Catechol Type Polyphenol Is a Potential Modifier of Protein Sulfhydryls: Development and Application of a New Probe for Understanding the Dietary Polyphenol Actions
Catechol Type Polyphenol Is a Potential Modifier of Protein Sulfhydryls: Development and Application of a New Probe for Understanding the Dietary Polyphenol Actions
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DOI：
10.1021/tx900148k
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发表时间：
2009-10-01
影响因子：
4.1
通讯作者：
Nakamura, Yoshimasa
中科院分区：
文献类型：
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作者：
Ishii, Takeshi;Ishikawa, Miki;Nakamura, Yoshimasa
具有儿茶酚结构的膳食多酚的氧化导致形成邻醌结构，其快速与巯基如谷胱甘肽和蛋白质半胱氨酸残基反应。这种修饰对于理解多酚对细胞功能的氧化还原调节可能是重要的。在这项研究中，研究的邻苯二酚修饰的蛋白质sulthydryls，我们使用3，4-二羟基苯基乙酸（DPA）作为模型儿茶酚化合物，并开发了一种新的探针，直接检测蛋白质修饰的儿茶酚型多酚使用生物素化的DPA（Bio-DPA）。DPA与肽巯基的氧化依赖性亲电反应性通过质谱法和核磁共振光谱法证实。当用Bio-DPA处理RL 34细胞时，通过Western印迹分析观察到Bio-DPA显著掺入到40 kDa蛋白中。质谱鉴定该条带为细胞骨架蛋白β-肌动蛋白。该鉴定通过使用抗β-肌动蛋白抗体的下拉测定来证实。为了检测儿茶酚型多酚如类黄酮对内源性β-肌动蛋白的反应性，将RL 34细胞共暴露于Bio-DPA和类黄酮槲皮素、（-）-表儿茶素和（-）-表儿茶素没食子酸酯。在类黄酮存在下将细胞暴露于Bio-DPA后，我们观察到DPA修饰的β-肌动蛋白显著减少。这些结果表明，β-肌动蛋白是儿茶酚型多酚修饰蛋白质的主要靶点之一，Bio-DPA是了解膳食多酚氧化还原调节的有用探针。此外，Keap 1，一个骨架蛋白的肌动蛋白细胞骨架控制细胞保护酶基因，也被确定为另一个合理的目标，儿茶酚型多酚的氧化修饰的细胞内巯基。这些结果提供了一种替代方法来理解儿茶酚型多酚是通过巯基修饰的氧化还原依赖性细胞事件的潜在修饰剂。
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 sulthydryls, 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.