K
authors.lastname: Uchida
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Nakamura
mesh_terms: D015102:3,4-Dihydroxyphenylacetic Acid / Q000737:chemistry / Q000633:toxicity; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002396:Catechols / Q000737:chemistry*; D002460:Cell Line; D005419:Flavonoids / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D010636:Phenols / Q000737:chemistry* / Q000633:toxicity; D059808:Polyphenols; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000199:Actins; D002396:Catechols; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; C486610:KEAP1 protein, rat; D000072019:Kelch-Like ECH-Associated Protein 1; D010455:Peptides; D010636:Phenols; D059808:Polyphenols; D011506:Proteins; D013438:Sulfhydryl Compounds; D015102:3,4-Dihydroxyphenylacetic Acid; D005978:Glutathione